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Marine fungal metabolite butyrolactone I prevents cognitive deficits by relieving inflammation and intestinal microbiota imbalance on aluminum trichloride-injured zebrafish

Marine fungal metabolite butyrolactone I prevents cognitive deficits by relieving inflammation... Background: Mounting evidences indicate that oxidative stress, neuroinflammation, and dysregulation of gut microbiota are related to neurodegenerative disorders (NDs). Butyrolactone I (BTL-I), a marine fungal metabolite, was previously reported as an in vitro neuroprotectant and inflammation inhibitor. However, little is known regarding its in vivo effects, whereas zebrafish (Danio rerio) could be used as a convenient in vivo model of toxicology and central nervous system (CNS) diseases. Methods: Here, we employed in vivo and in silico methods to investigate the anti-NDs potential of BTL-I. Specifi- cally, we established a cognitive deficit model in zebrafish by intraperitoneal (i.p.) injection of aluminum trichloride (AlCl ) (21 μg) and assessed their behaviors in the T-maze test. The proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α ( TNF-α) as well as acetylcholinesterase (AChE) activity or glutathione (GSH) levels were assayed 24 h after AlCl injection. The intestinal flora variation of the zebrafish was investigated by 16S rDNA high- throughput analysis. The marine fungal metabolite, butyrolactone I (BTL-I), was used to modulate zebrafish cognitive deficits evoked by AlCl and evaluated about its effects on the above inflammatory, cholinergic, oxidative stress, and gut floral indicators. Furthermore, the absorption, distribution, metabolism, excretion, and toxicity (ADMET ) and drug- likeness properties of BTL-I were studied by the in silico tool ADMETlab. Results: BTL-I dose-dependently ameliorated AlCl -induced cognitive deficits in zebrafish. While AlCl treatment 3 3 elevated the levels of central and peripheral proinflammatory cytokines, increased AChE activity, and lowered GSH in the brains of zebrafish, these effects, except GSH reduction, were reversed by 25–100 mg/kg BTL-I administra- tion. Besides, 16S rDNA high-throughput sequencing of the intestinal flora of zebrafish showed that AlCl decreased *Correspondence: hubeizhangyi@163.com College of Food Science and Technology, Guangdong Provincial Key Laboratory of Aquatic Product Processing and Safety, Guangdong Provincial Engineering Laboratory for Marine Biological Products, Research Institute for Marine Drugs and Nutrition, Shenzhen Institute of Guangdong Ocean University, Guangdong Ocean University, Zhanjiang 524088, China Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Nie et al. Journal of Neuroinflammation (2022) 19:39 Page 2 of 17 Gram-positive bacteria and increased proinflammatory Gram-negative bacteria, while BTL-I contributed to maintain- ing the predominance of beneficial Gram-positive bacteria. Moreover, the in silico analysis indicated that BTL-I exhibits acceptable drug-likeness and ADMET profiles. Conclusions: The present findings suggest that BTL-I is a potential therapeutic agent for preventing CNS deficits caused by inflammation, neurotoxicity, and gut flora imbalance. Keywords: Inflammation, Oxidative stress, Butyrolactone I, Neurodegenerative diseases, Acetylcholinesterase, Intestinal flora Background anti-AD drugs targeting orphan targets, such as Aβ and Neurodegenerative disorders (NDs), such as Alzheimer’s Tau, may be substantially related to malignant ampli- disease (AD) and Parkinson’s disease (PD), are chronic, fication induced by neuroinflammation and oxidative progressive, and severely debilitating neurological disor- stress. Thus, inhibiting inflammation and oxidative stress ders [1, 2]. NDs are characterized by cognitive and motor and regulating imbalanced GMB to protect neurons deficits, accompanied by neuronal apoptosis and reduced and intervene in the early stage of disease has become neurotransmission [3, 4]. Oxidative stress and inflamma - an important new strategy in developing novel anti- tion play critical roles in neuronal apoptosis [5–9] and NDs agents [20]. Aryl butyrolactones (BTLs), including may, thus, be considered potential risk factors for NDs. butyrolactone I (BTL-I, Fig.  1 inset), are characteristic Various factors, including peripheral or brain inflamma - natural products of fungi (e.g., Aspergillus sp. and Penicil- tion, β-amyloid peptide (Aβ), pathogenic infection and lium sp.) [21, 22]. Our previous studies have shown that toxins (e.g., aluminum), activate brain microglial cells BTL-I has strong in vitro anti-neuroinflammatory effects, [10–12]. The release of proinflammatory cytokines and inhibiting LPS-induced inflammatory proliferation of reactive oxygen species (ROS) trigger and amplify dam- microglia, the release of inflammatory mediators (NO age to neurons and astrocytes, whereas oxidative stress and IL-1β) and ROS, the expression of the inflammatory and inflammation further promote neuroinflammation, target enzyme cyclooxygenase-2 (COX-2), and intracel- which in turn activates microglia, eventually impairing lular migration of the signaling protein NF-κB p65 [23]. neurons and astrocytes [5, 7, 12–14]. Oxidative stress and Moreover, BTL-I plays a versatile anti-neurodegenerative inflammation are involved in cyclin-dependent kinase 5 role through multiple mechanisms, such as neuronal (CDK 5) activation-induced Tau hyperphosphorylation, nutrition and inhibition of neuronal injury [24–27]. resulting in neurofibrillary tangles, another important However, since previous BTL-I studies were limited to pathological marker of AD [5]. in vitro studies, its in vivo effects remain to be elucidated In addition, an increasing number of studies have as well as its influence on GMB. shown that intestinal microorganisms are closely related Due to their high genetic and physiological homology to the occurrence of NDs and their mechanism also to humans, zebrafish has long been used as a power - involves inflammation, oxidative stress and neurotrans - ful in  vivo model to assess anti-inflammatory drugs [28, mitters [15, 16]. Researchers revealed that the increase 29] and neuronal injury [30]. Zebrafish possess innate of proinflammatory gut microbiota (GMB) taxon (the and acquired immune systems similar to those of mam- producers of liposaccharides) and reduction of anti- mals [31] display well-characterized learning, memory, inflammatory GMB taxon are possibly associated with addiction and other behaviors that correspond to clinical the peripheral inflammation in patients with impaired phenotypes, and host rich GMB in their intestine [32], cognition and brain amyloidosis. The GMB may influence enabling them an ideal model for our purpose [33–36]. the cognition by changing gut permeability and inflam - AlCl causes AD-like pathology, aggravating neuro- matory-oxidative stress state, producing short-chain inflammation, oxidative stress and AChE activity in the fatty acids and neurotransmitters, promoting or reducing rodent brain [12, 37, 38]. The effect of AlCl on zebrafish beta-amyloid deposition and transfer to brain, etc. [17]. cognition remains unclear [39]. In the present study, we u Th s, GMB is becoming a new target of ND treatment. established a neurotoxic zebrafish model (induced by Notably, current clinical drugs for the prevention and AlCl ) to assess the potential effects of BTL-I on mem - treatment of NDs manifest limited efficacy. For exam - ory and cognitive impairment in vivo. The putative neu - ple, donepezil partially relieves symptoms of AD with- roprotective activity of BTL-I in zebrafish was further out reversing or preventing its progression [5]. The poor investigated by evaluating AChE activity and GSH levels understanding of NDs pathogenesis restricted new drug in the brain and by detecting the levels of the inflam - development [18, 19]. The repeated failure to develop matory cytokines IL-1β and TNF-α in both central and N ie et al. Journal of Neuroinflammation (2022) 19:39 Page 3 of 17 Fig. 1 A general diagram summarizing the experimental design used in the present study. Inset: the chemical structure of BTL-I peripheral tissues. The 16S rDNA high-throughput Briefly, following a 24-h fasting period, the fish were method was used to determine the structure and changes anesthetized and then received an i.p. injection. For of zebrafish intestinal flora. The in silico tool ADMETlab this purpose, eugenol was dissolved in 100  mL of anhy- was further applied to evaluate the absorption, distribu- drous ethanol to prepare a 1  mg/mL stock solution that tion, metabolism, excretion, and toxicity (ADMET) and was added to 5 L of water (28 ± 1 °C) and stirred evenly. drug-likeness properties of BTL-I. Zebrafish were then group-exposed to the anesthetic, and after stopping swimming (immobile > 2–3 min), they Methods were quickly injected with AlCl . After the injection, Animals and model development the animals recovered in a water-containing beaker and Adult wild-type AB zebrafish (approximately 6–8 months returned to the holding tanks once their normal swim- old; 50:50% male:female ratio) used in the present study ming resumed [41]. were obtained from a commercial supplier (Shanghai Jiayu Aquarium, Shanghai, China) and acclimatized in T‑maze behavioral testing a 50-L aquarium in the aquatic facility of Guangdong The aquatic T-maze was used for cognitive testing, as Ocean University for at least 2 weeks. The fish were kept described previously, with modifications [42, 43]. The on a 14  h:10  h light:dark cycle (lights on at 7 am) at a maze comprised a long vertical arm (50  cm) and two temperature of 25 ± 2 °C in a recirculating tank system. short horizontal arms (20  cm), with an arm width of The zebrafish were maintained according to standard 10 cm, a depth of 10 cm, and a water depth of 8 cm. The conditions [40] and fed Artemis larvae twice a day at 9 am right arm was connected to a rectangular water tank and 2 pm. (22  cm × 20  cm × 15  cm) with a black outer wall; sand As shown in Fig.  1, to establish an AlCl model, 75 and stones were added to the bottom of the tank, and bait zebrafish (3.0 ± 0.4 cm in length) were randomly divided was set inside the tank to provide an enriched chamber into control and AlCl - and BTL-I-treated groups (n = 15 (EC) (Fig.  1). During the final 4 days of treatment, 6 fish per group). BTL-I (25, 50 or 100 mg/kg/day) was admin- were randomly selected from each group, and the fish istered with food for 20  days. The control and AlCl were individually trained for 5 min daily to locate the EC groups were fed equal amounts of normal food. Twenty zone. If a fish did not enter the EC zone within the 5-min days later, the AlCl and BTL-I groups were anesthetized training session, it was guided into the EC zone and kept and injected (i.p.) with AlCl solution (4.2 mg/mL, 5 μL, there for > 30  s. Following 4  days of training, the trained pH = 5.0 ± 0.2) using a 10-μL gas phase injection needle fish were placed individually one day later into the start - with a 0.5-mm outer diameter. The control group was ing area of the long arm for behavioral testing, with injected with the same amount of saline. Memory testing scoring of the latency time (s) to enter the EC zone and was performed 24 h later. stay there for > 30  s. If a fish did not enter the EC zone Nie et al. Journal of Neuroinflammation (2022) 19:39 Page 4 of 17 Gut flora sequencing and data analysis within the 5-min test, the latency time was recorded as Genomic DNA was extracted by protease K lysis. The 300  s. Behavioral testing was performed between 10 am variable region of the 16S ribosomal RNA gene V3–V4 and 1  pm. A Microsoft LifeCam Studio 1080p HD cam- was amplified by PCR, and the specific primer sequences era was used to record videos with Apowersoft software were as follows: 357F 5′-ACT CCT ACG GRA GGC AGC (Apowersoft Co. Ltd., Hong Kong, China). Supersys soft- AG-3′ and 806R 5′-GGA CTA CHVGGG TWT CTAAT- ware was used for off-line video analyses (Shanghai Xin - 3′. Bidirectional sequencing was performed according ruan Information Technology Co. Ltd., Shanghai, China), to Illumina high-throughput sequencing requirements, assessing the latency of the first entry into the EC zone(s), and the library was constructed by two-step PCR ampli- the average swimming speed (cm/s), and the number of fication. The PCR conditions were 94 °C for 2 min, 94 °C EC entries. for 30 s, 56 °C for 30 s, 72 °C for 30 s (the primary PCR amplification 30 cycles, the secondary PCR amplification 8 cycles), 72 °C for 5 min, and a final extension at 10 °C. Reagents PCR amplification products were recovered by 2% aga - BTL-I was isolated for this study from the marine fun- rose gel electrophoresis. Recycling was performed using gus Aspergillus terreus C23-3 as described previously an AxyPrepDNA gel recovery kit from Axygen. [23]. The BCA protein, GSH and AChE assay kits, fish The PCR-amplified products of the zebrafish gut sam - IL-1β enzyme-linked immunosorbent assay (ELISA) kit ples were sequenced for 16S rDNA using the Illumina- and fish TNF-α ELISA kit were purchased from Nan - Misq high-throughput sequencing platform [TinyGene jing Jiancheng Bioengineering Institute (Nanjing, China). Bio-Tech (ShangHai) Co., Ltd, China], and the sequence Eugenol and AlCl were purchased from Huaxia Reagent length was 450 bp. The raw data obtained from sequenc - (Chengdu, China) and Xiya Reagent (Shandong, China), ing were evaluated for quality and optimized. Trimmo- respectively. matic was used for sequence filtration, and FLASH was used for splicing. Ambiguous, homologous and some chi- meras produced in the PCR process were subsequently Molecular biomarker assays screened using Mothur V.1.39.5 to obtain optimized Twenty-four hours after behavioral testing, the fish were sequences for subsequent operational taxonomic unit euthanized. Considering that the available assay kits (OTU) clustering and species information analysis. could not measure the small tissue samples of individual USEARCH was used to cluster OTUs of the above fish, the 15 fish in each group were randomly divided into treated sequences at 97% similarity. The representative 3 subgroups, and samples of brain, peripheral and intesti- OTU sequences were compared with the database Silva nal tract tissue from each subgroup (5 fish) were collected for species annotation (confidence threshold: 0.6). The and combined immediately and freeze-dried at −  80 °C. relative abundance percentages of each sample were All the samples (except for the gut samples) were homog- calculated at the phylum, class, order, family, genus and enized in phosphate-buffered solution (PBS) for further species levels. The rarefaction curve reflects the sequenc - assays. The supernatant was collected by centrifugation ing depth of the samples. The rank–abundance curve at 252 g at 4 °C for 15 min. Zebrafish brain sample super - explains species abundance and species evenness. natants were used to determine GSH levels and AChE A Venn diagram can be used to count the common and activity. Moreover, zebrafish brain samples and periph - unique OTU numbers of multiple samples, which can eral tissue supernatants were also used to determine the directly show the overlap and uniqueness in the OTU levels of IL-1β and TNF-α [44] following the manufactur- composition of different samples. er’s instructions. The results are expressed as U of AChE/ Alpha-diversity analysis reflected the richness and mg of protein and μmol of GSH/g of protein. Regression diversity of communities in the samples. Mothur (http:// equations for the IL-1β and TNF-α standard curves were w w w. mot h ur . or g/ w ik i/ S c hlo ss_ S OP# Alpha_ diver sity) calculated according to the OD value, and logistic curves was used to calculate the values of the Shannon, Simp- (4 parameters each) were used as the fitting models. son, Chao and ACE indices, and the R (3.4.1) language tool was used for graph plotting. Jaccard, Bray–Curtis, unweighted UniFrac, and Statistical analysis for behavioral and molecular weighted UniFrac were used to calculate the differences biomarkers between samples and conduct nonmetric multidimen- Statistical analysis was performed by one-way analysis sional scaling graphs (NMDS). Metastats (http:// metas of variance (ANOVA) followed by a post hoc Dunnett tats. cbcb. umd. edu/) was used for the comparison of the test. The results are expressed as the mean ± SD. P-val- features with different abundances between groups on ues < 0.05 indicated statistical significance for all tests. N ie et al. Journal of Neuroinflammation (2022) 19:39 Page 5 of 17 Eecfft of  AlCl on proinflammatory cytokines, AChE multiple taxonomic levels. The nonparametric facto - and GSH rial Kruskal–Wallis (KW) sum-rank test was applied to To further elucidate the effect of BTL-I on AlCl -induced determine the significant difference between the richness cognitive impairment in zebrafish, we assessed changes of the groups. LEfSe (LDA effect size) uses linear discri - in the levels of several biochemical indicators (includ- minant analysis (LDA) to estimate the impact of each ing IL-1β, TNF-α, GSH, AChE). The results showed that component (species) abundance on the difference effect AlCl treatment promoted the release of IL-1β from the of the groups. brain and peripheral tissue and TNF-α from the brain, and BTL-I supplementation inhibited the release of IL-1β In silico prediction of ADMET and drug‑likeness properties in the brain and periphery, as well as the release of TNF of BTL‑I in the brain [F (4, 10) = 20.64, P < 0.001 for brain IL-1β To evaluate the pharmacokinetic profile and toxicity of in Fig.  4a; F (4, 10) = 7.240, P < 0.01 for peripheral IL-1β BTL-I, we employed ADMETlab 2.0 (https:// admet mesh. in Fig.  4b; F (4, 10) = 11.75, P < 0.001 for brain TNF-α in scbdd. com/ pub/), which is a free online platform that Fig. 4c]. BTL-I also increased the peripheral TNF-α level enables researchers to predict the ADMET and drug- [F (4, 10) = 14.94, P < 0.001 in Fig. 4d]. likeness properties of a compound [45]. Furthermore, paralleling their cognitive deficits in the T-maze, zebrafish treated with AlCl exhibited higher Results brain AChE activity, whereas both moderate and high BTL‑I improved AlCl ‑induced memory impairment doses of BTL-I dose-dependently inhibited AChE activ- Overall, compared with the model group, the BTL-I- ity [F (4, 10) = 4.474, P < 0.05 in Fig.  4e]. This is consist - treated groups showed a significant treatment effect ent with the fact that excessive AChE activity is closely on cognitive performance in the T-maze test [F (4, related to memory deficits [46]. 25) = 40.60, P < 0.001 for the latency of first entry to the In addition, treatment with AlCl caused an oxidant– EC zone on day 5 in Fig. 2a, F (4, 25) = 9.029, P < 0.001 for antioxidant imbalance in the brain, and GSH, the key swimming speed in Fig. 2b, F (4, 25) = 31.65, P < 0.001 for nonenzymatic antioxidant in the body, has important the numbers of EC entries in Fig. 2c]. physiological functions, such as scavenging free radicals, Subsequent post hoc testing revealed that zebrafish in detoxifying, promoting iron absorption or maintaining the model group had an increased latency of first entry membrane integrity [47–51]. As GSH is a low-molecular- to the EC zone and reduced swimming speed and num- weight scavenger of O, H O and so on, its content is 2 2 2 ber of EC entries (Fig.  2b, c). The swimming tracks also an important indicator of the antioxidant capacity of the clearly showed the reduced preference of the model body [52]. Here, AlCl treatments decreased GSH levels group fish to the EC zone (Fig.  3) following the AlC l in the fish brain [(F (4, 10) = 18.90, P < 0.001 in Fig.  4f ]. injections. In contrast, pretreatment with medium and Compared with the model group, the BTL-I treatment high doses of BTL-I prevented these effects of AlCl . groups did not show antioxidant activity since no higher Fig. 2 Behavioral performance of zebrafish in the enriched chamber zone of the T-maze test. a The latency (s) of first entry into the EC zone of the # ## ### #### T-maze test. Day 5: *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, vs. the control group; P < 0.05, P < 0.01, P < 0.005, P < 0.001, vs. the model group. b Average swimming speed on the fifth day (n = 6). c The number of entries to the EC zone on the fifth day (n = 6). *P < 0.05, **P < 0.01, # ## ### #### ***P < 0.005, ****P < 0.001, vs. the control group; P < 0.05, P < 0.01, P < 0.005, P < 0.001, vs. the model group Nie et al. Journal of Neuroinflammation (2022) 19:39 Page 6 of 17 Fig. 3 Heatmaps of zebrafish activity in the T-maze on the fifth day. The X-axis and Y-axis in the figure represent the motion trajectory of zebrafish, while the Z-axis represents the residence time of zebrafish. The higher the Z-axis is, the longer the residence time of zebrafish at a certain point GSH level was observed. However, they dose-depend- 163 (Groups B1 and B2); 160 (Groups B1 and B3); ently increased GSH levels in zebrafish. 181 (Groups B1 and B4); and 179 (Groups B1 and B5) (Fig. 5b). To assess the rationality of intestinal flora sequenc - Results of intestinal flora diversity analysis ing of samples, we constructed rarefaction curves and Moreover, due to the close and inflammation-mediated ranked abundance curves of intestinal flora according relationship between gut microbiota (GMB) and NDs, to the OTU numbers at different sequencing depths. the change of intestinal flora of zebrafish is another The curve tends to be flat from 10,000 reads, indicat - important indicator for our determination. OTUs are ing that the sequencing data volume is adequate, and hypothetical computational taxa (e.g., strain, species, a greater data volume will only produce a small num- genus, group) that have been artificially established ber of new OTUs (Fig.  6a). A rank–abundance curve to facilitate the analysis of phylogenetic or population can be used to explain the abundance and evenness of genetics. Because of the conservatism of 16S rDNA, the species. In the horizontal direction, the abundance of sequence obtained by sequencing can represent a species. species is reflected by the width of the curve (i.e., the To understand the composition of a species in a popula- higher the abundance of species is, the wider the range tion sample, it is necessary to cluster the sequences. By of the curve is). The shape (smoothness) of the curve clustering, the sequence is divided into many groups reflects the evenness of species in the sample (i.e., the according to similarity, and one group is an OTU [53]. In flatter the curve, the more uniform the distribution of this study, 15 samples were investigated, and the number species). The results showed that the rank–abundance of OTUs received by each sample is shown in Fig. 5a. curve was smooth except for individual samples, indi- A Venn diagram was used to count the common cating that the species distribution of each sample was and unique OTU numbers of multiple samples, which even (Fig. 6b). can intuitively show the similarity and overlap of the Alpha-diversity can reflect the abundance and diver - OTU number composition of specific samples. Fig - sity of microbial communities, including the Chao ure  5b shows the differences in OTUs between the five index, Ace index, Shannon index, and Simpson index. groups. Different colors represent different groups, The Chao and Ace indices reflect the species rich - and the intersecting part is the OTU shared by adja- ness, i.e., the number of species in the sample, without cent groups. The OTUs in each group were as fol - considering the abundance of each species. Shannon lows: Group B1 (model) 235; Group B2 (control) 213; and Simpson indices reflect both species richness and Group B3 (25  mg/kg BTL-I + AlCl ) 233; Group B4 species evenness in the community. The comparison (50 mg/kg BTL-I + AlCl ) 229; and Group B5 (100 mg/ between all five groups showed that there was no sig - kg BTL-I + AlCl ) 216. The OTUs common between nificant total difference in bacterial diversity. However, the model group and the other groups were as follows: N ie et al. Journal of Neuroinflammation (2022) 19:39 Page 7 of 17 Fig. 4 The results of biochemical indices of zebrafish (n = 3). a GSH content in zebrafish brain tissue. b AChE activity in zebrafish brain tissue. c–f IL-1β and TNF-α content in zebrafish brain and peripheral tissue. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, vs. the control group; P < 0.05, ## ### #### P < 0.01, P < 0.005, P < 0.001, vs. the model group the Shannon and Simpson indices displayed relatively algorithms, Jaccard, Bray–Curtis, unweighted UniFrac, larger differences between the model and control and weighted UniFrac, were used for NMDS calculation. groups than between the control and administration The NMDS based on the Jaccard algorithm only consid - groups (Fig. 6c–f ). ers whether a specific OTU existed in the sample, not its Beta diversity analysis was performed to compare the abundance. The NMDS based on the Bray–Curtis algo - differences in species diversity of the paired samples. The rithm considers both OTU varieties and abundances in contents of each species in the samples were analyzed, samples. UniFrac analysis uses evolutionary informa- and the beta diversity values among different samples tion of sample sequences to compare whether the sam- were then calculated. ples have significant microbial community differences The NMDS method is a data analysis method that sim - in a particular evolutionary lineage. The unweighted plifies the research objects in multidimensional space UniFrac method only considers whether the specific to low-dimensional space for positioning, analysis and sequence appears in the community, not its abundance. classification while retaining the original relationship The weighted UniFrac method takes both existence between objects. The degree of difference between sam - and abundance into account. The results of the Jaccard, ples was reflected by the distance between points. Four unweighted UniFrac, and weighted UniFrac methods Nie et al. Journal of Neuroinflammation (2022) 19:39 Page 8 of 17 Fig. 5 Statistics of the OTUs. a Statistics of the OTUs of 15 samples. b Differences in the distribution of OTUs between groups using the Venn diagram. Group B1: model, Group B2: control, Group B3: 25 mg/kg BTL-I + AlCl , Group B4: 50 mg/kg BTL-I + AlCl , Group B5: 100 mg/kg BTL-I + AlCl 3 3 3 Fig. 6 Alpha-diversity. a The rarefaction curves. b The rank–abundance curves. c–f Alpha indices showed that there was no significant difference in OTU (Fig.  7a, c, d). However, the results of the Bray–Curtis varieties or evolutionary lineage between the experi- method showed that there were significant differences in mental groups, the control group and the model group OTU abundance between the model and control groups, N ie et al. Journal of Neuroinflammation (2022) 19:39 Page 9 of 17 Fig. 7 Beta diversity. a Jaccard algorithm (stress value = 0.137). Only the presence or absence of OTUs in the sample was considered, not the abundance. b Bray–Curtis algorithm (stress value = 0.096). Both the presence and absence of OTUs in the sample and the abundance were considered. c Unweighted-UniFrac algorithm (stress value = 0.064). It only considers whether the sequence was present in the community, not the abundance of the sequence. d Weighted-UniFrac algorithm (stress value = 0.069). It accounts for the abundance of sequences on the basis of unweighted UniFrac and was able to differentiate species abundance whereas there was no significant difference in OTU 100  mg/kg), the abundance of Firmicutes was signifi - abundance between the experimental groups (except for cantly increased, whereas those of Fusobacteria and the 50 mg/kg group) and the control group (Fig. 7b). Chlamydiae significantly decreased compared with that Microbial diversity analysis showed that the intestinal in the model group and basically returned to the same flora of zebrafish included the following 12 major phyla: level as that in the control group. However, in the BTL-I Proteobacteria, Firmicutes, Actinobacteria, Fusobacteria, treatment groups, there was almost no significant rever - Planctomycetes, Chlamydiae, Bacteroidetes, Chloroflexi, sal effect on the increase in Planctomycetes and Chloro - Tenericutes, Verrucomicrobia, Deinococcus-Thermus flexi abundance (Fig. 8c). and Saccharibacteria. Among them, Proteobacteria, At the genus level, a total of 30 major known taxa Firmicutes and Actinobacteria were the dominant bac- of intestinal flora were identified (Fig.  8b). The first 8 teria at the phylum level (Fig.  8a). The abundances of genera with intergroup abundance differences were Firmicutes in the gut of the model group were reduced, Bacillus, Bosea, Cetobacterium, Alpinimonas, Singuli- whereas those of Fusobacteria, Planctomycetes, Chlamy- sphaera, Phreatobacter, Mycobacterium, and Candida- diae and Chloroflexi significantly increased compared tus-Microthrix (Fig.  8d). Compared with the control with those observed in the control group. In the two group, the abundance of Bacillus in the model group experimental groups (administration of BTL-I 25 and significantly decreased, while those of the other seven Nie et al. Journal of Neuroinflammation (2022) 19:39 Page 10 of 17 Fig. 8 Relative abundance (a and b) and analysis of differential microorganisms (c and d). a and c Results at the phylum level. b and d Results at the # ## ### #### genus level. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, vs. the control group; P < 0.05, P < 0.01, P < 0.005, P < 0.001, vs. the model group genera mostly increased significantly. In the two BTL-I high-dose group (100 mg/kg BTL-I) (Gracilibacteraceae treatment groups (25 and 100  mg/kg), the abundance and Lutispora). of Bacillus was elevated to nearly normal levels, while those of Bosea and the other genera mostly decreased Prediction of ADMET and drug‑likeness properties significantly. Finally, the early evaluation of lead compound for its LEfSe was able to compare the taxonomic composi- potential to become a drug is critical step for drug devel- tion of multiple groups at different taxonomic levels, opment. To obtain more information on the pharmacoki- identify the taxa with significant intergroup differences netic profile of BTL-I and whether it has the potential to in abundance (i.e., biomarkers), and exhibit their line- become a drug, we used ADMETlab 2.0 [45] to predict age relationship. The results in Fig.  9 show the bio- its ADMET and drug-likeness properties. The corre - markers with significant effects (LDA scores > 2) in sponding predicted results are presented in Table  1, and each group, including 27 taxa in the model group (e.g., the physical properties of BTL-I are shown in Additional Cetobacterium in Fusobacteria, Bosea in Rhizobiales, file  1: Table S1. The results demonstrated that BTL-I pos - Chlamydiales, Candidatus-Microthrix, Mycobacte- sesses acceptable ADMET and drug-likeness properties rium), 11 taxa in the control group (e.g., Bacillus in Fir- in general. For example, the results showed that BTL-I micutes, Rhizobium rhizoryzae), 9 taxa in the low-dose is active in both human intestinal absorption (HIA) and group (25  mg/kg BTL-I) (e.g., Singulisphaera in Planc- blood–brain barrier (BBB) penetration. It has accept- tomycetes, Micromonospora), 10 taxa in the medium- able safety profiles, generally performing well on most dose group (50  mg/kg BTL-I) (e.g., Clostridiaceae, metrics (e.g., hERG blockers, Ames toxicity and carci- Chloroflexi, Pseudoxanthobacter), and 2 taxa in the nogenicity), and it is in harmony with the Lipinski rule N ie et al. Journal of Neuroinflammation (2022) 19:39 Page 11 of 17 Fig. 9 LDA effect size analysis. a Branching diagram of the evolution of different species between the control, model and experimental groups. b Bar graph of LDA values for different species Nie et al. Journal of Neuroinflammation (2022) 19:39 Page 12 of 17 Table 1 ADMET and drug-likeness properties of BTL-I through the online prediction tool ADMETlab 2.0 (the table is located below line 394) Property Value Decision Absorption Caco-2 permeability − 4.9 log cm/s Excellent Madin–Darby canine kidney cells (MDCK) permeability 2e-05 cm/s Excellent P-glycoprotein (Pgp)-inhibitor 0.023 Excellent P-glycoprotein (Pgp)-substrate 0.007 Excellent Human intestinal absorption (HIA) 0.008 Excellent 20% bioavailability (F20%) 0.059 Excellent Distribution Plasma protein binding (PPB) 0.987 Bad Volume distribution ( VD) 0.501 L/kg Excellent Blood–brain barrier (BBB) penetration 0.027 Excellent The fraction unbound in plasma (Fu) 0.012 Bad Metabolism CYP1A2-inhibitor 0.381 – CYP1A2-substrate 0.536 – CYP2C19-inhibitor 0.955 – CYP2C19-substrate 0.189 – CYP2C9-inhibitor 0.934 – CYP2C9-substrate 0.946 – CYP2D6-inhibitor 0.873 – CYP2D6-substrate 0.788 – CYP3A4-inhibitor 0.916 – CYP3A4-substrate 0.333 – Excretion Clearance 17.179 mL/min/kg Excellent The half-life ( T ) 0.371 – 1/2 Toxicity hERG blockers 0.023 Excellent Human hepatotoxicity (H-HT ) 0.402 Medium Drug-induced liver injury (DILI) 0.764 Bad Ames toxicity 0.106 Excellent Rat oral acute toxicity 0.559 Medium Maximum recommended daily dose (FDAMDD) 0.283 Excellent Skin sensitization 0.105 Excellent Carcinogenicity 0.162 Excellent Eye corrosion 0.003 Excellent Eye irritation 0.146 Excellent Respiratory toxicity 0.039 Excellent Drug-likeness MCE-18 [56] 68.839 Excellent Lipinski rule [53] Accepted (0 violation) Excellent Pfizer rule [54] Accepted (0 violation) Excellent Golden triangle [55] Accepted (0 violation) Excellent GSK rule [57] Rejected (1 violation) Bad N ie et al. Journal of Neuroinflammation (2022) 19:39 Page 13 of 17 [54] and others (such as the Pfizer rule [55] and golden activity against oxidative stress, neuroinflammation and triangle [56]), which indicates the drug-likeness proper- neuronal apoptosis, as well as in nerve growth without ties of a compound. Unfortunately, BTL-I displayed some inducing cytotoxicity [23–27]. Here, we found that BTL-I disadvantages, such as a high risk of inhibiting CYP2C19, also prevents cognitive deficits (induced in zebrafish by CYP2C9 and CYP3A4 and inducing liver injury. AlCl ) and exerts neuroprotective effects in this zebrafish model. In this study, we noted that the peripheral IL-1β level Discussion was suppressed while TNF-α level were elevated after Aluminum has been examined for its broad neurotoxic BTL-I administration. The different behaviors of the two effects and close relationship with AD, which promote inflammation markers are consistent with Tsarouchas’ tau hyperphosphorylation, aggregation, and neurofibril - report about the effects of spinal cord injury on zebrafish lary tangle formation in AD brains (by activating tau spinal axonal regeneration that increased TNF-α (mainly kinases CDK5 and GSK3β), accumulate in microglia produced by macrophage) and decreased IL-1β (mainly and induce proinflammatory cytokines, bind to Aβ and produced by neutrophil) both promote neurite regenera- induce its aggregation, stimulate iron-induced mem- tion in the periphery [65]. So, we presume that the upreg- brane lipid peroxidation and oxidative damage, decrease ulated peripheral TNF-α and downregulated IL-1β in the activity of antioxidant enzymes, and interact with BTL-I-treated zebrafish are actually consistent with the AChE on γ-peripheral sites to enhance enzymatic activ- improvement of cognition. However, further investiga- ity, resulting in reduced neurotransmission [12, 59, 60]. tion is necessary to reveal the exact mechanism. Furthermore, activated AChE can deteriorate Aβ aggre- Meanwhile, the inhibition of brain AChE elevates gation, decrease BDNF expression [59], and further pro- ACh levels and, hence, positively affects cognitive func - mote oxidative stress and neuroinflammation through a tion in rats [60, 66]. Subchronic exposure of zebrafish to ‘cholinergic anti-inflammatory pathway’ (CAIP) via α7 AlCl or i.p. injection of AlCl in mice enhances brain 3 3 nicotinic acetylcholine receptors [60, 61]. In addition, AChE activity [39]. Accordingly, our results show that in many reports on aluminum-induced AD or toxicity i.p. injection of AlCl also elevated AChE activity in the models, alterations in the host gut microbiota have been zebrafish brain, whereas BTL-I evoked neuroprotection observed [62, 63]. and lowered AChE activity (Fig.  4e). Because thin-layer Therefore, the present study established a zebrafish chromatography bioautography shows that BTL-I does model of subchronic inflammation induced by acute not inhibit AChE catalytic activity (data not shown), this i.p. AlCl administration, which resulted in subchronic compound seems to indirectly decrease zebrafish AChE peripheral and central inflammatory responses and activity here, likely involving other molecular pathways. enhanced oxidative stress and AChE activity in the brain. Furthermore, oxidative stress involves the excessive At the behavioral level, administration of AlCl strongly production of ROS and reactive nitrogen species (RNS) impaired the spatial and contextual memory of zebrafish [67] and may result in tissue damage. GSH is the most in the T-maze test. At the gut microbiota level, high- important nonenzymatic antioxidant, the neuroprotec- throughput sequencing results showed that the intesti- tive role of which in the brain is critical against oxidative nal flora of zebrafish was dramatically disturbed by acute damage caused by catecholamine oxidation or lipid per- AlCl administration. Collectively, these findings are oxidation [68]. In the present study, GSH levels markedly generally consistent with previous evidence that AlCl decreased 24  h after AlC l administration. With BTL-I induces memory deficits in both humans and animals, pretreatment at doses of 25 and 50 mg/kg, the GSH levels including zebrafish, and changes the intestinal flora [62– of the zebrafish were even lower than those of the model 64]. In contrast, BTL-I co-administration reversed these group. However, when the dose increased to 100 mg/kg, induced memory deficits and microbiota imbalances, the GSH content increased to the same level as that in the indicating the potential neuroprotective role of this drug. model group, although it was still lower than that in the In the present study, acute central and peripheral control group. BTL-I seemed to display doubtful antioxi- inflammation was characterized by the release of the dant effects via GSH. The lack of significant antioxidant proinflammatory cytokines IL-1β and TNF-α follow - effects of BTL-I relative to the model group may be due ing AlCl administration. Supplementation with BTL-I to the limited sample size and the resulting low statistical potently inhibited acute brain and peripheral inflam - power. However, it may also suggest that the antioxidant mation in AlCl -treated zebrafish. Mounting evidence mechanism of action of BTL-I may exist elsewhere. Fur- implicates BTL-I in multitargeted neuroprotective ther studies with larger sample sizes and better designs Nie et al. Journal of Neuroinflammation (2022) 19:39 Page 14 of 17 are warranted to test and explain this complicated phe- group, and the abundances of G bacteria were much nomenon to obtain a solid conclusion. lower. Additionally, in the low-dose group, another G In addition, the gut microbiota (GMB) plays a cru- bacterium, Micromonospora, in the phylum Actino- cial role in the stability and balance of the intestinal bacteria was recognized as a biomarker, suggesting its microecological environment, and the composition of possible positive role based on a report [16] on Actino- the human intestinal microbial community remains bacteria. Likewise, in the high-dose group, the G bac- basically stable after the age of 3  years [69]. In recent teria Lutispora in the family Gracilibacteriaceae and the years, a growing number of studies have shown that order Clostridiales were also biomarkers. Considering dysregulation of specific GMB is closely related to NDs the report on the strongly negative correlation of Clostri- [15, 16, 70]. Some reports have indicated that in the gut diaceae [16] with AD biomarkers in cerebrospinal fluid, of healthy humans or animals, there are higher popu- we speculate that Lutispora may also have some benefit lations of Gram-positive (G ) bacteria, including Fir- in neuroprotection. micutes and Actinobacteria, and lower populations of It is intriguing that the middle-dose group did not pos- Gram-negative (G ) bacteria, such as Bacteroidetes, at sess a high abundance of Bacillus. This discrepancy may the phylum level [15, 16]. At the family or genus level, be attributable to a nonlinear relationship of BTL-I and some G taxa, such as Bacillus, Eubacterium, Clostridi- Bacillus, and more concentration gradients of BTL-I will aceae in Firmicutes and Bifidobacterium in Actinobac - be set in future studies to explore this relationship. How- teria, show a higher abundance in healthy individuals ever, the G bacterium Clostridiaceae was found to be and benefit their hosts through different mechanisms, the key biomarker of this group; G bacterium Chloro- including reducing leakage of gut by the protection of flexi was another key biomarker, but bacteria in this phy - biofilms, inhibiting inflammation and antioxidation and lum have no LPS in their cell walls [72]. These findings reducing Aβ deposition and transfer from the gut to the may help to explain the behavioral improvement of this brain [15–17]. In contrast, some G taxa, such as Bac- group. teroides, Blautia, Escherichia coli, Shigella, Chlamydia, Our study suggests that administration of the marine and Fusobacterium, are closely and positively corre- fungal metabolite BTL-I prior to AlC l injection may aid lated with AD, mainly involving the activation of sys- in maintaining the predominance of beneficial G bacte- tematic inflammation by their enriched LPS in the cell ria in the gut of zebrafish to resist acute injury caused by wall and the invasion of proinflammatory cytokines, aluminum, related inflammation and AD pathology. We LPS, and even bacteria into the blood circulation sys- deduce that the stabilization of the GMB may contribute tem and brain, inducing Aβ deposition and tau phos- to the clearance of inflammation and lead to the improve - phorylation [15–17, 70]. ment of zebrafish’s cognition. However, the detailed In the present study, the control group zebrafish hosted mechanisms of intestinal flora regulation and the treat - a greater amount of G bacteria (Firmicutes at the phy- ment effect on an AlCl -induced chronic AD model need lum level and predominantly Bacillus at the genus level) to be further investigated for BTL-I in the future. than the AlC l -injured model group with memory Further, the early evaluation of ADMET and drug- impairment, while the model group zebrafish had much likeness properties of drug candidates are highly sig- fewer G bacteria than the control group zebrafish but nificant, as many drugs have been withdrawn in clinical significantly more G bacteria, including Cetobacterium trials and even in the marketing process due to unaccep- (in the family Fusobacteriaceae) and Chlamydiales (at the table pharmacokinetic properties [73–75]. Accordingly, order level). This result is highly consistent with previ - the prediction of ADMET and drug-likeness proper- ous studies, especially reports on the benefits of Bacil - ties of drug candidates has received extensive attention. lus subtilis in delaying neurodegeneration and behavior Numerous tools have been developed, such as ADMET- impairment in the AD model Caenorhabditis elegans and lab [76] admetSAR [77] and SwissADME [78]. In this reports on the negative effect of G bacteria, including study, in silico prediction with ADMETlab suggested Fusobacterium and Chlamydia [17, 70, 71]. that BTL-I caters to the majority of the ADMET proper- Generally, pretreatment with BTL-I maintained the ties and drug-likeness profiles, such as the typical Lipin - + − dominance of G bacteria vs. G bacteria in the gut of ski rule with 0 violation, and possesses good properties zebrafish when faced with aluminum exposure. How - in crossing the BBB. Such features render it a promis- ever, the dose levels exerted different influences. In the ing drug candidate for NDs since overcoming BBB pen- low- and high-dose groups, the abundances of Bacil- etration is essential for NDs drugs [79, 80]. It should be lus remained at high levels close to those of the control noted that BTL-I was predicted to be associated with a N ie et al. Journal of Neuroinflammation (2022) 19:39 Page 15 of 17 Zhanjiang City (XM-202008-01B1), Special Project in Key Fields of Guang- high risk of liver injury, which requires further confirma - dong Provincial Higher Education Institutions (Biomedicine and health care) tion and assessment and may provide clues for structural (2021ZDZX2064) and Guangdong Provincial Special Project in Science and optimization. In addition, it is predicted that CYP2C19, Technology (2021A50114). CYP2C9 and CYP3A4 may be inhibited by BTL-I and Availability of data and materials therefore co-administration with CYP2C19, CYP2C9 and All data generated or analyzed during this study are included in this published CYP3A4-substrate drugs may be avoided. article and its additional file. The datasets used and/or analyzed during the current study are available from the primary author on reasonable request. Conclusions Declarations This study showed that BTL-I dose-dependently ame - liorated AlCl -induced cognitive deficits in zebrafish, Ethics approval and consent to participate reversed the elevation of AlC l -induced central and The procedures were previously approved by the Animal Ethics Committee of Guangdong Ocean University, numbered 2019-11-23-2. peripheral proinflammatory cytokine levels and the increase in brain AChE activity, and contributed to main- Consent for publication taining the predominance of beneficial Gram-positive Not applicable. bacteria in the GMB of zebrafish, which was challenged Competing interests by AlCl . The in silico analysis indicated that BTL-I The authors declare that they have no competing interests. exhibits acceptable drug-likeness and ADMET profiles. Author details In summary, BTL-I has the potential as an intervention College of Food Science and Technology, Guangdong Provincial Key agent for preventing CNS deficits caused by inflamma - Laboratory of Aquatic Product Processing and Safety, Guangdong Provincial tion, neurotoxicity, and GMB imbalance. Engineering Laboratory for Marine Biological Products, Research Institute for Marine Drugs and Nutrition, Shenzhen Institute of Guangdong Ocean Uni- versity, Guangdong Ocean University, Zhanjiang 524088, China. Collaborative Innovation Center of Seafood Deep Processing, Dalian Polytechnic University, Abbreviations Dalian 116034, China. School of Pharmaceutical Science and Technology, AChE: Acetylcholinesterase; AD: Alzheimer’s disease; ADMET: Absorption, Tianjin University, Tianjin 30072, China. College of Pharmaceutical Sciences distribution, metabolism, excretion, and toxicity; AlCl : Aluminum trichloride; and Chinese Medicine, Southwest University, Chongqing 400715, China. Ural Aβ: β-Amyloid peptide; BTL-I: Butyrolactone I; BTLs: Butyrolactones; CAIP: Cho- Federal University, Ekaterinburg 620002, Russia. I nstitute of Translational linergic anti-inflammatory pathway; CDK 5: Cyclin-dependent kinase 5; CNS: Biomedicine, St. Petersburg State University, Saint Petersburg 199034, Russia. Central nervous system; COX-2: Cyclooxygenase-2; GMB: Gut microbiota; GSH: Glutathione; IL-1β: Interleukin-1β; KW: Kruskal–Wallis; LDA: Linear discriminant Received: 5 July 2021 Accepted: 20 January 2022 analysis; NDs: Neurodegenerative disorders; NMDS: Nonmetric multidimen- sional scaling graphs; PD: Parkinson’s disease; RNS: Reactive nitrogen species; ROS: Reactive oxygen species; TNF-α: Tumor necrosis factor-α. Supplementary Information References 1. Thomas B, Beal MF. Parkinson’s disease. Hum Mol Genet. 2007;16:R183–94. 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Marine fungal metabolite butyrolactone I prevents cognitive deficits by relieving inflammation and intestinal microbiota imbalance on aluminum trichloride-injured zebrafish

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Springer Journals
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1742-2094
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10.1186/s12974-022-02403-3
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Abstract

Background: Mounting evidences indicate that oxidative stress, neuroinflammation, and dysregulation of gut microbiota are related to neurodegenerative disorders (NDs). Butyrolactone I (BTL-I), a marine fungal metabolite, was previously reported as an in vitro neuroprotectant and inflammation inhibitor. However, little is known regarding its in vivo effects, whereas zebrafish (Danio rerio) could be used as a convenient in vivo model of toxicology and central nervous system (CNS) diseases. Methods: Here, we employed in vivo and in silico methods to investigate the anti-NDs potential of BTL-I. Specifi- cally, we established a cognitive deficit model in zebrafish by intraperitoneal (i.p.) injection of aluminum trichloride (AlCl ) (21 μg) and assessed their behaviors in the T-maze test. The proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α ( TNF-α) as well as acetylcholinesterase (AChE) activity or glutathione (GSH) levels were assayed 24 h after AlCl injection. The intestinal flora variation of the zebrafish was investigated by 16S rDNA high- throughput analysis. The marine fungal metabolite, butyrolactone I (BTL-I), was used to modulate zebrafish cognitive deficits evoked by AlCl and evaluated about its effects on the above inflammatory, cholinergic, oxidative stress, and gut floral indicators. Furthermore, the absorption, distribution, metabolism, excretion, and toxicity (ADMET ) and drug- likeness properties of BTL-I were studied by the in silico tool ADMETlab. Results: BTL-I dose-dependently ameliorated AlCl -induced cognitive deficits in zebrafish. While AlCl treatment 3 3 elevated the levels of central and peripheral proinflammatory cytokines, increased AChE activity, and lowered GSH in the brains of zebrafish, these effects, except GSH reduction, were reversed by 25–100 mg/kg BTL-I administra- tion. Besides, 16S rDNA high-throughput sequencing of the intestinal flora of zebrafish showed that AlCl decreased *Correspondence: hubeizhangyi@163.com College of Food Science and Technology, Guangdong Provincial Key Laboratory of Aquatic Product Processing and Safety, Guangdong Provincial Engineering Laboratory for Marine Biological Products, Research Institute for Marine Drugs and Nutrition, Shenzhen Institute of Guangdong Ocean University, Guangdong Ocean University, Zhanjiang 524088, China Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Nie et al. Journal of Neuroinflammation (2022) 19:39 Page 2 of 17 Gram-positive bacteria and increased proinflammatory Gram-negative bacteria, while BTL-I contributed to maintain- ing the predominance of beneficial Gram-positive bacteria. Moreover, the in silico analysis indicated that BTL-I exhibits acceptable drug-likeness and ADMET profiles. Conclusions: The present findings suggest that BTL-I is a potential therapeutic agent for preventing CNS deficits caused by inflammation, neurotoxicity, and gut flora imbalance. Keywords: Inflammation, Oxidative stress, Butyrolactone I, Neurodegenerative diseases, Acetylcholinesterase, Intestinal flora Background anti-AD drugs targeting orphan targets, such as Aβ and Neurodegenerative disorders (NDs), such as Alzheimer’s Tau, may be substantially related to malignant ampli- disease (AD) and Parkinson’s disease (PD), are chronic, fication induced by neuroinflammation and oxidative progressive, and severely debilitating neurological disor- stress. Thus, inhibiting inflammation and oxidative stress ders [1, 2]. NDs are characterized by cognitive and motor and regulating imbalanced GMB to protect neurons deficits, accompanied by neuronal apoptosis and reduced and intervene in the early stage of disease has become neurotransmission [3, 4]. Oxidative stress and inflamma - an important new strategy in developing novel anti- tion play critical roles in neuronal apoptosis [5–9] and NDs agents [20]. Aryl butyrolactones (BTLs), including may, thus, be considered potential risk factors for NDs. butyrolactone I (BTL-I, Fig.  1 inset), are characteristic Various factors, including peripheral or brain inflamma - natural products of fungi (e.g., Aspergillus sp. and Penicil- tion, β-amyloid peptide (Aβ), pathogenic infection and lium sp.) [21, 22]. Our previous studies have shown that toxins (e.g., aluminum), activate brain microglial cells BTL-I has strong in vitro anti-neuroinflammatory effects, [10–12]. The release of proinflammatory cytokines and inhibiting LPS-induced inflammatory proliferation of reactive oxygen species (ROS) trigger and amplify dam- microglia, the release of inflammatory mediators (NO age to neurons and astrocytes, whereas oxidative stress and IL-1β) and ROS, the expression of the inflammatory and inflammation further promote neuroinflammation, target enzyme cyclooxygenase-2 (COX-2), and intracel- which in turn activates microglia, eventually impairing lular migration of the signaling protein NF-κB p65 [23]. neurons and astrocytes [5, 7, 12–14]. Oxidative stress and Moreover, BTL-I plays a versatile anti-neurodegenerative inflammation are involved in cyclin-dependent kinase 5 role through multiple mechanisms, such as neuronal (CDK 5) activation-induced Tau hyperphosphorylation, nutrition and inhibition of neuronal injury [24–27]. resulting in neurofibrillary tangles, another important However, since previous BTL-I studies were limited to pathological marker of AD [5]. in vitro studies, its in vivo effects remain to be elucidated In addition, an increasing number of studies have as well as its influence on GMB. shown that intestinal microorganisms are closely related Due to their high genetic and physiological homology to the occurrence of NDs and their mechanism also to humans, zebrafish has long been used as a power - involves inflammation, oxidative stress and neurotrans - ful in  vivo model to assess anti-inflammatory drugs [28, mitters [15, 16]. Researchers revealed that the increase 29] and neuronal injury [30]. Zebrafish possess innate of proinflammatory gut microbiota (GMB) taxon (the and acquired immune systems similar to those of mam- producers of liposaccharides) and reduction of anti- mals [31] display well-characterized learning, memory, inflammatory GMB taxon are possibly associated with addiction and other behaviors that correspond to clinical the peripheral inflammation in patients with impaired phenotypes, and host rich GMB in their intestine [32], cognition and brain amyloidosis. The GMB may influence enabling them an ideal model for our purpose [33–36]. the cognition by changing gut permeability and inflam - AlCl causes AD-like pathology, aggravating neuro- matory-oxidative stress state, producing short-chain inflammation, oxidative stress and AChE activity in the fatty acids and neurotransmitters, promoting or reducing rodent brain [12, 37, 38]. The effect of AlCl on zebrafish beta-amyloid deposition and transfer to brain, etc. [17]. cognition remains unclear [39]. In the present study, we u Th s, GMB is becoming a new target of ND treatment. established a neurotoxic zebrafish model (induced by Notably, current clinical drugs for the prevention and AlCl ) to assess the potential effects of BTL-I on mem - treatment of NDs manifest limited efficacy. For exam - ory and cognitive impairment in vivo. The putative neu - ple, donepezil partially relieves symptoms of AD with- roprotective activity of BTL-I in zebrafish was further out reversing or preventing its progression [5]. The poor investigated by evaluating AChE activity and GSH levels understanding of NDs pathogenesis restricted new drug in the brain and by detecting the levels of the inflam - development [18, 19]. The repeated failure to develop matory cytokines IL-1β and TNF-α in both central and N ie et al. Journal of Neuroinflammation (2022) 19:39 Page 3 of 17 Fig. 1 A general diagram summarizing the experimental design used in the present study. Inset: the chemical structure of BTL-I peripheral tissues. The 16S rDNA high-throughput Briefly, following a 24-h fasting period, the fish were method was used to determine the structure and changes anesthetized and then received an i.p. injection. For of zebrafish intestinal flora. The in silico tool ADMETlab this purpose, eugenol was dissolved in 100  mL of anhy- was further applied to evaluate the absorption, distribu- drous ethanol to prepare a 1  mg/mL stock solution that tion, metabolism, excretion, and toxicity (ADMET) and was added to 5 L of water (28 ± 1 °C) and stirred evenly. drug-likeness properties of BTL-I. Zebrafish were then group-exposed to the anesthetic, and after stopping swimming (immobile > 2–3 min), they Methods were quickly injected with AlCl . After the injection, Animals and model development the animals recovered in a water-containing beaker and Adult wild-type AB zebrafish (approximately 6–8 months returned to the holding tanks once their normal swim- old; 50:50% male:female ratio) used in the present study ming resumed [41]. were obtained from a commercial supplier (Shanghai Jiayu Aquarium, Shanghai, China) and acclimatized in T‑maze behavioral testing a 50-L aquarium in the aquatic facility of Guangdong The aquatic T-maze was used for cognitive testing, as Ocean University for at least 2 weeks. The fish were kept described previously, with modifications [42, 43]. The on a 14  h:10  h light:dark cycle (lights on at 7 am) at a maze comprised a long vertical arm (50  cm) and two temperature of 25 ± 2 °C in a recirculating tank system. short horizontal arms (20  cm), with an arm width of The zebrafish were maintained according to standard 10 cm, a depth of 10 cm, and a water depth of 8 cm. The conditions [40] and fed Artemis larvae twice a day at 9 am right arm was connected to a rectangular water tank and 2 pm. (22  cm × 20  cm × 15  cm) with a black outer wall; sand As shown in Fig.  1, to establish an AlCl model, 75 and stones were added to the bottom of the tank, and bait zebrafish (3.0 ± 0.4 cm in length) were randomly divided was set inside the tank to provide an enriched chamber into control and AlCl - and BTL-I-treated groups (n = 15 (EC) (Fig.  1). During the final 4 days of treatment, 6 fish per group). BTL-I (25, 50 or 100 mg/kg/day) was admin- were randomly selected from each group, and the fish istered with food for 20  days. The control and AlCl were individually trained for 5 min daily to locate the EC groups were fed equal amounts of normal food. Twenty zone. If a fish did not enter the EC zone within the 5-min days later, the AlCl and BTL-I groups were anesthetized training session, it was guided into the EC zone and kept and injected (i.p.) with AlCl solution (4.2 mg/mL, 5 μL, there for > 30  s. Following 4  days of training, the trained pH = 5.0 ± 0.2) using a 10-μL gas phase injection needle fish were placed individually one day later into the start - with a 0.5-mm outer diameter. The control group was ing area of the long arm for behavioral testing, with injected with the same amount of saline. Memory testing scoring of the latency time (s) to enter the EC zone and was performed 24 h later. stay there for > 30  s. If a fish did not enter the EC zone Nie et al. Journal of Neuroinflammation (2022) 19:39 Page 4 of 17 Gut flora sequencing and data analysis within the 5-min test, the latency time was recorded as Genomic DNA was extracted by protease K lysis. The 300  s. Behavioral testing was performed between 10 am variable region of the 16S ribosomal RNA gene V3–V4 and 1  pm. A Microsoft LifeCam Studio 1080p HD cam- was amplified by PCR, and the specific primer sequences era was used to record videos with Apowersoft software were as follows: 357F 5′-ACT CCT ACG GRA GGC AGC (Apowersoft Co. Ltd., Hong Kong, China). Supersys soft- AG-3′ and 806R 5′-GGA CTA CHVGGG TWT CTAAT- ware was used for off-line video analyses (Shanghai Xin - 3′. Bidirectional sequencing was performed according ruan Information Technology Co. Ltd., Shanghai, China), to Illumina high-throughput sequencing requirements, assessing the latency of the first entry into the EC zone(s), and the library was constructed by two-step PCR ampli- the average swimming speed (cm/s), and the number of fication. The PCR conditions were 94 °C for 2 min, 94 °C EC entries. for 30 s, 56 °C for 30 s, 72 °C for 30 s (the primary PCR amplification 30 cycles, the secondary PCR amplification 8 cycles), 72 °C for 5 min, and a final extension at 10 °C. Reagents PCR amplification products were recovered by 2% aga - BTL-I was isolated for this study from the marine fun- rose gel electrophoresis. Recycling was performed using gus Aspergillus terreus C23-3 as described previously an AxyPrepDNA gel recovery kit from Axygen. [23]. The BCA protein, GSH and AChE assay kits, fish The PCR-amplified products of the zebrafish gut sam - IL-1β enzyme-linked immunosorbent assay (ELISA) kit ples were sequenced for 16S rDNA using the Illumina- and fish TNF-α ELISA kit were purchased from Nan - Misq high-throughput sequencing platform [TinyGene jing Jiancheng Bioengineering Institute (Nanjing, China). Bio-Tech (ShangHai) Co., Ltd, China], and the sequence Eugenol and AlCl were purchased from Huaxia Reagent length was 450 bp. The raw data obtained from sequenc - (Chengdu, China) and Xiya Reagent (Shandong, China), ing were evaluated for quality and optimized. Trimmo- respectively. matic was used for sequence filtration, and FLASH was used for splicing. Ambiguous, homologous and some chi- meras produced in the PCR process were subsequently Molecular biomarker assays screened using Mothur V.1.39.5 to obtain optimized Twenty-four hours after behavioral testing, the fish were sequences for subsequent operational taxonomic unit euthanized. Considering that the available assay kits (OTU) clustering and species information analysis. could not measure the small tissue samples of individual USEARCH was used to cluster OTUs of the above fish, the 15 fish in each group were randomly divided into treated sequences at 97% similarity. The representative 3 subgroups, and samples of brain, peripheral and intesti- OTU sequences were compared with the database Silva nal tract tissue from each subgroup (5 fish) were collected for species annotation (confidence threshold: 0.6). The and combined immediately and freeze-dried at −  80 °C. relative abundance percentages of each sample were All the samples (except for the gut samples) were homog- calculated at the phylum, class, order, family, genus and enized in phosphate-buffered solution (PBS) for further species levels. The rarefaction curve reflects the sequenc - assays. The supernatant was collected by centrifugation ing depth of the samples. The rank–abundance curve at 252 g at 4 °C for 15 min. Zebrafish brain sample super - explains species abundance and species evenness. natants were used to determine GSH levels and AChE A Venn diagram can be used to count the common and activity. Moreover, zebrafish brain samples and periph - unique OTU numbers of multiple samples, which can eral tissue supernatants were also used to determine the directly show the overlap and uniqueness in the OTU levels of IL-1β and TNF-α [44] following the manufactur- composition of different samples. er’s instructions. The results are expressed as U of AChE/ Alpha-diversity analysis reflected the richness and mg of protein and μmol of GSH/g of protein. Regression diversity of communities in the samples. Mothur (http:// equations for the IL-1β and TNF-α standard curves were w w w. mot h ur . or g/ w ik i/ S c hlo ss_ S OP# Alpha_ diver sity) calculated according to the OD value, and logistic curves was used to calculate the values of the Shannon, Simp- (4 parameters each) were used as the fitting models. son, Chao and ACE indices, and the R (3.4.1) language tool was used for graph plotting. Jaccard, Bray–Curtis, unweighted UniFrac, and Statistical analysis for behavioral and molecular weighted UniFrac were used to calculate the differences biomarkers between samples and conduct nonmetric multidimen- Statistical analysis was performed by one-way analysis sional scaling graphs (NMDS). Metastats (http:// metas of variance (ANOVA) followed by a post hoc Dunnett tats. cbcb. umd. edu/) was used for the comparison of the test. The results are expressed as the mean ± SD. P-val- features with different abundances between groups on ues < 0.05 indicated statistical significance for all tests. N ie et al. Journal of Neuroinflammation (2022) 19:39 Page 5 of 17 Eecfft of  AlCl on proinflammatory cytokines, AChE multiple taxonomic levels. The nonparametric facto - and GSH rial Kruskal–Wallis (KW) sum-rank test was applied to To further elucidate the effect of BTL-I on AlCl -induced determine the significant difference between the richness cognitive impairment in zebrafish, we assessed changes of the groups. LEfSe (LDA effect size) uses linear discri - in the levels of several biochemical indicators (includ- minant analysis (LDA) to estimate the impact of each ing IL-1β, TNF-α, GSH, AChE). The results showed that component (species) abundance on the difference effect AlCl treatment promoted the release of IL-1β from the of the groups. brain and peripheral tissue and TNF-α from the brain, and BTL-I supplementation inhibited the release of IL-1β In silico prediction of ADMET and drug‑likeness properties in the brain and periphery, as well as the release of TNF of BTL‑I in the brain [F (4, 10) = 20.64, P < 0.001 for brain IL-1β To evaluate the pharmacokinetic profile and toxicity of in Fig.  4a; F (4, 10) = 7.240, P < 0.01 for peripheral IL-1β BTL-I, we employed ADMETlab 2.0 (https:// admet mesh. in Fig.  4b; F (4, 10) = 11.75, P < 0.001 for brain TNF-α in scbdd. com/ pub/), which is a free online platform that Fig. 4c]. BTL-I also increased the peripheral TNF-α level enables researchers to predict the ADMET and drug- [F (4, 10) = 14.94, P < 0.001 in Fig. 4d]. likeness properties of a compound [45]. Furthermore, paralleling their cognitive deficits in the T-maze, zebrafish treated with AlCl exhibited higher Results brain AChE activity, whereas both moderate and high BTL‑I improved AlCl ‑induced memory impairment doses of BTL-I dose-dependently inhibited AChE activ- Overall, compared with the model group, the BTL-I- ity [F (4, 10) = 4.474, P < 0.05 in Fig.  4e]. This is consist - treated groups showed a significant treatment effect ent with the fact that excessive AChE activity is closely on cognitive performance in the T-maze test [F (4, related to memory deficits [46]. 25) = 40.60, P < 0.001 for the latency of first entry to the In addition, treatment with AlCl caused an oxidant– EC zone on day 5 in Fig. 2a, F (4, 25) = 9.029, P < 0.001 for antioxidant imbalance in the brain, and GSH, the key swimming speed in Fig. 2b, F (4, 25) = 31.65, P < 0.001 for nonenzymatic antioxidant in the body, has important the numbers of EC entries in Fig. 2c]. physiological functions, such as scavenging free radicals, Subsequent post hoc testing revealed that zebrafish in detoxifying, promoting iron absorption or maintaining the model group had an increased latency of first entry membrane integrity [47–51]. As GSH is a low-molecular- to the EC zone and reduced swimming speed and num- weight scavenger of O, H O and so on, its content is 2 2 2 ber of EC entries (Fig.  2b, c). The swimming tracks also an important indicator of the antioxidant capacity of the clearly showed the reduced preference of the model body [52]. Here, AlCl treatments decreased GSH levels group fish to the EC zone (Fig.  3) following the AlC l in the fish brain [(F (4, 10) = 18.90, P < 0.001 in Fig.  4f ]. injections. In contrast, pretreatment with medium and Compared with the model group, the BTL-I treatment high doses of BTL-I prevented these effects of AlCl . groups did not show antioxidant activity since no higher Fig. 2 Behavioral performance of zebrafish in the enriched chamber zone of the T-maze test. a The latency (s) of first entry into the EC zone of the # ## ### #### T-maze test. Day 5: *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, vs. the control group; P < 0.05, P < 0.01, P < 0.005, P < 0.001, vs. the model group. b Average swimming speed on the fifth day (n = 6). c The number of entries to the EC zone on the fifth day (n = 6). *P < 0.05, **P < 0.01, # ## ### #### ***P < 0.005, ****P < 0.001, vs. the control group; P < 0.05, P < 0.01, P < 0.005, P < 0.001, vs. the model group Nie et al. Journal of Neuroinflammation (2022) 19:39 Page 6 of 17 Fig. 3 Heatmaps of zebrafish activity in the T-maze on the fifth day. The X-axis and Y-axis in the figure represent the motion trajectory of zebrafish, while the Z-axis represents the residence time of zebrafish. The higher the Z-axis is, the longer the residence time of zebrafish at a certain point GSH level was observed. However, they dose-depend- 163 (Groups B1 and B2); 160 (Groups B1 and B3); ently increased GSH levels in zebrafish. 181 (Groups B1 and B4); and 179 (Groups B1 and B5) (Fig. 5b). To assess the rationality of intestinal flora sequenc - Results of intestinal flora diversity analysis ing of samples, we constructed rarefaction curves and Moreover, due to the close and inflammation-mediated ranked abundance curves of intestinal flora according relationship between gut microbiota (GMB) and NDs, to the OTU numbers at different sequencing depths. the change of intestinal flora of zebrafish is another The curve tends to be flat from 10,000 reads, indicat - important indicator for our determination. OTUs are ing that the sequencing data volume is adequate, and hypothetical computational taxa (e.g., strain, species, a greater data volume will only produce a small num- genus, group) that have been artificially established ber of new OTUs (Fig.  6a). A rank–abundance curve to facilitate the analysis of phylogenetic or population can be used to explain the abundance and evenness of genetics. Because of the conservatism of 16S rDNA, the species. In the horizontal direction, the abundance of sequence obtained by sequencing can represent a species. species is reflected by the width of the curve (i.e., the To understand the composition of a species in a popula- higher the abundance of species is, the wider the range tion sample, it is necessary to cluster the sequences. By of the curve is). The shape (smoothness) of the curve clustering, the sequence is divided into many groups reflects the evenness of species in the sample (i.e., the according to similarity, and one group is an OTU [53]. In flatter the curve, the more uniform the distribution of this study, 15 samples were investigated, and the number species). The results showed that the rank–abundance of OTUs received by each sample is shown in Fig. 5a. curve was smooth except for individual samples, indi- A Venn diagram was used to count the common cating that the species distribution of each sample was and unique OTU numbers of multiple samples, which even (Fig. 6b). can intuitively show the similarity and overlap of the Alpha-diversity can reflect the abundance and diver - OTU number composition of specific samples. Fig - sity of microbial communities, including the Chao ure  5b shows the differences in OTUs between the five index, Ace index, Shannon index, and Simpson index. groups. Different colors represent different groups, The Chao and Ace indices reflect the species rich - and the intersecting part is the OTU shared by adja- ness, i.e., the number of species in the sample, without cent groups. The OTUs in each group were as fol - considering the abundance of each species. Shannon lows: Group B1 (model) 235; Group B2 (control) 213; and Simpson indices reflect both species richness and Group B3 (25  mg/kg BTL-I + AlCl ) 233; Group B4 species evenness in the community. The comparison (50 mg/kg BTL-I + AlCl ) 229; and Group B5 (100 mg/ between all five groups showed that there was no sig - kg BTL-I + AlCl ) 216. The OTUs common between nificant total difference in bacterial diversity. However, the model group and the other groups were as follows: N ie et al. Journal of Neuroinflammation (2022) 19:39 Page 7 of 17 Fig. 4 The results of biochemical indices of zebrafish (n = 3). a GSH content in zebrafish brain tissue. b AChE activity in zebrafish brain tissue. c–f IL-1β and TNF-α content in zebrafish brain and peripheral tissue. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, vs. the control group; P < 0.05, ## ### #### P < 0.01, P < 0.005, P < 0.001, vs. the model group the Shannon and Simpson indices displayed relatively algorithms, Jaccard, Bray–Curtis, unweighted UniFrac, larger differences between the model and control and weighted UniFrac, were used for NMDS calculation. groups than between the control and administration The NMDS based on the Jaccard algorithm only consid - groups (Fig. 6c–f ). ers whether a specific OTU existed in the sample, not its Beta diversity analysis was performed to compare the abundance. The NMDS based on the Bray–Curtis algo - differences in species diversity of the paired samples. The rithm considers both OTU varieties and abundances in contents of each species in the samples were analyzed, samples. UniFrac analysis uses evolutionary informa- and the beta diversity values among different samples tion of sample sequences to compare whether the sam- were then calculated. ples have significant microbial community differences The NMDS method is a data analysis method that sim - in a particular evolutionary lineage. The unweighted plifies the research objects in multidimensional space UniFrac method only considers whether the specific to low-dimensional space for positioning, analysis and sequence appears in the community, not its abundance. classification while retaining the original relationship The weighted UniFrac method takes both existence between objects. The degree of difference between sam - and abundance into account. The results of the Jaccard, ples was reflected by the distance between points. Four unweighted UniFrac, and weighted UniFrac methods Nie et al. Journal of Neuroinflammation (2022) 19:39 Page 8 of 17 Fig. 5 Statistics of the OTUs. a Statistics of the OTUs of 15 samples. b Differences in the distribution of OTUs between groups using the Venn diagram. Group B1: model, Group B2: control, Group B3: 25 mg/kg BTL-I + AlCl , Group B4: 50 mg/kg BTL-I + AlCl , Group B5: 100 mg/kg BTL-I + AlCl 3 3 3 Fig. 6 Alpha-diversity. a The rarefaction curves. b The rank–abundance curves. c–f Alpha indices showed that there was no significant difference in OTU (Fig.  7a, c, d). However, the results of the Bray–Curtis varieties or evolutionary lineage between the experi- method showed that there were significant differences in mental groups, the control group and the model group OTU abundance between the model and control groups, N ie et al. Journal of Neuroinflammation (2022) 19:39 Page 9 of 17 Fig. 7 Beta diversity. a Jaccard algorithm (stress value = 0.137). Only the presence or absence of OTUs in the sample was considered, not the abundance. b Bray–Curtis algorithm (stress value = 0.096). Both the presence and absence of OTUs in the sample and the abundance were considered. c Unweighted-UniFrac algorithm (stress value = 0.064). It only considers whether the sequence was present in the community, not the abundance of the sequence. d Weighted-UniFrac algorithm (stress value = 0.069). It accounts for the abundance of sequences on the basis of unweighted UniFrac and was able to differentiate species abundance whereas there was no significant difference in OTU 100  mg/kg), the abundance of Firmicutes was signifi - abundance between the experimental groups (except for cantly increased, whereas those of Fusobacteria and the 50 mg/kg group) and the control group (Fig. 7b). Chlamydiae significantly decreased compared with that Microbial diversity analysis showed that the intestinal in the model group and basically returned to the same flora of zebrafish included the following 12 major phyla: level as that in the control group. However, in the BTL-I Proteobacteria, Firmicutes, Actinobacteria, Fusobacteria, treatment groups, there was almost no significant rever - Planctomycetes, Chlamydiae, Bacteroidetes, Chloroflexi, sal effect on the increase in Planctomycetes and Chloro - Tenericutes, Verrucomicrobia, Deinococcus-Thermus flexi abundance (Fig. 8c). and Saccharibacteria. Among them, Proteobacteria, At the genus level, a total of 30 major known taxa Firmicutes and Actinobacteria were the dominant bac- of intestinal flora were identified (Fig.  8b). The first 8 teria at the phylum level (Fig.  8a). The abundances of genera with intergroup abundance differences were Firmicutes in the gut of the model group were reduced, Bacillus, Bosea, Cetobacterium, Alpinimonas, Singuli- whereas those of Fusobacteria, Planctomycetes, Chlamy- sphaera, Phreatobacter, Mycobacterium, and Candida- diae and Chloroflexi significantly increased compared tus-Microthrix (Fig.  8d). Compared with the control with those observed in the control group. In the two group, the abundance of Bacillus in the model group experimental groups (administration of BTL-I 25 and significantly decreased, while those of the other seven Nie et al. Journal of Neuroinflammation (2022) 19:39 Page 10 of 17 Fig. 8 Relative abundance (a and b) and analysis of differential microorganisms (c and d). a and c Results at the phylum level. b and d Results at the # ## ### #### genus level. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, vs. the control group; P < 0.05, P < 0.01, P < 0.005, P < 0.001, vs. the model group genera mostly increased significantly. In the two BTL-I high-dose group (100 mg/kg BTL-I) (Gracilibacteraceae treatment groups (25 and 100  mg/kg), the abundance and Lutispora). of Bacillus was elevated to nearly normal levels, while those of Bosea and the other genera mostly decreased Prediction of ADMET and drug‑likeness properties significantly. Finally, the early evaluation of lead compound for its LEfSe was able to compare the taxonomic composi- potential to become a drug is critical step for drug devel- tion of multiple groups at different taxonomic levels, opment. To obtain more information on the pharmacoki- identify the taxa with significant intergroup differences netic profile of BTL-I and whether it has the potential to in abundance (i.e., biomarkers), and exhibit their line- become a drug, we used ADMETlab 2.0 [45] to predict age relationship. The results in Fig.  9 show the bio- its ADMET and drug-likeness properties. The corre - markers with significant effects (LDA scores > 2) in sponding predicted results are presented in Table  1, and each group, including 27 taxa in the model group (e.g., the physical properties of BTL-I are shown in Additional Cetobacterium in Fusobacteria, Bosea in Rhizobiales, file  1: Table S1. The results demonstrated that BTL-I pos - Chlamydiales, Candidatus-Microthrix, Mycobacte- sesses acceptable ADMET and drug-likeness properties rium), 11 taxa in the control group (e.g., Bacillus in Fir- in general. For example, the results showed that BTL-I micutes, Rhizobium rhizoryzae), 9 taxa in the low-dose is active in both human intestinal absorption (HIA) and group (25  mg/kg BTL-I) (e.g., Singulisphaera in Planc- blood–brain barrier (BBB) penetration. It has accept- tomycetes, Micromonospora), 10 taxa in the medium- able safety profiles, generally performing well on most dose group (50  mg/kg BTL-I) (e.g., Clostridiaceae, metrics (e.g., hERG blockers, Ames toxicity and carci- Chloroflexi, Pseudoxanthobacter), and 2 taxa in the nogenicity), and it is in harmony with the Lipinski rule N ie et al. Journal of Neuroinflammation (2022) 19:39 Page 11 of 17 Fig. 9 LDA effect size analysis. a Branching diagram of the evolution of different species between the control, model and experimental groups. b Bar graph of LDA values for different species Nie et al. Journal of Neuroinflammation (2022) 19:39 Page 12 of 17 Table 1 ADMET and drug-likeness properties of BTL-I through the online prediction tool ADMETlab 2.0 (the table is located below line 394) Property Value Decision Absorption Caco-2 permeability − 4.9 log cm/s Excellent Madin–Darby canine kidney cells (MDCK) permeability 2e-05 cm/s Excellent P-glycoprotein (Pgp)-inhibitor 0.023 Excellent P-glycoprotein (Pgp)-substrate 0.007 Excellent Human intestinal absorption (HIA) 0.008 Excellent 20% bioavailability (F20%) 0.059 Excellent Distribution Plasma protein binding (PPB) 0.987 Bad Volume distribution ( VD) 0.501 L/kg Excellent Blood–brain barrier (BBB) penetration 0.027 Excellent The fraction unbound in plasma (Fu) 0.012 Bad Metabolism CYP1A2-inhibitor 0.381 – CYP1A2-substrate 0.536 – CYP2C19-inhibitor 0.955 – CYP2C19-substrate 0.189 – CYP2C9-inhibitor 0.934 – CYP2C9-substrate 0.946 – CYP2D6-inhibitor 0.873 – CYP2D6-substrate 0.788 – CYP3A4-inhibitor 0.916 – CYP3A4-substrate 0.333 – Excretion Clearance 17.179 mL/min/kg Excellent The half-life ( T ) 0.371 – 1/2 Toxicity hERG blockers 0.023 Excellent Human hepatotoxicity (H-HT ) 0.402 Medium Drug-induced liver injury (DILI) 0.764 Bad Ames toxicity 0.106 Excellent Rat oral acute toxicity 0.559 Medium Maximum recommended daily dose (FDAMDD) 0.283 Excellent Skin sensitization 0.105 Excellent Carcinogenicity 0.162 Excellent Eye corrosion 0.003 Excellent Eye irritation 0.146 Excellent Respiratory toxicity 0.039 Excellent Drug-likeness MCE-18 [56] 68.839 Excellent Lipinski rule [53] Accepted (0 violation) Excellent Pfizer rule [54] Accepted (0 violation) Excellent Golden triangle [55] Accepted (0 violation) Excellent GSK rule [57] Rejected (1 violation) Bad N ie et al. Journal of Neuroinflammation (2022) 19:39 Page 13 of 17 [54] and others (such as the Pfizer rule [55] and golden activity against oxidative stress, neuroinflammation and triangle [56]), which indicates the drug-likeness proper- neuronal apoptosis, as well as in nerve growth without ties of a compound. Unfortunately, BTL-I displayed some inducing cytotoxicity [23–27]. Here, we found that BTL-I disadvantages, such as a high risk of inhibiting CYP2C19, also prevents cognitive deficits (induced in zebrafish by CYP2C9 and CYP3A4 and inducing liver injury. AlCl ) and exerts neuroprotective effects in this zebrafish model. In this study, we noted that the peripheral IL-1β level Discussion was suppressed while TNF-α level were elevated after Aluminum has been examined for its broad neurotoxic BTL-I administration. The different behaviors of the two effects and close relationship with AD, which promote inflammation markers are consistent with Tsarouchas’ tau hyperphosphorylation, aggregation, and neurofibril - report about the effects of spinal cord injury on zebrafish lary tangle formation in AD brains (by activating tau spinal axonal regeneration that increased TNF-α (mainly kinases CDK5 and GSK3β), accumulate in microglia produced by macrophage) and decreased IL-1β (mainly and induce proinflammatory cytokines, bind to Aβ and produced by neutrophil) both promote neurite regenera- induce its aggregation, stimulate iron-induced mem- tion in the periphery [65]. So, we presume that the upreg- brane lipid peroxidation and oxidative damage, decrease ulated peripheral TNF-α and downregulated IL-1β in the activity of antioxidant enzymes, and interact with BTL-I-treated zebrafish are actually consistent with the AChE on γ-peripheral sites to enhance enzymatic activ- improvement of cognition. However, further investiga- ity, resulting in reduced neurotransmission [12, 59, 60]. tion is necessary to reveal the exact mechanism. Furthermore, activated AChE can deteriorate Aβ aggre- Meanwhile, the inhibition of brain AChE elevates gation, decrease BDNF expression [59], and further pro- ACh levels and, hence, positively affects cognitive func - mote oxidative stress and neuroinflammation through a tion in rats [60, 66]. Subchronic exposure of zebrafish to ‘cholinergic anti-inflammatory pathway’ (CAIP) via α7 AlCl or i.p. injection of AlCl in mice enhances brain 3 3 nicotinic acetylcholine receptors [60, 61]. In addition, AChE activity [39]. Accordingly, our results show that in many reports on aluminum-induced AD or toxicity i.p. injection of AlCl also elevated AChE activity in the models, alterations in the host gut microbiota have been zebrafish brain, whereas BTL-I evoked neuroprotection observed [62, 63]. and lowered AChE activity (Fig.  4e). Because thin-layer Therefore, the present study established a zebrafish chromatography bioautography shows that BTL-I does model of subchronic inflammation induced by acute not inhibit AChE catalytic activity (data not shown), this i.p. AlCl administration, which resulted in subchronic compound seems to indirectly decrease zebrafish AChE peripheral and central inflammatory responses and activity here, likely involving other molecular pathways. enhanced oxidative stress and AChE activity in the brain. Furthermore, oxidative stress involves the excessive At the behavioral level, administration of AlCl strongly production of ROS and reactive nitrogen species (RNS) impaired the spatial and contextual memory of zebrafish [67] and may result in tissue damage. GSH is the most in the T-maze test. At the gut microbiota level, high- important nonenzymatic antioxidant, the neuroprotec- throughput sequencing results showed that the intesti- tive role of which in the brain is critical against oxidative nal flora of zebrafish was dramatically disturbed by acute damage caused by catecholamine oxidation or lipid per- AlCl administration. Collectively, these findings are oxidation [68]. In the present study, GSH levels markedly generally consistent with previous evidence that AlCl decreased 24  h after AlC l administration. With BTL-I induces memory deficits in both humans and animals, pretreatment at doses of 25 and 50 mg/kg, the GSH levels including zebrafish, and changes the intestinal flora [62– of the zebrafish were even lower than those of the model 64]. In contrast, BTL-I co-administration reversed these group. However, when the dose increased to 100 mg/kg, induced memory deficits and microbiota imbalances, the GSH content increased to the same level as that in the indicating the potential neuroprotective role of this drug. model group, although it was still lower than that in the In the present study, acute central and peripheral control group. BTL-I seemed to display doubtful antioxi- inflammation was characterized by the release of the dant effects via GSH. The lack of significant antioxidant proinflammatory cytokines IL-1β and TNF-α follow - effects of BTL-I relative to the model group may be due ing AlCl administration. Supplementation with BTL-I to the limited sample size and the resulting low statistical potently inhibited acute brain and peripheral inflam - power. However, it may also suggest that the antioxidant mation in AlCl -treated zebrafish. Mounting evidence mechanism of action of BTL-I may exist elsewhere. Fur- implicates BTL-I in multitargeted neuroprotective ther studies with larger sample sizes and better designs Nie et al. Journal of Neuroinflammation (2022) 19:39 Page 14 of 17 are warranted to test and explain this complicated phe- group, and the abundances of G bacteria were much nomenon to obtain a solid conclusion. lower. Additionally, in the low-dose group, another G In addition, the gut microbiota (GMB) plays a cru- bacterium, Micromonospora, in the phylum Actino- cial role in the stability and balance of the intestinal bacteria was recognized as a biomarker, suggesting its microecological environment, and the composition of possible positive role based on a report [16] on Actino- the human intestinal microbial community remains bacteria. Likewise, in the high-dose group, the G bac- basically stable after the age of 3  years [69]. In recent teria Lutispora in the family Gracilibacteriaceae and the years, a growing number of studies have shown that order Clostridiales were also biomarkers. Considering dysregulation of specific GMB is closely related to NDs the report on the strongly negative correlation of Clostri- [15, 16, 70]. Some reports have indicated that in the gut diaceae [16] with AD biomarkers in cerebrospinal fluid, of healthy humans or animals, there are higher popu- we speculate that Lutispora may also have some benefit lations of Gram-positive (G ) bacteria, including Fir- in neuroprotection. micutes and Actinobacteria, and lower populations of It is intriguing that the middle-dose group did not pos- Gram-negative (G ) bacteria, such as Bacteroidetes, at sess a high abundance of Bacillus. This discrepancy may the phylum level [15, 16]. At the family or genus level, be attributable to a nonlinear relationship of BTL-I and some G taxa, such as Bacillus, Eubacterium, Clostridi- Bacillus, and more concentration gradients of BTL-I will aceae in Firmicutes and Bifidobacterium in Actinobac - be set in future studies to explore this relationship. How- teria, show a higher abundance in healthy individuals ever, the G bacterium Clostridiaceae was found to be and benefit their hosts through different mechanisms, the key biomarker of this group; G bacterium Chloro- including reducing leakage of gut by the protection of flexi was another key biomarker, but bacteria in this phy - biofilms, inhibiting inflammation and antioxidation and lum have no LPS in their cell walls [72]. These findings reducing Aβ deposition and transfer from the gut to the may help to explain the behavioral improvement of this brain [15–17]. In contrast, some G taxa, such as Bac- group. teroides, Blautia, Escherichia coli, Shigella, Chlamydia, Our study suggests that administration of the marine and Fusobacterium, are closely and positively corre- fungal metabolite BTL-I prior to AlC l injection may aid lated with AD, mainly involving the activation of sys- in maintaining the predominance of beneficial G bacte- tematic inflammation by their enriched LPS in the cell ria in the gut of zebrafish to resist acute injury caused by wall and the invasion of proinflammatory cytokines, aluminum, related inflammation and AD pathology. We LPS, and even bacteria into the blood circulation sys- deduce that the stabilization of the GMB may contribute tem and brain, inducing Aβ deposition and tau phos- to the clearance of inflammation and lead to the improve - phorylation [15–17, 70]. ment of zebrafish’s cognition. However, the detailed In the present study, the control group zebrafish hosted mechanisms of intestinal flora regulation and the treat - a greater amount of G bacteria (Firmicutes at the phy- ment effect on an AlCl -induced chronic AD model need lum level and predominantly Bacillus at the genus level) to be further investigated for BTL-I in the future. than the AlC l -injured model group with memory Further, the early evaluation of ADMET and drug- impairment, while the model group zebrafish had much likeness properties of drug candidates are highly sig- fewer G bacteria than the control group zebrafish but nificant, as many drugs have been withdrawn in clinical significantly more G bacteria, including Cetobacterium trials and even in the marketing process due to unaccep- (in the family Fusobacteriaceae) and Chlamydiales (at the table pharmacokinetic properties [73–75]. Accordingly, order level). This result is highly consistent with previ - the prediction of ADMET and drug-likeness proper- ous studies, especially reports on the benefits of Bacil - ties of drug candidates has received extensive attention. lus subtilis in delaying neurodegeneration and behavior Numerous tools have been developed, such as ADMET- impairment in the AD model Caenorhabditis elegans and lab [76] admetSAR [77] and SwissADME [78]. In this reports on the negative effect of G bacteria, including study, in silico prediction with ADMETlab suggested Fusobacterium and Chlamydia [17, 70, 71]. that BTL-I caters to the majority of the ADMET proper- Generally, pretreatment with BTL-I maintained the ties and drug-likeness profiles, such as the typical Lipin - + − dominance of G bacteria vs. G bacteria in the gut of ski rule with 0 violation, and possesses good properties zebrafish when faced with aluminum exposure. How - in crossing the BBB. Such features render it a promis- ever, the dose levels exerted different influences. In the ing drug candidate for NDs since overcoming BBB pen- low- and high-dose groups, the abundances of Bacil- etration is essential for NDs drugs [79, 80]. It should be lus remained at high levels close to those of the control noted that BTL-I was predicted to be associated with a N ie et al. Journal of Neuroinflammation (2022) 19:39 Page 15 of 17 Zhanjiang City (XM-202008-01B1), Special Project in Key Fields of Guang- high risk of liver injury, which requires further confirma - dong Provincial Higher Education Institutions (Biomedicine and health care) tion and assessment and may provide clues for structural (2021ZDZX2064) and Guangdong Provincial Special Project in Science and optimization. In addition, it is predicted that CYP2C19, Technology (2021A50114). CYP2C9 and CYP3A4 may be inhibited by BTL-I and Availability of data and materials therefore co-administration with CYP2C19, CYP2C9 and All data generated or analyzed during this study are included in this published CYP3A4-substrate drugs may be avoided. article and its additional file. The datasets used and/or analyzed during the current study are available from the primary author on reasonable request. Conclusions Declarations This study showed that BTL-I dose-dependently ame - liorated AlCl -induced cognitive deficits in zebrafish, Ethics approval and consent to participate reversed the elevation of AlC l -induced central and The procedures were previously approved by the Animal Ethics Committee of Guangdong Ocean University, numbered 2019-11-23-2. peripheral proinflammatory cytokine levels and the increase in brain AChE activity, and contributed to main- Consent for publication taining the predominance of beneficial Gram-positive Not applicable. bacteria in the GMB of zebrafish, which was challenged Competing interests by AlCl . The in silico analysis indicated that BTL-I The authors declare that they have no competing interests. exhibits acceptable drug-likeness and ADMET profiles. Author details In summary, BTL-I has the potential as an intervention College of Food Science and Technology, Guangdong Provincial Key agent for preventing CNS deficits caused by inflamma - Laboratory of Aquatic Product Processing and Safety, Guangdong Provincial tion, neurotoxicity, and GMB imbalance. Engineering Laboratory for Marine Biological Products, Research Institute for Marine Drugs and Nutrition, Shenzhen Institute of Guangdong Ocean Uni- versity, Guangdong Ocean University, Zhanjiang 524088, China. Collaborative Innovation Center of Seafood Deep Processing, Dalian Polytechnic University, Abbreviations Dalian 116034, China. School of Pharmaceutical Science and Technology, AChE: Acetylcholinesterase; AD: Alzheimer’s disease; ADMET: Absorption, Tianjin University, Tianjin 30072, China. College of Pharmaceutical Sciences distribution, metabolism, excretion, and toxicity; AlCl : Aluminum trichloride; and Chinese Medicine, Southwest University, Chongqing 400715, China. Ural Aβ: β-Amyloid peptide; BTL-I: Butyrolactone I; BTLs: Butyrolactones; CAIP: Cho- Federal University, Ekaterinburg 620002, Russia. I nstitute of Translational linergic anti-inflammatory pathway; CDK 5: Cyclin-dependent kinase 5; CNS: Biomedicine, St. Petersburg State University, Saint Petersburg 199034, Russia. Central nervous system; COX-2: Cyclooxygenase-2; GMB: Gut microbiota; GSH: Glutathione; IL-1β: Interleukin-1β; KW: Kruskal–Wallis; LDA: Linear discriminant Received: 5 July 2021 Accepted: 20 January 2022 analysis; NDs: Neurodegenerative disorders; NMDS: Nonmetric multidimen- sional scaling graphs; PD: Parkinson’s disease; RNS: Reactive nitrogen species; ROS: Reactive oxygen species; TNF-α: Tumor necrosis factor-α. Supplementary Information References 1. Thomas B, Beal MF. Parkinson’s disease. Hum Mol Genet. 2007;16:R183–94. 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Journal of NeuroinflammationSpringer Journals

Published: Feb 7, 2022

Keywords: Inflammation; Oxidative stress; Butyrolactone I; Neurodegenerative diseases; Acetylcholinesterase; Intestinal flora

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