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Indole alkaloids of Alstonia scholaris (L.) R. Br. alleviated nonalcoholic fatty liver disease in mice fed with high-fat diet

Indole alkaloids of Alstonia scholaris (L.) R. Br. alleviated nonalcoholic fatty liver disease in... Alstonia scholaris (L.) R. Br (Apocynaceae) is a well‑ documented medicinal plant for treating respiratory diseases, liver diseases and diabetes traditionally. The current study aimed to investigate the effects of TA on non‑alcoholic fatty liver disease (NAFLD). A NAFLD model was established using mice fed a high‑fat diet (HFD) and administered with TA (7.5, 15 and 30 mg/kg) orally for 6 weeks. The biochemical parameters, expressions of lipid metabolism‑related genes or proteins were analyzed. Furthermore, histopathological examinations were evaluated with Hematoxylin–Eosin and MASSON staining. TA treatment significantly decreased the bodyweight of HFD mice. The concentrations of low‑ density lipoprotein (LDL), triglyceride ( TG), aspartate aminotransferase (AST ) and alanine aminotransferase (ALT ) were also decreased significantly in TA‑treated mice group, accompanied by an increase in high‑ density lipoprotein (HDL). Furthermore, TA alleviated hepatic steatosis injury and lipid droplet accumulation of liver tissues. The liver mRNA levels involved in hepatic lipid synthesis such as sterol regulatory element‑binding protein 1C (SREBP-1C), regulators of liver X receptor α (LXRα), peroxisome proliferator activated receptor (PPAR)γ, acetyl‑ CoA carboxylase (ACC1) and stearyl coenzyme A dehydrogenase‑1 (SCD1), were markedly decreased, while the expressions involved in the regulation of fatty acid oxidation, PPARα, carnitine palmitoyl transterase 1 (CPT1A), and acyl coenzyme A oxidase 1 (ACOX1) were increased in TA‑treated mice. TA might attenuate NAFLD by regulating hepatic lipogenesis and fatty acid oxidation. Keywords: Hepatic disease, Hepatic lipogenesis, Fatty acid oxidation 1 Introduction and hepatic parenchymal cell steatosis that is not the Non-alcoholic fatty liver disease (NAFLD) is a chronic result of excessive alcohol consumption [2]. Obesity and hepatic damaging disease and includes fatty liver, non- an excessively high-fat diet (HFD) are important factors alcoholic steatohepatitis (NASH), related cirrhosis and involved in NAFLD pathogenesis [3]. The prevalence of hepatocellular carcinoma [1]. NFALD is characterized by NAFLD in relation to a HFD is as high as 90% in severely serious lipid accumulation, irregular hepatic vein array obese individuals [4]. NAFLD is not only associated with metabolic diseases, such as insulin resistance, obe- sity, diabetes, and hyperlipidemia [5, 6], but also closely *Correspondence: wenxue_wang@163.com; xdluo@mail.kib.ac.cn; Jiawei_ related to cardiovascular disease [7], which can seriously Geng@kmust.edu.cn Shui‑Fen Sun and Hui‑ Jie Zhong contributed equally to this work harm the health of patients. It is also closely related to Department of Infectious Disease and Hepatic Disease, First People’s hepatocyte injury, which is clinically evaluated via aspar- Hospital of Yunnan Province, Affiliated Hospital of Kunming University tate aminotransferase (AST) and alanine aminotrans- of Science and Technology, Kunming 650032, Yunnan, China State Key Laboratory of Phytochemistry and Plant Resources in West ferase (ALT) levels [8]. China, Kunming Institute of Botany, Chinese Academy of Sciences, Lipid accumulation in the livers of patients with Kunming 650201, People’s Republic of China NAFLD usually originates from an imbalance between 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/. Sun et al. Natural Products and Bioprospecting (2022) 12:14 Page 2 of 11 lipogenesis and lipolysis [9]. Therapeutic drugs for (RT = 44.915  min), picrinine (RT = 74.29  min), and the NAFLD sufferers not only remain scarce, but also show contents were 5.26%, 1.13%, 13.91%, 17.39%, respectively. some side effects. For example, pioglitazone, a PPARγ ligand, induces weight gain, fluid retention, osteopenia 2.2 TA treatment decreased HFD‑induced weight‑gaining and increased fracture risk, especially in older women After 8 weeks of HFD administration, the HFD-fed mice [10, 11]. Obeticholic acid, a synthetic ligand that activat- (31.92 ± 0.62  g) were obviously overweight with the ing farnesoid X receptor (FXR), causes both pruritus and growth rate of 21.46% compared to the normal diet-fed moderate increases in low-density lipoprotein (LDL) cho- mice (26.28 ± 0.45  g, Table  1). However, HFD-induced lesterol in 25 mg per day doses (NCT02548351) [10, 11]. weight gain was significantly attenuated in TA (30) dose u Th s, exploring novel drugs for NAFLD management by group (27.88 ± 0.59  g, P < 0.01) and the increase ampli- inhibiting the activity of transcription factors related to tude was 12.65%. Silymarin, a routinely used clinical drug lipid synthesis has considerable therapeutic potential. for fatty liver and hepatitis, also improved HFD-induced Traditional Chinese medicines for NAFLD has weight-gaining. attracted increasing attention in recent decades due to their few adverse effects, proven curative effect and ther - 2.3 TA treatment improved plasma lipid profiles apeutic mechanisms or benefits [12, 13]. Alstonia schola- of HFD‑fed mice ris is traditionally used to treat respiratory diseases, liver As shown in Table  2, HFD-fed mice showed higher TG diseases and diabetes in China and Malaysia [14, 15]. The (from 137.60 ± 24.01 to 222.20 ± 41.27  μg/mL) and LDL chemical components of different parts of the herb were (from 96.39 ± 8.18 to 113.70 ± 7.02  μg/mL) levels com- intensively investigated by our research group [16–37]. pared with the control mice. However, TA (30) treatment More than 100 indole alkaloids were reported, and TA significantly reduced TG (133.50 ± 21.04  μg/mL) and was proved to be the major pharmacological constituents LDL (84.62 ± 2.82  μg/mL) levels in HFD-fed mice. Fur- of A. scholaris in preventing respiratory diseases by us thermore, HDL levels of serum in mice were significantly [38–48]. Both preclinical investigation [49–51] and clini- decreased after 8 weeks HFD intaking (from 90.72 ± 2.08 cal trials [52, 53] have confirmed that TA was safe for fur - to 73.89 ± 5.34 μg/mL, P < 0.05), which increased partially ther clinical trials. Besides, the metabolism of TA in rats after TA (30) treatment (83.53 ± 1.87  μg/mL). The treat - indicated that scholaricine-type alkaloids could get into ment of silymarin recovered LDL levels (90.01 ± 13.13 μg/ circulation more readily than the other types [54]. Hou mL) and decreased HDL (75.28 ± 2.61  μg/mL), but had et  al., reported akuammidine, (E)-alstoscholarine, and no significant on TG (232.10 ± 21.50  μg/mL, P > 0.05). (Z)-alstoscholarine from TA with nuclear factor-kappa B These results suggest that TA treatment can restore nor - inhibition [55]. And Shang et al., verified the anti-inflam - mal blood lipid profiles in mice with HFD-intaking. Of mation activity of TA in mice [56]. Furtherly, Zhao et al., note, the reduction of TG in TA (30) group was better indicated that TA had an inhibitory effect on airway than that in the positive control group (P < 0.05). inflammation via regulating the balance of oxidation and anti-oxidation [42]. In addition, A. scholaris decoction is 2.4 TA treatment improves plasma aminotransferase used for treating diabetes, hypertension and malaria [14]. profiles in HFD‑fed mice The treatment of NAFLD, a disease related to inflam - Following, the plasma levels of AST and ALT were tested mation and oxidation assumed that TA might have a to investigate the effects of TA on liver function in mice therapeutic effect on NAFLD, together with its folk use in with HFD. As shown in Table 3, the ALT levels increased treating hepatopathy [57–59], then we undertook a phar- significantly from 0.05 ± 0.01 ng/mL of the control group macological evaluation of TA on NAFLD in mice induced to 0.33 ± 0.04  ng/mL of the HFD group (P < 0.01). Simi- by high fat diet and explored the potential molecular larly, the AST levels also increased significantly from mechanism. 0.09 ± 0.02  ng/mL to 0.13 ± 0.02  ng/mL (P < 0.01). How- ever, these increases were significantly attenuated by the TA treatment (P < 0.05/0.01). Interestingly, TA (30) dose- induced improvement in ALT (0.06 ± 0.01  ng/mL) was 2 Results better than that of silymarin (0.11 ± 0.01 ng/mL, P < 0.05), 2.1 HPL C profile and main constituent contents of TA which was an excellent elicitor of liver function repair in The main chemical constituents in the TA were separated clinical practice. Interestingly, TA-induced improvement and identified by high-performance liquid chromatog - (0.09 ± 0.03  ng/mL) in AST also exceeded that induced raphy. Briefly, four chemical components (Fig.  1) were by silymarin (0.10 ± 0.01 ng/mL). Therefore, these results identified as the major medicinal agents in TA, includ - suggest that TA treatment has a significant impact on ing scholaricine (retention time [RT] = 22.057  min), liver injury induced by HFD. 19-epischolarine (RT = 23.667  min), vallesamine Sun  et al. Natural Products and Bioprospecting (2022) 12:14 Page 3 of 11 Fig. 1 Quantification and identification of TA by HPLC analysis. A Structures of four major alkaloids from A. scholaris; B HPLC chromatographic profile of total alkaloids and UV spectra of four major alkaloids Table 1 Eec ff ts of TA on body weight (BW) in HFD ‑fed mice at liver tissues of HFD-fed mice showed an obviously rough six weeks of treatment surface, whereas the liver tissues of normal mice showed a grayish red and glossy surface (Fig.  2B). Interestingly, Group Weight (average ± SEM) high-dose TA (30) treatment improved the HFD-induced roughness and redness of the liver surface. Control 26.28 ± 0.45 We also performed H&E staining to further confirm the ▲▲ HFD 31.92 ± 0.62 TA-induced histopathological improvement of the livers. HFD + TA (7.5) 29.48 ± 0.53 As seen in Fig.  2C, normal diet mice showed complete HFD + TA (15) 31.16 ± 0.28 hepatic lobule structures, regular hepatic vein array, and **/# HFD + TA (30) 27.88 ± 0.59 clear cellular structure profiles. In HFD-fed mice, how - HFD + Silymarin 21.12 ± 1.05 ever, the hepatic lobule structure disappeared entirely, Note: BW was measured in g. Control: mice were fed a normal diet and treated hepatic vein array was irregular, and the cell shape and with 0.5% CMC-Na (solvent) via gavage; HFD: mice were fed HFD; HFD + TA (7.5), nucleus were blurry. Such liver damage could contribute HFD + TA (15), and HFD + TA (30): mice were fed HFD and treated with TA (7.5, 15, and 30 mg/kg, respectively) via gavage; HFD + Silymarin: mice were fed HFD to the loss of normal lipid metabolic function including ▲▲ ** and treated with silymarin via gavage. P < 0.01 vs. Control; P < 0.01 vs. HFD # lipid synthesis and lipolysis. Furthermore, inflamma - group. P < 0.05 vs. HFD + Silymarin group tory cells penetrated the hepatocyte intervals. Of note, whereas, TA treatment partially repaired the hepatic lob- 2.5 TA treatment alleviates HFD‑induced liver injury ule structure and vein array, especially in the high-dose After HFD administration, mice showed obvious signs of TA (30) group. Thus, high-dose TA (30) administration obesity, i.e., 21.46% overweight (Table  1) and greasy hair not only repaired the hepatic lobule structure and vein (Fig. 2A). Of note, TA treatment markedly improved hair array, but also induced lipid droplets to disappear. These condition, especially in the high-dose TA (30) group. The TA-induced improvements were comparable to those Sun et al. Natural Products and Bioprospecting (2022) 12:14 Page 4 of 11 Table 2 Eec ff ts of TA treatment on blood lipid profiles in HFD ‑fed mice Group TG (average ± SEM, μg/mL) HDL (average ± SEM, μg/mL) LDL (average ± SEM, μg/mL) Control 137.60 ± 24.01 90.72 ± 2.08 96.39 ± 8.18 ▲▲ ▲ ▲ HFD 222.20 ± 41.27 73.89 ± 5.34 113.70 ± 7.02 HFD + TA (7.5) 193.90 ± 18.88 65.72 ± 5.33 98.35 ± 7.55 HFD + TA (15) 207.00 ± 8.88 75.86 ± 7.55 94.47 ± 0.60 **/# * HFD + TA (30) 133.50 ± 21.04 83.53 ± 1.87 84.62 ± 2.82 ▲ ▲ HFD + Silymarin 232.10 ± 21.50 75.28 ± 2.61 90.01 ± 13.13 TG, HDL, and LDL were measured in μg/mL. Control: mice were fed a normal diet and treated with 0.5%CMC-Na (solvent) via gavage; HFD: mice were fed HFD; HFD + TA (7.5), HFD + TA (15), and HFD + TA (30) mice were fed HFD and treated with TA (7.5, 15, and 30 mg/kg, respectively) via gavage; HFD + Silymarin: mice were ▲/▲▲ */** # fed HFD and treated with silymarin via gavage. P < 0.05/0.01 vs. Control; P < 0.05/0.01 vs. HFD group. P < 0.05 vs. HFD + Silymarin group Table 3 Eec ff ts of TA treatment on blood aminotransferase potential [42, 47, 56]. Therefore, we speculated that levels in HFD‑fed mice TA may have a positive effect on NAFLD and prevent disease progression. As expected, TA treatment sig- Group ALT (average ± SEM, AST ng/mL) (average ± SEM, nificantly attenuated the expression levels of SREBP- ng/mL) 1C, ACC1, and SCD1 in a dose-dependent manner (Fig.  3A–C). Interestingly, TA-induced attenuations of Control 0.05 ± 0.01 0.09 ± 0.02 ▲▲ ▲▲ ACC1 and SCD1 were more significant, compared with HFD 0.33 ± 0.04 0.13 ± 0.02 * * silymarin (Fig.  3B, C, P < 0.01/0.001). Other contribu- HFD + TA (7.5) 0.19 ± 0.04 0.09 ± 0.01 ** * tors to lipid synthesis, namely PPARγ and LXRα, also HFD + TA (15) 0.13 ± 0.02 0.08 ± 0.02 showed a significant decrease in mRNA levels, even **/# * HFD + TA (30) 0.06 ± 0.01 0.09 ± 0.03 not in a dose-dependent manner. These results con- ** HFD + Silymarin 0.11 ± 0.01 0.10 ± 0.01 firm that TA treatment reduces lipid synthesis molec- ALT and AST were measured in ng/mL. Control: mice were fed a normal diet ular signaling, especially under high dose conditions and treated with 0.5%CMC-Na (solvent) via gavage; HFD: mice were fed HFD; HFD + TA (7.5), HFD + TA (15), and HFD + TA (30) mice were fed HFD and treated (30 mg/kg). with TA (7.5, 15, and 30 mg/kg, respectively) via gavage; HFD + Silymarin: mice PPARα, ACOX1, and CPT1A maintain lipid metabo- ▲▲ */** were fed HFD and treated with silymarin via gavage. P < 0.01 vs. Control; lism and restrain excessive accumulation of lipids in P < 0.05/0.01 vs. HFD group. P < 0.05 vs. HFD + Silymarin group the liver [62–65]. Here, the mRNA levels of PPARα and ACOX1 decreased markedly following HFD; how- of silymarin (Fig.  2C). We also applied Masson staining ever, TA treatment restored the mRNA expression lev- to liver tissue to assess the effects of TA treatment on els dose dependently (Fig. 3F, G). Although TA (30) did hepatic fibrosis. As expected, TA treatment remarkably not show the best performance, all doses of TA induced decreased HFD-induced collagen accumulation (Fig. 2D). recovery of CPT1A mRNA expression. Of note, the These results strongly suggest that TA possesses consid - positive control silymarin did not rescue mRNA levels erable potential in clinical treatment of NFALD. of PPARα and CPT1A (Fig. 3F and H, P > 0.05). The dif - ferent performance between silymarin and TA in liver 2.6 TA treatment alters mRNA levels of hepatic fatty acid lipid metabolism suggests they may regulate NFALD metabolism‑regulating genes in HFD‑fed mice progression via different molecular mechanisms. As shown in Fig.  3, HFD administration induced high To confirm the TA-induced molecular signaling of mRNA levels of SREBP-1C, ACC1 SCD1 and PPARγ, lipid metabolism, we applied protein immunoblotting which are key mediators of lipid synthesis [5, 60, 61]. In to examine the protein levels of SREBP-1C and PPARα addition, LXRα, a key mediator of lipid transport, also genes (Fig.  3I). As expected, TA treatment inhibited showed a high mRNA level in HFD-fed mice (Fig.  3E). the expressions of SREBP-1C protein and increased The expressions of signaling proteins, including PPARα, respectively, that both were induced by HFD PPARα, ACOX1 and CPT1A, were decreased in clinical administration (Fig.  3I). Our investigations revealed NAFLD samples. We observed similar results in the a mRNA-matched protein levels of SREBP-1C and NAFLD mouse model (Fig. 3F-H), thus supporting the PPARα genes, that further confirmed TA management reliability of our model. We previously reported that regulates lipid metabolism-molecular signaling during TA possesses anti-pulmonary fibrosis and pneumonia NFALD progression. Sun  et al. Natural Products and Bioprospecting (2022) 12:14 Page 5 of 11 Fig. 2 Eec ff t of TA on general observation and histopathologic examination. A general observation of mice and livers (B), HE (C) and Masson (D) staining of liver tissues. Control: mice were fed anormal diet and treated with 0.5%CMC‑Na (solvent) via gavage; HFD: mice were fed HFD; HFD + TA (7.5), HFD + TA (15), and HFD + TA (30) mice were fed a HFD and treated with TA (7.5, 15, and 30 mg/kg, respectively) via gavage; HFD + Silymarin: mice were fed HFD and treated with silymarin via gavage 3 Discussion and significantly increased the accumulation of lipid A diet high in fat is considered the main cause of NAFLD, droplets in the liver. Using this animal model, we inves- and then HFD-fed animal models are often used for stud- tigated whether TA treatment can improve HFD-induced ies on NAFLD [66]. In the current research, the body- NAFLD and which lipid metabolism signaling was weight of mice increased significantly after 8  weeks of involved in the disease progression. HFD administration. The HFD not only increased plasma Lipid metabolism disorders can cause excessive TG content, but also caused lipid metabolism disorders hepatic lipid accumulation. Previous studies have Sun et al. Natural Products and Bioprospecting (2022) 12:14 Page 6 of 11 Fig. 3 Eec ff t of TA on Lipid metabolism‑related gene and protein expressions in liver tissues of HFD ‑fed mice. mRNA levels of SREBP-1C (A), ACC1 (B), SCD1 (C), PPARγ (D), LXRα (E), PPARα (F), ACOX1 (G), and CPT1A (H); I Protein expressions of SREBP-1C and PPARα. Control: mice were fed a normal diet and treated with 0.5%CMC‑Na (solvent) via gavage; HFD: mice were fed HFD; HFD + TA (7.5), HFD + TA (15), and HFD + TA (30) mice were fed HFD and treated with TA (7.5, 15, and 30 mg/kg, respectively) via gavage; HFD + Silymarin: mice were fed HFD and treated with silymarin via gavage. ▲/▲▲/▲▲▲ */**/*** #/##/### P < 0.05/0.01/0.001 vs. control group; P < 0.05/0.01/0.001 vs. HFD group; P < 0.05/0.01/0.001 vs. HFD + Silymarin group shown that lipid synthesis and uptake-related genes are SCD1 are not only significantly less obese than their up-regulated, while genes involved in lipid degrada- control counterparts, but also lean and hypermetabolic tion and secretion are down-regulated in NAFLD [62, [5]. In this study, the HFD increased the mRNA levels 65, 67]. Sterol regulatory element-binding protein 1C of LXRα, SREBP-1C, ACC1, and SCD1 and the SREBP- (SREBP-1C), a key transcription factor of lipogenesis, 1C protein in liver tissues. These results are consistent is activated by upstream regulators of liver X receptor with other studies that describe SREBP activation as α (LXRα), acetyl-CoA carboxylase (ACC1) and fatty essential for hypertriglyceridemia [68]. Our research acid synthase (FAS). In NAFLD patients, successive revealed that TA obtained from the leaves of A. scho- SREBP-1C activation originating from ACC1 and FAS laris not only inhibited the synthesis of TG, but also can induce hepatic lipid accumulation [61]. Hepatic decreased the mRNA levels of LXRα, SREBP-1C, ACC1, stearoyl-CoA desaturase 1 (SCD1) catalyzes the bio- and SCD1 and protein level of SREBP-1C in a dose- synthesis of monounsaturated fatty acids. Mice lacking dependent manner. Sun  et al. Natural Products and Bioprospecting (2022) 12:14 Page 7 of 11 Furthermore, high levels of PPAR gamma (PPARγ), preventing cardiovascular diseases [73]. We found that a transcriptional modulator of adipocyte development HFD significantly increased the plasma concentrations in all types of adipose tissue, promote lipid accumula- of TG and LDL and decreased the plasma concentration tion [60]. As such, we tested the effects of TA on PPARγ of HDL, which were, in turn, effectively controlled by expression and found that HFD-induced PPARγ acti- TA treatment. Therefore, our research indicates that TA vation was also inhibited. Collectively, these results administration may decrease the risk of cardiovascular suggest that TA alleviates hepatic fat accumulation in disease in obese people. NAFLD by restraining liver lipid synthesis and adipocyte In conclusion, our study demonstrated that TA treat- development. ment can successfully ameliorate NAFLD by reducing the Maintaining liver lipid homeostasis, such as up- expression of the key transcriptional factors SREBP-1C as regulating fatty acid oxidation (FAO), is essential for well as the lipogenic enzymes ACC-1, PPARγ, LXRα and reducing liver damage resulting from redundant lipid SCD-1. Meanwhile, it upregulated the expression of the accumulation [69]. Fatty acid oxidation mainly occurs in lipolytic enzyme CPT1A, PPARα and ACOX1, which are the mitochondria with the involvement of peroxisome involved in fatty acid oxidation in liver tissues. Therefore, proliferator-activated receptor α (PPARα) [70]. Previous TA have a therapeutic effect on NAFLD through regulat - studies have shown that PPARα knockout mice exhibit ing hepatic lipogenesis and fatty acid oxidation. severe hepatic steatosis accompanied by a decrease in fatty acid uptake and oxidation [71]. The translocation of 4 Materials and methods fatty acid into the mitochondria is dependent on carni- 4.1 Preparation of total alkaloids tine palmitoyl transferase 1A (CPT1A), which is located A. scholaris leaves were collected in 2018 in Pu’er city in the mitochondrial outer membrane [63]. Clinical and (Yunnan Province, China) and identified by Dr. Xiao- animal studies have confirmed that PPARα , acyl coen- Dong Luo, Kunming Institute of Botany, Chinese Acad- zyme A oxidase 1 (ACOX1), and CPT1A are significantly emy of Sciences (Kunming, China). A voucher specimen down-regulated in NAFLD liver [62, 64, 65]. In the cur- (Luo20180105) was deposited in the State Key Laboratory rent study, we observed that a HFD altered the expres- of Phytochemistry and Plant Resources in West China, sion levels of PPARα and downstream targets CPT1A and Chinese Academy of Sciences, Kunming, China. The ACOX1. Compared with the control group, the HFD- dried and powdered leaves of A. scholaris were extracted fed mice showed a significant decrease in the expres - with 90% EtOH under reflux conditions (3  h X 4), and sion levels of lipid synthesis-inhibiting genes, including the solvent was evaporated in vacuo to obtain ethanolic PPARα, CPT1A, and ACOX1. These results suggest that extract. Next, the ethanolic extract was dissolved in 0.3% lipid oxidation is blocked in HFD-fed mice. When HFD aqueous HCl solution and filtered. The acidic solution group mice were treated with TA, the expression levels of was adjusted to pH 9–10 with 10% aqueous ammonia PPARα, CPT1A and ACOX1 increased dose dependently. and was extracted with EtOAc to obtain TA fraction, in These results indicate that TA may repair lipid metabo - which picrinine (17.39%), vallesamine (13.91%), scholari- lism balance in liver tissues of HFD-fed mice by unlock- cine (5.26%), and 19-epischolaricine (1.13%) were quanti- ing lipid oxidation. fied by HPLC with four standard compounds. Chronic lipid accumulation eventually triggers oxi- dative stress and hepatic injury, which are normally 4.2 Chemicals described by AST and ALT values in clinical diagnosis Silymarin was purchased from Madaus AG (Cologne, [8]. Our previous studies confirmed that the alkaloid Germany). Enzyme-linked immunosorbent assay fractions of A. scholaris exhibits excellent anti-inflam - (ELISA) reagents of TG, HDL, LDL, AST, and ALT matory and antioxidant activities, and efficiently inhibits were purchased from Suzhou Calvin Biotechnology Co., lipid peroxidation [39, 41, 42]. In the present study, the Ltd. (Suzhou, China). RNAiso Plus was purchased from HFD significantly increased the plasma levels of ALT and Takara Biotechnology Co., Ltd. (Dalian, China). All prim- AST, which were successfully inhibited by TA adminis- ers were synthesized by Sangon Biotech Co., Ltd. (Shang- tration. These results suggest that TA displays the antiox - hai, China). GO-Script Reverse Transcription Mix and idant and anti-inflammatory effect protecting liver from Eastep qPCR Master Mix were purchased from Pro- damage during HFD-induced NFALD progression. mega (Madison, WI, USA). The SDS-PAGE Gel Quick NAFLD increases the risk of cardiovascular disease, Preparation Kit and Bicinchoninic Acid (BCA) Protein and excessive accumulation of TG and LDL can cause Assay Kit were purchased from the Beyotime Institute of atherosclerosis [72]. Furthermore, HDL promotes the Biotechnology (Jiangsu, China). Antibodies of SREBP-1C induction of anti-atherosclerotic lipoproteins through and PPARα were purchased from Abcam (Cambridge, the reverse transport of cholesterol, thereby effectively MA, USA). The GAPDH antibody and horseradish Sun et al. Natural Products and Bioprospecting (2022) 12:14 Page 8 of 11 peroxidase (HRP)-conjugated secondary antibodies were was collected and stored at -80  °C for later analysis. procured from the Proteintech Group Inc. (Chicago, All enzyme-linked immunosorbent assay (ELISA) rea- IL, USA) and Thermo Fisher Scientific (Waltham, MA, gent sets were purchased from Suzhou Calvin Bio- USA), respectively. High-signal ECL Western Blotting technology Co. Ltd (Suzhou, China), including total Substrate was purchased from Tanon (Shanghai, China). triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), aspartate aminotrans- ferase (AST) and alanine aminotransferase (ALT). Both 4.3 Animals and procedures ALT and AST measurements utilize the Alanine Ami- Six-week-old C57BL/6  N mice (male) were purchased notransferase (ALTP5P) / Aspartate Aminotransferase from Charles River Laboratories (Beijing, China). The (ASTP5P) method respectively. The measurement of high-fat diet (No. D12492) was obtained from Research TG was based on the Fossati three-step enzymatic reac- Diets Inc. (Middlesex County, NJ, USA), and consisted tion with a Trinder endpoint. The calculated LDL was of protein (26.2%), carbohydrate (26.3%), fat (34.9%). determined by subtracting the determined HDL and All experimental procedures were performed in accord- one-fifth of the triglycerides measured from the total ance with the National Institute of Health Guide for the cholesterol. Care and Use of Laboratory Animals. The protocol was approved by the Laboratory Animal Ethics Committee of Kunming University of Science and Technology with 4.6 Determination of hepatic gene expression based approval numbers of 2018GJ512. on real‑time quantitative polymerase chain reaction Mice were randomly divided into five experimental (qRT‑PCR) groups (10 mice/group). The control group was fed a nor - Total RNA was extracted from liver tissues using Trizol mal chow diet; the HFD group was fed a HFD; and the reagent according to the manufacturer’s protocols. The TA (7.5), TA (15), and TA (30) groups were fed a HFD. concentrations and purities of the RNA samples were After two weeks of HFD, mice in the TA groups were then measured. Reverse transcription was performed administered (gavage) A. scholaris-obtained TA [sus- using the GO-Script Reverse Transcription Mix, Oligo pended in 0.5% carboxymethylcellulose sodium (CMC- (dT). A SYBR Green I Real-Time PCR Kit was used Na)] at doses of 7.5, 15, and 30 mg/kg body weight (BW), for quantification of PPARα , PPARγ, CPT1A, ACOX1, respectively. The positive group mice were fed a HFD, SREBP-1C, ACC1, SCD-1, LXRα, and GAPDH mRNA then administered with silymarin by gavage at a dose of levels using an ABI PRISM 7500 Real-Time System. The 47.8  mg/kg.BW. Both TA and silymarin were adminis- amplification reaction conditions were: 95  °C for 5  min, tered six days a week for six weeks. After treatment, the and 35 cycles at 95 °C for 15 s, 60 °C for 30 s, and 72 °C mice were fasted for 12 h before sacrifice. Serum was col - for 1 min. Target gene mRNA levels were compared with lected from blood obtained by extirpating the eyeballs. GAPDH as a reference gene, and the relative quantifi - Liver tissues were collected and frozen at -80 °C for anal- −ΔΔCt cation of mRNA levels was performed using the 2 ysis of gene and protein expression or fixed in 10% for - method. Primer sequences used for real-time quantita- malin for further histopathological analysis. tive PCR are listed in Table 4. 4.4 Histological analysis Liver histology was assessed using hematoxylin and 4.7 Western blot analysis eosin (H&E) and Masson stains. Liver tissues were fixed Protein was extracted from liver tissues using RIPA in 10% formalin. The fixed tissues were cut into 5  μm lysate containing 1% PMSF and quantitated using a BCA pieces and stained with hematoxylin and eosin (H&E) Protein Assay Kit. Protein samples were separated by using standard commercially kits. Masson’s stains in par- SDS-PAGE and transferred to polyvinylidene difluoride affin-embedded sections were furtherly performed using membranes (PVDF). The membranes were first incu - established methodology. Steatohepatitis was defined by bated with 5% fat-free milk at room temperature for 3 h, the presence of steatosis and inflammation. The sever - then incubated with antibodies against mouse SREBP- ity of steatosis and lobular inflammation were scored 1C (1:5 000), PPARα (1:5 000), and GAPDH (1:5 000) at using the NASH-Clinical Research Network criteria [3]. 4 °C overnight, and finally incubated with corresponding Stained samples were observed and photographed using HRP-conjugated secondary antibodies for 1  h at room an optical microscope. temperature. Specific bands were visualized by enhanced chemiluminescence (ECL) detection and quantified using 4.5 ELISA ImageJ software. The housekeeping protein GAPDH was Blood was collected via retro-orbital bleeding and cen- analyzed for normalization. trifuged for 15  min at 1,500  g at 4  °C, then the serum Sun  et al. Natural Products and Bioprospecting (2022) 12:14 Page 9 of 11 Authors’ contributions Table 4 The primer sequences SS, HZ carried out the experiment and drafted the original. YZ performed data Gene Primer 5′–3′sequence curation and revised the manuscript. XM and JL completed serum collection and measurement. LZ and YZ carried out gene and protein analysis. WW, XL LXRα ForwardGGG TTG CTT TAG GGA TAG G and JG contributed to the conception, methodology, review and funding acquisition. All authors read and approved the final manuscript. ReverseCAT AGC GTG CTC CCT TGA T SREBP-1C ForwardTTT GCA GAC CCT GGT GAG CG Declarations ReverseGCA AGA CGG CGG ATT TAT TCA ACC1 ForwardTCT GTA TGA GAA AGG CTA TG Competing interests No potential conflict of interest was reported by the author(s). ReverseAAG AGG TTA GGG AAG TCA T SCD1 ForwardGCT CTA CAC CTG CCT CTT C Author details ReverseCGT GCC TTG TAA GTT CTG TG Department of Infectious Disease and Hepatic Disease, First People’s Hospital of Yunnan Province, Affiliated Hospital of Kunming University of Science PPARγ ForwardGCC CTT TAC CAC AGT TGA and Technology, Kunming 650032, Yunnan, China. School of Medicine, ReverseACA GAC TCG GCA CTC AAT Kunming University of Science and Technology, Kunming 650500, Yunnan, PPARα ForwardCAA GTG CCT GTC TGT CGG China. Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, Yunnan, China. State Key Laboratory ReverseGCG GGT TGT TGC TGG TCT of Phytochemistry and Plant Resources in West China, Kunming Institute ACOX1 ForwardCTA CGC CCA GAC GGA GAT of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic ReverseACG GAT AGG GAC AAC AAA of China. CPT1A ForwardGGT GTC CAA GTA TCT GGC AGTC Received: 17 February 2022 Accepted: 17 March 2022 ReverseTCA GGG TAT TTC TCA AAG TCAA GAPDH ForwardGAG TGT TTC CTC GTC CCG ReverseATG GCA ACA ATC TCC ACT TT References 1. Michelotti GA, Machado MV, Diehl AM. NAFLD, NASH and liver cancer. Nat Rev Gastroenterol Hepatol. 2013;10:656–65. 2. Angulo P. Medical progress ‑ Nonalcoholic fatty liver disease. N Engl J 4.8 Statistical analysis Med. 2002;346:1221–31. 3. Asgharpour A, Cazanave SC, Pacana T, Seneshaw M, Vincent R, Banini Results are presented as mean ± standard error of the BA, Kumar DP, Daita K, Min H‑K, Mirshahi F, Bedossa P, Sun X, Hoshida Y, mean (SEM). Statistical analyses were performed using Koduru SV, Contaifer D Jr, Warncke O, Wijesinghe DS, Sanyal AJ. A diet‑ one-way analysis of variance (ANOVA), followed by induced animal model of non‑alcoholic fatty liver disease and hepatocel‑ lular cancer. J Hepatol. 2016;65:579–88. Tukey’s post-hoc test using SPSS 15 software. Dif- 4. 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Indole alkaloids of Alstonia scholaris (L.) R. Br. alleviated nonalcoholic fatty liver disease in mice fed with high-fat diet

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Abstract

Alstonia scholaris (L.) R. Br (Apocynaceae) is a well‑ documented medicinal plant for treating respiratory diseases, liver diseases and diabetes traditionally. The current study aimed to investigate the effects of TA on non‑alcoholic fatty liver disease (NAFLD). A NAFLD model was established using mice fed a high‑fat diet (HFD) and administered with TA (7.5, 15 and 30 mg/kg) orally for 6 weeks. The biochemical parameters, expressions of lipid metabolism‑related genes or proteins were analyzed. Furthermore, histopathological examinations were evaluated with Hematoxylin–Eosin and MASSON staining. TA treatment significantly decreased the bodyweight of HFD mice. The concentrations of low‑ density lipoprotein (LDL), triglyceride ( TG), aspartate aminotransferase (AST ) and alanine aminotransferase (ALT ) were also decreased significantly in TA‑treated mice group, accompanied by an increase in high‑ density lipoprotein (HDL). Furthermore, TA alleviated hepatic steatosis injury and lipid droplet accumulation of liver tissues. The liver mRNA levels involved in hepatic lipid synthesis such as sterol regulatory element‑binding protein 1C (SREBP-1C), regulators of liver X receptor α (LXRα), peroxisome proliferator activated receptor (PPAR)γ, acetyl‑ CoA carboxylase (ACC1) and stearyl coenzyme A dehydrogenase‑1 (SCD1), were markedly decreased, while the expressions involved in the regulation of fatty acid oxidation, PPARα, carnitine palmitoyl transterase 1 (CPT1A), and acyl coenzyme A oxidase 1 (ACOX1) were increased in TA‑treated mice. TA might attenuate NAFLD by regulating hepatic lipogenesis and fatty acid oxidation. Keywords: Hepatic disease, Hepatic lipogenesis, Fatty acid oxidation 1 Introduction and hepatic parenchymal cell steatosis that is not the Non-alcoholic fatty liver disease (NAFLD) is a chronic result of excessive alcohol consumption [2]. Obesity and hepatic damaging disease and includes fatty liver, non- an excessively high-fat diet (HFD) are important factors alcoholic steatohepatitis (NASH), related cirrhosis and involved in NAFLD pathogenesis [3]. The prevalence of hepatocellular carcinoma [1]. NFALD is characterized by NAFLD in relation to a HFD is as high as 90% in severely serious lipid accumulation, irregular hepatic vein array obese individuals [4]. NAFLD is not only associated with metabolic diseases, such as insulin resistance, obe- sity, diabetes, and hyperlipidemia [5, 6], but also closely *Correspondence: wenxue_wang@163.com; xdluo@mail.kib.ac.cn; Jiawei_ related to cardiovascular disease [7], which can seriously Geng@kmust.edu.cn Shui‑Fen Sun and Hui‑ Jie Zhong contributed equally to this work harm the health of patients. It is also closely related to Department of Infectious Disease and Hepatic Disease, First People’s hepatocyte injury, which is clinically evaluated via aspar- Hospital of Yunnan Province, Affiliated Hospital of Kunming University tate aminotransferase (AST) and alanine aminotrans- of Science and Technology, Kunming 650032, Yunnan, China State Key Laboratory of Phytochemistry and Plant Resources in West ferase (ALT) levels [8]. China, Kunming Institute of Botany, Chinese Academy of Sciences, Lipid accumulation in the livers of patients with Kunming 650201, People’s Republic of China NAFLD usually originates from an imbalance between 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/. Sun et al. Natural Products and Bioprospecting (2022) 12:14 Page 2 of 11 lipogenesis and lipolysis [9]. Therapeutic drugs for (RT = 44.915  min), picrinine (RT = 74.29  min), and the NAFLD sufferers not only remain scarce, but also show contents were 5.26%, 1.13%, 13.91%, 17.39%, respectively. some side effects. For example, pioglitazone, a PPARγ ligand, induces weight gain, fluid retention, osteopenia 2.2 TA treatment decreased HFD‑induced weight‑gaining and increased fracture risk, especially in older women After 8 weeks of HFD administration, the HFD-fed mice [10, 11]. Obeticholic acid, a synthetic ligand that activat- (31.92 ± 0.62  g) were obviously overweight with the ing farnesoid X receptor (FXR), causes both pruritus and growth rate of 21.46% compared to the normal diet-fed moderate increases in low-density lipoprotein (LDL) cho- mice (26.28 ± 0.45  g, Table  1). However, HFD-induced lesterol in 25 mg per day doses (NCT02548351) [10, 11]. weight gain was significantly attenuated in TA (30) dose u Th s, exploring novel drugs for NAFLD management by group (27.88 ± 0.59  g, P < 0.01) and the increase ampli- inhibiting the activity of transcription factors related to tude was 12.65%. Silymarin, a routinely used clinical drug lipid synthesis has considerable therapeutic potential. for fatty liver and hepatitis, also improved HFD-induced Traditional Chinese medicines for NAFLD has weight-gaining. attracted increasing attention in recent decades due to their few adverse effects, proven curative effect and ther - 2.3 TA treatment improved plasma lipid profiles apeutic mechanisms or benefits [12, 13]. Alstonia schola- of HFD‑fed mice ris is traditionally used to treat respiratory diseases, liver As shown in Table  2, HFD-fed mice showed higher TG diseases and diabetes in China and Malaysia [14, 15]. The (from 137.60 ± 24.01 to 222.20 ± 41.27  μg/mL) and LDL chemical components of different parts of the herb were (from 96.39 ± 8.18 to 113.70 ± 7.02  μg/mL) levels com- intensively investigated by our research group [16–37]. pared with the control mice. However, TA (30) treatment More than 100 indole alkaloids were reported, and TA significantly reduced TG (133.50 ± 21.04  μg/mL) and was proved to be the major pharmacological constituents LDL (84.62 ± 2.82  μg/mL) levels in HFD-fed mice. Fur- of A. scholaris in preventing respiratory diseases by us thermore, HDL levels of serum in mice were significantly [38–48]. Both preclinical investigation [49–51] and clini- decreased after 8 weeks HFD intaking (from 90.72 ± 2.08 cal trials [52, 53] have confirmed that TA was safe for fur - to 73.89 ± 5.34 μg/mL, P < 0.05), which increased partially ther clinical trials. Besides, the metabolism of TA in rats after TA (30) treatment (83.53 ± 1.87  μg/mL). The treat - indicated that scholaricine-type alkaloids could get into ment of silymarin recovered LDL levels (90.01 ± 13.13 μg/ circulation more readily than the other types [54]. Hou mL) and decreased HDL (75.28 ± 2.61  μg/mL), but had et  al., reported akuammidine, (E)-alstoscholarine, and no significant on TG (232.10 ± 21.50  μg/mL, P > 0.05). (Z)-alstoscholarine from TA with nuclear factor-kappa B These results suggest that TA treatment can restore nor - inhibition [55]. And Shang et al., verified the anti-inflam - mal blood lipid profiles in mice with HFD-intaking. Of mation activity of TA in mice [56]. Furtherly, Zhao et al., note, the reduction of TG in TA (30) group was better indicated that TA had an inhibitory effect on airway than that in the positive control group (P < 0.05). inflammation via regulating the balance of oxidation and anti-oxidation [42]. In addition, A. scholaris decoction is 2.4 TA treatment improves plasma aminotransferase used for treating diabetes, hypertension and malaria [14]. profiles in HFD‑fed mice The treatment of NAFLD, a disease related to inflam - Following, the plasma levels of AST and ALT were tested mation and oxidation assumed that TA might have a to investigate the effects of TA on liver function in mice therapeutic effect on NAFLD, together with its folk use in with HFD. As shown in Table 3, the ALT levels increased treating hepatopathy [57–59], then we undertook a phar- significantly from 0.05 ± 0.01 ng/mL of the control group macological evaluation of TA on NAFLD in mice induced to 0.33 ± 0.04  ng/mL of the HFD group (P < 0.01). Simi- by high fat diet and explored the potential molecular larly, the AST levels also increased significantly from mechanism. 0.09 ± 0.02  ng/mL to 0.13 ± 0.02  ng/mL (P < 0.01). How- ever, these increases were significantly attenuated by the TA treatment (P < 0.05/0.01). Interestingly, TA (30) dose- induced improvement in ALT (0.06 ± 0.01  ng/mL) was 2 Results better than that of silymarin (0.11 ± 0.01 ng/mL, P < 0.05), 2.1 HPL C profile and main constituent contents of TA which was an excellent elicitor of liver function repair in The main chemical constituents in the TA were separated clinical practice. Interestingly, TA-induced improvement and identified by high-performance liquid chromatog - (0.09 ± 0.03  ng/mL) in AST also exceeded that induced raphy. Briefly, four chemical components (Fig.  1) were by silymarin (0.10 ± 0.01 ng/mL). Therefore, these results identified as the major medicinal agents in TA, includ - suggest that TA treatment has a significant impact on ing scholaricine (retention time [RT] = 22.057  min), liver injury induced by HFD. 19-epischolarine (RT = 23.667  min), vallesamine Sun  et al. Natural Products and Bioprospecting (2022) 12:14 Page 3 of 11 Fig. 1 Quantification and identification of TA by HPLC analysis. A Structures of four major alkaloids from A. scholaris; B HPLC chromatographic profile of total alkaloids and UV spectra of four major alkaloids Table 1 Eec ff ts of TA on body weight (BW) in HFD ‑fed mice at liver tissues of HFD-fed mice showed an obviously rough six weeks of treatment surface, whereas the liver tissues of normal mice showed a grayish red and glossy surface (Fig.  2B). Interestingly, Group Weight (average ± SEM) high-dose TA (30) treatment improved the HFD-induced roughness and redness of the liver surface. Control 26.28 ± 0.45 We also performed H&E staining to further confirm the ▲▲ HFD 31.92 ± 0.62 TA-induced histopathological improvement of the livers. HFD + TA (7.5) 29.48 ± 0.53 As seen in Fig.  2C, normal diet mice showed complete HFD + TA (15) 31.16 ± 0.28 hepatic lobule structures, regular hepatic vein array, and **/# HFD + TA (30) 27.88 ± 0.59 clear cellular structure profiles. In HFD-fed mice, how - HFD + Silymarin 21.12 ± 1.05 ever, the hepatic lobule structure disappeared entirely, Note: BW was measured in g. Control: mice were fed a normal diet and treated hepatic vein array was irregular, and the cell shape and with 0.5% CMC-Na (solvent) via gavage; HFD: mice were fed HFD; HFD + TA (7.5), nucleus were blurry. Such liver damage could contribute HFD + TA (15), and HFD + TA (30): mice were fed HFD and treated with TA (7.5, 15, and 30 mg/kg, respectively) via gavage; HFD + Silymarin: mice were fed HFD to the loss of normal lipid metabolic function including ▲▲ ** and treated with silymarin via gavage. P < 0.01 vs. Control; P < 0.01 vs. HFD # lipid synthesis and lipolysis. Furthermore, inflamma - group. P < 0.05 vs. HFD + Silymarin group tory cells penetrated the hepatocyte intervals. Of note, whereas, TA treatment partially repaired the hepatic lob- 2.5 TA treatment alleviates HFD‑induced liver injury ule structure and vein array, especially in the high-dose After HFD administration, mice showed obvious signs of TA (30) group. Thus, high-dose TA (30) administration obesity, i.e., 21.46% overweight (Table  1) and greasy hair not only repaired the hepatic lobule structure and vein (Fig. 2A). Of note, TA treatment markedly improved hair array, but also induced lipid droplets to disappear. These condition, especially in the high-dose TA (30) group. The TA-induced improvements were comparable to those Sun et al. Natural Products and Bioprospecting (2022) 12:14 Page 4 of 11 Table 2 Eec ff ts of TA treatment on blood lipid profiles in HFD ‑fed mice Group TG (average ± SEM, μg/mL) HDL (average ± SEM, μg/mL) LDL (average ± SEM, μg/mL) Control 137.60 ± 24.01 90.72 ± 2.08 96.39 ± 8.18 ▲▲ ▲ ▲ HFD 222.20 ± 41.27 73.89 ± 5.34 113.70 ± 7.02 HFD + TA (7.5) 193.90 ± 18.88 65.72 ± 5.33 98.35 ± 7.55 HFD + TA (15) 207.00 ± 8.88 75.86 ± 7.55 94.47 ± 0.60 **/# * HFD + TA (30) 133.50 ± 21.04 83.53 ± 1.87 84.62 ± 2.82 ▲ ▲ HFD + Silymarin 232.10 ± 21.50 75.28 ± 2.61 90.01 ± 13.13 TG, HDL, and LDL were measured in μg/mL. Control: mice were fed a normal diet and treated with 0.5%CMC-Na (solvent) via gavage; HFD: mice were fed HFD; HFD + TA (7.5), HFD + TA (15), and HFD + TA (30) mice were fed HFD and treated with TA (7.5, 15, and 30 mg/kg, respectively) via gavage; HFD + Silymarin: mice were ▲/▲▲ */** # fed HFD and treated with silymarin via gavage. P < 0.05/0.01 vs. Control; P < 0.05/0.01 vs. HFD group. P < 0.05 vs. HFD + Silymarin group Table 3 Eec ff ts of TA treatment on blood aminotransferase potential [42, 47, 56]. Therefore, we speculated that levels in HFD‑fed mice TA may have a positive effect on NAFLD and prevent disease progression. As expected, TA treatment sig- Group ALT (average ± SEM, AST ng/mL) (average ± SEM, nificantly attenuated the expression levels of SREBP- ng/mL) 1C, ACC1, and SCD1 in a dose-dependent manner (Fig.  3A–C). Interestingly, TA-induced attenuations of Control 0.05 ± 0.01 0.09 ± 0.02 ▲▲ ▲▲ ACC1 and SCD1 were more significant, compared with HFD 0.33 ± 0.04 0.13 ± 0.02 * * silymarin (Fig.  3B, C, P < 0.01/0.001). Other contribu- HFD + TA (7.5) 0.19 ± 0.04 0.09 ± 0.01 ** * tors to lipid synthesis, namely PPARγ and LXRα, also HFD + TA (15) 0.13 ± 0.02 0.08 ± 0.02 showed a significant decrease in mRNA levels, even **/# * HFD + TA (30) 0.06 ± 0.01 0.09 ± 0.03 not in a dose-dependent manner. These results con- ** HFD + Silymarin 0.11 ± 0.01 0.10 ± 0.01 firm that TA treatment reduces lipid synthesis molec- ALT and AST were measured in ng/mL. Control: mice were fed a normal diet ular signaling, especially under high dose conditions and treated with 0.5%CMC-Na (solvent) via gavage; HFD: mice were fed HFD; HFD + TA (7.5), HFD + TA (15), and HFD + TA (30) mice were fed HFD and treated (30 mg/kg). with TA (7.5, 15, and 30 mg/kg, respectively) via gavage; HFD + Silymarin: mice PPARα, ACOX1, and CPT1A maintain lipid metabo- ▲▲ */** were fed HFD and treated with silymarin via gavage. P < 0.01 vs. Control; lism and restrain excessive accumulation of lipids in P < 0.05/0.01 vs. HFD group. P < 0.05 vs. HFD + Silymarin group the liver [62–65]. Here, the mRNA levels of PPARα and ACOX1 decreased markedly following HFD; how- of silymarin (Fig.  2C). We also applied Masson staining ever, TA treatment restored the mRNA expression lev- to liver tissue to assess the effects of TA treatment on els dose dependently (Fig. 3F, G). Although TA (30) did hepatic fibrosis. As expected, TA treatment remarkably not show the best performance, all doses of TA induced decreased HFD-induced collagen accumulation (Fig. 2D). recovery of CPT1A mRNA expression. Of note, the These results strongly suggest that TA possesses consid - positive control silymarin did not rescue mRNA levels erable potential in clinical treatment of NFALD. of PPARα and CPT1A (Fig. 3F and H, P > 0.05). The dif - ferent performance between silymarin and TA in liver 2.6 TA treatment alters mRNA levels of hepatic fatty acid lipid metabolism suggests they may regulate NFALD metabolism‑regulating genes in HFD‑fed mice progression via different molecular mechanisms. As shown in Fig.  3, HFD administration induced high To confirm the TA-induced molecular signaling of mRNA levels of SREBP-1C, ACC1 SCD1 and PPARγ, lipid metabolism, we applied protein immunoblotting which are key mediators of lipid synthesis [5, 60, 61]. In to examine the protein levels of SREBP-1C and PPARα addition, LXRα, a key mediator of lipid transport, also genes (Fig.  3I). As expected, TA treatment inhibited showed a high mRNA level in HFD-fed mice (Fig.  3E). the expressions of SREBP-1C protein and increased The expressions of signaling proteins, including PPARα, respectively, that both were induced by HFD PPARα, ACOX1 and CPT1A, were decreased in clinical administration (Fig.  3I). Our investigations revealed NAFLD samples. We observed similar results in the a mRNA-matched protein levels of SREBP-1C and NAFLD mouse model (Fig. 3F-H), thus supporting the PPARα genes, that further confirmed TA management reliability of our model. We previously reported that regulates lipid metabolism-molecular signaling during TA possesses anti-pulmonary fibrosis and pneumonia NFALD progression. Sun  et al. Natural Products and Bioprospecting (2022) 12:14 Page 5 of 11 Fig. 2 Eec ff t of TA on general observation and histopathologic examination. A general observation of mice and livers (B), HE (C) and Masson (D) staining of liver tissues. Control: mice were fed anormal diet and treated with 0.5%CMC‑Na (solvent) via gavage; HFD: mice were fed HFD; HFD + TA (7.5), HFD + TA (15), and HFD + TA (30) mice were fed a HFD and treated with TA (7.5, 15, and 30 mg/kg, respectively) via gavage; HFD + Silymarin: mice were fed HFD and treated with silymarin via gavage 3 Discussion and significantly increased the accumulation of lipid A diet high in fat is considered the main cause of NAFLD, droplets in the liver. Using this animal model, we inves- and then HFD-fed animal models are often used for stud- tigated whether TA treatment can improve HFD-induced ies on NAFLD [66]. In the current research, the body- NAFLD and which lipid metabolism signaling was weight of mice increased significantly after 8  weeks of involved in the disease progression. HFD administration. The HFD not only increased plasma Lipid metabolism disorders can cause excessive TG content, but also caused lipid metabolism disorders hepatic lipid accumulation. Previous studies have Sun et al. Natural Products and Bioprospecting (2022) 12:14 Page 6 of 11 Fig. 3 Eec ff t of TA on Lipid metabolism‑related gene and protein expressions in liver tissues of HFD ‑fed mice. mRNA levels of SREBP-1C (A), ACC1 (B), SCD1 (C), PPARγ (D), LXRα (E), PPARα (F), ACOX1 (G), and CPT1A (H); I Protein expressions of SREBP-1C and PPARα. Control: mice were fed a normal diet and treated with 0.5%CMC‑Na (solvent) via gavage; HFD: mice were fed HFD; HFD + TA (7.5), HFD + TA (15), and HFD + TA (30) mice were fed HFD and treated with TA (7.5, 15, and 30 mg/kg, respectively) via gavage; HFD + Silymarin: mice were fed HFD and treated with silymarin via gavage. ▲/▲▲/▲▲▲ */**/*** #/##/### P < 0.05/0.01/0.001 vs. control group; P < 0.05/0.01/0.001 vs. HFD group; P < 0.05/0.01/0.001 vs. HFD + Silymarin group shown that lipid synthesis and uptake-related genes are SCD1 are not only significantly less obese than their up-regulated, while genes involved in lipid degrada- control counterparts, but also lean and hypermetabolic tion and secretion are down-regulated in NAFLD [62, [5]. In this study, the HFD increased the mRNA levels 65, 67]. Sterol regulatory element-binding protein 1C of LXRα, SREBP-1C, ACC1, and SCD1 and the SREBP- (SREBP-1C), a key transcription factor of lipogenesis, 1C protein in liver tissues. These results are consistent is activated by upstream regulators of liver X receptor with other studies that describe SREBP activation as α (LXRα), acetyl-CoA carboxylase (ACC1) and fatty essential for hypertriglyceridemia [68]. Our research acid synthase (FAS). In NAFLD patients, successive revealed that TA obtained from the leaves of A. scho- SREBP-1C activation originating from ACC1 and FAS laris not only inhibited the synthesis of TG, but also can induce hepatic lipid accumulation [61]. Hepatic decreased the mRNA levels of LXRα, SREBP-1C, ACC1, stearoyl-CoA desaturase 1 (SCD1) catalyzes the bio- and SCD1 and protein level of SREBP-1C in a dose- synthesis of monounsaturated fatty acids. Mice lacking dependent manner. Sun  et al. Natural Products and Bioprospecting (2022) 12:14 Page 7 of 11 Furthermore, high levels of PPAR gamma (PPARγ), preventing cardiovascular diseases [73]. We found that a transcriptional modulator of adipocyte development HFD significantly increased the plasma concentrations in all types of adipose tissue, promote lipid accumula- of TG and LDL and decreased the plasma concentration tion [60]. As such, we tested the effects of TA on PPARγ of HDL, which were, in turn, effectively controlled by expression and found that HFD-induced PPARγ acti- TA treatment. Therefore, our research indicates that TA vation was also inhibited. Collectively, these results administration may decrease the risk of cardiovascular suggest that TA alleviates hepatic fat accumulation in disease in obese people. NAFLD by restraining liver lipid synthesis and adipocyte In conclusion, our study demonstrated that TA treat- development. ment can successfully ameliorate NAFLD by reducing the Maintaining liver lipid homeostasis, such as up- expression of the key transcriptional factors SREBP-1C as regulating fatty acid oxidation (FAO), is essential for well as the lipogenic enzymes ACC-1, PPARγ, LXRα and reducing liver damage resulting from redundant lipid SCD-1. Meanwhile, it upregulated the expression of the accumulation [69]. Fatty acid oxidation mainly occurs in lipolytic enzyme CPT1A, PPARα and ACOX1, which are the mitochondria with the involvement of peroxisome involved in fatty acid oxidation in liver tissues. Therefore, proliferator-activated receptor α (PPARα) [70]. Previous TA have a therapeutic effect on NAFLD through regulat - studies have shown that PPARα knockout mice exhibit ing hepatic lipogenesis and fatty acid oxidation. severe hepatic steatosis accompanied by a decrease in fatty acid uptake and oxidation [71]. The translocation of 4 Materials and methods fatty acid into the mitochondria is dependent on carni- 4.1 Preparation of total alkaloids tine palmitoyl transferase 1A (CPT1A), which is located A. scholaris leaves were collected in 2018 in Pu’er city in the mitochondrial outer membrane [63]. Clinical and (Yunnan Province, China) and identified by Dr. Xiao- animal studies have confirmed that PPARα , acyl coen- Dong Luo, Kunming Institute of Botany, Chinese Acad- zyme A oxidase 1 (ACOX1), and CPT1A are significantly emy of Sciences (Kunming, China). A voucher specimen down-regulated in NAFLD liver [62, 64, 65]. In the cur- (Luo20180105) was deposited in the State Key Laboratory rent study, we observed that a HFD altered the expres- of Phytochemistry and Plant Resources in West China, sion levels of PPARα and downstream targets CPT1A and Chinese Academy of Sciences, Kunming, China. The ACOX1. Compared with the control group, the HFD- dried and powdered leaves of A. scholaris were extracted fed mice showed a significant decrease in the expres - with 90% EtOH under reflux conditions (3  h X 4), and sion levels of lipid synthesis-inhibiting genes, including the solvent was evaporated in vacuo to obtain ethanolic PPARα, CPT1A, and ACOX1. These results suggest that extract. Next, the ethanolic extract was dissolved in 0.3% lipid oxidation is blocked in HFD-fed mice. When HFD aqueous HCl solution and filtered. The acidic solution group mice were treated with TA, the expression levels of was adjusted to pH 9–10 with 10% aqueous ammonia PPARα, CPT1A and ACOX1 increased dose dependently. and was extracted with EtOAc to obtain TA fraction, in These results indicate that TA may repair lipid metabo - which picrinine (17.39%), vallesamine (13.91%), scholari- lism balance in liver tissues of HFD-fed mice by unlock- cine (5.26%), and 19-epischolaricine (1.13%) were quanti- ing lipid oxidation. fied by HPLC with four standard compounds. Chronic lipid accumulation eventually triggers oxi- dative stress and hepatic injury, which are normally 4.2 Chemicals described by AST and ALT values in clinical diagnosis Silymarin was purchased from Madaus AG (Cologne, [8]. Our previous studies confirmed that the alkaloid Germany). Enzyme-linked immunosorbent assay fractions of A. scholaris exhibits excellent anti-inflam - (ELISA) reagents of TG, HDL, LDL, AST, and ALT matory and antioxidant activities, and efficiently inhibits were purchased from Suzhou Calvin Biotechnology Co., lipid peroxidation [39, 41, 42]. In the present study, the Ltd. (Suzhou, China). RNAiso Plus was purchased from HFD significantly increased the plasma levels of ALT and Takara Biotechnology Co., Ltd. (Dalian, China). All prim- AST, which were successfully inhibited by TA adminis- ers were synthesized by Sangon Biotech Co., Ltd. (Shang- tration. These results suggest that TA displays the antiox - hai, China). GO-Script Reverse Transcription Mix and idant and anti-inflammatory effect protecting liver from Eastep qPCR Master Mix were purchased from Pro- damage during HFD-induced NFALD progression. mega (Madison, WI, USA). The SDS-PAGE Gel Quick NAFLD increases the risk of cardiovascular disease, Preparation Kit and Bicinchoninic Acid (BCA) Protein and excessive accumulation of TG and LDL can cause Assay Kit were purchased from the Beyotime Institute of atherosclerosis [72]. Furthermore, HDL promotes the Biotechnology (Jiangsu, China). Antibodies of SREBP-1C induction of anti-atherosclerotic lipoproteins through and PPARα were purchased from Abcam (Cambridge, the reverse transport of cholesterol, thereby effectively MA, USA). The GAPDH antibody and horseradish Sun et al. Natural Products and Bioprospecting (2022) 12:14 Page 8 of 11 peroxidase (HRP)-conjugated secondary antibodies were was collected and stored at -80  °C for later analysis. procured from the Proteintech Group Inc. (Chicago, All enzyme-linked immunosorbent assay (ELISA) rea- IL, USA) and Thermo Fisher Scientific (Waltham, MA, gent sets were purchased from Suzhou Calvin Bio- USA), respectively. High-signal ECL Western Blotting technology Co. Ltd (Suzhou, China), including total Substrate was purchased from Tanon (Shanghai, China). triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), aspartate aminotrans- ferase (AST) and alanine aminotransferase (ALT). Both 4.3 Animals and procedures ALT and AST measurements utilize the Alanine Ami- Six-week-old C57BL/6  N mice (male) were purchased notransferase (ALTP5P) / Aspartate Aminotransferase from Charles River Laboratories (Beijing, China). The (ASTP5P) method respectively. The measurement of high-fat diet (No. D12492) was obtained from Research TG was based on the Fossati three-step enzymatic reac- Diets Inc. (Middlesex County, NJ, USA), and consisted tion with a Trinder endpoint. The calculated LDL was of protein (26.2%), carbohydrate (26.3%), fat (34.9%). determined by subtracting the determined HDL and All experimental procedures were performed in accord- one-fifth of the triglycerides measured from the total ance with the National Institute of Health Guide for the cholesterol. Care and Use of Laboratory Animals. The protocol was approved by the Laboratory Animal Ethics Committee of Kunming University of Science and Technology with 4.6 Determination of hepatic gene expression based approval numbers of 2018GJ512. on real‑time quantitative polymerase chain reaction Mice were randomly divided into five experimental (qRT‑PCR) groups (10 mice/group). The control group was fed a nor - Total RNA was extracted from liver tissues using Trizol mal chow diet; the HFD group was fed a HFD; and the reagent according to the manufacturer’s protocols. The TA (7.5), TA (15), and TA (30) groups were fed a HFD. concentrations and purities of the RNA samples were After two weeks of HFD, mice in the TA groups were then measured. Reverse transcription was performed administered (gavage) A. scholaris-obtained TA [sus- using the GO-Script Reverse Transcription Mix, Oligo pended in 0.5% carboxymethylcellulose sodium (CMC- (dT). A SYBR Green I Real-Time PCR Kit was used Na)] at doses of 7.5, 15, and 30 mg/kg body weight (BW), for quantification of PPARα , PPARγ, CPT1A, ACOX1, respectively. The positive group mice were fed a HFD, SREBP-1C, ACC1, SCD-1, LXRα, and GAPDH mRNA then administered with silymarin by gavage at a dose of levels using an ABI PRISM 7500 Real-Time System. The 47.8  mg/kg.BW. Both TA and silymarin were adminis- amplification reaction conditions were: 95  °C for 5  min, tered six days a week for six weeks. After treatment, the and 35 cycles at 95 °C for 15 s, 60 °C for 30 s, and 72 °C mice were fasted for 12 h before sacrifice. Serum was col - for 1 min. Target gene mRNA levels were compared with lected from blood obtained by extirpating the eyeballs. GAPDH as a reference gene, and the relative quantifi - Liver tissues were collected and frozen at -80 °C for anal- −ΔΔCt cation of mRNA levels was performed using the 2 ysis of gene and protein expression or fixed in 10% for - method. Primer sequences used for real-time quantita- malin for further histopathological analysis. tive PCR are listed in Table 4. 4.4 Histological analysis Liver histology was assessed using hematoxylin and 4.7 Western blot analysis eosin (H&E) and Masson stains. Liver tissues were fixed Protein was extracted from liver tissues using RIPA in 10% formalin. The fixed tissues were cut into 5  μm lysate containing 1% PMSF and quantitated using a BCA pieces and stained with hematoxylin and eosin (H&E) Protein Assay Kit. Protein samples were separated by using standard commercially kits. Masson’s stains in par- SDS-PAGE and transferred to polyvinylidene difluoride affin-embedded sections were furtherly performed using membranes (PVDF). The membranes were first incu - established methodology. Steatohepatitis was defined by bated with 5% fat-free milk at room temperature for 3 h, the presence of steatosis and inflammation. The sever - then incubated with antibodies against mouse SREBP- ity of steatosis and lobular inflammation were scored 1C (1:5 000), PPARα (1:5 000), and GAPDH (1:5 000) at using the NASH-Clinical Research Network criteria [3]. 4 °C overnight, and finally incubated with corresponding Stained samples were observed and photographed using HRP-conjugated secondary antibodies for 1  h at room an optical microscope. temperature. Specific bands were visualized by enhanced chemiluminescence (ECL) detection and quantified using 4.5 ELISA ImageJ software. The housekeeping protein GAPDH was Blood was collected via retro-orbital bleeding and cen- analyzed for normalization. trifuged for 15  min at 1,500  g at 4  °C, then the serum Sun  et al. Natural Products and Bioprospecting (2022) 12:14 Page 9 of 11 Authors’ contributions Table 4 The primer sequences SS, HZ carried out the experiment and drafted the original. YZ performed data Gene Primer 5′–3′sequence curation and revised the manuscript. XM and JL completed serum collection and measurement. LZ and YZ carried out gene and protein analysis. WW, XL LXRα ForwardGGG TTG CTT TAG GGA TAG G and JG contributed to the conception, methodology, review and funding acquisition. All authors read and approved the final manuscript. ReverseCAT AGC GTG CTC CCT TGA T SREBP-1C ForwardTTT GCA GAC CCT GGT GAG CG Declarations ReverseGCA AGA CGG CGG ATT TAT TCA ACC1 ForwardTCT GTA TGA GAA AGG CTA TG Competing interests No potential conflict of interest was reported by the author(s). ReverseAAG AGG TTA GGG AAG TCA T SCD1 ForwardGCT CTA CAC CTG CCT CTT C Author details ReverseCGT GCC TTG TAA GTT CTG TG Department of Infectious Disease and Hepatic Disease, First People’s Hospital of Yunnan Province, Affiliated Hospital of Kunming University of Science PPARγ ForwardGCC CTT TAC CAC AGT TGA and Technology, Kunming 650032, Yunnan, China. School of Medicine, ReverseACA GAC TCG GCA CTC AAT Kunming University of Science and Technology, Kunming 650500, Yunnan, PPARα ForwardCAA GTG CCT GTC TGT CGG China. Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, Yunnan, China. State Key Laboratory ReverseGCG GGT TGT TGC TGG TCT of Phytochemistry and Plant Resources in West China, Kunming Institute ACOX1 ForwardCTA CGC CCA GAC GGA GAT of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic ReverseACG GAT AGG GAC AAC AAA of China. CPT1A ForwardGGT GTC CAA GTA TCT GGC AGTC Received: 17 February 2022 Accepted: 17 March 2022 ReverseTCA GGG TAT TTC TCA AAG TCAA GAPDH ForwardGAG TGT TTC CTC GTC CCG ReverseATG GCA ACA ATC TCC ACT TT References 1. Michelotti GA, Machado MV, Diehl AM. NAFLD, NASH and liver cancer. Nat Rev Gastroenterol Hepatol. 2013;10:656–65. 2. Angulo P. Medical progress ‑ Nonalcoholic fatty liver disease. N Engl J 4.8 Statistical analysis Med. 2002;346:1221–31. 3. Asgharpour A, Cazanave SC, Pacana T, Seneshaw M, Vincent R, Banini Results are presented as mean ± standard error of the BA, Kumar DP, Daita K, Min H‑K, Mirshahi F, Bedossa P, Sun X, Hoshida Y, mean (SEM). Statistical analyses were performed using Koduru SV, Contaifer D Jr, Warncke O, Wijesinghe DS, Sanyal AJ. A diet‑ one-way analysis of variance (ANOVA), followed by induced animal model of non‑alcoholic fatty liver disease and hepatocel‑ lular cancer. J Hepatol. 2016;65:579–88. Tukey’s post-hoc test using SPSS 15 software. Dif- 4. 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Natural Products and Bioprospecting – Springer Journals

Published: Dec 1, 2022

Keywords: Hepatic disease; Hepatic lipogenesis; Fatty acid oxidation

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