PuRenDan alleviates type 2 diabetes mellitus symptoms by modulating the gut microbiota and its metabolites.

ETHNOPHARMACOLOGICAL RELEVANCE: PuRenDan (PRD) is a traditional Chinese medicine formula comprising five herbs that have been traditionally used to treat type 2 diabetes mellitus (T2DM). While PRD has been shown to be effective in treating T2DM in clinical and animal studies, the mechanisms by which it works on the gut microbiome and metabolites related to T2DM are not well understood. AIM OF THE STUDY: The objective of this study was to partially elucidate the mechanism of PRD in treating T2DM through analyses of the gut microbiota metagenome and metabolome. MATERIALS AND METHODS: Sprague-Dawley rats were fed high-fat diets (HFDs) and injected with low-dose streptozotocin (STZ) to replicate T2DM models. Then the therapeutic effects of PRD were evaluated by measuring clinical markers such as blood glucose, insulin resistance (IR), lipid metabolism biomarkers (total cholesterol, low-density lipoprotein, non-esterified fatty acids, and triglycerides), and inflammatory factors (tumor necrosis factor alpha, interleukin-6 [IL-6], interferon gamma, and IL-1ß). Colon contents were collected, and metagenomics, combined with ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry metabolic profiling, was performed to evaluate the effects of T2DM and PRD on gut microbiota and its metabolites in rats. Spearman analysis was used to calculate the correlation coefficient among different microbiota, clinical indices, and metabolites. RESULTS: PRD exhibited significant improvement in blood glucose and IR, and reduced serum levels of lipid metabolism biomarkers and inflammatory factors. Moreover, the diversity and abundance of gut microbiota undergo significant changes in rats with T2DM that PRD was able to reverse. The gut microbiota associated with T2DM including Rickettsiaceae bacterium 4572_127, Psychrobacter pasteurii, Parabacteroides sp. CAG409, and Paludibacter propionicigenes were identified. The gut microbiota most closely related to PRD were Prevotella sp. 10(H), Parabacteroides sp. SN4, Flavobacteriales bacterium, Bacteroides massiliensis, Alistipes indistinctus, and Ruminococcus flavefaciens. Additionally, PRD regulated the levels of gut microbiota metabolites including pantothenic acid, 1-Methylhistamine, and 1-Methylhistidine; these affected metabolites were involved in pantothenate and coenzyme A biosynthesis, histidine metabolism, and secondary bile acid biosynthesis. Correlation analysis illustrated a close relationship among gut microbiota, its metabolites, and T2DM-related indexes. CONCLUSION: Our study provides insights into the gut microbiota and its metabolites of PRD therapy for T2DM. It clarifies the role of gut microbiota and the metabolites in the pathogenesis of T2DM, highlighting the potential of PRD for the treatment of this disease.

The role of Huidouba in regulating skeletal muscle metabolic disorders in prediabetic mice through AMPK/PGC-1α/PPARα pathway.

Prediabetes is a transitional state between normal blood glucose levels and diabetes, but it is also a reversible process. At the same time, as one of the most important tissues in the human body, the metabolic disorder of skeletal muscle is closely related to prediabetes. Huidouba (HDB) is a clinically proven traditional Chinese medicine with significant effects in regulating disorders of glucose and lipid metabolism. Our study aimed to investigate the efficacy and mechanism of HDB in prediabetic model mice from the perspective of skeletal muscle. C57BL/6J mice (6 weeks old) were fed a high-fat diet (HFD) for 12 weeks to replicate the prediabetic model. Three concentrations of HDB were treated with metformin as a positive control. After administration, fasting blood glucose was measured as an indicator of glucose metabolism, as well as lipid metabolism indicators such as total triglyceride (TG), low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C), free fatty acid (FFA), and lactate dehydrogenase (LDH). Muscle fat accumulation and glycogen accumulation were observed. The protein expression levels of p-AMPK, AMPK, PGC-1α, PPAR-α, and GLUT-4 were detected. After HDB treatment, fasting blood glucose was significantly improved, and TG, LDL-C, FFA, and LDH in serum and lipid accumulation in muscle tissue were significantly reduced. In addition, HDB significantly upregulated the expression levels of p-AMPK/AMPK, PGC-1α, PPAR-α, and GLUT-4 in muscle tissue. In conclusion, HDB can alleviate the symptoms of prediabetic model mice by promoting the AMPK/PGC-1α/PPARα pathway and upregulating the expression of GLUT-4 protein.

Functional and binding studies of gallic acid showing platelet aggregation inhibitory effect as a thrombin inhibitor.

Objective: This study was devoted to identifying natural thrombin inhibitors from traditional Chinese medicine (TCM) and evaluating its biological activity in vitro and binding characteristics. Methods: A combination strategy containing molecular docking, thrombin inhibition assay, surface plasmon resonance (SPR) and molecular dynamics simulation were applied to verify the study result. Results: Gallic acid was confirmed as a direct thrombin inhibitor with IC50 of 9.07 µmol/L and showed a significant inhibitory effect on thrombin induced platelet aggregation. SPR-based binding studies demonstrated that gallic acid interacted with thrombin with a KD value of 8.29 µmol/L. Molecular dynamics and binding free energy analysis revealed that thrombin-gallic acid system attained equilibrium rapidly with very low fluctuations, the calculated binding free energies was -14.61 kcal/mol. Ala230, Glu232, Ser235, Gly258 and Gly260 were the main amino acid residues responsible for thrombin inhibition by gallic acid, providing a mechanistic basis for further optimization. Conclusion: This study proved that gallic acid is a direct thrombin inhibitor with platelet aggregation inhibitory effect, which could provide a basis for the follow-up research and development for novel thrombin inhibitors.

The therapeutic mechanism of PuRenDan for the treatment of diabetic nephropathy: Network pharmacology and experimental verification.

ETHNOPHARMACOLOGICAL RELEVANCE: Purendan (PRD), as a Chinese medicinal formula, behaves remarkable therapeutic effects on diabetes and complications in clinical and experimental research. However, the underlying pharmacological mechanism in the treatment of diabetic nephropathy (DN) is still unclear. AIMS: To investigate the therapeutical effects of PRD on DN and to explore its pharmacological mechanisms using network pharmacology and experimental verification. MATERIALS AND METHODS: The active compounds and putative targets in PRD, and disease-related targets of DN were extracted from public databases. The key targets were identified through the protein-protein interaction (PPI) network and module analysis. The GO and KEGG enrichment analysis were performed to discover potential pharmacological mechanisms. The expression of the key targets was detected in kidney tissue in Gene Expression Omnibus (GEO) dataset. The affinity between key proteins and corresponding compounds was evaluated by molecular docking and validated by the surface plasmon resonance (SPR) assay. The indicators on major pathways and hub genes were verified by in vivo experiments. RESULTS: In network pharmacology, 137 common targets in PRD for DN treatment were screened. The key targets and main signaling pathways including AGE-RAGE and lipid pathways were identified. The statistical difference in the expression of the key targets was verified in GSE96804 database, confirming the association with DN. The docking scores obtained from molecular docking illustrated good binding force between hub proteins and active compounds. And the good component-protein affinities were validated by SPR assay. Furthermore, the results of animal experiment indicated that PRD could ameliorate the level of serum glucose and renal function in rat model. It could regulate the expression of hub targets (AKT1, MAPK3, and STAT3) and improve indicators related with oxidative stress and lipid metabolism. CONCLUSION: The key targets and major signaling pathways in the treatment of PRD on DN were identified. The mechanism might relate to regulation of oxidative stress and lipid metabolism.

Purendan alleviates non-alcoholic fatty liver disease in aged type 2 diabetic rats via regulating mTOR/S6K1/SREBP-1c signaling pathway.

Older people are more likely to develop insulin resistance and lipid metabolism disorders. Purendan (PRD) is a clinically verified traditional Chinese medicine compound, which plays an obvious role in regulating lipid metabolism disorder and improving insulin sensitivity. Our study aimed to investigate the efficacy and mechanism of PRD on aged type 2 diabetes mellitus (T2DM) complicated with non-alcoholic fatty liver disease (NAFLD) rats. Sprague-Dawley rats (13 months) were fed with high-fat diet (HFD) and injected with low-dose STZ to replicate T2DM model. PRD was treated at three concentrations with metformin as a positive control. After administration, blood and liver tissue samples were collected to measure glucose metabolism indexes such as serum glucose and insulin, as well as lipid metabolism indexes such as TC, TG, LDL, HDL and FFA. Liver fat accumulation was observed by HE staining and oil red O staining. And protein expression levels of mTOR, p-mTOR, S6K1, p-S6K1 and SREBP-1c were detected by western blot. After PRD treatment, not only the insulin sensitivity and insulin resistance were significantly improved, but also the TC, TG, LDL, FFA, AST and ALT in serum and the lipid accumulation in liver tissue were significantly decreased. Moreover, PRD significantly down-regulated the expression of p-mTOR, p-S6K1 and SREBP-1c in liver tissues. In conclusion, PRD can alleviate NAFLD in aged T2DM rats by inhibiting the mTOR /S6K1/ SREBP-1c pathway.

Hypoglycemic effects of Tibetan medicine Huidouba in STZ-induced diabetic mice and db/db mice.

Objective: Huidouba (HDB) is a Chinese folk medicine used to treat diabetes in Sichuan Province, China. Therefore, we investigated the anti-diabetic effects of HDB and its underlying mechanisms. We hypothesized that HDB treatment could enhance glucose tolerance and insulin sensitivity, and thus prevent a hyperglycemia state. Methods: To test the hypothesis, streptozotocin (STZ)-induced diabetic mice and db/db mice, widely used models of hyperglycemia and insulin-resistant diabetes, were either treated with HDB, metformin, or acarbose. Blood glucose, oral glucose tolerance test, insulin tolerance test, pancreatic histopathology and serum biochemistry were detected to assess the hypoglycemic effect of HDB. Results: HDB treatments were found to show the effect in reducing glucose levels. HDB also resulted in a significant reduction in body weight and food intake in the STZ-induced diabetic mouse model. Furthermore, it significantly improved glucose and insulin tolerance in the two diabetic mouse models. Importantly, insulin, glucagon, pancreatic polypeptide, and somatostatin immunohistochemistry revealed that HDB treatment improved the function and the location of the cells in the islets compared with the other two treatments. HDB treatment resulted in significant restoration of islet function. Our results illustrated the underlying mechanism of HDB in the progression of diabetes, and HDB can be an effective agent for the treatment of diabetes. Conclusion: The results of this study suggested that HDB can reduce blood glucose levels in STZ-induced hyperglycemic mice and db/db mice.

Huidouba Improved Podocyte Injury by Down-Regulating Nox4 Expression in Rats With Diabetic Nephropathy.

Diabetic nephropathy (DN), as the most common microvascular complication of diabetes mellitus (DM), has become one of the leading causes of end-stage renal disease (ESRD). Numerous studies have indicated that podocyte loss plays an important role in the development of DN and can even cause proteinuria in the early stage of DN. In the study, we found that Huidouba (HDB) significantly decreased the level of fasting blood glucose (FBG), the ratio of microalbumin to urine creatine (mAlb/Ucr), serum creatine (Scr), serum urea nitrogen (BUN), and malondialdehyde (MDA) in the kidney and downregulated the expression of Nox4 predominantly located in glomerular tissue while upregulating nephrin and WT1 expression in DN rats. In addition, HDB could also reduce podocyte damage and glomerular basement membrane (GBM) pathologic changes, as shown by transmission electron microscopy (TEM). In vitro study showed that HDB could inhibit high glucose (HG)-induced Reactive Oxygen Species (ROS) production and protect against podocyte apoptosis by downregulated Nox4 expression in podocytes. These results may provide a scientific basis for developing HDB as a potential folk medicine for the treatment of DN.

The effect of Chinese medicine Pu-Ren-Dan on pancreatic angiogenesis in high fat diet/streptozotocin-induced diabetic rats.

OBJECTIVES: The islet vascular system is critical for ß-cell function. This study investigated the antidiabetic effect of the Chinese Pu-Ren-Dan (PRD) recipe by regulating the pancreatic angiogenic factors in T2DM rats. MATERIALS METHODS: High fat diet/streptozotocin-induced obese type-2 diabetes mellitus rats were developed and treated with PRD for 4 weeks. Then glucolipid metabolism, insulin secretion, pancreatic blood flow, ultrastructure of islet ß-cell, histological changes of islet and protein expressions of pancreatic angiogenic factors were investigated. RESULTS: PRD-reduced T2DM rats' body weight and blood glucose level resisted the lipid metabolism disturbance, and ameliorated the insulin resistance and ß-cell function. In addition, the histological and morphological studies proved that PRD could maintain the normal distribution of endocrine cell in islet and normal ultrastructure of ß cell. An increased pancreatic blood flow was observed after the PRD treatment. In the investigation of pancreatic angiogenic factors, PRD inhibited the decreased expression of VEGF and Ang-1, and reversed the reduction of VEGFR2 and Tie2 phosphorylation in T2DM rats; the Ang-2 and TGFß expression were up-regulated by PRD while PKC was activated; endostatin and angiostatin were down-regulated by PRD. CONCLUSIONS: The results suggest that increasing VEGF expression, regulating VEGF/VEGFR2 signaling, stimulating Ang-1/Tie-2 signaling pathway, and inhibiting PKC-TGFß signaling and antiangiogenic factors might be the underlying mechanism of PRD's antidiabetic effect.

Mechanisms involved in the cytotoxic effects of berberine on human colon cancer HCT-8 cells

Berberine, a constituent of some traditional Chinese medicinal plants, has been reported to have cytotoxicity effects on different human cancer cell lines. There is no available information about the effects and mechanism of action of berberine on human colon cancer cell line HCT-8. In this paper, the cytotoxicity of berberine on HCT-8 cancer cells was investigated by MTT assay, fluorescence microscopy and flow cytometry analysis. Our data revealed that berberine could significantly inhibit the growth of HCT-8 cells in a dose- and time-dependent manner. Morphology of apoptotic cells was studied with acridine orange/ethidium bromide staining. The concentrations of lactate dehydrogenase and both acid and alkaline phosphatases were significantly increased in cell supernatants after berberine treatment, suggesting cell death. Furthermore, flow cytometry analysis showed that berberine could arrest HCT-8 cells at S phase in a time-dependent manner. To further investigate the apoptotic molecular mechanism, reverse transcription-polymerase chain reaction (RT-PCR) and western blotting methods were used. The up-regulated mRNA and/or protein expressions of Fas, FasL, TNF-a, caspase-3 and down-regulation of pro-caspase-3 suggest that the death receptor pathway may be involved in the apoptotic pathway induced by berberine. Decrease of Bcl-2 and increase of Bax in mRNA and/or protein expressions showed that the Bcl-2 family proteins were involved in berberine-induced apoptosis. We also found that berberine-induced apoptosis was associated with an up-regulated expressions of p53 and prohibitin (PHB), and decreased vimentin expression. These results suggest that berberine can suppress cell growth and reduce cell survival by arresting the cell-cycle and by inducing apoptosis of HCT-8 cells.(AU)

Mechanisms involved in the cytotoxic effects of berberine on human colon cancer HCT-8 cells

Berberine, a constituent of some traditional Chinese medicinal plants, has been reported to have cytotoxicity effects on different human cancer cell lines. There is no available information about the effects and mechanism of action of berberine on human colon cancer cell line HCT-8. In this paper, the cytotoxicity of berberine on HCT-8 cancer cells was investigated by MTT assay, fluorescence microscopy and flow cytometry analysis. Our data revealed that berberine could significantly inhibit the growth of HCT-8 cells in a dose- and time-dependent manner. Morphology of apoptotic cells was studied with acridine orange/ethidium bromide staining. The concentrations of lactate dehydrogenase and both acid and alkaline phosphatases were significantly increased in cell supernatants after berberine treatment, suggesting cell death. Furthermore, flow cytometry analysis showed that berberine could arrest HCT-8 cells at S phase in a time-dependent manner. To further investigate the apoptotic molecular mechanism, reverse transcription-polymerase chain reaction (RT-PCR) and western blotting methods were used. The up-regulated mRNA and/or protein expressions of Fas, FasL, TNF-a, caspase-3 and down-regulation of pro-caspase-3 suggest that the death receptor pathway may be involved in the apoptotic pathway induced by berberine. Decrease of Bcl-2 and increase of Bax in mRNA and/or protein expressions showed that the Bcl-2 family proteins were involved in berberine-induced apoptosis. We also found that berberine-induced apoptosis was associated with an up-regulated expressions of p53 and prohibitin (PHB), and decreased vimentin expression. These results suggest that berberine can suppress cell growth and reduce cell survival by arresting the cell-cycle and by inducing apoptosis of HCT-8 cells.

Identification of molecular target proteins in berberine-treated cervix adenocarcinoma HeLa cells by proteomic and bioinformatic analyses.

In this study, the apoptosis of HeLa cells induced by berberine was investigated. Fifty-one differentially expressed proteins were identified before and after berberine treatment by a proteomic method, which either interacted with each other directly or through one intermediate protein to form a connected protein interaction sub-network. Nine of them were selected and validated. Compared with the cells in the control group, the expressions of 14-3-3σ and lamin-A/C of the cells treated by berberine for 48 h increased by 94.12 and 5.24 times, respectively, and the expressions of annexin A5, cytokeratin 17, prohibitin, heat shock cognate 71 kDa protein (HSPA8), programmed cell death 6 and vimentin decreased by 4.1, 1.34, 23.8, 11.85, 4.63 and 5.24 times, respectively. In addition, tubulin-ß decreased from 9537 to 6908 ng/L. Furthermore, the inverse dock program (INVDOCK) was used to predict the possible drug-target of berberine's anticancer activity, and the results showed that HSPA8 and annexin A5 might be the drug targets. This study suggests that the anticancer effect of berberine on HeLa cells was a complex process based on affecting multiple protein expression and acting on an interaction network. Our work could be helpful to elucidate the mechanism of berberine's anticancer activity on HeLa cells.

Mechanisms involved in the cytotoxic effects of berberine on human colon cancer HCT-8 cells.

Berberine, a constituent of some traditional Chinese medicinal plants, has been reported to have cytotoxicity effects on different human cancer cell lines. There is no available information about the effects and mechanism of action of berberine on human colon cancer cell line HCT-8. In this paper, the cytotoxicity ofberberine on HCT-8 cancer cells was investigated by MTT assay, fluorescence microscopy and flow cytometry analysis. Our data revealed that berberine could significantly inhibit the growth of HCT-8 cells in a dose- and time-dependent manner. Morphology of apoptotic cells was studied with acridine orange/ethidium bromide staining. The concentrations of lactate dehydrogenase and both acid and alkaline phosphatases were significantly increased in cell supernatants after berberine treatment, suggesting cell death. Furthermore, flow cytometry analysis showed that berberine could arrest HCT-8 cells at S phase in a time-dependent manner. To further investigate the apoptotic molecular mechanism, reverse transcription-polymerase chain reaction (RT-PCR) and western blotting methods were used. The up-regulated mRNA and/or protein expressions of Fas, FasL, TNF-alpha, caspase-3 and down-regulation of pro-caspase-3 suggest that the death receptor pathway may be involved in the apoptotic pathway induced by berberine. Decrease of Bcl-2 and increase of Bax in mRNA and/or protein expressions showed that the Bcl-2 family proteins were involved in berberine-induced apoptosis. We also found that berberine-induced apoptosis was associated with an upregulated expressions of p53 and prohibitin (PHB), and decreased vimentin expression. These results suggest that berberine can suppress cell growth and reduce cell survival by arresting the cell-cycle and by inducing apoptosis of HCT-8 cells.

Application of proteomic and bioinformatic techniques for studying the hepatoprotective effect of dioscin against CCl4-induced liver damage in mice.

In this study, the significant hepatoprotective effect of dioscin against CCl4-induced acute liver damage in mice was first discovered, and the effect produced by dioscin at the dose of 100 mg/kg was equal to the action produced by silymarin at the dose of 200 mg/kg. Then, 1-dimension gel electrophoresis was used to separate the liver proteins, and five differentially expressed bands were selected. After in-gel digestion, 71 proteins were identified by nano-RP-HPLC-ESI-MS/MS/MS. Further network analysis suggested that the identified proteins formed a connected protein interaction subnetwork. Ten functional categories were selected to demonstrate the distribution of the proteins by Gene Ontology (GO) enrichment analysis. Six of the proteins, heat shock protein 5 (HSPA5), annexin 6 (ANXA6), isovaleryl-CoA dehydrogenase (IVD), ribosomal protein S6 (RPS6), cytoglobin (Cygb), and nucleoside diphosphate kinase A (NDPK-A), were validated by Western blotting assay. They might be involved in the hepatoprotective effect of dioscin, and their investigation could be useful, together with the determination of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) levels, as well as the liver histopathologic study, for the elucidation of the action mechanisms of dioscin against CCl4-induced liver injury. Our work shows that the validated proteins should be considered as biomarkers for the investigation of acute liver injury, and its results should contribute to the therapy of liver damage by dioscin in the future.

Simple and reliable methods for the determination of sixteen marker components for quality control of Daochi pill by HPLC coupled with diode array detection.

INTRODUCTION: Traditional Chinese medicine plays a very important role in the healthcare system of China and thus the quality control of medicinal herb products is of paramount concern. OBJECTIVE: To establish a simple and effective high-performance liquid chromatography (HPLC) method to evaluate the quality of Daochi pill. METHODOLOGY: Two HPLC methods were developed for the determination of 16 marker components in Daochi pills. In method A, the analytes were separated on a Lichrosorb C(18) column using a gradient elution of methanol and 1% aqueous phosphoric acid (pH 2.9, adjusted by triethylamine). In method B, the separation was achieved on an Agilent Eclipse Plus C(18) column using a gradient elution of methanol and 3% aqueous phosphoric acid (pH 2.0, adjusted by triethylamine) in a gradient elution mode. RESULTS: Methods were linear over the range 0.27-500 microg/mL (r(2) >or= 0.9995). Accuracy, precision and repeatability were all within the required limits. The mean recoveries measured at three concentrations were higher than 95% with RSD

Simultaneous determination of 11 active components in two well-known traditional Chinese medicines by HPLC coupled with diode array detection for quality control.

A simple and sensitive high-performance liquid chromatography coupled with diode array detection (HPLC-DAD) method was investigated for simultaneous determination of 11 components (chlorogenic acid, coptisine, epiberberine, jatrorrhizine, berberine, palmatine, baicalin, wogonoside, baicalein, wogonin and chrysin) in Qinhuanghouzheng (QHHZ) capsule and Xiaoerqingre (XEQR) tablet, for quality control of these two well-known traditional Chinese medicines (TCMs). The method was established using an Eclipse Plus C(18) (150 mm x 4.6 mm i.d., 5 microm) column. The mobile phase comprising methanol (A) 3% phosphoric acid (B) (pH 2.0, adjusted by triethylamine) was used to elute the targets in gradient elution mode. Flow rate and detection wavelength were set at 0.8 mL/min and 270 nm, respectively. All calibration curves showed good linearity with R(2) > 0.9995. Inter- and intra-day precisions for all investigated components expressed as relative standard deviation (R.S.D.) ranged from 0.26% to 1.77%. Recoveries measured at three concentrations were in the range of 95.0-103.0% with R.S.D. < or = 3%. The validated method is simple, reliable, and successfully applied to determine the contents of the selected compounds in QHHZ capsule and XEQR tablet for quality evaluation and control. The 11 main active marker compounds measured occur only in 2 or 3 plant species out of 7-10 species comprising the two TCMs. Additional procedures need to be developed for the quality control of plant materials other than Coptis chinensis Franch, Scutellaria baicalensis Georgi and Phellodendron amurense Rupr.