Exploration of the underlying mechanism of Astragaloside III in attenuating immunosuppression via network pharmacology and vitro/vivo pharmacological validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragalus mongholicus Bunge (AM, recorded in http://www.worldfloraonline.org, 2023-08-03) is a kind of medicine food homology plant with a long medicinal history in China. Astragaloside III (AS-III) has immunomodulatory effects and is one of the most active components in AM. However, its underlying mechanism of action is still not fully explained. AIM OF THE STUDY: The research was designed to discuss the protective effects of AS-III on immunosuppression and to elucidate its prospective mechanism. MATERIALS AND METHODS: Molecular docking methods and network pharmacology analysis were used to comprehensively investigate potential targets and relative pathways for AS-III and immunosuppression. In order to study and verify the pharmacological activity and mechanism of AS-III in alleviating immunosuppression, immunosuppression mouse model induced by cyclophosphamide (CTX) in vivo and macrophage RAW264.7 cell model induced by hypoxia/lipopolysaccharide (LPS) in vitro were used. RESULTS: A total of 105 common targets were obtained from the AS-III-related and immunosuppression-related target networks. The results of network pharmacology and molecular docking demonstrate that AS-III may treat immunosuppression through by regulating glucose metabolism-related pathways such as regulation of lipolysis in adipocytes, carbohydrate digestion and absorption, cGMP-PKG signaling pathway, central carbon metabolism in cancer together with HIF-1 pathway. The results of molecular docking showed that AS-III has good binding relationship with LDHA, AKT1 and HIF1A. In CTX-induced immunosuppressive mouse model, AS-III had a significant protective effect on the reduction of body weight, immune organ index and hematological indices. It can also protect immune organs from damage. In addition, AS-III could significantly improve the expression of key proteins involved in energy metabolism and serum inflammatory factors. To further validate the animal results, an initial inflammatory/immune response model of macrophage RAW264.7 cells was constructed through hypoxia and LPS. AS-III improved the immune function of macrophages, reduced the release of NO, TNF-α, IL-1ß, PDHK-1, LDH, lactate, HK, PK and GLUT-1, and restored the decrease of ATP caused by hypoxia. Besides, AS-III was also demonstrated that it could inhibit the increase of HIF-1α, PDHK-1 and LDH by adding inhibitors and agonists. CONCLUSIONS: In this study, the main targets of AS-III for immunosuppressive therapy were initially analyzed. AS-III was systematically confirmed to attenuates immunosuppressive state through the HIF-1α/PDHK-1 pathway. These findings offer an experimental foundation for the use of AS-III as a potential candidate for the treatment of immunosuppression.

Biological activities, Molecular mechanisms, and Clinical application of Naringin in Metabolic syndrome.

Metabolic syndrome has become major health problems in recent decades, and natural compounds receive considerable attention in the management of metabolic syndrome. Among them, naringin is abundant in citrus fruits and tomatoes. Many studies have investigated the therapeutic effects of naringin in metabolic syndrome. This review discusses in vitro and in vivo studies on naringin and implications for clinical trials on metabolic syndrome such as diabetes mellitus, obesity, nonalcoholic fatty liver disease, dyslipidemia, and hypertension over the past decades, overviews the molecular mechanisms by which naringin targets metabolic syndrome, and analyzes possible correlations between the different mechanisms. This review provides a theoretical basis for the further application of naringin in the treatment of metabolic syndrome.

Exploring and Verifying the Mechanism and Targets of Shenqi Pill in the Treatment of Nonalcoholic Steatohepatitis via Network Pharmacology and Experiments.

Shenqi pill (SQP), a famous traditional Chinese medicine (TCM) herbal formula derived from Jinguiyaolue (Synopsis of Prescriptions of the Golden Chamber), has long been used to treat kidney yang deficiency syndrome. According to the TCM treatment principle that the liver and kidney are homologies, the clinical use of SQP in the treatment of nonalcoholic steatohepatitis (NASH) has achieved a good effect. However, the active targeted genes and underlying mechanism remain unclear. In this study, we aimed to explore the treatment mechanism of SQP in NASH rats, which may further contribute to the in-depth exploration of SQP in clinical applications. Network pharmacology analysis was used to screen the target genes of SQP for NASH treatment based on public databases. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and protein-protein interaction (PPI) analysis were used to search for crucial target genes and mechanisms. UPLC-MS/MS was used to verify the active compounds of the SQP screened. The hepatic pathology and biochemical indicators of rats were used to judge the modeling results and the curative effect of SQP. Western blotting and qRT-PCR were used to verify the expression of crucial target genes at the protein and RNA levels, respectively. Network pharmacology analysis and bioinformatics analysis showed that PTGS2, JUN, MYC, and CDKN1A might be crucial target genes in the primary mechanism of SQP in treating NASH and improving the inflammatory response. The UPLC-MS/MS results confirmed that the hub active compound, quercetin, screened out through the TCMSP database, is indeed present in SQP. Hepatic injury and lipid metabolism indicators of NASH rats were significantly improved after SQP treatment. The results of WB and qRT-PCR showed that the expression of PTGS2, JUN, MYC, and CDKN1A was higher in NASH rats than in normal rats and decreased after SQP treatment. The expression of inflammatory cytokines (IL-1ß, IL-6, TNF-α) was reduced after SQP treatment, which confirmed that SQP could improve hepatic inflammation in rats. These results suggested that SQP could ameliorate NASH in rats, and that quercetin may be the critical active compound that exerts the therapeutic effect.

Yiqi-Bushen-Tiaozhi Recipe Attenuated High-Fat and High-Fructose Diet Induced Nonalcoholic Steatohepatitis in Mice via Gut Microbiota.

Aim: To investigate the treating effect of Yiqi-Bushen-Tiaozhi (YBT) recipe on nonalcoholic steatohepatitis (NASH) mice, determine whether the outcome was associated with gut microbiota, and clarify the regulating mechanism. Methods: NASH mice were induced by high-fat and high-fructose diets (HFFD). In the fifth week, mice in the YBT group were orally administrated YBT (22.12g·kg-1·d-1) daily for 12 weeks. Fresh stool of mice was collected at the 16th week for fecal 16S rDNA analysis. Hepatic pathology and biochemical indicators were used to reflect the improvement of YBT on hepatic inflammation and lipid metabolism in NASH mice. Quantitative real-time PCR (qRT-PCR) was used to verify the results of PICRUSt analysis. Results: Results of the pathological and biochemical index showed that YBT could improve NASH mice. Compared with improving inflammation and hepatocyte damage, YBT may be more focused on enhancing metabolic disorders in mice, such as increasing HDL-c level. The diversity and richness of the gut microbiota of NASH mice induced by HFFD are significantly different from the normal control (NC) group. After YBT treatment, the diversity and richness of the mice microbiota will be increased to similar NC mice. Intestinimonas, Acetatifactor, Alistipes, Intestinimonas, Acetatifactor, and Alistipes have the most significant changes in the class level. PICRUSt analysis was performed to predict genomic functions based on the 16S rDNA results and reference sequencing. The efficacy of YBT in the treatment of NASH can be achieved by regulating the diversity and richness of gut microbiota. PICRUSt analysis results showed that the most relevant function of the microbiota construction variations is α- Linolenic acid (ALA) metabolism. Results of qRT-PCR showed significant differences between groups in the expression of Fatty acid desaturase 1 (FADS1), Fatty acid desaturase 2 (FADS2), Acyl-CoA Oxidase 1 (ACOX1), and Acyl-CoA Oxidase 2 (ACOX2) related to ALA metabolism. The expression of the above genes will be inhibited in the liver and small intestine of the HFFD group mice, and the expression can be restored after YBT treatment. Conclusion: YBT could treat NASH mice by improving the diversity and richness of gut microbiota and further the improvement of ALA metabolism.

Bioactive components, pharmacological effects, and drug development of traditional herbal medicine Rubus chingii Hu (Fu-Pen-Zi).

Rubus chingii Hu (Chinese Raspberry), known as Fu-Pen-Zi in Chinese, a woody perennial plant of the genus Rubus in the Rosaceae family, has specific nutritional and medicinal values, which is considered food-medicine herb in China for thousands of years to treat impotence, premature ejaculation, enuresis, frequent urination, and other diseases. This review aims to summarize recent advances in the bioactive components, pharmacological effects, and drug development and utilization of Rubus chingii Hu, hoping to provide useful support for its further research and clinical application. The bioactive components in Rubus chingii Hu contain mainly terpenoids, flavonoids, alkaloids, phenolic acids, polysaccharides, and steroids. The main pharmacological effects are their anti-oxidant, anti-inflammatory, and anti-tumor capacity on human health. Rubus chingii Hu is a very valuable food-medicine herb. The development of Rubus chingii Hu-related drugs is relatively single, which is limited to traditional Chinese medicine and prescriptions. Therefore, it is vital to pay interest to Rubus chingii Hu and its bioactive components in the future and extend its scientific application.

Predicting Target Genes of San-Huang-Chai-Zhu Formula in Treating ANIT-Induced Acute Intrahepatic Cholestasis Rat Model via Bioinformatics Analysis Combined with Experimental Validation.

BACKGROUND: San-Huang-Chai-Zhu formula (SHCZF) has been used to improve cholestasis for many years. This study aims to predict the possible gene targets of SHCZF in treating acute intrahepatic cholestasis (AIC) in rats. MATERIALS AND METHODS: Eighteen SD rats were randomly assigned to the normal group, ANIT group, and ANIT + SHCZF group. Alpha-naphthylisothiocyanate (ANIT) was used to induce AIC. Serum biochemical indexes were detected in each group. After treatment, the livers were collected and used to extract RNA. The library was constructed by TruSeq RNA, sequenced by Illumina, and analyzed by various bioinformatics methods. qRT-PCR was used to verify the target genes related to the efficacy of SHCZF. RESULTS: Serum ALT, AST, ALP, and TBIL were significantly higher in the ANIT group than in the normal group. Serum ALT and AST levels in the ANIT + SHCZF group were substantially lower than those in the ANIT group. A total of 354 intersected genes were screened by expression level correlation and PCA analysis, GO and KEGG pathway enrichment analysis, and WGCNA and STEM analysis. Then, 4 overlapping genes were found by pathway/BP/gene network construction. SHCZF reversed the downregulation of expression of CYP4A1 and HACL1 and the upregulation of expression of DBI and F11R induced by ANIT. In addition, the qRT-PCR result showed that mRNA expression of CYP4A1, HACL1, and F11R genes in the liver was consistent with the prediction result of bioinformatics analysis. CONCLUSION: CYP4A1, HACL1, and F11R are genes related to the occurrence of ANIT-induced AIC in rats and may be considered as targets of SHCZF for the treatment of AIC.

Natural Compounds: A Potential Treatment for Alcoholic Liver Disease?

Excessive alcohol intake is a direct cause of alcoholic liver disease (ALD). ALD usually manifests as fatty liver in the initial stage and then develops into alcoholic hepatitis (ASH), fibrosis and cirrhosis. Severe alcoholism induces extensive hepatocyte death, liver failure, and even hepatocellular carcinoma (HCC). Currently, there are few effective clinical means to treat ALD, except for abstinence. Natural compounds are a class of compounds extracted from herbs with an explicit chemical structure. Several natural compounds, such as silymarin, quercetin, hesperidin, and berberine, have been shown to have curative effects on ALD without side effects. In this review, we pay particular attention to natural compounds and developing clinical drugs based on natural compounds for ALD, with the aim of providing a potential treatment for ALD.

Pure total flavonoids from citrus attenuate non-alcoholic steatohepatitis via regulating the gut microbiota and bile acid metabolism in mice.

BACKGROUND: Our previous studies found that Pure total flavnoids from citrus (PTFC) can effectively improve non-alcoholic steatohepatitis (NASH) in mice. Here, we discuss on the mechanism of PTFC in treating NASH with focus on the regulation of the gut microbiota and bile acid metabolism. METHODS: C57BL/6 J mice were randomly divided into three groups: normal diet group (Normal), high-fat diet group (HFD) and high-fat + PTFC treatment group (PTFC). Mice in the Normal group were fed chow diet, while the other groups were fed high fat diet (HFD) for 16 weeks. In the 5th week, the mice in the PTFC group were treated with 50 mg/kg/day PTFC for an additional twelve weeks. After sacrifice, histopathology of the liver was assessed, and the gut microbial composition was analyzed by 16S rDNA gene sequencing. Bile Acid profiles in serum were determined by ultraperformance liquid chromatography (UPLC-MS/MS). RESULTS: PTFC intervention significantly attenuated HFD-induced NASH symptoms compared with the HFD group in mice. 16S rDNA sequencing showed that PTFC treatment increased the phylogenetic diversity of the HFD-induced microbiota dysbiosis. PTFC intervention significantly increased the relative abundances of Bacteroidaceae and Christensenellaceae. Furthermore, PTFC reduced the content of toxic bile acids, such as TDCA, DCA, TCA, CA and increased the ratio of secondary to primary bile acids. FXR and TGR5 deficiency were significantly alleviated. CONCLUSION: PTFC can improve NASH via the the gut microbiota and bile acid metabolism.

Berberine attenuates non-alcoholic steatohepatitis by regulating chemerin/CMKLR1 signalling pathway and Treg/Th17 ratio.

To observe the therapeutic effect of berberine (BBR) on non-alcoholic steatohepatitis (NASH) in rats and the underlying mechanism. A rat model of NASH was established by a high-fat diet, and BBR was used as treatment. Haematoxylin-eosin staining and Oil Red O staining were used to observe the pathological changes in the liver tissue. Western blotting and real-time PCR were used to measure the mRNA and protein levels in the liver. Flow cytometry was performed to detect the number of intrahepatic lymphocyte subtypes. The expression of pro-inflammatory cytokines in the peripheral blood was measured by ELISA. An automatic biochemical method was used to examine the level of blood lipids in the blood. Compared with the rats in the model group, the rats in the BBR group showed significantly improved liver histopathology and serum pro-inflammatory cytokines and free fatty acid (FFA) levels. Moreover, the protein and mRNA expression of chemerin, CMKLR1 and CCR2 in the liver were obviously reduced by BBR treatment. In addition, the high-fat diet remarkably reduced the intrahepatic Treg/Th17 ratio, which could be recovered by BBR treatment. Berberine can ameliorate non-alcoholic steatohepatitis, and its mechanism may be related to restoring the Treg/Th17 ratio, regulating the chemerin/CMKLR1 signalling pathway to reduce liver inflammation and reducing lipid deposition.

The Target MicroRNAs and Potential Underlying Mechanisms of Yiqi-Bushen-Tiaozhi Recipe against-Non-Alcoholic Steatohepatitis.

MicroRNAs (miRNAs) have emerged as potential therapeutic targets for non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH). Traditional Chineses Medicine (TCM) plays an important role in the prevention or treatment of NAFLD/NASH. However, miRNA targets of TCM against NASH still remain largely unknown. Here, we showed that Yiqi-Bushen-Tiaozhi (YBT) recipe effectively attenuated diet-induced NASH in C57BL/6 mice. To identify the miRNA targets of YBT and understand the potential underlying mechanisms, we performed network pharmacology using miRNA and mRNA deep sequencing data combined with Ingenuity Pathway Analysis (IPA). Mmu-let-7a-5p, mmu-let-7b-5p, mmu-let-7g-3p and mmu-miR-106b-3p were screened as the main targets of YBT. Our results suggested that YBT might alleviate NASH by regulating the expression of these miRNAs that potentially modulate inflammation/immunity and oxidative stress. This study provides useful information for guiding future studies on the mechanism of YBT against NASH by regulating miRNAs.

Regulation of pure total flavonoids from Citrus on TH17/Treg balance in mice with NASH.

This study aimed to investigate the involved immunologic mechanism of pure total flavonoids from Citrus (PTFC) on the development of non-alcoholic steatohepatitis (NASH). C57BL/6 mice were fed with high .fat diet for 16 weeks to induce the NASH model, and from the 7th week three dosages (25, 50 and 100 mg x kg(-1) x d(-1)) of PTFC were administrated intragastric for 10 weeks respectively. Serum TG, CHOL, ALT, AST were determined by biochemical assay, histopathological changes of the liver tissue were observed by HE staining, expression of RORyt and Foxp3 mRNA of the liver tissue was detected by Real-time PCR, and serum IL-17, IL-6, IL-10 and IL-4 were determined by.Cytometric Beads Array. As a result, we find that after the administration of PTFC, the in- flammation of the liver tissue of NASH mice was attenuated, liver function was improved, and the expression of RORgammat mRNA was higher in the liver tissue while which was lower of Foxp3 mRNA, the level of proinflammatory cytokines IL-17 and IL-6 decreased and the level of suppressive cytokines IL-10 and IL-4 increased. These data show that PTFC protects the development of NASH through regulating the Th17/Treg balance and attenuating inflammation.

[Experimental study on intervention effect of Grifola frondosa on nonalcoholic steatohepatitis].

To study the preventive effect of Grifola frondosa on nonalcoholic steatohepatitis (NASH). The rat model of NASH was established by feeding high-fat diets for 12 weeks and intervened with 0.5 g · kg(-1) · d(-1) and 1.0 g · kg(-1) · d(-1) of C. frondosa powder suspensions. The degrees of hepatocyte fatty degeneration and inflammation were observed under the optical microscope with routine HE staining. The NAFLD activity scores (NAS) were calculated. Serum ALT, AST and hepatic TG and CHOL were tested by the biochemical method. The hepatic MDA was examined by thiobarbituric acid method. The hepatic SOD was tested by the xanthine oxidase test. The hepatic GSH-PX activity was determined by the dithio-nitrobenzoic acid method. Hepatic TNF-α and IL-6 were detected by the enzyme-linked immunosorbent assay (ELISA). The NASH model group induced by high-fat diets showed higher hepatic NAS, ser- um ALT, AST, CHOL and hepatic TG, CHOL, MDA, TNF-α, IL-6 (P < 0.01 or P < 0.05) and lower serum TG and hepatic SOD, GSH-PX (P < 0.01, P < 0.05) than the normal control group. After being intervened with different doses of G. frondosa, the NASH group revealed significantly lower hepatic NAS, serum ALT and hepatic TG, CHOL, MDA, TNF-α and IL-6 (P < 0.05) and higher hepatic SOD, GSH-PX (P < 0.05) than the model group. G. frondosa may prevent the further development of NASH by improving the disorder of lipid metabolism in rats with NASH induced by high-fat diets, relieving the level of oxidative stress and reducing the generation of inflammatory cytokines.

[Effect of pure total flavonoids from citrus on hepatic SIRT1/PGC-1alpha pathway in mice with NASH].

OBJECTIVE: To observe the effect of pure total flavonoids from Citrus (PTFC) on the hepatic fatty degeneration, inflammation, oxidative stress and SIRT1/PGC-1alpha expressions in mice with non-alcohol steatohepatitis (NASH), and discuss the action mechanism of PTFC on NASH. METHOD: Mice were given high-fat diet for 16 weeks to induce the NASH model. Since the seventh week after the model establishment, the mice were intervened with 100, 50 and 25 mg x kg(-1) x d(-1) PTFC for 10 weeks. The pathologic changes in hepatic tissues were observed with HE staining. The contents of TG, CHOL in hepatic tissue, as well as the levels of AST, ALT in serum were detected by using the biochemical process. The expression of SIRT1, PGC-1alpha and MnSOD mRNA in hepatic tissues were detected with Real-time PCR assay. SIRT1, PGC-1alpha protein and 8-OHdG expressions were determined with the immunohistochemical method. The SOD level in hepatic tissues was tested by the xanthine oxidase method. The MDA content in hepatic tissues was examined by the thiobarbituric acid method. RESULT: The contents of TG, CHOL, NAFLD activity scores and ALT level in serum in hepatic tissues of mice in the model induced by fat-rich diet were obviously higher than that of the normal group (P < 0.010. The SIRT1, PGC-1alpha, MnSOD mRNA and protein expression in hepatic tissues were significantly lower than that of the normal group (P < 0.01). The expression of 8-OHdG and the content of MDA in hepatic tissues were obviously higher than that of the normal group (P < 0.01). After the intervention with different doses of PTFC, the NAFLD activity scores, the content of TG and the level of AST in serum were notably lower than that of the normal group (P < 0.01, P < 0.05); whereas the SIRT1, PGC-1alpha, MnSOD mRNA and protein expression were obviously higher than that of the normal group (P < 0.01, P < 0.05), with the significant decrease in the expression of 8-OHdG and the content of MDA (P < 0.01). CONCLUSION: Oxidative stress/lipid peroxidation enhancement in in NASH mice induced by high-fat diet may be related to the changes in SIRT1/PGC-1alpha signal transduction pathway. PTFC could enhance the anti-oxidant capacity in liver, relieve the damage of reactive oxygen during the fatty acid metabolic process, and prevent NASH from the occurrence and development by regulating the SIRT1/PGC-1alpha signal pathway.

[Effect of total flavones of hawthorn leafon (TFHL) on expression of UCP2 in liver of NASH rats].

OBJECTIVE: To study the expression of uncoupling protein 2 (UCP2) in liver of rats with nonalcoholic steatohepatitis (NASH) induced by fat-rich diet, and the effect of total flavones of hawthorn leafon (TFHL) on UCP2. METHOD: The NASH model of rat was induced by 12 weeks of fat-rich diet. Subsequently the rats were administrated with TFHL in accordance with 250, 125 mg x kg(-1) x d(-1) and the Essentiale N with 195.4 mg x kg(-1) x d(-1). The change of liver pathological. The levels of serum ALT and AST, the content of TG, CHOL, MDA and T-AOC activity of liver and were evaluated. The UCP2mRNA expression in liver was detected with RT-PCR, and the contents of UCP2 were examined with ELISA. RESULT: There are severe steatosis, inflammatory cellular infiltration in the liver of the NASH models. The levels of serum ALT, AST and the contents of TG, CHOL, MDA and UCP2 in the model group were higher than those of in the normal groop. The expression of UCP2mRNA was obviously enhanced and the activity of T-AOC decreased. The expression of UCP2 mRNA of rats was positively correlation with the contents of MDA, TNF-alpha. The inflammation activity in rat liver, the contents of MDA and UCP2, the expression of UCP2 mRNA in the administrated groups were obviously lower than those in the model group, while the activity of T-AOC was higher than that of model. CONCLUSION: TFHL may alleviate liver injury by means of the suppression of Oxidative stress/lipid peroxidation reaction and the overexpression of UCP2 in liver, which could prevent the further development of NASH.