Tangerine Peel-Derived Exosome-Like Nanovesicles Alleviate Hepatic Steatosis Induced by Type 2 Diabetes: Evidenced by Regulating Lipid Metabolism and Intestinal Microflora.

Purpose: Non-alcoholic fatty liver disease (NAFLD) represents a significant global health burden, exhibiting a strong correlation with insulin resistance, obesity, and type 2 diabetes (T2DM). Despite the severity of hepatic steatosis in T2DM patients, no specific drugs have been approved for clinical treatment of the disease. Tangerine peel is one kind of popular functional food and reported to possess hypoglycemic and lipid-lowering potential. In this study, we investigated the effects of Tangerine-peel-derived exosome-like nanovesicles (TNVs) on hepatic lipotoxicity associated with T2DM. Methods: The TNVs was prepared by differential centrifugation of the aqueous extract of Tangerine and chemical properties were characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and LC-MS/MS. The hypoglycemic and lipid-lowering potential of TNVs were possessed by biochemical measurement, RT-PCR, 16S rRNA sequencing, GC/MS, UHPLC-MS/MS, in vivo small animal imaging assay and HE staining. Subsequently, effects of TNVs on lipid accumulation and glycolysis were investigated on 3T3-L1 and AML-12 cells. Results: TNVs significantly inhibited insulin resistance, reduced hepatic lipid accumulation, facilitate intestinal mucosal repair, rescued gut microbiota dysbiosis, regulated colonic SCFA and liver bile acid metabolism in db/db mice. Furthermore, TNVs restored the expression of key genes in glucose and lipid metabolism (ACC, AMPK, CD36, LXRα, PPAR-γ, SREBP-1) while activating the expression of genes related to glycolysis (G6Pase, GLUT2, PCK1, PEPCK) in db/db mice. Further cell-based mechanistic studies revealed that TNVs reduced lipid accumulation in 3T3-L1 and AML-12 cells via regulation of glucose and lipid metabolism-related genes (UCP1, FGFR4, PRDM16, PGC-1α, Tmem26, Cpt1, Cpt2 and PPAR-α). Conclusion: We for the first time demonstrated that TNVs could significantly improve glucose and lipid metabolism via activating the expression of genes related to fatty acid ß-oxidation and glycolysis.

Gastrointestinal metabolism of Astragalus membranaceus polysaccharides and its related hypoglycemic mechanism based on gut microbial transformation.

Astragalus membranaceus polysaccharides (AMP) was reported to exhibit hypoglycemic potential in diabetic host. However, the metabolic fate of AMP in gastrointestinal tract and its underlying hypoglycemic mechanisms remained unclear. Our current study aimed to reveal the structure alteration of AMP in gastrointestinal tract and its hypoglycemic mechanism from the perspective of microbial transformation. Caco-2 monolayer cell model revealed that AMP exhibited poor intestinal absorption. The in-vitro digestion and fermentation study revealed that AMP remained intact after gastrointestinal digestion while it could be degraded and utilized by gut microbiota with increased SCFA formation and decreased levels of all the monosaccharides in AMP except for mannose. Additionally, diversity of gut microbiota was improved with the increased abundance of Dubosiella and Monoglobus and decreased abundance of Escherichia-Shigella and Acinetobacter after fermentation of AMP. Further hypoglycemic mechanism study for the first time revealed that both AMP and its potential microbial metabolites, SCFA salt mixture, could enhance intestinal integrity significantly on LPS induced Caco-2 cell model, while only SCFA salt mixture rather than AMP could significantly stimulate GLP-1 secretion in NCI-H716 cell model possibly via promoting GPCR43 expression. Such findings provided insights into the hypoglycemic mechanism of AMP from the perspective of microbial transformation.

Dendrobium officinale regulate lipid metabolism in diabetic mouse liver via PPAR-RXR signaling pathway: Evidence from an integrated multi-omics analysis.

Dendrobium officinale (DEN) is recognized as a kind of functional food that can effectively ameliorate endocrine and metabolic disruptions. This study delved into the pharmacological mechanism of DEN on hepatic lipotoxicity associated with Type II diabetes mellitus (T2DM). In vivo study experiments on db/db mice indicated that DEN treatment notably enhanced liver function, decreased blood lipid levels, and improved insulin sensitivity. Non-targeted metabolomics analysis revealed that DEN significantly ameliorated metabolism pathways, including lipoic acid, linoleic acid, bile secretion, and the alanine/aspartate/glutamate metabolism, as well as taurine and hypotaurine metabolism. Transcriptomics analysis demonstrated DEN treatment could modulate the expression of genes such as Cpt1b, Scd1, G6pc2, Fos, Adrb2, Atp2a1, Ppp1r1b, and Cyp7a1. Furthermore, Proteomics analysis indicated that the beneficial effect of DEN on lipid metabolism was linked to pathways like AMPK and PPAR signaling. The integrative analysis of multi-omics revealed that the PPAR-RXR signaling was critical to the therapeutic effect of DEN on T2DM-induced fatty liver. Additionally, in vitro study on AML-12 cells confirmed that DEN counteract PA-induced lipid accumulation by activating the PPAR-RXR pathway. Overall, these findings suggested that DEN exhibited the potential to mitigate T2DM-induced hepatic lipo-toxicity and manage lipid imbalances in T2DM.

Role of gut microbiota on regulation potential of Dendrobium officinale Kimura & Migo in metabolic syndrome: In-vitro fermentation screening and in-vivo verification in db/db mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Dendrobium officinale Kimura & Migo (DEN) is a traditional medicine in China since Han dynasty. Decoction of its stem is often used in the treatment of Type-II diabetes (T2D), which is a typical metabolic disease accompanied with the impaired metabolic function of blood glucose and lipid. AIM OF THE STUDY: Our study aimed to investigate the role of gut microbiota in differentiating DEN from different sources and its related pathway in the alleviation of metabolic syndromes induced by T2D. MATERIALS AND METHODS: The aqueous extracts of four commercially available Dendrobium (DEN-1∼4) were prepared and screened through an in-vitro fermentation system. Based on their alterations in monosaccharide composition and short chain fatty acids (SCFA) formation during fermentation with db/db faecal fluid, one DEN extract was selected for further in vivo verification. The selected Dendrobium (DEN-4) was orally administered to db/db mice for 16 days once daily at the dosage of 200 mg/kg followed by evaluating its effect on blood glucose level, liver function and intestinal microenvironment including alterations of intestinal integrity and gut microbiota composition. In addition, liver metabolomics analysis was employed to reveal the related metabolic pathways. RESULTS: Different extent of SCFA formation and utilization of monosaccharides were observed for the extracts of four DEN from different sources with a negative correlation between SCFA level and the ratio of Utilized glucose/Utilized mannose observed in the in-vitro fermentation system with db/db faecal fluid. DEN-4 with the highest SCFA formation during the in-vitro fermentation was selected and exhibited significantly hypoglycaemic effect in db/db mice with the alleviation of hepatic steatosis and impaired lipid homeostasis. Further mechanistic studies revealed that orally administered DEN-4 could improve the intestinal integrity of db/db mice via elevating their tight junction protein (ZO-1 and Occludin) expression in the colon and improve the diversity of gut microbiota with enhanced formation of SCFA. Moreover, metabolomics and KEGG pathway analysis of liver tissues suggested that the alleviated metabolic syndrome in db/db mice by DEN-4 might possibly be achieved through activation of PPAR pathway. CONCLUSION: Our current study not only revealed the potential of gut microbiota in differentiating DEN from different sources, but also demonstrated that DEN exhibited its beneficial effect on the T2D induced metabolic syndrome possibly through enhancement of intestinal integrity and activation of PPAR pathway via gut-liver axis in db/db mice.

Elucidation of Carboxylesterase Mediated Pharmacokinetic Interactions between Irinotecan and Oroxylin A in Rats via Physiologically Based Pharmacokinetic Modeling.

PURPOSE: Our previous screening studies identified Oroxylin A (OXA) as a strong inhibitor on the carboxyolesterase mediated hydrolysis of irinotecan to SN-38. The current study employed a whole-body physiologically based pharmacokinetic (PBPK) modeling approach to investigate the underlying mechanisms of the carboxylesterase-mediated pharmacokinetics interactions between irinotecan and OXA in rats. METHODS: Firstly, rats received irinotecan intravenous treatment at 35 µmol/kg without or with oral OXA pretreatment (2800 µmol/kg) daily for 5 days. On day 5, blood and tissues were collected for analyses of irinotecan/SN-38 concentrations and carboxylesterase expression. In addition, effects of OXA on the enzyme kinetics of irinotecan hydrolysis and unbound fractions of irinotecan and SN-38 in rat plasma, liver and intestine were also determined. Finally, a PBPK model that integrated the physiological parameters, enzyme kinetics, and physicochemical properties of irinotecan and OXA was developed. RESULTS: Our PBPK model could accurately predict the pharmacokinetic profiles of irinotecan/SN-38, with AUC0-6h and Cmax values within ±27% of observed values. When OXA was included as a carboxylesterase inhibitor, the model could also predict the irinotecan/SN-38 plasma concentrations within twofold of those observed. In addition, the PBPK model indicated inhibition of carboxylesterase-mediated hydrolysis of irinotecan in the intestinal mucosa as the major underlying mechanism for the pharmacokinetics interactions between irinotecan and OXA. CONCLUSION: A whole-body PBPK model was successfully developed to not only predict the impact of oral OXA pretreatment on the pharmacokinetics profiles of irinotecan but also reveal its inhibition on the intestinal carboxylesterase as the major underlying mechanism.

Gut microbiota mediated hypoglycemic effect of Astragalus membranaceus polysaccharides in db/db mice.

Gut microbiota has been reported to be closely associated with Type-II diabetes. Restoration of disordered gut microbiota ecosystem has been developed into a therapeutic strategy and gradually applied on Type-II diabetes treatment with both western drugs and herbal polysaccharides. Although Astragalus membranaceus polysaccharides (AMP) have also been used to treat Type-II diabetes, no study investigated correlations between gut microbiota regulation and its hypoglycemic effect. In the present study, the role of gut microbiota on the hypoglycemic effect of AMP in db/db mice was investigated for the first time. Sixteen days treatment of AMP at the dosage of 600 mg/kg in db/db mice not only alleviated its diabetic symptoms significantly but also restored its gut microbiota community with increased production of fecal short chain fatty acids (SCFA). Our further Pearson correlation analyses revealed that the relative abundance of two intestinal bacteria, Akkermansia and Faecalibaculum, were significantly positively correlated with the hypoglycemic effect of AMP as well as fecal SCFA production. It was also noted that treatment of AMP resulted in increased secretion of glucagon-like peptide-1 (GLP-1) in serum and enhanced intestinal integrity. Further mechanistic study revealed that the increased SCFA after AMP treatment could stimulate GLP-1 secretion and improve intestinal integrity via enhancing the expression of G protein-coupled receptors 41/43 and tight junction proteins (Occudin and ZO-1), respectively, leading to the alleviation of diabetic symptoms in db/db mice.

Idiosyncratic liver injury induced by bolus combination treatment with emodin and 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucopyranoside in rats.

Polygoni Multiflori Radix (PMR) is a commonly used traditional Chinese medicine in clinical practice, while adverse effects of hepatotoxicity related to PMR have been frequently reported. The clinical case reports indicated that PMR hepatotoxicity could occur under both overdose medication/long-term exposure and low doses with short-duration (idiosyncratic) conditions. The combination treatment with emodin and 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucopyranoside (TSG), two major PMR components, was reported to contribute to PMR hepatotoxicity after long-term treatment. However, the role of the combination treatment of these two components in PMR-induced idiosyncratic liver injury has not been clearly clarified. In this study, the LPS-mediated inflammatory stress model rats were adopted to explore the idiosyncratic liver injury induced by the bolus combination treatment with emodin and TSG. After a bolus oral administration with TSG (165 mg/kg), emodin (5 mg/kg) or their combination in both normal and LPS-mediated inflammatory stress model rats, the systemic/hepatic concentrations of emodin, emodin glucuronides and bile acids were determined; the hepatotoxicity assessments were conducted via monitoring histopathological changes and liver injury biomarkers (ALT and AST). Moreover, the protein expressions of bile acid homeostasis- and apoptosis-related proteins were examined. No liver damage was observed in the normal rats after a bolus dose with the individual or combination treatment, while the bolus combination treatment with emodin and TSG induced liver injury in the LPS-mediated inflammatory stress model rats, evidenced by the elevated plasma levels of alanine aminotransferase (∼66%) and aspartate aminotransferase (∼72%) accompanied by severe inflammatory cell infiltration and apoptotic hepatocytes in liver tissue. Moreover, such combination treatment at a bolus dose in the LPS-mediated inflammatory stress model rats could significantly elevate the hepatic TBA levels by about 45% via up-regulating the hepatic protein expression levels of bile acid synthesis enzymes and inhibiting that of bile acid efflux transporters and the expression levels of apoptosis-related proteins. Our study for the first time proved the major contribution of the combination treatment with emodin and TSG in PMR-induced idiosyncratic liver injury.

Inhibition of Radix Scutellariae flavones on carboxylesterase mediated activations of prodrugs.

AIMS: Carboxylesterase (CES) plays an essential role in the hydrolysis of ester prodrugs. Our study explored the inhibitions of Radix Scutellariae flavones, including baicalein (B), baicalin (BG), wogonin (W), wogonoside (WG), oroxylin A (OXA) and oroxylin A-7-O-glucuronide (OAG), on CES-mediated hydrolysis of seven prodrugs (capecitabine, clopidogrel, mycophenolate mofetil, dabigatran etexilate, acetylsalicylic acid, prasugrel and irinotecan). MAIN METHODS: In vitro screenings were developed by incubating the flavones with prodrugs in rat plasma, intestine S9 and liver S9. Docking simulations were conducted using AMDock v1.5.2. In vivo evaluations were performed in rats co-administered with the selected flavone and prodrug via oral gavage/intravenous administration for five consecutive days. KEY FINDINGS: The in vitro investigation showed that B and OXA demonstrated strongest inhibitions on the hydrolysis of irinotecan followed by dabigatran in rat plasma, intestine S9 and liver S9. Consistent results showed in the molecular docking analyses. Additionally, in rats receiving irinotecan, B/OXA intravenous and oral pre-treatments both led to reduction trends on the active metabolite SN-38 formation in plasma. Besides, significant decreases of SN-38/irinotecan plasma concentration ratios were found in the B/OXA oral pre-treatment group with quicker and stronger inhibition potential in OXA pre-treatment than that from B pre-treatment. OXA oral pre-treatment was also found to be able to significantly inhibit intestinal CES2 activities at 0.5 h and 5 h after irinotecan administration. SIGNIFICANCE: Our current findings for the first time alert on potential CES-mediated HDIs between RS flavones and prodrugs, which provide a constructive information referring to rational drug combinations in clinical practice.

2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucopyranoside enhances the hepatotoxicity of emodin in vitro and in vivo.

Herb-induced liver injury results from the interplay between the herb and host with the herbal components serving as the major origin for hepatotoxicity. Although Polygoni Multiflori Radix (PMR) has been frequently reported to induce liver injury, contributions of its major components such as emodin, emodin-8-O-ß-D-glucopyranoside, physcion and 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucopyranoside (TSG) towards its hepatotoxicity have not been clearly identified. Our initial cytotoxicity screenings of the major PMR components using rat hepatocytes identified emodin as the most toxic. Subsequently, the bile acid homeostasis-related mechanisms of emodin and its combination treatment with TSG in PMR-associated liver injury were explored in sandwich-cultured rat hepatocytes (SCRH) and verified in rats. In SCRH, emodin was found to be able to induce total bile acid accumulation in a dose-dependent manner. In both SCRH and rats, the presence of TSG significantly enhanced the hepatotoxicity of emodin via i) increasing its hepatic exposure by inhibiting its glucuronidation mediated metabolism; ii) enhancing its disruption on bile acid homeostasis through amplifying its inhibition on bile acid efflux transporters and its up-regulation on bile acids synthesis enzymes; iii) enhancing its apoptosis. Our study for the first time demonstrated the critical role of the combination treatment with emodin and TSG in PMR-induced liver injury.

Optimized flash extraction and UPLC-MS analysis on antioxidant compositions of Nitraria sibirica fruit.

The fruit of Nitraria sibirica Pall. (N. sibirica) is a common folk medicine in the Northwest China and contains plentiful antioxidant compositions. In the study, a rapid flash extraction method (extracting for 3.3 min with 53% ethanol at the solvent-sample ratio of 34 mL/g) for the antioxidant compositions in N. sibirica fruit was optimized by response surface methodology. Twenty-seven compounds of the extract were recognized by ultra-high performance liquid chromatography combined with ion trap mass spectrometry (UPLC-IT/MS), including twelve flavonoids, twelve alkaloids and three phenolic acids. Four compounds among them were first reported in Nitraria genus. Moreover, eight main antioxidant compounds in eight batches of N. sibirica fruit collected from different regions were quantified by a rapid and reliable method of ultra-high performance liquid chromatography combined with triple quadrupole tandem mass spectrometry (UPLC-QQQ/MS). Chemometric study further revealed that the contents of two compounds, tryptophan and alcesefoliside, were highly correlated with the antioxidant activity, which provided a reference for the quality evaluation of N. sibirica fruit.