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The effect of a novel anticonvulsant chemical Q808 on gut microbiota and hippocampus neurotransmitters in pentylenetetrazole-induced seizures in rats

The effect of a novel anticonvulsant chemical Q808 on gut microbiota and hippocampus... Background: The gut microbiota can modulate brain function and behavior and is increasingly recognized as an important factor in mediating the risk of epilepsy and the effects of seizure interventions. Drug therapy is one of the factors that influence the composition of the intestinal microbiota. Q808 is an innovative chemical with strong anti‑ convulsant activity and low neurotoxicity. However, studies evaluating the effect of Q808 on gut microbial communi‑ ties are lacking. In this study, we aimed to evaluate the anticonvulsant activity of Q808 on a pentylenetetrazol (PTZ)— induced seizure model and analyze and compare the intestinal microbiota composition of non‑PTZ vehicle control group, the PTZ‑induced seizure model rats with and without Q808, through 16S rDNA sequencing. Neurotransmitter levels in the hippocampus were quantitatively estimated using HPLC–MS. Results: The results suggest that Q808 effectively alleviates seizures in chronic PTZ ‑kindled model rats. Additionally, based on the analyzed abundance of the gut microbiota, dysbacteriosis of model rats was found to be corrected after Q808 treatment at the phylum level. The unique bacterial taxa (e.g., Lactobacillus) that are associated with acetylcho‑ line production, were significantly increased. Several short ‑ chain fatty acids (SCFAs)‑producing bacteria, including Roseburia, Alloprevptella, Prevotellaceae_NK3B31_group, Prevotellaceae_UCG-001, and Prevotella_9, were enriched. In the hippocampus, the contents of acetylcholine increased, whereas the levels of 3‑methoxytyramine, glutamine, and 5‑hydroxyindole acetic acid (5‑HIAA) decreased after Q808 treatment. Conclusions: This study demonstrates that Q808 can be used to remodel the dysbiosis of the gut microbiome and influence neurotransmitter levels in the hippocampus of PTZ ‑induced seizure model rats. We hope that these novel findings prompt further research on the interaction between gut microbiota and seizures and the mechanism of Q808. Keywords: Seizure, Gut microbiota, Neurotransmitter, Q808 Background Epilepsy is a severe neurological disease that affects more than 50 million people of all ages worldwide, and an esti- *Correspondence: chenxjluedu@163.com; liwei611201@163.com mated 2.4 million patients are diagnosed with epilepsy Department of Pharmacology, College of Basic Medical Sciences, Jilin every year [1]. Epilepsy often requires lifelong medication University, Changchun, Jilin, China and places an enormous burden on individuals and soci- Academy of Chinese Medical Sciences of Jilin Province, Changchun, Jilin, China ety [2]. Full list of author information is available at the end of the article © The Author(s) 2022. 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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. Li et al. BMC Neuroscience (2022) 23:7 Page 2 of 9 Recent researchers have showed that dysbiosis is often anticonvulsant drugs and the gut microbiota. Lamo- found in conjunction with central nervous system (CNS) trigine, a traditional anticonvulsant drug, exhibits anti- diseases, such as Parkinson’s disease, multiple sclerosis, bacterial activity against gram-positive bacteria, such and epilepsy [3]. At the same time, there is mounting as Bacillus subtilis and Streptococcus faecalis [16]. Val- evidence that gut microbiota could affect the occurrence proate significantly increases the levels of Clostridium and development of CNS disease. For example, depletion sensu stricto 1 and Ruminiclostridium 5 and decreases of the gut microbiome caused by antibiotic treatment the relative abundance of S24-7 uncultbact [17]. A few or germ-free mice increases susceptibility to seizures cohort studies have reported the mild effects of antisei - and is associated with alterations in memory, sociabil- zure treatments, such as carbamazepine, on gut microbes ity, and cognition [4]. These behavioral alterations can be [18]. These studies highlight the need to consider drug- restored by recolonizing the complete microbiota or spe- induced changes in the gut microbiota. cific microbes [5]. Transferring the gut microbiota of a Q808 [6-(4-chlorophenoxy)-tetrazolo(5,1-a)phthala- person suffering from a CNS disease to animals through zine] is an innovative compound that has exhibited stool transplantation can facilitate the transfer of disease potent anticonvulsant activity in a maximal electroshock symptoms, such as depression [6]. Thus, utilizing the mouse seizure model and has been shown to provide gut microbiota to maintain brain function and behav- resistance against seizures induced by PTZ, ISO, THIO, ior is promising. Emerging studies have indicated that and 3-MP [19]. The chemical structure of Q808 is shown reconstruction of the gut microbiota can have a positive in Fig.  1. Our previous study showed that Q808 can effect on epileptic seizures [7]. For example, some stud - increase GABA levels in the hippocampus [20]. However, ies showed an almost 50% reduction in seizure frequency the level of other neurotransmitters and changing in the in patients with refractory epilepsy after probiotic treat- gut microbiota induced by Q808 remain unknown. The ment or ketogenic diet, which poses a significant effect current study aimed to sequence the 16S rDNA in the on imbalanced gut microbiota [8, 9]. Other studies sug- fecal contents of a PTZ-induced seizure model in rats to gested a beneficial effect of healthy donor fecal micro - understand the effects of Q808. Moreover, seizure lev - biota transplantation (FMT) in disease symptoms and els and neurotransmitter contents in the hippocampi of pathogenesis in epilepsy [10]. These animal studies and PTZ-induced model rats with and without Q808 were clinical cases have suggested a potential associations determined. between gut microbiome and epileptic seizures. Neurotransmitters play an important role in epilepsy. Methods For example, an imbalance of excitatory and inhibitory Experimental animals and drugs neurotransmitters may lead to epileptic seizures [11]. Gut A total of 50 male Wistar rats (body weight 180–200  g; microbes regulate central neurotransmitter metabolism age 7–8  weeks; SPF level) were purchased from Beijing directly or indirectly through host biosynthetic pathways Vital River Laboratory Animal Technology Co., Ltd., and can influence CNS diseases [12]. In a recent study, China. Two rats were housed per polycarbonate cage in the enrichment of Akkermansia and Parabacteroides were shown to affect hippocampal γ-aminobutyric acid (GABA)/glutamate ratios and restore protection from seizures [4]. Additionally, chronic treatment with Lacto- bacillus rhamnosus JB1 changes GABA mRNA expres- sion in the mouse brain [13], and acetate, the main metabolite produced by microbes, changes the contents of glutamine, glutamate, and GABA in the hypothalamus [14]. Overall, factors that can modify the construction of gut microbiota may have the potential to regulate neuro- transmitter levels in the brain and influence CNS disease. Pharmacological treatment, diet, and other external factors, such as infection and psychological and physi- cal stressors, can disturb the gut microbiota, thus can be associated with the risk of epilepsy [1]. A study of 1197 medications showed that nearly a quarter of non- antibiotic drugs across all classes inhibit the growth of at least one of the 40 known bacterial strains [15]. To date, Fig. 1 The chemical structure of Q808 few studies have investigated the relationship between Li  et al. BMC Neuroscience (2022) 23:7 Page 3 of 9 an SPF laboratory animal room under controlled condi- hippocampal samples were stored at − 80 °C for further tions (temperature: 25 ± 2 °C; humidity: 50–60%). All ani- analysis. mals were fed a standard diet and water ad libitum. After a 1-week adaptation period, the mice were randomly DNA isolation and quality measurement allocated to different research groups. Q808 was gifted to Total genomic DNA was extracted from the samples (wet us from the Academy of Chinese Medical Sciences of Jilin weight 120  mg) using a DNA Extraction Kit (Tiangen Province. The other drugs and reagents were purchased Company, Beijing, China) following the manufacturer’s from Sigma-Aldrich Chemical Company (St. Louis, MO, instructions. Quality and quantity control of DNA was USA). detected using a Nanodrop and agarose gel. PTZ kindling and seizure scoring PCR and 16S rDNA gene sequencing PTZ was freshly diluted in 0.9% NaCl, and a sub-convul- Extracted DNA was diluted to a concentration of 1  ng/ sive dose (35  mg/kg, intraperitoneal) was injected every μL and used as a template for PCR amplification of the other day for 28  days. One week after the last injection bacterial 16S rDNA gene with barcoded primers and of PTZ, it was again administered to the rats for another Takara Ex Taq. The primers 343F (5′-3′ TAC GGR AGG five consecutive days. Rat behaviors were evaluated CAG CAG) and 798R (5′-3′ AGG GTA TCT AAT CCT) blindly by observation for 30 min after the PTZ injection were used to amplify the V3-V4 variable region of the and scored according to Racine’s scale evaluation as fol- 16S rDNA genes. The PCR products of sterile water were lows: Phase 0: no evidence of convulsive activity; Phase used as negative controls for 16S rDNA sequencing. PCR 1: mouth and facial movements; Phase 2: head nodding; was performed using Bio-Rad. Cycling was performed Phase 3: facial twitching, forelimb clonus; Phase 4: gen- using the following parameters: 5 min at 94 °C, followed eralized clonic convulsions; Phase 5: falling and loss of by 26 cycles of denaturation (94  °C for 30  s), annealing righting reflex. The animals that were scored as phase (56 °C for 30 s), and extension (72 °C for 20 s), and a final 3, 4, or 5 for at least three consecutive evaluations were extension at 72 °C for 5 min. The samples were identified considered to be completely kindled [21]. by electrophoresis on a 1% agarose gel. The amplicons were sequenced using an Illumina MiSeq System (Illu- Drug administration and experimental design mina, California, USA). The rats were divided into two groups. The ten rats in the vehicle control group received saline. The remaining forty 16S rDNA microbial community analysis rats were injected intraperitoneally with 35  mg/kg PTZ Raw sequencing data were in FASTQ format. Paired- to establish an epileptic seizure model. After the model end reads were then obtained using Trimmomatic soft- was successfully established (success rate, 60%), the rats ware to detect and remove ambiguous bases, and were were randomly allocated into either the PTZ + vehicle merged using FLASH software. Sequence denoising was group or the PTZ + Q808 group (12 rats per group). The performed using QIIME software (version 1.8.0) with PTZ + Q808 group received Q808 over the following ambiguous reads, and homologous sequences, putative 28  days via oral gavage daily. The vehicle control group chimeric sequences, and sequences with < 200bps were and PTZ + vehicle group received the solvents of Q808 at removed. The sequences based on distance had a clus - the same time and same route as the PTZ + Q808 group. tering structure to generate operational taxonomic units Q808 drug solutions were freshly prepared with Tween- (OTUs) using Vsearch software according to 97% similar- 80 and 0.5% CMC-Na, and 30  mg/kg was administered ity. The representative reads of each OTU were selected to the rats via oral gavage daily. The body weights of the using the QIIME package. All representative reads were experimental animals were measured every other day. annotated and blasted against the Silva database Version 123 using the RDP classifier (70% confidence threshold). Fecal and hippocampal sample collection After the behavioral tests, fresh feces were collected from Estimation of neurotransmitter each animal. A sterile filter paper was placed on the test Levels of the main neurotransmitters, including GABA, bench. Rat was caught gently and feces were snapped glutamine, glutamate, 5-HIAA, normetanephrine, dopa- frozen in liquid nitrogen before storing at −  80  °C. The mine, histamine, norepinephrine, 3-methoxytyramine, animals were then humanely euthanized by CO inhala- acetylcholine, DOPA, epinephrine, 5-Hydroxy-l-tryp - tion. The animals were decapitated, and the heads were tophan, tyramine, and serotonin in the hippocampus transferred to dry ice. The hippocampi were immedi - were estimated using the LC–MS method with Agilent ately dissected and homogenized in nine volumes of cold 1290 Infinity LC (Agilent, USA) and 5500 QTRAP (AB saline to prepare 10% cerebral homogenates. All fecal and SCIEX, USA). A BEH C18 column (particle size 1.7  μm; Li et al. BMC Neuroscience (2022) 23:7 Page 4 of 9 Results 2.1 mm × 100 mm i.d.; Waters; Milford, USA) was used at Scores of PTZ kindling seizure model 45 °C. Samples were injected and chromatographic sepa- PTZ-induced seizure model rats were successfully estab- ration was achieved with a mobile phase that consisted of lished by repetitive administration of a sub-convulsive two solvent (A and B). Solvent A contained formic acid dose of PTZ for 28  days. The kindled rats were then (0.1%) and ammonium formate (25  mM), and solvent B administered Q808. PTZ was then injected again for consisted of acetonitrile containing 0.1% formic acid. 5  days, seizure intensity was scored according to the The gradient elution of solvent B was performed using Racine’s scale evaluation, and mean seizure phases were the following program: 0–18 min, 90–40%; 18–18.1 min, calculated (Fig.  2A). The data demonstrated that Q808 40%–90%; 18.1–23  min, 90%. The flow rate was set at significantly reduced the mean seizure score compared to 300 μL/min, and the injection volume was approximately the model group, indicating that Q808 had an excellent 2 μL. Mass spectrometry signals were collected using the anticonvulsant effect on the PTZ-induced seizure model. positive ion (ESI +) scanning mode. The main parameters No significant changes in the rat’s body weights were were as follows: source temperature, 450  °C; ion source observed during the PTZ injection and drug administra- gas 1 (Gas1), 60; ion source gas 2 (Gas2), 60; curtain gas, tion process (Fig. 2B). 30; and ion spray voltage floating, 5000  V. The multiple reaction monitoring mode was used. Statistical analysis Within‑sample microbial diversity in seizure models Prism version 5.00 (GraphPad Software, Inc., USA) was with and without Q808 used for analysis. The continuous variables such as body An average length of 411.81 reads per samples was weight was presented as the mean ± SEM values and obtained after filtering. Then all remaining reads were compared between groups using one-way ANOVA fol- clustered into 2988 OTUs with 97% sequence similarity. lowed by Tukey’s test. The neurotransmitter contents The Venn diagram shows 1567 OTUs in all groups, with were presented as the mean ± SEM values and analyzed 213, 201, and 232 OTUs unique to the control, model, using the Kruskal–Wallis and Dunn’s non-parametric and Q808 groups, respectively (Fig. 3A). Most rarefaction test. Seizure score (a discrete variable) was analyzed with curves, as shown in Additional file  1: Fig. S1, tended to Mann–Whitney U test. Indexes of α-diversity (Chao 1 reach the saturation plateau, indicating that the sequenc- index) were analyzed by the Kruskal–Wallis test. Differ - ing depth was sufficient to cover the entire bacterial ences in relative abundances of OTUs were calculated diversity. Specifically, microbiota profiles from the model using Tukey’s honest significance test by R package. group exhibited lower α-diversity than those from the β-diversity was calculated by the pMANOVA analysis control group when measured using the Chao index for based on Bray Curtis distances. Statistical significance evenness, and α-diversity was significantly elevated after was set at p < 0.05. treatment with Q808 (Fig. 3B). Fig. 2 Eec ff ts of Q808 on PTZ kindling‑induced A seizure stage and B body weight. Q808 effectively alleviates seizures in PTZ ‑kindling model rats (p = 0.003). The body weight of rats in each group have no difference. These data were shown as mean ± SEM and seizure score was analyzed with Mann–Whitney U test. **p < 0.01 Li  et al. BMC Neuroscience (2022) 23:7 Page 5 of 9 Fig. 3 A Venn diagram and B α‑ diversity analysis for control, model, and Q808 groups (n = 6). Venn diagram illustrates the average unique and common OTU numbers in three groups. The α‑ diversity (Chao 1 index) showed that the diversity of the gut microbial community was significantly decreased in model group compared with control (p = 0.021) and significantly increased in Q808 group compared with model group (p = 0.001). Differences in relative abundances of OTUs were calculated using Tukey’s honest significance test by R package. Indexes of α‑ diversity (Chao 1 index) were analyzed by the Kruskal–Wallis test. *p < 0.05, **p < 0.01 Alterations in gut microbiota composition Neurotransmitters in the hippocampus in the PTZ‑induced epileptic model before and after Q808 The level of 15 neurotransmitters were identified by treatment based on the 16S rDNA data LC–MS analysis. The extracted ion chromatograms 16S rDNA sequencing revealed distinct fecal microbiota of the neurotransmitter standards are shown in Addi- alterations depending on the presence of Q808 treat- tional file  1: Fig. S2. The metabolites were separated by ment. Unweighted UniFrac analysis, which focuses on chromatography, and each chromatographic peak was the diversity of gut microbiota (β-diversity), was used sharp and symmetrical, and could thus be used for mass to evaluate the differences in species complexity among spectrometry. the groups. As shown in Fig.  4A, the three-dimensional As shown in Fig.  5, the content of GABA decreased plots of unweighted UniFrac analysis showed an obvious significantly in the PTZ group compared with that difference in the gut microbial community composition in the vehicle control group. Acetylcholine, 3-meth- among the three groups. At the phylum level, reduced oxytyramine, glutamine, and 5-HIAA levels drastically relative abundance of Bacteroides, Epsilonbacteraeota, changed after Q808 administration. 3-methoxytyramine, and Proteobacteria and elevated abundance of Firmi- glutamine, and 5-HIAA levels decreased compared with cutes and Actinobacteria were observed in the epileptic those in the vehicle control group, whereas acetylcholine model group compared with the control group. However, levels increased compared with that in the PTZ group. dysbiosis of the gut microbiota in the epileptic model No significant differences were observed in the levels was reconstructed after treatment with Q808 (Fig.  4B). of normetanephrine, norepinephrine, glutamate, dopa- In other words, the levels of Bacteroides, Epsilonbacte- mine, histamine, and serotonin among the three groups. raeota, and Proteobacteria increased, whereas those of The contents of DOPA, epinephrine, tyramine, and Firmicutes and Actinobacteria decreased after the admin- 5-hydroxy-l-tryptophan were not detected. istration of Q808 for 28 days. Compared with the control group, the bacterial community profiles at the genus level Discussion showed that Ruminococcaceae_UCG-014 and Parasutte- The gut microbiota can modulate brain function and rella were highly enriched in the seizure model, whereas behaviors through the “microbiota-gut-brain” axis and Prevotella_9, Alloprevotella, Lactobacillus, and Roseburia may thus influence the onset of CNS diseases. Differ - were lower in the model group. After treatment with ent interventions, such as pharmacological treatments, Q808, the relative abundance of Prevotella_9, Alloprevo- can exert considerable effects on the gut microbiota. In tella, Lactobacillus, and Roseburia increased, whereas this paper, we reported alterations in the gut microbiota the levels of Ruminococcaceae_UCG-014 and Parasut- of PTZ-induced model rats before and after treatment terella significantly decreased (Fig.  4C, Additional file  1: with the novel anticonvulsant drug, Q808. The chemical Table S1). kindling of seizures triggered by PTZ has been used to Li et al. BMC Neuroscience (2022) 23:7 Page 6 of 9 Fig. 4 Alterations in the gut microbiota of PTZ‑induced seizure model rats depending on Q808 treatment. A Two ‑ dimensional principal coordinate analysis (PCoA) of the control, model, and Q808 groups. Each sample is marked by a dot. Samples that are more similar to one another are closer together. B Distribution of gut microbiota in the control, model, and Q808 groups at the phylum level. C Distribution of gut microbiota in the control, model, and Q808 groups at the genus level (n = 6). β‑ diversity was calculated by the pMANOVA analysis based on Bray Curtis distances reflect the pathogenesis of human epilepsy and is con - [1]. Supplementation with probiotics is frequently used sidered an appropriate model for drug-resistant epilepsy in clinical practice [23]. Therefore, normalization of the [21]. The effect of Q808 on other models of epilepsy, such intestinal microbiota is considered a novel treatment as kainic acid model, needs to be verified in the future. strategy for epileptic seizures. At the genus level, the 16S rDNA gene sequencing, a deep sequencing technol- abundance of different microbes changed in the model ogy, was used to analyze gut microbial communities. Our group. In a future study, we plan to determine whether rat model of PTZ-kindled seizures showed a decrease changes in the abundance of specific microbes in the in the relative abundance of Bacteroidetes and Proteo- model group can increase susceptibility to seizures and bacteria and an increase in the abundance of Firmicutes whether these changes are biomarkers of epilepsy. In and Actinobacteria, consistent with clinical reports on addition, the effect of discontinuation Q808 on seizures drug resistant epilepsy [22]. These four phyla are the should be investigated in a future study. main classes of microbiota, and an imbalance between Increasing evidence showed gut–brain interactions their compositions is associated with epileptogenesis in neurological diseases [24], but only few studies have Li  et al. BMC Neuroscience (2022) 23:7 Page 7 of 9 Fig. 5 Normalized intensity of neurotransmitters in the hippocampi of the rats in three groups (n = 6). In the seizure model group, the content of GABA was drastically decreased compared with that in control group (p = 0.011). After Q808 treatment, the content of acetylcholine increased (p = 0.002 compared with the PTZ group), whereas the levels of 3‑methoxytyramine, glutamine, and 5‑HIAA decreased (p = 0.003, p = 0.001, and p = 0.0004, separately, compared with the control group). No significant differences were observed in the levels of normetanephrine, norepinephrine, glutamate, dopamine, histamine, and serotonin among the three groups. Data were means ± SEM and analyzed using the Kruskal– Wallis and Dunn’s non‑parametric test. *p < 0.05, **p < 0.01, ***p < 0.001 deeply focused on the relationship between gut micro- bacteria, including Lactobacillus [12], were enriched. In biota and epilepsy. Q808 could be an effective scientific a recent study, probiotics such as Lactobacillus rham- tool to help determine the role of gut microbiota in epi- nosus GG (LGG) were found to positively affect intesti - lepsy. Q808 is an innovative anticonvulsant chemical nal permeability [26]. However, whether the use of Q808 with an international patent and is currently approved can restore intestinal permeability needs to be further for clinical trials. After the administration of Q808, PTZ- explored. Asano’s group have reported that the norepi- induced epileptic seizures were effectively controlled, and nephrine levels of the cecal contents were lower in the GF the composition of the gut microbiota were nearly nor- mice than in the SPF mice, suggesting that gut microbes mal at the phylum level. In other words, Q808 showed are a likely source for gut luminal norepinephrine [27]. anticonvulsant activity and restored the balance of the In this study, Q808 decreased the norepinephrine levels gut microbiota. The relative abundance of some probi - in the hippocampus, and low levels of norepinephrine is otics, such as Prevotella_9, Lactobacillus, and Roseburia associated with depression [28]. 5-HIAA is a metabolite increased after treatment with Q808. Whether Q808 of serotonin and is also associated with depression [29], exerts an antiepileptic effect on gut microbiota and and levels decreased after Q808 gavage in rats. These through the “microbiota-gut-brain” axis should be veri- findings suggest that the use of Q808 may increase sus - fied using the FMT method. ceptibility to depression. Therefore, the combination of Recent studies have reported that the gut microbiota Q808 and serotonin-norepinephrine reuptake inhibi- participates in the synthesis and metabolism of a wide tors may be a reasonable option for clinical use. In addi- range of neurotransmitters, including GABA and ace- tion to the above neurotransmitters, 3-methoxytyramine tylcholine [25], and neurotransmitters play an impor- (3-MT) and glutamine levels were altered after Q808 tant role in maintaining normal brain activity. In this treatment. 3-MT is a metabolite of dopamine and a novel study, we showed that the level of acetylcholine increases neuromodulator involved in movement control [30], and after treatment with Q808, and acetylcholine-producing glutamine is the precursor of glutamate [31]. The gut Li et al. BMC Neuroscience (2022) 23:7 Page 8 of 9 microbiome of patients with schizophrenia modulates Supplementary Information glutamine levels and schizophrenia-relevant behaviors in The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s12868‑ 022‑ 00690‑3. mice [32]. However, the specific gut microbes that pro - duce these neurotransmitters in epilepsy is unknown. Additional file 1: Fig. S1. The Shannon and Simpson indices. The major ‑ The associations between gut microbial structure and ity of the rarefaction curves tended to approach the saturation plateau, hippocampal neurotransmitter levels should be clarified suggesting that the sequencing depth of the gut microbiome was by FMT or specific microbe supplementation in future sufficient for each group. Fig. S2. Extracted ion chromatograms of the 15 neurotransmitter standards. The metabolites were separated by chroma‑ studies. Gut microbes may not be the only influence on tography, and each chromatographic peak was sharp and symmetrical. the response to neurotransmitter changes in the brain, Table S1. 16S rDNA sequencing results of rats in vehicle control, PTZ + although they are likely involved [12]. However, the rela- vehicle, and PTZ + Q808 group. tionship between gut microbiota and neurotransmitters after Q808 treatment remains unknown and needs to be Acknowledgements further explored. Not applicable. Metabolites contained within the transplant material, Authors’ contributions rather than the microbiome itself, can play a direct or XL and WL conceived the study, conceptualized the experimental design. XC indirect role in host behavior. SCFAs are the main metab- and WL provided the institutional infrastructure. QW, DW and DWZ conducted the experiments. DW, SCL and SWZ generated the figures and analyzed the olites produced by intestinal bacteria and are known to data. XL wrote the first draft and prepared the manuscript. All authors pro ‑ influence neuropsychiatric disorders. Animal studies vided critical revisions. All authors read and approved the final manuscript. have shown that SCFAs can cross the blood–brain bar- Funding rier and alter the levels of neurotransmitters, including This work was supported by China Postdoctoral Science Foundation glutamate, glutamine, and GABA, and enzymes involved (No. 2020M681051) and Education Department of Jilin Province (No. in the synthesis of neurotransmitters, and induce the JJKH20211224KJ ). transcription of neurotransmitters [33]. Interestingly, our Availability of data and materials results showed that the abundance of SCFA-producing The datasets used and/or analyzed during the current study are available from bacteria, such as Roseburia, Alloprevptella, Prevotella_9, the corresponding author on reasonable request. Prevotellaceae_NK3B31_group, and Prevotellaceae_ UCG-001 increased in the Q808 group. SCFAs exert an Declarations effect on appetite suppression through the vagus nerve, Ethics approval and consent to participate mediated by fatty acid receptor 3 [34]. Vagus nerve stim- This study was approved by the Institutional Animal Care and Use Committee ulation is an effective method to treat epilepsy. Thus, of the College of Basic Medical Science of Jilin University in accordance with international regulations. The study was carried out in compliance with the whether SCFAs provide protection against epilepsy ARRIVE guidelines. should be explored. Q808 may be a good medium for elu- cidating the role of SCFAs in epilepsy. Consent for publication Not applicable. Competing interests Conclusions The authors declare that they have no competing interests. In this study, we provided seminal evidence that Q808 Author details can reconstruct the composition of gut microbiota and Department of Pharmacology, College of Basic Medical Sciences, Jilin increase probiotic levels in the gut. The levels of neuro - University, Changchun, Jilin, China. Academy of Chinese Medical Sciences transmitters in the hippocampus changed after Q808 of Jilin Province, Changchun, Jilin, China. Jilin Cancer Hospital, Changchun, Jilin, China. treatment. Moreover, we found that microbial changes after treatment with Q808 were closely related to neu- Received: 10 August 2021 Accepted: 28 January 2022 rotransmitter production. Our findings provide a novel way to explore the mechanism of Q808 and provide novel insights into the relationship among microbiota, epileptic seizure, and anticonvulsant drugs. References 1. Lum GR, Olson CA, Hsiao EY. Emerging roles for the intestinal microbiome in epilepsy. Neurobiol Dis. 2020;135: 104576. Abbreviations 2. Lee H, Lee S, Lee DH, Kim DW. A comparison of the gut microbiota PTZ: Pentylenetetrazol; SCFAs: Short‑ chain fatty acids; CNS: Central nervous among adult patients with drug‑responsive and drug‑resistant epilepsy: system; GABA: γ‑Aminobutyric acid; 5‑HIAA: 5‑Hydroxyindole acetic acid. an exploratory study. Epilepsy Res. 2021;172: 106601. Li  et al. BMC Neuroscience (2022) 23:7 Page 9 of 9 3. Ma Q, Xing C, Long W, Wang HY, Liu Q, Wang RF. Impact of microbiota on with acute gastroenteritis treated with Lactobacillus rhamnosus GG: central nervous system and neurological diseases: the gut‑brain axis. J a randomized, double‑blind, placebo ‑ controlled trial. Clin Infect Dis. Neuroinflammation. 2019;16(1):53. 2014;58(8):1107–15. 4. Olson CA, Vuong HE, Yano JM, Liang QY, Nusbaum DJ, Hsiao EY. The gut 27. Asano Y, Hiramoto T, Nishino R, Aiba Y, Kimura T, Yoshihara K, et al. Criti‑ microbiota mediates the anti‑seizure effects of the ketogenic diet. Cell. cal role of gut microbiota in the production of biologically active, free 2018;173(7):1728‑41 e13. catecholamines in the gut lumen of mice. Am J Physiol Gastrointest Liver 5. Iannone LF, Preda A, Blottiere HM, Clarke G, Albani D, Belcastro V, et al. Physiol. 2012;303(11):G1288–95. Microbiota‑ gut brain axis involvement in neuropsychiatric disorders. 28. Wang YQ, Li R, Zhang MQ, Zhang Z, Qu WM, Huang ZL. The neurobiologi‑ Expert Rev Neurother. 2019;19(10):1037–50. cal mechanisms and treatments of REM sleep disturbances in depression. 6. Kelly JR, Borre Y, O’Brien C, Patterson E, El Aidy S, Deane J, et al. Transfer‑ Curr Neuropharmacol. 2015;13(4):543–53. ring the blues: depression‑associated gut microbiota induces neurobe ‑ 29. Dorelle LS, Da Cuña RH, Rey Vázquez G, Höcht C, Shimizu A, Genovese G, havioural changes in the rat. J Psychiatr Res. 2016;82:109–18. et al. The SSRI fluoxetine exhibits mild effects on the reproductive axis in 7. Arulsamy A, Tan QY, Balasubramaniam V, O’Brien TJ, Shaikh MF. Gut micro‑ the cichlid fish Cichlasoma dimerus ( Teleostei, Cichliformes). Chemos‑ biota and epilepsy: a systematic review on their relationship and possible phere. 2017;171:370–8. therapeutics. ACS Chem Neurosci. 2020;11(21):3488–98. 30. Ma SR, Yu JB, Fu J, Pan LB, Yu H, Han P, et al. Determination and applica‑ 8. Gomez‑Eguilaz M, Ramon‑ Trapero JL, Perez‑Martinez L, Blanco JR. The tion of nineteen monoamines in the gut microbiota targeting phenyla‑ beneficial effect of probiotics as a supplementary treatment in drug‑ lanine, tryptophan, and glutamic acid metabolic pathways. Molecules. resistant epilepsy: a pilot study. Benef Microbes. 2018;9(6):875–81. 2021. https:// doi. org/ 10. 3390/ molec ules2 60513 77. 9. Xie G, Zhou Q, Qiu CZ, Dai WK, Wang HP, Li YH, et al. Ketogenic diet poses 31. Pan L, Han P, Ma S, Peng R, Wang C, Kong W, et al. Abnormal metabo‑ a significant effect on imbalanced gut microbiota in infants with refrac‑ lism of gut microbiota reveals the possible molecular mechanism of tory epilepsy. World J Gastroenterol. 2017;23(33):6164–71. nephropathy induced by hyperuricemia. Acta pharmaceutica Sinica B. 10. Vendrik KEW, Ooijevaar RE, de Jong PRC, Laman JD, van Oosten BW, van 2020;10(2):249–61. Hilten JJ, et al. Fecal microbiota transplantation in neurological disorders. 32. Zheng P, Zeng B, Liu M, Chen J, Pan J, Han Y, et al. The gut microbiome Front Cell Infect Microbiol. 2020;10:98. from patients with schizophrenia modulates the glutamate‑ glutamine‑ 11. Akyuz E, Polat AK, Eroglu E, Kullu I, Angelopoulou E, Paudel YN. Revisiting GABA cycle and schizophrenia‑relevant behaviors in mice. Sci Adv. the role of neurotransmitters in epilepsy: an updated review. Life Sci. 2019;5(2):eaau8317. 2021;265: 118826. 33. Dalile B, Van Oudenhove L, Vervliet B, Verbeke K. The role of short‑ chain 12. Strandwitz P. Neurotransmitter modulation by the gut microbiota. Brain fatty acids in microbiota‑ gut‑brain communication. Nat Rev Gastroen‑ Res. 2018;1693(Pt B):128–33. terol Hepatol. 2019;16(8):461–78. 13. Bravo JA, Forsythe P, Chew MV, Escaravage E, Savignac HM, Dinan TG, 34. Bonaz B, Bazin T, Pellissier S. The vagus nerve at the interface of the et al. Ingestion of Lactobacillus strain regulates emotional behavior and microbiota‑ gut‑brain axis. Front Neurosci. 2018;12:49. central GABA receptor expression in a mouse via the vagus nerve. Proc Natl Acad Sci USA. 2011;108(38):16050–5. Publisher’s Note 14. Frost G, Sleeth ML, Sahuri‑Arisoylu M, Lizarbe B, Cerdan S, Brody L, et al. Springer Nature remains neutral with regard to jurisdictional claims in pub‑ The short‑ chain fatty acid acetate reduces appetite via a central homeo‑ lished maps and institutional affiliations. static mechanism. Nat Commun. 2014;5:3611. 15. Maier L, Pruteanu M, Kuhn M, Zeller G, Telzerow A, Anderson EE, et al. Extensive impact of non‑antibiotic drugs on human gut bacteria. Nature. 2018;555(7698):623–8. 16. Stokes JM, Davis JH, Mangat CS, Williamson JR, Brown ED. Discovery of a small molecule that inhibits bacterial ribosome biogenesis. Elife. 2014;3: e03574. 17. Barks A, Fretham SJB, Georgieff MK, Tran PV. Early‑life neuronal‑specific iron deficiency alters the adult mouse hippocampal transcriptome. J Nutr. 2018;148(10):1521–8. 18. Gomes IB, Simoes LC, Simoes M. The effects of emerging environmental contaminants on Stenotrophomonas maltophilia isolated from drinking water in planktonic and sessile states. Sci Total Environ. 2018;643:1348–56. 19. Günzel D, Yu AS. Claudins and the modulation of tight junction perme‑ ability. Physiol Rev. 2013;93(2):525–69. 20. Li X, Zhang HJ, Wang Q, Zhang DW, Wu D, Li W, et al. Current study of the mechanism of action of the potential anti‑ epileptic agent Q808. Molecules. 2017. https:// doi. org/ 10. 3390/ molec ules2 20711 34. 21. Löscher W. Animal models of seizures and epilepsy: past, present, and future role for the discovery of antiseizure drugs. Neurochem Res. 2017;42(7):1873–88. 22. Lindefeldt M, Eng A, Darban H, Bjerkner A, Zetterstrom CK, Allander T, Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : et al. The ketogenic diet influences taxonomic and functional composi‑ tion of the gut microbiota in children with severe epilepsy. NPJ Biofilms fast, convenient online submission Microbiomes. 2019;5:5. thorough peer review by experienced researchers in your field 23. Bagheri S, Heydari A, Alinaghipour A, Salami M. Eec ff t of probiotic supple ‑ mentation on seizure activity and cognitive performance in PTZ‑induced rapid publication on acceptance chemical kindling. Epilepsy Behav. 2019;95:43–50. support for research data, including large and complex data types 24. Cryan JF, O’Riordan KJ, Cowan CSM, Sandhu KV, Bastiaanssen • gold Open Access which fosters wider collaboration and increased citations TFS, Boehme M, et al. The microbiota‑ gut‑brain axis. Physiol Rev. 2019;99(4):1877–2013. maximum visibility for your research: over 100M website views per year 25. Borre YE, O’Keeffe GW, Clarke G, Stanton C, Dinan TG, Cryan JF. Microbiota and neurodevelopmental windows: implications for brain disorders. At BMC, research is always in progress. Trends Mol Med. 2014;20(9):509–18. Learn more biomedcentral.com/submissions 26. Sindhu KN, Sowmyanarayanan TV, Paul A, Babji S, Ajjampur SS, Priya‑ darshini S, et al. Immune response and intestinal permeability in children http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Neuroscience Springer Journals

The effect of a novel anticonvulsant chemical Q808 on gut microbiota and hippocampus neurotransmitters in pentylenetetrazole-induced seizures in rats

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Abstract

Background: The gut microbiota can modulate brain function and behavior and is increasingly recognized as an important factor in mediating the risk of epilepsy and the effects of seizure interventions. Drug therapy is one of the factors that influence the composition of the intestinal microbiota. Q808 is an innovative chemical with strong anti‑ convulsant activity and low neurotoxicity. However, studies evaluating the effect of Q808 on gut microbial communi‑ ties are lacking. In this study, we aimed to evaluate the anticonvulsant activity of Q808 on a pentylenetetrazol (PTZ)— induced seizure model and analyze and compare the intestinal microbiota composition of non‑PTZ vehicle control group, the PTZ‑induced seizure model rats with and without Q808, through 16S rDNA sequencing. Neurotransmitter levels in the hippocampus were quantitatively estimated using HPLC–MS. Results: The results suggest that Q808 effectively alleviates seizures in chronic PTZ ‑kindled model rats. Additionally, based on the analyzed abundance of the gut microbiota, dysbacteriosis of model rats was found to be corrected after Q808 treatment at the phylum level. The unique bacterial taxa (e.g., Lactobacillus) that are associated with acetylcho‑ line production, were significantly increased. Several short ‑ chain fatty acids (SCFAs)‑producing bacteria, including Roseburia, Alloprevptella, Prevotellaceae_NK3B31_group, Prevotellaceae_UCG-001, and Prevotella_9, were enriched. In the hippocampus, the contents of acetylcholine increased, whereas the levels of 3‑methoxytyramine, glutamine, and 5‑hydroxyindole acetic acid (5‑HIAA) decreased after Q808 treatment. Conclusions: This study demonstrates that Q808 can be used to remodel the dysbiosis of the gut microbiome and influence neurotransmitter levels in the hippocampus of PTZ ‑induced seizure model rats. We hope that these novel findings prompt further research on the interaction between gut microbiota and seizures and the mechanism of Q808. Keywords: Seizure, Gut microbiota, Neurotransmitter, Q808 Background Epilepsy is a severe neurological disease that affects more than 50 million people of all ages worldwide, and an esti- *Correspondence: chenxjluedu@163.com; liwei611201@163.com mated 2.4 million patients are diagnosed with epilepsy Department of Pharmacology, College of Basic Medical Sciences, Jilin every year [1]. Epilepsy often requires lifelong medication University, Changchun, Jilin, China and places an enormous burden on individuals and soci- Academy of Chinese Medical Sciences of Jilin Province, Changchun, Jilin, China ety [2]. 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. Li et al. BMC Neuroscience (2022) 23:7 Page 2 of 9 Recent researchers have showed that dysbiosis is often anticonvulsant drugs and the gut microbiota. Lamo- found in conjunction with central nervous system (CNS) trigine, a traditional anticonvulsant drug, exhibits anti- diseases, such as Parkinson’s disease, multiple sclerosis, bacterial activity against gram-positive bacteria, such and epilepsy [3]. At the same time, there is mounting as Bacillus subtilis and Streptococcus faecalis [16]. Val- evidence that gut microbiota could affect the occurrence proate significantly increases the levels of Clostridium and development of CNS disease. For example, depletion sensu stricto 1 and Ruminiclostridium 5 and decreases of the gut microbiome caused by antibiotic treatment the relative abundance of S24-7 uncultbact [17]. A few or germ-free mice increases susceptibility to seizures cohort studies have reported the mild effects of antisei - and is associated with alterations in memory, sociabil- zure treatments, such as carbamazepine, on gut microbes ity, and cognition [4]. These behavioral alterations can be [18]. These studies highlight the need to consider drug- restored by recolonizing the complete microbiota or spe- induced changes in the gut microbiota. cific microbes [5]. Transferring the gut microbiota of a Q808 [6-(4-chlorophenoxy)-tetrazolo(5,1-a)phthala- person suffering from a CNS disease to animals through zine] is an innovative compound that has exhibited stool transplantation can facilitate the transfer of disease potent anticonvulsant activity in a maximal electroshock symptoms, such as depression [6]. Thus, utilizing the mouse seizure model and has been shown to provide gut microbiota to maintain brain function and behav- resistance against seizures induced by PTZ, ISO, THIO, ior is promising. Emerging studies have indicated that and 3-MP [19]. The chemical structure of Q808 is shown reconstruction of the gut microbiota can have a positive in Fig.  1. Our previous study showed that Q808 can effect on epileptic seizures [7]. For example, some stud - increase GABA levels in the hippocampus [20]. However, ies showed an almost 50% reduction in seizure frequency the level of other neurotransmitters and changing in the in patients with refractory epilepsy after probiotic treat- gut microbiota induced by Q808 remain unknown. The ment or ketogenic diet, which poses a significant effect current study aimed to sequence the 16S rDNA in the on imbalanced gut microbiota [8, 9]. Other studies sug- fecal contents of a PTZ-induced seizure model in rats to gested a beneficial effect of healthy donor fecal micro - understand the effects of Q808. Moreover, seizure lev - biota transplantation (FMT) in disease symptoms and els and neurotransmitter contents in the hippocampi of pathogenesis in epilepsy [10]. These animal studies and PTZ-induced model rats with and without Q808 were clinical cases have suggested a potential associations determined. between gut microbiome and epileptic seizures. Neurotransmitters play an important role in epilepsy. Methods For example, an imbalance of excitatory and inhibitory Experimental animals and drugs neurotransmitters may lead to epileptic seizures [11]. Gut A total of 50 male Wistar rats (body weight 180–200  g; microbes regulate central neurotransmitter metabolism age 7–8  weeks; SPF level) were purchased from Beijing directly or indirectly through host biosynthetic pathways Vital River Laboratory Animal Technology Co., Ltd., and can influence CNS diseases [12]. In a recent study, China. Two rats were housed per polycarbonate cage in the enrichment of Akkermansia and Parabacteroides were shown to affect hippocampal γ-aminobutyric acid (GABA)/glutamate ratios and restore protection from seizures [4]. Additionally, chronic treatment with Lacto- bacillus rhamnosus JB1 changes GABA mRNA expres- sion in the mouse brain [13], and acetate, the main metabolite produced by microbes, changes the contents of glutamine, glutamate, and GABA in the hypothalamus [14]. Overall, factors that can modify the construction of gut microbiota may have the potential to regulate neuro- transmitter levels in the brain and influence CNS disease. Pharmacological treatment, diet, and other external factors, such as infection and psychological and physi- cal stressors, can disturb the gut microbiota, thus can be associated with the risk of epilepsy [1]. A study of 1197 medications showed that nearly a quarter of non- antibiotic drugs across all classes inhibit the growth of at least one of the 40 known bacterial strains [15]. To date, Fig. 1 The chemical structure of Q808 few studies have investigated the relationship between Li  et al. BMC Neuroscience (2022) 23:7 Page 3 of 9 an SPF laboratory animal room under controlled condi- hippocampal samples were stored at − 80 °C for further tions (temperature: 25 ± 2 °C; humidity: 50–60%). All ani- analysis. mals were fed a standard diet and water ad libitum. After a 1-week adaptation period, the mice were randomly DNA isolation and quality measurement allocated to different research groups. Q808 was gifted to Total genomic DNA was extracted from the samples (wet us from the Academy of Chinese Medical Sciences of Jilin weight 120  mg) using a DNA Extraction Kit (Tiangen Province. The other drugs and reagents were purchased Company, Beijing, China) following the manufacturer’s from Sigma-Aldrich Chemical Company (St. Louis, MO, instructions. Quality and quantity control of DNA was USA). detected using a Nanodrop and agarose gel. PTZ kindling and seizure scoring PCR and 16S rDNA gene sequencing PTZ was freshly diluted in 0.9% NaCl, and a sub-convul- Extracted DNA was diluted to a concentration of 1  ng/ sive dose (35  mg/kg, intraperitoneal) was injected every μL and used as a template for PCR amplification of the other day for 28  days. One week after the last injection bacterial 16S rDNA gene with barcoded primers and of PTZ, it was again administered to the rats for another Takara Ex Taq. The primers 343F (5′-3′ TAC GGR AGG five consecutive days. Rat behaviors were evaluated CAG CAG) and 798R (5′-3′ AGG GTA TCT AAT CCT) blindly by observation for 30 min after the PTZ injection were used to amplify the V3-V4 variable region of the and scored according to Racine’s scale evaluation as fol- 16S rDNA genes. The PCR products of sterile water were lows: Phase 0: no evidence of convulsive activity; Phase used as negative controls for 16S rDNA sequencing. PCR 1: mouth and facial movements; Phase 2: head nodding; was performed using Bio-Rad. Cycling was performed Phase 3: facial twitching, forelimb clonus; Phase 4: gen- using the following parameters: 5 min at 94 °C, followed eralized clonic convulsions; Phase 5: falling and loss of by 26 cycles of denaturation (94  °C for 30  s), annealing righting reflex. The animals that were scored as phase (56 °C for 30 s), and extension (72 °C for 20 s), and a final 3, 4, or 5 for at least three consecutive evaluations were extension at 72 °C for 5 min. The samples were identified considered to be completely kindled [21]. by electrophoresis on a 1% agarose gel. The amplicons were sequenced using an Illumina MiSeq System (Illu- Drug administration and experimental design mina, California, USA). The rats were divided into two groups. The ten rats in the vehicle control group received saline. The remaining forty 16S rDNA microbial community analysis rats were injected intraperitoneally with 35  mg/kg PTZ Raw sequencing data were in FASTQ format. Paired- to establish an epileptic seizure model. After the model end reads were then obtained using Trimmomatic soft- was successfully established (success rate, 60%), the rats ware to detect and remove ambiguous bases, and were were randomly allocated into either the PTZ + vehicle merged using FLASH software. Sequence denoising was group or the PTZ + Q808 group (12 rats per group). The performed using QIIME software (version 1.8.0) with PTZ + Q808 group received Q808 over the following ambiguous reads, and homologous sequences, putative 28  days via oral gavage daily. The vehicle control group chimeric sequences, and sequences with < 200bps were and PTZ + vehicle group received the solvents of Q808 at removed. The sequences based on distance had a clus - the same time and same route as the PTZ + Q808 group. tering structure to generate operational taxonomic units Q808 drug solutions were freshly prepared with Tween- (OTUs) using Vsearch software according to 97% similar- 80 and 0.5% CMC-Na, and 30  mg/kg was administered ity. The representative reads of each OTU were selected to the rats via oral gavage daily. The body weights of the using the QIIME package. All representative reads were experimental animals were measured every other day. annotated and blasted against the Silva database Version 123 using the RDP classifier (70% confidence threshold). Fecal and hippocampal sample collection After the behavioral tests, fresh feces were collected from Estimation of neurotransmitter each animal. A sterile filter paper was placed on the test Levels of the main neurotransmitters, including GABA, bench. Rat was caught gently and feces were snapped glutamine, glutamate, 5-HIAA, normetanephrine, dopa- frozen in liquid nitrogen before storing at −  80  °C. The mine, histamine, norepinephrine, 3-methoxytyramine, animals were then humanely euthanized by CO inhala- acetylcholine, DOPA, epinephrine, 5-Hydroxy-l-tryp - tion. The animals were decapitated, and the heads were tophan, tyramine, and serotonin in the hippocampus transferred to dry ice. The hippocampi were immedi - were estimated using the LC–MS method with Agilent ately dissected and homogenized in nine volumes of cold 1290 Infinity LC (Agilent, USA) and 5500 QTRAP (AB saline to prepare 10% cerebral homogenates. All fecal and SCIEX, USA). A BEH C18 column (particle size 1.7  μm; Li et al. BMC Neuroscience (2022) 23:7 Page 4 of 9 Results 2.1 mm × 100 mm i.d.; Waters; Milford, USA) was used at Scores of PTZ kindling seizure model 45 °C. Samples were injected and chromatographic sepa- PTZ-induced seizure model rats were successfully estab- ration was achieved with a mobile phase that consisted of lished by repetitive administration of a sub-convulsive two solvent (A and B). Solvent A contained formic acid dose of PTZ for 28  days. The kindled rats were then (0.1%) and ammonium formate (25  mM), and solvent B administered Q808. PTZ was then injected again for consisted of acetonitrile containing 0.1% formic acid. 5  days, seizure intensity was scored according to the The gradient elution of solvent B was performed using Racine’s scale evaluation, and mean seizure phases were the following program: 0–18 min, 90–40%; 18–18.1 min, calculated (Fig.  2A). The data demonstrated that Q808 40%–90%; 18.1–23  min, 90%. The flow rate was set at significantly reduced the mean seizure score compared to 300 μL/min, and the injection volume was approximately the model group, indicating that Q808 had an excellent 2 μL. Mass spectrometry signals were collected using the anticonvulsant effect on the PTZ-induced seizure model. positive ion (ESI +) scanning mode. The main parameters No significant changes in the rat’s body weights were were as follows: source temperature, 450  °C; ion source observed during the PTZ injection and drug administra- gas 1 (Gas1), 60; ion source gas 2 (Gas2), 60; curtain gas, tion process (Fig. 2B). 30; and ion spray voltage floating, 5000  V. The multiple reaction monitoring mode was used. Statistical analysis Within‑sample microbial diversity in seizure models Prism version 5.00 (GraphPad Software, Inc., USA) was with and without Q808 used for analysis. The continuous variables such as body An average length of 411.81 reads per samples was weight was presented as the mean ± SEM values and obtained after filtering. Then all remaining reads were compared between groups using one-way ANOVA fol- clustered into 2988 OTUs with 97% sequence similarity. lowed by Tukey’s test. The neurotransmitter contents The Venn diagram shows 1567 OTUs in all groups, with were presented as the mean ± SEM values and analyzed 213, 201, and 232 OTUs unique to the control, model, using the Kruskal–Wallis and Dunn’s non-parametric and Q808 groups, respectively (Fig. 3A). Most rarefaction test. Seizure score (a discrete variable) was analyzed with curves, as shown in Additional file  1: Fig. S1, tended to Mann–Whitney U test. Indexes of α-diversity (Chao 1 reach the saturation plateau, indicating that the sequenc- index) were analyzed by the Kruskal–Wallis test. Differ - ing depth was sufficient to cover the entire bacterial ences in relative abundances of OTUs were calculated diversity. Specifically, microbiota profiles from the model using Tukey’s honest significance test by R package. group exhibited lower α-diversity than those from the β-diversity was calculated by the pMANOVA analysis control group when measured using the Chao index for based on Bray Curtis distances. Statistical significance evenness, and α-diversity was significantly elevated after was set at p < 0.05. treatment with Q808 (Fig. 3B). Fig. 2 Eec ff ts of Q808 on PTZ kindling‑induced A seizure stage and B body weight. Q808 effectively alleviates seizures in PTZ ‑kindling model rats (p = 0.003). The body weight of rats in each group have no difference. These data were shown as mean ± SEM and seizure score was analyzed with Mann–Whitney U test. **p < 0.01 Li  et al. BMC Neuroscience (2022) 23:7 Page 5 of 9 Fig. 3 A Venn diagram and B α‑ diversity analysis for control, model, and Q808 groups (n = 6). Venn diagram illustrates the average unique and common OTU numbers in three groups. The α‑ diversity (Chao 1 index) showed that the diversity of the gut microbial community was significantly decreased in model group compared with control (p = 0.021) and significantly increased in Q808 group compared with model group (p = 0.001). Differences in relative abundances of OTUs were calculated using Tukey’s honest significance test by R package. Indexes of α‑ diversity (Chao 1 index) were analyzed by the Kruskal–Wallis test. *p < 0.05, **p < 0.01 Alterations in gut microbiota composition Neurotransmitters in the hippocampus in the PTZ‑induced epileptic model before and after Q808 The level of 15 neurotransmitters were identified by treatment based on the 16S rDNA data LC–MS analysis. The extracted ion chromatograms 16S rDNA sequencing revealed distinct fecal microbiota of the neurotransmitter standards are shown in Addi- alterations depending on the presence of Q808 treat- tional file  1: Fig. S2. The metabolites were separated by ment. Unweighted UniFrac analysis, which focuses on chromatography, and each chromatographic peak was the diversity of gut microbiota (β-diversity), was used sharp and symmetrical, and could thus be used for mass to evaluate the differences in species complexity among spectrometry. the groups. As shown in Fig.  4A, the three-dimensional As shown in Fig.  5, the content of GABA decreased plots of unweighted UniFrac analysis showed an obvious significantly in the PTZ group compared with that difference in the gut microbial community composition in the vehicle control group. Acetylcholine, 3-meth- among the three groups. At the phylum level, reduced oxytyramine, glutamine, and 5-HIAA levels drastically relative abundance of Bacteroides, Epsilonbacteraeota, changed after Q808 administration. 3-methoxytyramine, and Proteobacteria and elevated abundance of Firmi- glutamine, and 5-HIAA levels decreased compared with cutes and Actinobacteria were observed in the epileptic those in the vehicle control group, whereas acetylcholine model group compared with the control group. However, levels increased compared with that in the PTZ group. dysbiosis of the gut microbiota in the epileptic model No significant differences were observed in the levels was reconstructed after treatment with Q808 (Fig.  4B). of normetanephrine, norepinephrine, glutamate, dopa- In other words, the levels of Bacteroides, Epsilonbacte- mine, histamine, and serotonin among the three groups. raeota, and Proteobacteria increased, whereas those of The contents of DOPA, epinephrine, tyramine, and Firmicutes and Actinobacteria decreased after the admin- 5-hydroxy-l-tryptophan were not detected. istration of Q808 for 28 days. Compared with the control group, the bacterial community profiles at the genus level Discussion showed that Ruminococcaceae_UCG-014 and Parasutte- The gut microbiota can modulate brain function and rella were highly enriched in the seizure model, whereas behaviors through the “microbiota-gut-brain” axis and Prevotella_9, Alloprevotella, Lactobacillus, and Roseburia may thus influence the onset of CNS diseases. Differ - were lower in the model group. After treatment with ent interventions, such as pharmacological treatments, Q808, the relative abundance of Prevotella_9, Alloprevo- can exert considerable effects on the gut microbiota. In tella, Lactobacillus, and Roseburia increased, whereas this paper, we reported alterations in the gut microbiota the levels of Ruminococcaceae_UCG-014 and Parasut- of PTZ-induced model rats before and after treatment terella significantly decreased (Fig.  4C, Additional file  1: with the novel anticonvulsant drug, Q808. The chemical Table S1). kindling of seizures triggered by PTZ has been used to Li et al. BMC Neuroscience (2022) 23:7 Page 6 of 9 Fig. 4 Alterations in the gut microbiota of PTZ‑induced seizure model rats depending on Q808 treatment. A Two ‑ dimensional principal coordinate analysis (PCoA) of the control, model, and Q808 groups. Each sample is marked by a dot. Samples that are more similar to one another are closer together. B Distribution of gut microbiota in the control, model, and Q808 groups at the phylum level. C Distribution of gut microbiota in the control, model, and Q808 groups at the genus level (n = 6). β‑ diversity was calculated by the pMANOVA analysis based on Bray Curtis distances reflect the pathogenesis of human epilepsy and is con - [1]. Supplementation with probiotics is frequently used sidered an appropriate model for drug-resistant epilepsy in clinical practice [23]. Therefore, normalization of the [21]. The effect of Q808 on other models of epilepsy, such intestinal microbiota is considered a novel treatment as kainic acid model, needs to be verified in the future. strategy for epileptic seizures. At the genus level, the 16S rDNA gene sequencing, a deep sequencing technol- abundance of different microbes changed in the model ogy, was used to analyze gut microbial communities. Our group. In a future study, we plan to determine whether rat model of PTZ-kindled seizures showed a decrease changes in the abundance of specific microbes in the in the relative abundance of Bacteroidetes and Proteo- model group can increase susceptibility to seizures and bacteria and an increase in the abundance of Firmicutes whether these changes are biomarkers of epilepsy. In and Actinobacteria, consistent with clinical reports on addition, the effect of discontinuation Q808 on seizures drug resistant epilepsy [22]. These four phyla are the should be investigated in a future study. main classes of microbiota, and an imbalance between Increasing evidence showed gut–brain interactions their compositions is associated with epileptogenesis in neurological diseases [24], but only few studies have Li  et al. BMC Neuroscience (2022) 23:7 Page 7 of 9 Fig. 5 Normalized intensity of neurotransmitters in the hippocampi of the rats in three groups (n = 6). In the seizure model group, the content of GABA was drastically decreased compared with that in control group (p = 0.011). After Q808 treatment, the content of acetylcholine increased (p = 0.002 compared with the PTZ group), whereas the levels of 3‑methoxytyramine, glutamine, and 5‑HIAA decreased (p = 0.003, p = 0.001, and p = 0.0004, separately, compared with the control group). No significant differences were observed in the levels of normetanephrine, norepinephrine, glutamate, dopamine, histamine, and serotonin among the three groups. Data were means ± SEM and analyzed using the Kruskal– Wallis and Dunn’s non‑parametric test. *p < 0.05, **p < 0.01, ***p < 0.001 deeply focused on the relationship between gut micro- bacteria, including Lactobacillus [12], were enriched. In biota and epilepsy. Q808 could be an effective scientific a recent study, probiotics such as Lactobacillus rham- tool to help determine the role of gut microbiota in epi- nosus GG (LGG) were found to positively affect intesti - lepsy. Q808 is an innovative anticonvulsant chemical nal permeability [26]. However, whether the use of Q808 with an international patent and is currently approved can restore intestinal permeability needs to be further for clinical trials. After the administration of Q808, PTZ- explored. Asano’s group have reported that the norepi- induced epileptic seizures were effectively controlled, and nephrine levels of the cecal contents were lower in the GF the composition of the gut microbiota were nearly nor- mice than in the SPF mice, suggesting that gut microbes mal at the phylum level. In other words, Q808 showed are a likely source for gut luminal norepinephrine [27]. anticonvulsant activity and restored the balance of the In this study, Q808 decreased the norepinephrine levels gut microbiota. The relative abundance of some probi - in the hippocampus, and low levels of norepinephrine is otics, such as Prevotella_9, Lactobacillus, and Roseburia associated with depression [28]. 5-HIAA is a metabolite increased after treatment with Q808. Whether Q808 of serotonin and is also associated with depression [29], exerts an antiepileptic effect on gut microbiota and and levels decreased after Q808 gavage in rats. These through the “microbiota-gut-brain” axis should be veri- findings suggest that the use of Q808 may increase sus - fied using the FMT method. ceptibility to depression. Therefore, the combination of Recent studies have reported that the gut microbiota Q808 and serotonin-norepinephrine reuptake inhibi- participates in the synthesis and metabolism of a wide tors may be a reasonable option for clinical use. In addi- range of neurotransmitters, including GABA and ace- tion to the above neurotransmitters, 3-methoxytyramine tylcholine [25], and neurotransmitters play an impor- (3-MT) and glutamine levels were altered after Q808 tant role in maintaining normal brain activity. In this treatment. 3-MT is a metabolite of dopamine and a novel study, we showed that the level of acetylcholine increases neuromodulator involved in movement control [30], and after treatment with Q808, and acetylcholine-producing glutamine is the precursor of glutamate [31]. The gut Li et al. BMC Neuroscience (2022) 23:7 Page 8 of 9 microbiome of patients with schizophrenia modulates Supplementary Information glutamine levels and schizophrenia-relevant behaviors in The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s12868‑ 022‑ 00690‑3. mice [32]. However, the specific gut microbes that pro - duce these neurotransmitters in epilepsy is unknown. Additional file 1: Fig. S1. The Shannon and Simpson indices. The major ‑ The associations between gut microbial structure and ity of the rarefaction curves tended to approach the saturation plateau, hippocampal neurotransmitter levels should be clarified suggesting that the sequencing depth of the gut microbiome was by FMT or specific microbe supplementation in future sufficient for each group. Fig. S2. Extracted ion chromatograms of the 15 neurotransmitter standards. The metabolites were separated by chroma‑ studies. Gut microbes may not be the only influence on tography, and each chromatographic peak was sharp and symmetrical. the response to neurotransmitter changes in the brain, Table S1. 16S rDNA sequencing results of rats in vehicle control, PTZ + although they are likely involved [12]. However, the rela- vehicle, and PTZ + Q808 group. tionship between gut microbiota and neurotransmitters after Q808 treatment remains unknown and needs to be Acknowledgements further explored. Not applicable. Metabolites contained within the transplant material, Authors’ contributions rather than the microbiome itself, can play a direct or XL and WL conceived the study, conceptualized the experimental design. XC indirect role in host behavior. SCFAs are the main metab- and WL provided the institutional infrastructure. QW, DW and DWZ conducted the experiments. DW, SCL and SWZ generated the figures and analyzed the olites produced by intestinal bacteria and are known to data. XL wrote the first draft and prepared the manuscript. All authors pro ‑ influence neuropsychiatric disorders. Animal studies vided critical revisions. All authors read and approved the final manuscript. have shown that SCFAs can cross the blood–brain bar- Funding rier and alter the levels of neurotransmitters, including This work was supported by China Postdoctoral Science Foundation glutamate, glutamine, and GABA, and enzymes involved (No. 2020M681051) and Education Department of Jilin Province (No. in the synthesis of neurotransmitters, and induce the JJKH20211224KJ ). transcription of neurotransmitters [33]. Interestingly, our Availability of data and materials results showed that the abundance of SCFA-producing The datasets used and/or analyzed during the current study are available from bacteria, such as Roseburia, Alloprevptella, Prevotella_9, the corresponding author on reasonable request. Prevotellaceae_NK3B31_group, and Prevotellaceae_ UCG-001 increased in the Q808 group. SCFAs exert an Declarations effect on appetite suppression through the vagus nerve, Ethics approval and consent to participate mediated by fatty acid receptor 3 [34]. Vagus nerve stim- This study was approved by the Institutional Animal Care and Use Committee ulation is an effective method to treat epilepsy. Thus, of the College of Basic Medical Science of Jilin University in accordance with international regulations. The study was carried out in compliance with the whether SCFAs provide protection against epilepsy ARRIVE guidelines. should be explored. Q808 may be a good medium for elu- cidating the role of SCFAs in epilepsy. Consent for publication Not applicable. Competing interests Conclusions The authors declare that they have no competing interests. In this study, we provided seminal evidence that Q808 Author details can reconstruct the composition of gut microbiota and Department of Pharmacology, College of Basic Medical Sciences, Jilin increase probiotic levels in the gut. The levels of neuro - University, Changchun, Jilin, China. Academy of Chinese Medical Sciences transmitters in the hippocampus changed after Q808 of Jilin Province, Changchun, Jilin, China. Jilin Cancer Hospital, Changchun, Jilin, China. treatment. Moreover, we found that microbial changes after treatment with Q808 were closely related to neu- Received: 10 August 2021 Accepted: 28 January 2022 rotransmitter production. Our findings provide a novel way to explore the mechanism of Q808 and provide novel insights into the relationship among microbiota, epileptic seizure, and anticonvulsant drugs. References 1. Lum GR, Olson CA, Hsiao EY. Emerging roles for the intestinal microbiome in epilepsy. Neurobiol Dis. 2020;135: 104576. Abbreviations 2. Lee H, Lee S, Lee DH, Kim DW. A comparison of the gut microbiota PTZ: Pentylenetetrazol; SCFAs: Short‑ chain fatty acids; CNS: Central nervous among adult patients with drug‑responsive and drug‑resistant epilepsy: system; GABA: γ‑Aminobutyric acid; 5‑HIAA: 5‑Hydroxyindole acetic acid. an exploratory study. Epilepsy Res. 2021;172: 106601. Li  et al. BMC Neuroscience (2022) 23:7 Page 9 of 9 3. Ma Q, Xing C, Long W, Wang HY, Liu Q, Wang RF. Impact of microbiota on with acute gastroenteritis treated with Lactobacillus rhamnosus GG: central nervous system and neurological diseases: the gut‑brain axis. J a randomized, double‑blind, placebo ‑ controlled trial. Clin Infect Dis. Neuroinflammation. 2019;16(1):53. 2014;58(8):1107–15. 4. Olson CA, Vuong HE, Yano JM, Liang QY, Nusbaum DJ, Hsiao EY. The gut 27. Asano Y, Hiramoto T, Nishino R, Aiba Y, Kimura T, Yoshihara K, et al. Criti‑ microbiota mediates the anti‑seizure effects of the ketogenic diet. Cell. cal role of gut microbiota in the production of biologically active, free 2018;173(7):1728‑41 e13. catecholamines in the gut lumen of mice. Am J Physiol Gastrointest Liver 5. Iannone LF, Preda A, Blottiere HM, Clarke G, Albani D, Belcastro V, et al. Physiol. 2012;303(11):G1288–95. Microbiota‑ gut brain axis involvement in neuropsychiatric disorders. 28. Wang YQ, Li R, Zhang MQ, Zhang Z, Qu WM, Huang ZL. The neurobiologi‑ Expert Rev Neurother. 2019;19(10):1037–50. cal mechanisms and treatments of REM sleep disturbances in depression. 6. Kelly JR, Borre Y, O’Brien C, Patterson E, El Aidy S, Deane J, et al. Transfer‑ Curr Neuropharmacol. 2015;13(4):543–53. ring the blues: depression‑associated gut microbiota induces neurobe ‑ 29. Dorelle LS, Da Cuña RH, Rey Vázquez G, Höcht C, Shimizu A, Genovese G, havioural changes in the rat. J Psychiatr Res. 2016;82:109–18. et al. The SSRI fluoxetine exhibits mild effects on the reproductive axis in 7. Arulsamy A, Tan QY, Balasubramaniam V, O’Brien TJ, Shaikh MF. Gut micro‑ the cichlid fish Cichlasoma dimerus ( Teleostei, Cichliformes). Chemos‑ biota and epilepsy: a systematic review on their relationship and possible phere. 2017;171:370–8. therapeutics. ACS Chem Neurosci. 2020;11(21):3488–98. 30. Ma SR, Yu JB, Fu J, Pan LB, Yu H, Han P, et al. Determination and applica‑ 8. Gomez‑Eguilaz M, Ramon‑ Trapero JL, Perez‑Martinez L, Blanco JR. The tion of nineteen monoamines in the gut microbiota targeting phenyla‑ beneficial effect of probiotics as a supplementary treatment in drug‑ lanine, tryptophan, and glutamic acid metabolic pathways. Molecules. resistant epilepsy: a pilot study. Benef Microbes. 2018;9(6):875–81. 2021. https:// doi. org/ 10. 3390/ molec ules2 60513 77. 9. Xie G, Zhou Q, Qiu CZ, Dai WK, Wang HP, Li YH, et al. Ketogenic diet poses 31. Pan L, Han P, Ma S, Peng R, Wang C, Kong W, et al. Abnormal metabo‑ a significant effect on imbalanced gut microbiota in infants with refrac‑ lism of gut microbiota reveals the possible molecular mechanism of tory epilepsy. World J Gastroenterol. 2017;23(33):6164–71. nephropathy induced by hyperuricemia. Acta pharmaceutica Sinica B. 10. Vendrik KEW, Ooijevaar RE, de Jong PRC, Laman JD, van Oosten BW, van 2020;10(2):249–61. Hilten JJ, et al. Fecal microbiota transplantation in neurological disorders. 32. Zheng P, Zeng B, Liu M, Chen J, Pan J, Han Y, et al. The gut microbiome Front Cell Infect Microbiol. 2020;10:98. from patients with schizophrenia modulates the glutamate‑ glutamine‑ 11. Akyuz E, Polat AK, Eroglu E, Kullu I, Angelopoulou E, Paudel YN. Revisiting GABA cycle and schizophrenia‑relevant behaviors in mice. Sci Adv. the role of neurotransmitters in epilepsy: an updated review. Life Sci. 2019;5(2):eaau8317. 2021;265: 118826. 33. Dalile B, Van Oudenhove L, Vervliet B, Verbeke K. The role of short‑ chain 12. Strandwitz P. Neurotransmitter modulation by the gut microbiota. Brain fatty acids in microbiota‑ gut‑brain communication. Nat Rev Gastroen‑ Res. 2018;1693(Pt B):128–33. terol Hepatol. 2019;16(8):461–78. 13. Bravo JA, Forsythe P, Chew MV, Escaravage E, Savignac HM, Dinan TG, 34. Bonaz B, Bazin T, Pellissier S. The vagus nerve at the interface of the et al. Ingestion of Lactobacillus strain regulates emotional behavior and microbiota‑ gut‑brain axis. Front Neurosci. 2018;12:49. central GABA receptor expression in a mouse via the vagus nerve. Proc Natl Acad Sci USA. 2011;108(38):16050–5. Publisher’s Note 14. Frost G, Sleeth ML, Sahuri‑Arisoylu M, Lizarbe B, Cerdan S, Brody L, et al. Springer Nature remains neutral with regard to jurisdictional claims in pub‑ The short‑ chain fatty acid acetate reduces appetite via a central homeo‑ lished maps and institutional affiliations. static mechanism. Nat Commun. 2014;5:3611. 15. Maier L, Pruteanu M, Kuhn M, Zeller G, Telzerow A, Anderson EE, et al. Extensive impact of non‑antibiotic drugs on human gut bacteria. Nature. 2018;555(7698):623–8. 16. Stokes JM, Davis JH, Mangat CS, Williamson JR, Brown ED. Discovery of a small molecule that inhibits bacterial ribosome biogenesis. Elife. 2014;3: e03574. 17. Barks A, Fretham SJB, Georgieff MK, Tran PV. Early‑life neuronal‑specific iron deficiency alters the adult mouse hippocampal transcriptome. J Nutr. 2018;148(10):1521–8. 18. Gomes IB, Simoes LC, Simoes M. The effects of emerging environmental contaminants on Stenotrophomonas maltophilia isolated from drinking water in planktonic and sessile states. Sci Total Environ. 2018;643:1348–56. 19. Günzel D, Yu AS. Claudins and the modulation of tight junction perme‑ ability. Physiol Rev. 2013;93(2):525–69. 20. Li X, Zhang HJ, Wang Q, Zhang DW, Wu D, Li W, et al. Current study of the mechanism of action of the potential anti‑ epileptic agent Q808. Molecules. 2017. https:// doi. org/ 10. 3390/ molec ules2 20711 34. 21. Löscher W. Animal models of seizures and epilepsy: past, present, and future role for the discovery of antiseizure drugs. Neurochem Res. 2017;42(7):1873–88. 22. Lindefeldt M, Eng A, Darban H, Bjerkner A, Zetterstrom CK, Allander T, Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : et al. The ketogenic diet influences taxonomic and functional composi‑ tion of the gut microbiota in children with severe epilepsy. NPJ Biofilms fast, convenient online submission Microbiomes. 2019;5:5. thorough peer review by experienced researchers in your field 23. Bagheri S, Heydari A, Alinaghipour A, Salami M. Eec ff t of probiotic supple ‑ mentation on seizure activity and cognitive performance in PTZ‑induced rapid publication on acceptance chemical kindling. Epilepsy Behav. 2019;95:43–50. support for research data, including large and complex data types 24. Cryan JF, O’Riordan KJ, Cowan CSM, Sandhu KV, Bastiaanssen • gold Open Access which fosters wider collaboration and increased citations TFS, Boehme M, et al. The microbiota‑ gut‑brain axis. Physiol Rev. 2019;99(4):1877–2013. maximum visibility for your research: over 100M website views per year 25. Borre YE, O’Keeffe GW, Clarke G, Stanton C, Dinan TG, Cryan JF. Microbiota and neurodevelopmental windows: implications for brain disorders. At BMC, research is always in progress. Trends Mol Med. 2014;20(9):509–18. Learn more biomedcentral.com/submissions 26. Sindhu KN, Sowmyanarayanan TV, Paul A, Babji S, Ajjampur SS, Priya‑ darshini S, et al. Immune response and intestinal permeability in children

Journal

BMC Neuroscience – Springer Journals

Published: Feb 3, 2022

Keywords: Seizure; Gut microbiota; Neurotransmitter; Q808

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