Integrated fecal microbiota and metabolomics analysis of the orlistat intervention effect on polycystic ovary syndrome rats induced by letrozole combined with a high-fat diet.

BACKGROUND: This study aimed to compare the characteristics of the gut microbiota and their metabolite profiles between polycystic ovary syndrome (PCOS) and orlistat-treated PCOS rats (ORL-PCOS), which could help to better understand the underlying mechanism of the effect of orlistat on PCOS. METHODS: PCOS rat models were established using letrozole combined with a high-fat diet. Ten rats were randomly selected as a PCOS control group (PCOS). The other three groups (n = 10/group) were additionally supplemented with different doses of orlistat (low, medium, high). Then, fecal samples of the PCOS and ORL-PCOS groups were analysed by 16S rRNA gene sequencing and untargeted metabolomics. Blood samples were collected to detect serum sex hormones and lipids. RESULTS: The results showed that orlistat attenuated the body weight gain, decreased the levels of T, LH, the LH/FSH ratio, TC, TG and LDL-C; increased the level of E2; and improved estrous cycle disorder in PCOS rats. The bacterial richness and diversity of the gut microbiota in the ORL-PCOS group were higher than those in the PCOS group. The ratio of Firmicutes to Bacteroidetes was decreased with orlistat treatment. Moreover, orlistat treatment led to a significant decrease in the relative abundance of Ruminococcaceae and Lactobacillaceae, and increases in the abundances of Muribaculaceae and Bacteroidaceae. Metabolic analysis identified 216 differential fecal metabolites in total and 6 enriched KEGG pathways between the two groups, including steroid hormone biosynthesis, neuroactive ligand-receptor interaction and vitamin digestion and absorption. Steroid hormone biosynthesis was the pathway with the most significant enrichment. The correlations between the gut microbiota and differential metabolites were calculated, which may provide a basis for understanding the composition and function of microbial communities. CONCLUSIONS: Our data suggested that orlistat exerts a PCOS treatment effect, which may be mediated by modifying the structure and composition of the gut microbiota, as well as the metabolite profiles of PCOS rats.

Shouhui Tongbian Capsules induce regression of inflammation to improve intestinal barrier in mice with constipation by targeted binding to Prkaa1: With no obvious toxicity.

Constipation arising from the poor bowel movement is a rife enteric health problem. Shouhui Tongbian Capsule (SHTB) is a traditional Chinese medicine (TCM) which effectively improve the symptoms of constipation. However, the mechanism has not been fully evaluated. The purpose of this study was to evaluate the effect of SHTB on the symptoms and intestinal barrier of mice with constipation. Our data showed that SHTB effectively improved the constipation induced by diphenoxylate, which was confirmed by shorter first defecation time, higher internal propulsion rate and fecal water content. Additionally, SHTB improved the intestinal barrier function, which was manifested by inhibiting the leakage of Evans blue in intestinal tissues and increasing the expression of occludin and ZO-1. SHTB inhibited NLRP3 inflammasome signaling pathway and TLR4/NF-κB signaling pathway, reduced the number of proinflammatory cell subsets and increased the number of immunosuppressive cell subsets to relieve inflammation. The photochemically induced reaction coupling system combined with cellular thermal shift assay and central carbon metabolomics technology confirmed that SHTB activated AMPKα through targeted binding to Prkaa1 to regulate Glycolysis/Gluconeogenesis and Pentose Phosphate Pathway, and finally inhibited intestinal inflammation. Finally, no obvious toxicity related to SHTB was found in a repeated drug administration toxicity test for consecutive 13 weeks. Collectively, we reported SHTB as a TCM targeting Prkaa1 for anti-inflammation to improve intestinal barrier in mice with constipation. These findings broaden our knowledge of Prkaa1 as a druggable target protein for inflammation inhibition, and open a new avenue to novel therapy strategy for constipation injury.

[Mechanism of pathogenesis of Jingfang Mixture in intervention of chronic spontaneous urticaria based on serum metabolomics].

This study aims to clarify the effect of Jingfang Mixture on the treatment of chronic urticarial and its mechanism, and investigate the regulatory effect of chronic urticaria on the metabolic disorder of endogenous metabolites in the blood. The mice were randomly divided into normal group, model group, and Jingfang Mixture group, and modeling and administration continued for 21 d. The changes in endogenous small molecules in rat serum were determined by ultra-high performance liquid chromatography-electrospray ionization-Q Exactive-Orbitrap-mass spectrometry(UHPLC-ESI-QE-Orbitrap-MS) metabolomics technology. The change trend of endogenous metabolites in rat serum was analyzed to find potential biomarkers. The results showed that Jingfang Mixture regulate 16 biomarkers, mainly including taurine, glutamate, succinic acid, docosahexaenoic acid, and arachidonic acid. Metabolic pathway analysis was carried out by MetaboAnalyst, and P<0.01 was taken as the potential key metabolic pathway. Ten metabolic pathways were closely related to the treatment of chronic urticarial by Jingfang Mixture, mainly involved in the glutamate metabolism, taurine and hypotaurine metabolism, arginine and proline metabolism, arachidonic acid metabolism, tricarboxylic acid cycle, unsaturated fatty acid biosynthesis, glutathione metabolism, phenylalanine metabolism, alanine, aspartic acid, and glutamate metabolism, and butyric acid metabolism. Glutamate metabolism and butyric acid metabolism involved more metabolic pathways than others. Therefore, it was speculated that Jingfang Mixture had a balanced regulating effect on the related metabolic pathways which caused the serum disorder in the rats with urticaria, and tended to regulate the metabolic differential to the normal level in the rats with urticaria. This paper provides references for studying the mechanism of Jingfang Mixture from the perspective of endogenous metabolites and metabolic pathways in vivo. At the same time, the endogenous substances explored in this paper can be used as important biomarkers for the prevention of urticaria.

[Proteomic study of Jingfang Mixture on urticaria based on label-free quantitative proteomics technology].

This study aims to explore the effect of Jingfang Mixture on the protein expression of urticaria in mice and explain the mechanism of Jingfang Mixture in the treatment of urticaria. Twenty-seven male Kunming mice were randomly divided into a normal group, a model group and a Jingfang Mixture group according to body weight. Except for the normal group, mice in the model group and the Jingfang Mixture group were injected with the mixture of ovalbumin and Al(OH)_3 gel for the first immunization, and the second immunization was performed on the 10 th day to induce the urticaria model. Mice in the Jingfang Mixture group started to be administered on the 6 th day after the initial immunization, and was administered continuously for 21 days. The normal group and the model group were replaced with the same amount of purified water. Twenty-four hours after the last administration, an appropriate amount of skin was taken, and label-free quantitative proteomics technology was used to detect the differences in protein expression in skin tissue. The signaling pathways involved in the differential proteins was further analyzed. The results of proteomics indicated that seventy-six proteins were involved in the intervention of Jingfang Mixture on mice with urticaria, and the differential proteins were mainly enriched in biological process(BP), molecular function(MF), and cellular component(CC). Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis showed that the signaling pathways regulated by Jingfang Mixture mainly involved carbon metabolism, metabolic pathways, glucagon signaling pathway, glycolysis/gluconeogenesis, pentose phosphate pathway, hypoxia inducible factor-1(HIF-1) signaling pathway, purine metabolism, adherens junction, calcium signaling pathway, leukocyte transendothelial migration, and inflammatory mediator regulation of transient receptor potential(TRP) channels, which were involved in skin tissue energy metabolism and immune regulation. The findings of this study showed that the protective effect of Jingfang Mixture on mice with urticaria was closely related to the regulation of immune disorders, and the regulatory effect on immune system may be achieved through the regulation of energy metabolism by Jingfang Mixture.

[Therapeutic effect and mechanism of Shouhui Tongbian Capsules on slow transit constipation model mice].

Shouhui Tongbian Capsules was used to explore the therapeutic effect and potential mechanism on slow transit constipation model mice induced by loperamide hydrochloride. In the experiment, loperamide hydrochloride-induced ICR mice were used as the model of slow transit constipation. Fifty ICR mice were divided into the blank group, model group and high, medium and low dose groups of Shouhui Tongbian Capsules extract(100, 200 and 400 mg·kg~(-1)). The model group and the administration groups were then modeled using loperamide hydrochloride intragastrically to obtain slow transit constipation. After successful modeling, high, medium and low doses of drugs were given to each drug group by intragastric administration. After 14 days of administration, the first defecation time, 6 h defecation grain number, 6 h defecation wet weight and dry weight, black feces discharged within 6 h and the fecal water content were measured. Intestinal tissues were taken for c-Kit and SCF immunohistochemical sections to detect the expression of c-Kit and SCF in the blank group, model group and high, medium and low dose groups of the medicinal extract of Shouhui Tongbian Capsules. The tissue changes in the intestinal wall of mice were detected by HE staining. At the same time, partial intestinal tissues were taken to test the activity of ATP synthase and isocitrate dehydrogenase in intestinal tissues of mice. RESULTS:: showed that Shouhui Tongbian Capsules effectively improved the symptoms of slow transit constipation in ICR mice and promoted intestinal movement. Shouhui Tongbian Capsules obviously shortened the time of discharging black stool for the first time, improved the intestinal propulsion rate, increased the water content and amount of feces, and improved the constipation symptoms. Mechanism study revealed that Shouhui Tongbian Capsules increased ATP synthase activity and mitochondrial isocitrate dehydrogenase activity in intestinal tissue, and up-regulated c-Kit/SCF signaling pathway to promote interstitial Cajal cells proliferation, intestinal nerve transmission, intestinal motility and transport capacity.

Essential oil from Euphorbia esula inhibits proliferation and induces apoptosis in HepG2 cells via mitochondrial dysfunction

Hepatocellular carcinoma is one of the most prevalent malignancies and a leading cause of cancer-related mortality worldwide. However, the therapies to prevent hepatocellular carcinoma are still limited and the emergence of drug resistance leads to the development of new anti-cancer drugs and combinational chemotherapy regimens. Our study was aimed to explore the anticancer effects of the essential oil extract (EEEO) from Euphorbia esula which has been widely used in traditional Chinese folk medicine and possessed potential cytotoxic effects in several human tumor cells. However, the mechanisms of EEEO-induced anti-proliferation and apoptosis have not been completely elucidated. In this study, EEEO was prepared by hydro-distillation and the main chemical component of EEEO was identified by GC-MS. HepG2 cells were treated with EEEO in vitro and then evaluated with respect to proliferation, apoptosis, and levels of reactive oxygen species (ROS) and apoptotic proteins. Our studies showed that EEEO decreased cell viability, elevated ROS levels, and induced apoptosis of HepG2 cells in a concentration- and time-dependent manner. Furthermore, Bcl-2 was down-regulated, while Bax was up-regulated in HepG2 after EEEO treatment. These results suggest that EEEO induced apoptosis of HepG2 cells and indicate that this apoptosis might be mediated by the mitochondrial pathway.

Essential Oil from Carpesium abrotanoides L. Induces Apoptosis via Activating Mitochondrial Pathway in Hepatocellular Carcinoma Cells.

The effects of essential oil from Carpesium abrotanoides L. (CAEO) on the proliferation and apoptosis of human hepatic cancer cells were investigated in this study. MTT assays indicated that CAEO inhibited the proliferation of HCC cells with the IC50 values ranging from 41.28±3.06 to 130.36±20.79 µg/mL. Moreover, many obviously nuclear morphological changes of apoptotic cells in CAEO-treated HepG2 cells were detected by Hoechst 33258 staining and fluorescence microscopy. Flow cytometry was used to detect cell apoptosis and cell cycle, and noticeable findings showed that CAEO arrested cell-cycle at S and G2/M phases. The decreased Bcl-2/Bax protein ratio and the activation of caspase-3, caspase-9 were also detected by Western blotting. All results suggested that CAEO is a potential agent to fight against liver cancer, and the mitochondria-mediated intrinsic apoptotic pathway could be involved in CAEO-mediated apoptosis of human liver carcinoma cells.

Icaritin Improves Antibody-Induced Thrombocytopenia in a Mouse Model by Regulating T-cell Polarization.

Previous studies have shown that icaritin (ICT) has significant protective effects on immune thrombocytopenia (ITP), and the present study aimed to discuss the mechanism of this protective effect from the aspect of regulating T-cell polarization by an antibody-induced ITP mice model. Mice were given rat anti-mouse CD41 antibody (MWReg30) by intraperitoneal injection for 7 d to produce ITP model. At the same time, ICT was administrated at 10 mg/kg/d orally for 9 d. Peripheral blood platelets were counted by hematology analyzer. Spleen index was also tested. Spleen T-helper cell (Th), cytotoxic T-cell (CTL), Th1, Th2, Th17, regulatory T-cell (Treg), and follicular helper T-cell (Tfh) were quantified by flow cytometry. Serum Th1/Th2/Th17 cytokines were tested by mouse Th1/Th2/Th17 cytometric bead array (CBA) kit and transforming growth factor beta (TGF-ß) were analyzed by enzyme-linked immunosorbent assay (ELISA) kit. The results indicated that ICT (10 mg/kg) protected against MWReg30-induced ITP, as evidenced by increased blood platelets and decreased spleen index. In addition, the imbalance of Th/CTL in ITP mice spleen was regulated by ICT. Meanwhile, ICT inhibited Th1, Th17, and Tfh and improved Th2 and Treg in ITP mice spleen. Furthermore, the results of CBA and ELISA suggested that ICT decreased serum Th1- and Th17-related cytokines and increased Th2 cytokines, as well as promoted the release of TGF-ß. These results demonstrated that the protective effect of ICT on ITP was mediated by regulating T-cell polarization.

Essential oil from Siegesbeckia pubescens induces apoptosis through the mitochondrial pathway in human HepG2 cells.

Siegesbeckia pubescens (SP) has been used as a traditional medicine for the treatment of and inflammatory diseases. However, the activities of SP against hepatocellular carcinoma and the related mechanisms remain unclear. The present study aimed to examine the effects of the essential oil of SP (SPEO) on the proliferation of hepatocellular carcinoma cells and the possible mechanisms. The growth inhibition of HepG2 cells was analyzed by MTT assay. Hoechst 33258 and fluorescence microscopy were utilized to observe the nuclear morphological changes of apoptotic cells. Flow cytometry was used to detect cell apoptosis and cell cycle. The expressions of the target proteins were detected by Western blotting. The results showed that SPEO obviously inhibited the proliferation of HepG2 cells in a dose-dependent manner. SPEO activated a series of apoptotic proteins in HepG2 cells, increasing expression levels of Bax, caspase-3 and caspase-9, and decreasing the bcl-2 expression level. SPEO displayed promising anti-hepatocellular carcinoma activities in vitro, partly by inducing apoptosis in HepG2 cells through activating the mitochondrial pathway.

Essential Oil from Siegesbeckia pubescens Induces Apoptosis through the Mitochondrial Pathway in Human HepG2 Cells / 华中科技大学学报（医学）（英德文版）

Siegesbeckia pubescens (SP) has been used as a traditional medicine for the treatment of and inflammatory diseases.However,the activities of SP against hepatocellular carcinoma and the related mechanisms remain unclear.The present study aimed to examine the effects of the essential oil of SP (SPEO) on the proliferation of hepatocellular carcinoma cells and the possible mechanisms.The growth inhibition of HepG2 cells was analyzed by MTT assay.Hoechst 33258 and fluorescence microscopy were utilized to observe the nuclear morphological changes of apoptotic cells.Flow cytometry was used to detect cell apoptosis and cell cycle.The expressions of the target proteins were detected by Western blotting.The results showed that SPEO obviously inhibited the proliferation of HepG2 cells in a dose-dependent manner.SPEO activated a series of apoptotic proteins in HepG2 cells,increasing expression levels of Bax,caspase-3 and caspase-9,and decreasing the bcl-2 expression level.SPEO displayed promising anti-hepatocellular carcinoma activities in vitro,partly by inducing apoptosis in HepG2 cells through activating the mitochondrial pathway.

Inhibitory effect of arctigenin on lymphocyte activation stimulated with PMA/ionomycin.

This study investigated the effect of arctigenin (Arc) on the cell activation, cytokines expression, proliferation, and cell-cycle distribution of mouse T lymphocytes. Mouse lymphocytes were prepared from lymph node and treated with Phorbol-12-myristate-13-acetate (PMA)/Ionimycin (Ion) and/or Arc. CD69, CD25, cytokines, proliferation and cell cycle were assayed by flow cytometry. The results showed that, at concentrations of less than 1.00 micromol x L(-1), Arc expressed non-obvious cell damage to cultured lymphocytes, however, it could significantly down-regulate the expression of CD69 and CD25, as well as TNF-alpha, IFN-gamma, IL-2, IL-4, IL-6 and IL-10 on PMA/Ion stimulated lymphocytes. At the same time, Arc could also inhibit the proliferation of PMA/Ion-activated lymphocytes and exhibited lymphocyte G 0/G1 phase cycle arrest. These results suggest that Arc possesses significant anti-inflammatory effects that may be mediated through the regulation of cell activation, cytokines expression and cell proliferation.