Moutan Cortex and Paeoniae Radix Rubra reverse high-fat-diet-induced metabolic disorder and restore gut microbiota homeostasis

Chinese Journal of Natural Medicines 2017, 15(3): 02100219

Chinese Journal of Natural Medicines

Moutan Cortex and Paeoniae Radix Rubra reverse high-fat-diet-induced metabolic disorder and restore gut microbiota homeostasis ZHONG Ling-Jun 1, XIE Zhi-Sheng 1, YANG Hua 1, LI Ping 1*, XU Xiao-Jun 1, 2* 1

State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China; Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, China Pharmaceutical University, Nanjing 210009, China

2

Available online 20 Mar., 2017

[ABSTRACT] The present study was designed to investigate the therapeutic effcts of Moutan Cortex (CM, root bark of Paeonia suffruticosa Andr) and Paeoniae Radix Rubra (PR, root of Paeonia veitchii Lynch) on metabolic disorders, focusing on the infuence of CM and PR on the obesity-related gut microbiota homeostasis. The diet-induced obese (DIO) mouse model was used to test the therapeutic effects of CM and PR. The mice were orally administered with CM and PR for 6 weeks, and oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed to evaluate the insulin sensitivity of the mice. Sterol-regulatory element binding proteins (SREBPs) and their target genes were measured by quantitative RT-PCR. High-throughput 16S ribosomal RNA (16S rRNA) gene sequencing technology was used to determine the composition of gut microbiota, and the metabolites in serum were analyzed by GC-MS. Our results indicated that CM and PR combination alleviated obese and insulin resistance in the DIO mice, leading to increased glucose uptake and gene expression in muscle and liver, and down-regulated SREBPs and their target genes in liver. Interesting, neither the CM-PR extracts, nor the major components of CM and PR did not affect SREBPs activity in cultured cells. Meanwhile, CM and PR significantly modulated the gut microbiota of the high-fat diet (HFD) treated mice, similar to metformin, and CM-PR reversed the overall microbiota composition similar to the normal chow diet (NCD) treated mice. In conclusion, our results provide novel mechanisms of action for the effects of CM and PR in treating DIO-induced dysregulation of sugar and lipid metabolism. [KEY WORDS] Moutan Cortex; Paeoniae Radix Rubra; Insulin resistance; Gut microbiota

[CLC Number] R965

[Document code] A

[Article ID] 2095-6975(2017)03-0210-10

Introduction Type 2 diabetes (T2D) is a complex disease caused by genetic factors and environmental stress [1]. The main [Received on] 17-Aug.-2016 [Research funding] This work was supported by the Fund for Creative Research Groups of China, National Natural Science Foundation of China (No. 81421005), the Specialized Research Fund of the Doctoral Program of Higher Education (SRFDP) and Research Council Earmarked Research Grants (RCERG) (No. 201300 96140001), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). This work was also partially supported by the National Natural Science Foundation of China (No. 81274159). XU Xiao-Jun was supported by New Century Excellent Talents in University (NCET-12-0976). [*Corresponding authors] Tel/Fax: 86-25-83271379, E-mail: [email protected] (LI Ping); Tel: 86-25-83271382, E-mail: xiaojunxu2000@ 163.com (XU Xiao-Jun). These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved

symptom of T2D is long-term hyperglycemia, accompanied by polydipsia, polyphagia, and polyuria. With the abundance of nutrients and less physical activity, energy intake exceeds expenditure, leading to increased body mass, especially fat mass, and becoming obese [2]. Since obesity is the primary risk factor for T2D, nutrients management is essential for preventing obesity and diabetes [3]. The gastrointestinal tract is an important digestive organ system, where food is broken down and nutrients are absorbed. Besides the enzymes secreted by the organs, microbial community colonized in gut also play important roles in food digestion, especially for those dietary fibers which cannot be digested by humans [4]. High-fat diet (HFD) can alter gut microbiota physiology [5-6] and is subsequently associated with different physiological states [7-8]. More interestingly, transferring the gut microbiota from lean to obese individuals can change the obese and insulin sensitive phenotypes [9-12]. These studies have brought new strategy of treating obesity and T2D. Although Moutan Cortex (CM, root bark of Paeonia

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suffruticosa Andr.) and Paeoniae Radix Rubra (PR, root of Paeonia veitchii Lynch) are commonly used for anti-diabetic treatment [13-15], the molecular mechanisms of action, especially in vivo, have not been systematically investigated [15]. There are lines of evidence supporting that many traditional herbal medicines exhibit their anti-diabetic effects through modulating gut microbiota [16-17]. CM has been shown to suppress a broad range of bacteria, such as Escherichia coli, Streptococcus sanguis, and Cholera vibrio [18-19]. PR has been reported to have antimicrobial activity against Helicobacter pylori, Clostridium difficile, Clostridium perfringens, and Bacillus subtilis [20-21]. Whether CM and PR can remodel the whole intestinal microbiota and subsequently alleviate diabetic phenotypes remains elusive. In the present study, we combined 16S rRNA gene sequencing and mass spectrometry-based metabolomics to determine the effects of Moutan Cortex and Paeoniae Radix Rubra on remodeling the intestinal microbiota in HFD mice, leading to alleviated diabetic phenotypes.

Material and Methods Preparation of CM and PR extracts CM and PR were purchased from Baicaotang (Nanjing, China) and collected form Henan and Yunnan, China. They were authenticated by Professor LI Ping, and confirmed authenticity by HPLC [22] (Figs. 5A and 5B). CM and PR were extracted and analyzed as previously reported [22]. Animal models and treatment Obese fruit flies were prepared by HFD feeding. After treatment of feeing with vehicle, metformin, CM, PR, or CM-PR in food, and biochemistry assays were performed as previously reported [22-23]. Six-week-old, male C57BL/6J mice weighing 18−20 g were purchased from Yangzhou University (Yangzhou, China), fed with HFD [22] for 10 weeks. The animals were randomly divided into six groups (n = 10 for each group): High fat diet (HFD, fat accounting for 45% of the calories), metformin (Met, 200 mg·kg−1, as an anti-diabetic drug control), CM (0.4 g crude drug per kg), PR (0.4 g crude drug per kg), CM-PR-L (CM and PR each 0.4 g crude drug per kg), and CM-PR-H (CM and PR each 0.8 g crude drug per kg). Age-matched male C57BL/6J mice fed with normal chow diet [22] were used as normal diet control. The drugs or vehicle were given by oral gavage daily for 6 weeks. OGTT and ITT were carried as described previously [24]. All the animal care and experimental procedures were approved by the Science and Technology Department of Jiangsu Province (license number: SYXK (Su) 2012-0005) and conducted in accordance with the Provision and General Recommendation of Chinese Experimental Animals Administration Legislation. RNA analysis The liver tissue samples were collected, flash frozen in liquid nitrogen, and stored at −80 °C until analysis. Total RNA was extracted using TRIzol® reagent (Invitrogen,

Carlsbad, California, USA), according to the manufacturer’s protocol, and subjected to reverse transcription. First-strand cDNA synthesis was performed using RevertAid™ Reverse Transcriptase kit (Fermentas, Carlsbad, California, USA) and an Oligo (dT) 18 primer. SYBR Green RT-PCR Kit (Roche, Mannheim, Germany) was utilized to perform qRT-PCR on a real-time PCR system (Roche LC96). GAPDH was used as a normalizer to measure the relative expression levels of the target genes using the 2-ΔΔCt formula. Specific primers used for PCR amplification were reported before [25]. Blood chemistry and histopathology The blood samples were collected by retro-orbital bleeding at the end of the experiments. Plasma biochemical analyses for insulin, total cholesterol (TC), triglycerides (TG), high density lipoprotein-cholesterol (HDL-c), and low density lipoprotein-cholesterol (LDL-c) were carried with the commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Other metabolites were detected using GC-MS methods on a Shimadzu GCMSQP2010 Ultra system (Shimadzu, Tokyo, Japan) equipped with Rtx-5MS capillary column (30.0 m × 0.25 mm ID, 0.25 μm) (Shimadzu, Tokyo, Japan) [22]. The liver and epididymal fat tissues were collected, fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), and embedded in paraffin. Sections of 5 μm were made and stained with haematoxylin and eosin (H&E) or Periodic Acid-Schiff (PAS) staining. Cell culture The HL-7702 cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China), and maintainedin Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g·L−1 glucose (Corning, New York, USA), containing 10% fetal bovine serum (FBS), 100 units/mL of penicillin, and 100 μg·mL−1 of streptomycin, in a humidified atmosphere containing 5% CO2 at 37 °C. The HL-7702 cells were plated at a density of 500 000 cells per well in 6-well plates and incubated until 70%−80% confluency, when cells were treated with DMSO, 25-hydroxycholesterol (25-hc, 1 μmol·L−1), and paeonol, paeoniflorin, oxypaeoniflorin, benzoylpaeoniflorin, gallic acid, β-sitosterol or benzoic acid at 2 μmol·L−1 in Medium E (DMEM : F-12k, 1 : 1) containing 5% Lean project Delivery System (Kalen Biomedical, MD, USA) and 50 μmol·L−1 of mevastatin (TCI, Tokyo, Japan). After drug treatment for 24 h, the cells were collected and prepared for Western blot and mRNAs analyses. The primers were used form primer bank (http://pga.mgh.harvard.edu/primerbank/). Western blot analysis The cultured cells were lysed in RIPA lysis buffer (Beyotime, Shanghai, China) containing protease inhibitors (Roche) [22] . The protein concentrations were determined using BCA assay Kit (Pierce, Rockford, USA). The samples were blotted using SREBP1 (ab3259, abcam, Cambridge, UK), SREBP2 (ab30682, abcam, Cambridge, UK),  -Actin (Cell Signaling Technology, Boston, USA) antibodies.

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Gut microbiota analysis Mice feces were collected, snap-frozen in liquid nitrogen, and stored at −80 °C until further processing. Total DNA was extracted using TIANamp Stool DNA Kit (TIANGEN Biotech, Beijing, China) according to the manufacturer’s instructions. 16S rRNA V4-V6 regions were amplified and sequenced on an Illumina MiSeq sequencer as reported before [26]. After quality filtering and deduplication, each sample contained more than 10 000 reads, which were subjected to operational taxonomic units (OTU) analysis with the cutoff similarity of 97% identity. The obtained OUT sequences were blasted to the database in Ribosomal Database Project (RDP), grouped at genus level, and then subjected to phylogenetic analysis as described previously [27]. To analyze the correlation between blood metabolites and fecal bacterial species, Spearman’s rank correlation coefficient was calculated [28]. Statistical Analysis Values were expressed as the mean ± SEM. The Unpaired Student’s t-test was used for comparison between two groups. For multiple group comparisons, the data were analyzed using one-way ANOVA with Dunnett’s multiple comparisons test. The independent experiments were performed for at least three times. P < 0.05 was considered statistically significant.

treated mice (Fig. 1D). Insulin tolerance test (ITT) demonstrated that after CM, PR or CM-PR treatment, the mice exhibited more sensitive response after intraperitoneal injection of 1.5 IU·kg−1 of insulin (Fig. 1E).

Results CM-PR treatment protects animals from obesity and insulin resistance To test whether CM and PR protects animals from obesity and insulin resistance, we used two different model animals in the present study. In high calories diet fed fruit flies, CM and PR did not affect the food intake at the concentration of less than 1% (SFig. 1A). However, administration of CM and PR reversed the hypertriglyceridemia and hyperglycemia phenotypes caused by either high fat diet (HFD) (SFigs. 1B and 1C) or high glucose diet (HGD) (SFigs. 1D and 1E). In mammalian study, the C57BL/6J mice were fed with 45% more calories HFD for 10 weeks, and then treated with vehicle, metformin, CM, PR or CM-PR combination for additional 6 weeks [29]. As shown in Fig. 1A, CM-PR treatment reduced body weight gain significantly after 3 weeks of treatment. In fasted mice, plasma insulin concentration increased to 360 pmol·L−1 after HFD challenge, compared with 180 pmol·L−1 in the NCD control (Fig. 1B). CM, PR, and CM-PR dramatically reduced fasting plasma insulin concentration. HFD also drastically increased fasting blood glucose level to 9.8 mmol·L−1 from about 6.3 mmol·L−1, while CM-PR treatment reduced plasma glucose level to around 8.0 mmol·L−1 (Fig. 1C). Oral glucose tolerance test (OGTT) was used to test glucose utilization after HFD feeding. It was revealed that CM, PR, and CM-PR treatment significantly reduced blood glucose concentration after oral gavage of 2 g·kg−1 glucose, compared to HFD fed vehicle

Fig. 1 CM-PR treatment improves insulin sensitivity and glucose tolerance in DIO mice. (A) Body weight in each group. (B) Fasting blood insulin concentration in each group. (C) Oral glucose tolerance test (OGTT) in the CM-PR treated group (n = 8 per group). (D) Quantification of the area under the curve (AUC) from the OGTT in (C). (E) Insulin tolerance test in each treatment group (n = 8 per group). (F) Quantification of the AUC of the ITT in (E). Data are presented as Mean ± SEM, *P < 0.05, **P < 0.01 vs HFD

CM-PR treatment ameliorates hyperlipidemia in HFDinduced obese mice To observe the effects of CM-PR on blood lipids in HFD-induced obese mice, plasma samples were collected in mice fed with 45% more calories HFD for 10 weeks. HFD induced around 2 fold of increases in both serum TG and TC levels; CM or PR alone significantly reduced the serum TG andslightly reduced serum TC, while the combination of CM and PR showed a further decrease in TG and TC, indicative of possible synergistic effect (Figs. 2A and 2B). We further measured HDL-c and LDL-c in plasma. HFD increased around 4 fold of LDL-c, and CM or PR alone did not show significant decrease of LDL-c, but when CM was combined

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Fig. 2 CM-PR treatment ameliorate lipids in blood of DIO mice. Plasma TG (A), TC (B), LDL-c (C) and HDL-c (D) were measured accordingly (n = 6 per group). Data are presented as Mean ± SEM, *P < 0.05, **P < 0.01 vs HFD

with PR, the inhibitory effect on LDL-c was notable (Fig. 2C) HDL-c increased slightly upon HFD treatment. CM or PR alone caused significant increase in HDL-c, but the combination of CM and PR exhibited slightly better effect (Fig. 2D). These data indicated that CM or PR alone exhibited beneficial effects on plasma lipid parameters. When combined together, CM and PR showed synergistic effects. In liver and fat tissues, CM alone only slightly reduced fat storage, while PR or CM-PR effectively decreased fat storage in liver and fat tissues (Figs. 3A and 3C). We also detected liver glycogen deposition using PAS staining method. It turned out that CM, PR, and CM-PR all effectively increased glycogen accumulation in the liver (Fig. 3B). CM-PR treatment affects the expressions of glucose and fat metabolic genes in muscle and liver tissues In muscle and liver tissues, glucose transporter protein 4 (Glut-4) transports glucose from blood to muscle or liver cells, thus reducing blood glucose concentration. Afterwards, glucose is either degraded to produce energy or stored as glycogen. Glucokinase (GK) catalyzes the first step of both glycogen synthesis and glycolysis [30]. In both muscle and liver tissues, CM-PR increased the expression of Glut-4 and GK (Figs. 4A and 4B), indicative of increased glucose uptake and mobilization. Meanwhile, insulin receptor substrate 1 and 2 (IRS-1 and IRS-2), two essential genes contributing to maintaining glucose homeostasis [31], were also increased upon the treatment of CM-PR (Fig. 4A). Phosphoenolpyruvate carboxykinase (PEPCK) is a key enzyme regulating gluconeogenesis, which was notably down-regulated in muscle and liver tissue, suggesting a reduced gluconeogenesis.

Glucose-6-phosphate dehydrogenase (G6PD) is one of the rate-limiting enzymes involved in pentose phosphate pathway (PPP). Upon CM-PR treatment, G6PD expression was dramatically reduced in the muscle tissues, while increased in the liver tissues (Figs. 4A and 4B), indicative of an increased PPP in liver to catabolize glucose. Taken together, these data suggested that CM-PR treatment changed glucose metabolic features, in favor of glucose uptake, catabolism, and storage, thus reducing blood glucose concentration. Meanwhile, we observed a significant reduced SREBP1c and SREBP-2 expression in the liver tissues of the CM-PR treated mice. SREBP-1c regulates a variety of rate-limiting genes in free fatty acid synthesis, such as Acetyl-CoA carboxylase (ACC), Fatty acid synthase (FASN), and acetyl-CoA synthetase (ACS), which were all significantly reduced in the liver tissues of the CM-PR treated mice (Fig. 4C). Similar effects were observed with SREBP-2 expression and SREBP-2 target 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) expression (Fig. 4C). SREBPs are master regulators controlling fatty acid and cholesterol de novo synthesis [32]. Since CM-PR showed notable effects of hypotriglyceridemia and hypocholesterolemia and reduced the expressions of SREBP-1c, SREBP-2, and their transcription target genes, we further tested whether CM-PR exhibited similar effects in cultured liver cells. First, we analyzed the composition of CM-PR by HPLC (Figs. 5A and 5B). Then we tested the inhibitory activity using HL-7702 liver cells carrying a SREBP reporter plasmid. As shown in Fig. 5C, 25-hc nicely inhibited SREBPs reporter activity as reported previously [25]. However, the extract of

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Fig. 3 Histological analysis of liver and adipose tissue in DIO mice. (A) Representative images of H&E staining of liver sections with different treatment. (B) Glycogen deposition was indicated by PAS staining in liver sections with different treatment. (C) Representative images of H&E staining of visceral fat with different treatment

CM-PR did not show any inhibitory effects on the SREBPs reporter (Fig. 5C). Furthermore, none of the major components of CM-PR, paeonol, paeoniflorin, oxypaeoniflorin, benzoylpaeoniflorin, gallic acid, and β-sitosterol, benzoic acid, exhibited inhibitory effects on SREBPs (Figs. 5D−5F). These

data indicated that the inhibitory effects of SREBPs were not led directly by the components in CM-PR extracts. CM-PR affects gut microbiota and overall metabolites Due to the discrepancy of the in vitro and in vivo data obtained in the present study, we reasoned the hypotrigly

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Anaerotruncus and Flavonifractor [34, 38]. Some other genus of bacteria were also reversed by CM-PR, such as Alistipes and Oscillibacter (Fig. 6 and SFig. 2), but their relation to the obesity phenotypes is still elusive [39-40]. Collectively, these results showed that CM and/or PR significantly modulated the gut microbiota of the HFD treated mice, similar to metformin, restoring the overall microbiota composition to the level similar to the NCD-fed mice. We next characterized the metabolites in the HFD fed mice treated with metformin, CM, PR or CM-PR, using GC-MS. Significant correlations between blood metabolites and bacterial species are summarized in Fig. 7. For example, branched-chain amino acids (BCAAs) were positively correlated with Mucispirillum, Sutterella, Helicobacter, but negatively correlated with Oscillibacter and Oscillospira. On the contrary, D-galactose and pyridine were negatively correlated with Mucispirillum, Sutterella, and Helicobacter, but positively correlated with Oscillibacter and Oscillospira (Fig. 7). Anaerotruncus, Ruminococcus and Oscillibacter were negatively correlated with cholesterol, while Allobaculum and Dorea were positively correlated with cholesterol (Fig. 7). These data indicated that the composition of bacteria affected endogenous metabolites significantly and specifically.

Discussion

Fig. 4 CM-PR treatment regulate mRNAs in DIO mice. (A) CM-PR treatment regulate glucose metabolism in muscle. (B) CM-PR treatment regulate glucose metabolism in liver. (C) CM-PR treatment regulate lipid metabolism in liver (n = 4 per group). Data are presented as Mean ± SEM, *P < 0.05, **P < 0.01 vs HFD

ceridemia and hypocholesterolemia efficacy of CM-PR was accomplished indirectly in vivo. Recently, growing evidence indicates that obesity and associated metabolic disorders are closely correlated with the changes in the composition of the gut microbiota [28, 33]. Therefore, we checked the gut microbial population using high-throughput 16S ribosomal RNA (16S rRNA) gene sequencing technology. HFD challenge caused disrupted gut microbiota composition (Fig. 6 and SFig. 2). Compared to the NCD-fed mice, HFD feeding greatly altered general operational taxonomic units (OTUs). Bacteria positively related obesity or type 2 diabetes, such as Bacteroides, Parabacteroides, Akkermansia, and Mucispirillu [34-37], were found enhanced by HFD, which were all reversed by CM-PR treatment. It seemed that PR exerted more potent inhibitory effects on these bacteria, while CM did not show significant inhibition (Fig. 6 and SFig. 2). For the regulation of Mucispirillum, CM-PR was even better than metformin. Notably, many bacteria negatively related with obesity were found decreased in the HFD feeding mice, which were reversed by CM-PR treatment. These bacteria include

CM and PR are common herbs used in traditional Chinese medicine for anti-diabetic treatment. It has been shown that CM inhibits glucose uptake in intestine, but increases glucose uptake in hepatocytes and adiopocytes [13, 15]. PR has anti-hyperglycemic effect by suppression of PEPCK transcription. However, the effects of CM and PR on the regulation of fat metabolism are rarely reported. In the present study, we used HFD to induce obese and insulin resistance in mice and found that CM and PR alleviated hyperlipidemia phenotypes, indicating that CM and PR modulated fat metabolism. Meanwhile, our results revealed that CM and PR modulated the obesity related gut microbiota. In recent years, growing evidences support that gut microbiota and/or their metabolites contribute to many chronic diseases, such as obesity [28, 41], diabetes [36, 42], and cancer [27]. Fecal transfer of lean donors may cause reduced weight gain in obese acceptors [31, 34] and vise versa [43], and therefore modulating gut microbiota becomes a new therapeutic approach [12, 44] for obesity and diabetes. However, transferring bacteria into human body is still quite risky. Therefore, dietary or compounds modulating disease related microbiota would be of great interest for the development of effective and safe therapeutics. In a modern science point of view, human health is maintained by the homeostasis of human genome and environment interactions [45]. As microbiota composition as well as the blood metabolites are dynamically regulated and the changes occur significantly [46] , maintaining the homeostasis of gut microbiota is essential for the host’s health.

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Fig. 5 The HPLC chromatogram of CM/PR and the effects of CM/PR components on SREBPs activity in HL-7702 cells. (A) The HPLC chromatogram of CM. (B) The HPLC chromatogram of PR. The SREBPs inhibitory effects of CM and PR extracts (C) and their major components (D) by mRNAs. (F) The SREBPs inhibitory effects of the major components from CM and PR by western blot

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Fig. 6 CM and PR alters microbiota composition in DIO mice. Mean relative abundance of OTUs classified at the genera level for the most abundant taxa. The bacterial taxonomic profiling of the most abundant 28 species from different mous

Fig. 7 Associations of gut bacteria abundance and serum metabolites. Spearman’s rank correlation coefficients and P values for the correlations of serum metabolites and cecal bacterial species-level taxa are calculated

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Many traditional Chinese medicine (TCM) have been shown to modulate HFD induced abnormity of gut microbiota [34, 41, 47], and therefore, TCM may provide rich resources for discovery and development of new drugs modulating gut microbiota. In the present study, we found that CM-PR treatment significantly reduced SREBPs activity in liver of the HFD fed mice, but neither the CM-PR extracts, nor the major components of CM and PR affected SREBPs activity in cultured cells. CM and PR treatment also affected blood metabolites. BCAAs have been shown to increase SREBP-1c expression [48]. These data indicated that the regulation of SREBPs in vivo by CM and PR was via the microbiota-blood metabolites-liver axis. These cases may be in consistent with the concept of TCM, i.e., TCM holistically regulates both the body and the outer world. Akkermansia is reversely correlated with obesity and insulin resistance in mice [49-50]. CM increased Akkermansia abundance after HFD treatment, but PR could fully inhibit this trend when combined with CM. These data indicated that, when using herbal medicine to regulate microbiota, the correlation between components and bacterial abundance should be comprehensively studied. Thus, in our future studies, we will profile the chemical constituents of CM and PR and their corresponding activity on disease-related bacteria.

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Cite this article as: ZHONG Ling-Jun, XIE Zhi-Sheng, YANG Hua, LI Ping, XU Xiaojun. Moutan Cortex and Paeoniae Radix Rubra reverse high-fat-diet-induced metabolic disorder and restore gut microbiota homeostasis [J]. Chin J Nat Med, 2017, 15(3): 210-219.

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