Berberine promotes the degradation of phenylacetic acid to prevent thrombosis by modulating gut microbiota.

BACKGROUND: Berberine is the main bioactive constituent of Coptis chinensis, a quaternary ammonium alkaloid. While berberine's cardiovascular benefits are well-documented, its impact on thrombosis remains not fully understood. PURPOSE: This study investigates the potential of intestinal microbiota as a novel target for preventing thrombosis, with a focus on berberine, a natural compound known for its effectiveness in managing cardiovascular conditions. METHODS: Intraperitoneal injection of carrageenan induces the secretion of chemical mediators such as histamine and serotonin from mast cells to promote thrombosis. This model can directly and visually observe the progression of thrombosis in a time-dependent manner. Thrombosis was induced by intravenous injection of 1 % carrageenan solution (20 mg/kg) to all mice except the vehicle control group. Quantitative analysis of gut microbiota metabolites through LC/MS. Then, the gut microbiota of mice was analyzed using 16S rRNA sequencing to assess the changes. Finally, the effects of gut microbiota on thrombosis were explored by fecal microbiota transplantation. RESULTS: Our research shows that berberine inhibits thrombosis by altering intestinal microbiota composition and related metabolites. Notably, berberine curtails the biosynthesis of phenylacetylglycine, a thrombosis-promoting coproduct of the host-intestinal microbiota, by promoting phenylacetic acid degradation. This research underscores the significance of phenylacetylglycine as a thrombosis-promoting risk factor, as evidenced by the ability of intraperitoneal phenylacetylglycine injection to reverse berberine's efficacy. Fecal microbiota transplantation experiment confirms the crucial role of intestinal microbiota in thrombus formation. CONCLUSION: Initiating our investigation from the perspective of the gut microbiota, we have, for the first time, unveiled that berberine inhibits thrombus formation by promoting the degradation of phenylacetic acid, consequently suppressing the biosynthesis of PAG. This discovery further substantiates the intricate interplay between the gut microbiota and thrombosis. Our study advances the understanding that intestinal microbiota plays a crucial role in thrombosis development and highlights berberine-mediated intestinal microbiota modulation as a promising therapeutic approach for thrombosis prevention.

Biotransformation of antioxidant eriocitrin into characteristic metabolites by the gut microbiota.

Eriocitrin is a flavonoid glycoside with strong antioxidant capacity that has a variety of pharmacological activities, such as hypolipidemic, anticancer and anti-inflammatory effects. We found that the gut microbiota could rapidly metabolize eriocitrin. By using LC/MSn-IT-TOF, we identified three metabolites of eriocitrin metabolized in the intestinal microbiota: eriodictyol-7-O-glucoside, eriodictyol, and dihydrocaffeic acid. By comparing these two metabolic pathways of eriocitrin (the gut microbiota and liver microsomes), the intestinal microbiota may be the primary metabolic site of eriocitrin metabolism. These findings provide a theoretical foundation for the study of pharmacologically active substances.

Metabolites analysis of plantamajoside based on gut microbiota-drug interaction.

BACKGROUND: Plantaginis Herba (Plantago asiatica L.) has the effects of clearing heat and diuresis, oozing wet and drenching. As the main active components of Plantaginis Herba (Plantago asiatica L.), plantamajoside have a wide range of antitumor activities but very low bioavailability. The process of interacting between plantamajoside and gut microbiota remains unclear. PURPOSE: To illustrate the process of interacting between plantamajoside and gut microbiota based on high-resolution mass spectrometry and targeted metabolomics methods. STUDY DESIGN AND METHODS: This experiment was divided into two parts. First, metabolites produced from plantamajoside by gut microbiota were identified and quantified based on high-resolution mass spectrometry and LC-MS/MS. Additionally, stimulation of plantamajoside on gut microbiota-derived metabolites was determined by targeted metabolomics and gas chromatography. RESULTS: We first found that plantamajoside was rapidly metabolized by gut microbiota. Then, we identified metabolites of plantamajoside by high-resolution mass spectrometry and speculated that plantamajoside was metabolized into five metabolites including calceolarioside A, dopaol glucoside, hydroxytyrosol, 3-(3-hydroxyphenyl) propionic acid (3-HPP) and caffeic acid. Among them, we quantitatively analyzed four possible metabolites based on LC‒MS/MS and found that hydroxytyrosol and 3-HPP were final products by the gut microbiota. In addition, we studied whether plantamajoside could affect the short-chain fatty acid (SCFA) and amino acid metabolites. We found that plantamajoside could inhibit the acetic acid, kynurenic acid (KYNA) and kynurenine (KN) produced by intestinal bacteria and promote the indole propionic acid (IPA) and indole formaldehyde (IALD) produced by intestinal bacteria. CONCLUSION: An interaction between plantamajoside and gut microbiota was revealed in this study. Unlike the traditional metabolic system, the special metabolic characteristics of plantamajoside in gut microbiota was found. Plantamajoside was metabolized into the following active metabolites: calceolarioside A, dopaol glucoside, hydroxytyrosol, caffeic acid and 3-HPP. Besides, plantamajoside could affect SCFA and tryptophan metabolism by gut microbiota. Especially, the exogenous metabolites hydroxytyrosol, caffeic acid and endogenous metabolites IPA may have potential association with the antitumor activity of plantamajoside.

Gut microbiota-based metabolites of Xiaoyao Pills (a typical Traditional Chinese medicine) ameliorate depression by inhibiting fatty acid amide hydrolase levels in brain.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicines (TCMs) are often prepared in oral dosage forms, making TCMs interact with gut microbiota after oral administration, which could affect the therapeutic effect of TCM. Xiaoyao Pills (XYPs) are a commonly used TCM in China to treat depression. The biological underpinnings, however, are still in its infancy due to its complex chemical composition. AIM OF THE STUDY: The study aims to explore XYPs' underlying antidepressant mechanism from both in vivo and in vitro. MATERIALS AND METHODS: XYPs were composed of 8 herbs, including the root of Bupleurum chinense DC., the root of Angelica sinensis (Oliv.) Diels, the root of Paeonia lactiflora Pall., the sclerotia of Poria cocos (Schw.) Wolf, the rhizome of Glycyrrhiza uralensis Fisch., the leaves of Mentha haplocalyx Briq., the rhizome of Atractylis lancea var. chinensis (Bunge) Kitam., and the rhizome of Zingiber officinale Roscoe, in a ratio of 5:5:5:5:4:1:5:5. The chronic unpredictable mild stress (CUMS) rat models were established. After that, the sucrose preference test (SPT) was carried out to evaluate if the rats were depressed. After 28 days of treatment, the forced swimming test and SPT were carried out to evaluate the antidepressant efficacy of XYPs. The feces, brain and plasma were taken out for 16SrRNA gene sequencing analysis, untargeted metabolomics and gut microbiota transformation analysis. RESULTS: The results revealed multiple pathways affected by XYPs. Among them, the hydrolysis of fatty acids amide in brain decreased most significant via XYPs treatment. Moreover, the XYPs' metabolites which mainly derived from gut microbiota (benzoic acid, liquiritigenin, glycyrrhetinic acid and saikogenin D) were found in plasma and brain of CUMS rats and could inhibit the levels of FAAH in brain, which contributed to XYPs' antidepressant effect. CONCLUSIONS: The potential antidepressant mechanism of XYPs by untargeted metabolomics combined with gut microbiota-transformation analysis was revealed, which further support the theory of gut-brain axis and provide valuable evidence of the drug discovery.

Hydroxyurea ameliorates atherosclerosis in ApoE-/- mice by potentially modulating Niemann-Pick C1-like 1 protein through the gut microbiota.

Rationale: The efficacy and mechanism of hydroxyurea in the treatment of atherosclerosis have rarely been reported. The goal of this study was to investigate the efficacy of hydroxyurea in high-fat diet-fed ApoE-/- mice against atherosclerosis and examine the possible mechanism underlying treatment outcomes. Methods: ApoE-/- mice were fed a high-fat diet for 1 month and then administered hydroxyurea by gavage continuously for 2 months. Aortic root hematoxylin-eosin (H&E) staining and oil red O staining were used to verify the efficacy of hydroxyurea; biochemical methods and ELISA were used to detect changes in relevant metabolites in serum. 16S rRNA was used to detect composition changes in the intestinal bacterial community of animals after treatment with hydroxyurea. Metabolomics methods were used to identify fecal metabolites and their changes. Immunohistochemical staining and ELISA were used for the localization and quantification of intestinal NPC1L1. Results: We showed that aortic root HE staining and oil red O staining determined the therapeutic efficacy of hydroxyurea in the treatment of atherosclerosis in high-fat diet-fed ApoE-/- mice. Serological tests verified the ability of hydroxyurea to lower total serum cholesterol and LDL cholesterol. The gut microbiota was significantly altered after HU treatment and was significantly different from that after antiplatelet and statin therapy. Meanwhile, a metabolomic study revealed that metabolites, including stearic acid, palmitic acid and cholesterol, were significantly enriched in mouse feces. Further histological and ELISAs verified that the protein responsible for intestinal absorption of cholesterol in mice, NPC1L1, was significantly reduced after hydroxyurea treatment. Conclusions: In high-fat diet-fed ApoE-/- mice, hydroxyurea effectively treated atherosclerosis, lowered serum cholesterol, modulated the gut microbiota at multiple levels and affected cholesterol absorption by reducing NPC1L1 in small intestinal epithelial cells.

Gut microbiota-derived metabolites in inflammatory diseases based on targeted metabolomics.

The gut microbiota plays an important role in inflammatory diseases. Metabolites in the three metabolic pathways of tryptophan (Trp), histidine (His), and phenylalanine (Phe) can affect various inflammatory conditions, such as obesity, diabetes, arthritis, colitis, atherosclerosis, and neuroinflammation. We established an LC-MS/MS method to measure 17 metabolites-Trp, 3-indole-acetic acid (Iaa), 3-indole-lactate (Ila), 3-indole-propionic acid (Ipa), 3-indole formaldehyde (Iald), kynurenine (Kn), kynurenic acid (Kyna), 3-Hydroxyanthranilic acid (3-Haa), His, 3-methylhistidine (3-Mhis), histamine (Hist), imidazole propionic acid (Imp), 4-imidazoacetic acid (Imaa), urocanic acid (Ua), Phe, phenylethylamine (Pea), and hippuric acid (Ha)-in the three metabolic pathways. The method exhibited high sensitivity and good selectivity, linearity, accuracy, precision, stability; and recovery rate; all met the requirements of biological sample analysis. By establishing a rheumatoid arthritis (RA) model of Sprague-Dawley rats and performing 16S rRNA sequencing on their feces, it was found that there was dysbiosis, including changes in phylum level, genus level, and α biodiversity of gut bacteria. The contents of the microbiota metabolites Iaa and Ipa in the model group were significantly decreased, and those of Iald, Kn, Kyna, Ha, and Imp were significantly increased. The common therapeutic drugs Tripterygium glycosides, total glucosides of peony, and their main active ingredients were screened by in vitro incubation with gut bacteria: it was found that Tripterygium glycosides and their active ingredients could lead to a variation in metabolites in the Trp and Phe pathways. Total glucosides and active components of peony could lead to a variation in metabolites in the Phe pathway of the gut microbiota.

Metabolites Analysis of Anti-Myocardial Ischemia Active Components of Saussurea involucrata Based on Gut Microbiota-Drug Interaction.

Saussurea involucrata has been reported to have potential therapeutic effects against myocardial ischemia. The pharmacological effects of oral natural medicines may be influenced by the participation of gut microbiota. In this study, we aimed to investigate the bidirectional regulation of gut microbiota and the main components of Saussurea involucrata. We first established a quantitative method for the four main components (chlorogenic acid, syringin, acanthoside B, rutin) which were chosen by fingerprint using liquid chromatography tandem mass spectrometry (LC-MS/MS), and found that gut microbiota has a strong metabolic effect on them. Meanwhile, we identified five major rat gut microbiota metabolites (M1-M5) using liquid chromatography tandem time-of-flight mass spectrometry (LC/MSn-IT-TOF). The metabolic properties of metabolites in vitro were preliminarily elucidated by LC-MS/MS for the first time. These five metabolites of Saussurea involucrata may all have potential contributions to the treatment of myocardial ischemia. Furthermore, the four main components (10 µg/mL) can significantly stimulate intestinal bacteria to produce short chain fatty acids in vitro, respectively, which can further contribute to the effect in myocardial ischemia. In this study, the therapeutic effect against myocardial ischemia of Saussurea involucrata was first reported to be related to the intestinal flora, which can be useful in understanding the effective substances of Saussurea involucrata.

Biotransformation of Liquiritigenin into Characteristic Metabolites by the Gut Microbiota.

The bioavailability of flavonoids is generally low after oral administration. The metabolic transformation of flavonoids by the gut microbiota may be one of the main reasons for this, although these metabolites have potential pharmacological activities. Liquiritigenin is an important dihydroflavonoid compound found in Glycyrrhiza uralensis that has a wide range of pharmacological properties, such as antitumor, antiulcer, anti-inflammatory, and anti-AIDS effects, but its mechanism of action remains unclear. This study explored the metabolites of liquiritigenin by examining gut microbiota metabolism and hepatic metabolism in vitro. Using LC-MS/MS and LC/MSn-IT-TOF techniques, three possible metabolites of liquiritigenin metabolized by the gut microbiota were identified: phloretic acid (M3), resorcinol (M4), and M5. M5 is speculated to be davidigenin, which has antitumor activity. By comparing these two metabolic pathways of liquiritigenin (the gut microbiota and liver microsomes), this study revealed that there are three main metabolites of liquiritigenin generated by intestinal bacteria, which provides a theoretical basis for the study of pharmacologically active substances in vivo.

Construction of A New Random Short Hairpin RNA Library Based on microRNA Context.

Objective To build an efficient random short hairpin RNA(shRNA)library. Methods shRNA expression vector was constructed with enhanced green fluorescent protein(EGFP)in the upstream of shRNA,driven by pol Ⅱ promoter(CMV).After the constructs were transfected into cells,the proteins were collected.The inhibition efficiency of shRNA was determined by Western blot and dual luciferase reporter system.After the shRNA expression vector was constructed with EGFP in the upstream of shRNA,driven by pol Ⅱ promoter(CMV),shRNA was further embedded into microRNA(miRNA)context.The constructs were transfected into cells,and then the inhibition efficiency of shRNA against target genes was evaluated by quantificational real-time polymerase chain reaction.According to the result of quantificational real-time polymerase chain reaction,a new random shRNA library was constructed based on miRNA context. Results shRNA downstream of a large transcript was transcripted efficiently by pol Ⅱ promoter(CMV).The efficiency of shRNA interference on target gene was improved when shRNA was embedded into miRNA context.Thus,we constructed a new random shRNA library sized 1.8×107 based on miRNA context.Conclusion We successfully constructed a new large random shRNA library.