Study On The Hepatoprotective And Choleretic Effects Of Key Pharmacological Components Of Gentiana Scabra Bge.on Alcoholic And Non-alcoholic Hepatobiliary Injury

Gentiana scabra Bge.(Long-Dan)is a traditional Chinese medicine with the effect of laxing the actual fire of liver and gall bladder.At present,the material basis for quality evaluation of Long-Dan herbs is content determination with a single gentiopicroside as the index,which cannot reflect the overall quality of Chinese Long-Dan for the time being and seriously hinders the effective quality control of Long-Dan.Secondly,the study of biological activity and conformational relationship is relatively superficial.Objective: This study was conducted from the chemical composition of the key pharmacodynamic components of Long-Dan,the material basis of the active components,the mechanism of action and the pharmacodynamic relationship,screening the key pharmacodynamic components of Long-Dan,revealing the chemical composition of the key pharmacodynamic components of Long-Dan,laying the foundation for clarifying the quality markers of Long-Dan and constructing scientific and reasonable quality standards;and elucidating the mechanisms of the key pharmacodynamic components of Long-Dan based on cellular and animal models of alcoholic and non-alcoholic liver injury,providing a scientific basis for resource utilization and clinical research.We also aim to elucidate the mechanism of liver-protective and bile-protective effects of the key components of Long-Dan based on cellular and animal models of alcoholic and non-alcoholic liver injury,and to provide a scientific basis for resource utilization and clinical research of Long-Dan.Methods: 1.In order to clarify the quality markers for the quality evaluation of LongDan as early as possible,scientific studies were conducted on the key pharmacodynamic components of Long-Dan.Firstly,the different active ingredients of Long-Dan were obtained using the process-optimized microporous resin column separation technique;the key pharmacodynamic components of Long-Dan were identified by combining the yield,active ingredient content and cellular activity.Secondly,the chemical composition of the key pharmacodynamic components of Long-Dan has analyzed by the LC-Q-TOF-MS technique to clarify the chemical composition of the key pharmacodynamic components of Long-Dan.2.To explore the activity and mechanism of key pharmacodynamic components of Long Dan at the cellular level.Using 2 cellular models as the basis of the study,lipopolysaccharide(LPS)-induced macrophages(RAW264.7)as an inflammatory model and free fatty acid(FFA)-induced hepatocellular carcinoma cells(Hep G2)as a fatty liver model,the effects on cell survival were investigated in the concentration range of 0-8.0 mg-m L-1,respectively,using the CCK-8 method,with liver function alanine aminotransferase(ALT),aspartate aminotransferase(AST)levels and levels of inflammatory factors interleukin 1β(IL-1β),interleukin 6(IL-6),tumor necrosis factor α(TNF-α),oil red O staining method and TG content were investigated to evaluate the anti-inflammatory,hepatoprotective and hypolipidemic activities of the key pharmacological components of Long Dan.Then,the m RNA levels of cellular Toll-like receptor 4(TLR4)and nuclear transcription factor-kappa B(NF-κB)were measured by fluorescence quantitative PCR(q PCR)to examine the gene expression,and protein blotting(Western Blot)was used to determine NF-κB,phosphorylated NF-κB(p-NF-κB)and nuclear factor-κB inhibitor α(IκBα)protein The expression of NF-κB,pNF-κB and nuclear factor κB inhibitor α(IκBα)was determined by Western blotting to find the molecular mechanisms of anti-inflammatory,lipid-lowering and hepatoprotective activities at cellular level.3.To reveal the hepatoprotective effects and mechanisms of key pharmacodynamic components of Long Dan in a mouse model.(1)Using alcohol-induced alcoholic liver injury model in mice,the expression levels of enzymes,genes and proteins in serum and liver tissues were evaluated by hematoxylin-eosin staining(HE staining method),microplate method,enzyme-linked immunoassay(Elisa method),PCR method and Western blot method as assays to clarify the expression levels in TLR4/NF-κB and phosphorylated p38 mitogen-activated protein kinase(p38-MAPK)signaling pathway,and explore the potential mechanisms in the protective effects of Long Dan fractions against alcoholic liver injury(2)Using a high-fat diet-induced fatty liver model in mice,we measured lipid metabolismrelated indexes using HE staining,oil red O staining,microplate method and Elisa method;using PCR The expression levels of liver-related genes and proteins were measured by PCR and Western blot,based on TLR4/NF-κB,sterol regulatory element binding protein 1c(SREBP-1c)/acetyl coenzyme A carboxylase 1(ACC1)/fatty acid synthase(FAS),peroxisome proliferator-activated receptor α(PPARα)/carnitine palmitoyltransferase 1(CPT-1)and adenylate-activated protein kinase(AMPK)signaling pathways to explore the protective effects and potential mechanisms of key pharmacodynamic components of Long Dan in non-alcoholic liver injury.4.To elucidate the mechanism of hepatoprotective and cholestatic effects of key medicinal components of gentian.Using a mouse model of alcoholic and non-alcoholic hepatobiliary injury and bile injury,we observed the morphological changes of gallbladder by microscopy,and measured the contents of bile and serum indicators alkaline phosphatase(ALP),glutamyl transferase(GGT),total bilirubin(TBIL),cholesterol(TC)and total bile acids(TBA)by microplate and Elisa assay,and measured the m RNA of Farnesoid X receptor(FXR),fibroblast growth factor receptor-4(FGFR4),cholesterol 7α-hydroxylase(CYP7A1),etc.The m RNA and protein expression of Farnesoid X receptor(FXR),fibroblast growth factor receptor-4(FGFR4)and cholesterol 7α-hydroxylase(CYP7A1)were also measured to reveal the mechanism of hepatoprotective and biliary effects based on FXR/FGFR4/CYP7A1 signaling pathway.5.Using LC-MS and 16 S r DNA high-throughput sequencing techniques to identify metabolic differentials and analyze the composition of intestinal flora,we explored the effects of key pharmacodynamic components of gentian on the regulation of metabolism and intestinal flora in NAFLD.Results: 1.The extraction,separation and identification of gentian herbs revealed that the 30% ethanol eluate had moderate yield among the eluted flow fractions in the macroporous resin column,and the highest quality evaluation index of gentian bitter glycoside content compared with other fractions,and had the best hepatoprotective,antiinflammatory and hypolipidemic activities after cellular activity screening.The chemical composition of the key potent components of gentian was analyzed by LC-Q-TOF-MS,and a total of 63 compounds were identified,with 17 and 46 compounds identified as positive and negative ions,respectively.Moreover,after screening various influencing factors of the macroporous adsorption resin,the separation and purification process was optimized to prepare the key pharmacodynamic fractions of gentian.The preparation process is simple,stable and feasible,which provides material guarantee for the subsequent pharmacodynamic research and theoretical guidance for the industrial production.2.Cellular activity screening revealed that in the LPS-induced RAW264.7 cells as an inflammation model,a classical inflammation model was successfully constructed,and the levels of inflammatory factors IL-1β,IL-6 and TNF-α after treatment with gentian key potent fraction suggested that gentian key potent fraction had anti-inflammatory activity.In the FFA-induced Hep G2 cells as a fatty liver model,the optimal FFA modeling concentration of 0.5 m M was screened by CCK-8,and the optimal concentration of gentian key efficacy fraction was 0.5-2.0 g-m L-1.The level of inflammatory factors showed that the key potent fraction of gentian had significant anti-inflammatory activity.3.In the ethanol-induced alcoholic liver injury model,the serum levels of ALT,AST,IL-1β,IL-6 and TNF-α in the alcohol model group were significantly higher than those in the normal group,and the values in the gentian key efficacy component administration group decreased to different degrees compared with those in the alcohol model group(P<0.05);the results of liver pathology showed that the hepatic cords in the alcohol model group were disordered and hepatocytes Compared with the normal group,the expression levels of TLR4,NF-κB p65,p-F-κB p65 and p38 MAPK proteins in liver tissues of mice in the alcohol model group were increased to different degrees(P<0.05),while the expression levels of TLR4,NF-κB p65,p-F-κB p65 and p38 MAPK proteins in the gentian key efficacy component administration group were reduced to different degrees(P<0.05).The expression levels of p65 and p38 MAPK proteins were increased to different degrees(P<0.05),and all of them were decreased to different degrees(P<0.05)after the intervention of the gentian key potency components.In the mouse fatty liver model induced using high-fat feed,HE staining method with oil red O staining revealed a large amount of inflammatory infiltrates and accumulation of lipid droplets compared to the normal group,which was significantly improved by the treatment with gentian key potent fraction.Compared with the model group,the TG content,AST,ALT,IL-1β,IL-6,TNF-α levels,TLR4,NF-κB,SREBP-1c,FAS,and other m RNA expression levels in the key pharmacodynamic fractions of gentian were significantly reduced(P<0.05);TLR4,NF-κB,SREBP-1c,ACC1,FAS,PPARαAMP,K and NF-κB phosphorylated protein levels were significantly reduced(P<0.05 or P<0.01),and CPT-1 and IκBα protein expression levels were significantly increased(P<0.01).4.In the mouse alcoholic and non-alcoholic hepatobiliary injury and bile injury model,the morphological changes of the gallbladder were observed by microscopic observation,and it was found that the bile in the gallbladder of mice in the model group was brown and turbid compared with the normal group,and flocculent precipitation was visible to the naked eye.Compared with the normal group,the serum levels of ALP,GGT and TBIL were significantly increased in the model group(P<0.05);the TC content in bile was significantly increased(P<0.01)and the TBA content was significantly decreased(P<0.01);the m RNA and protein expression of liver FGFR4 were significantly increased(P<0.05)and the m RNA expression of CYP7A1 was significantly decreased(P<0.05),and protein expression tended to decrease significantly,while these indexes were improved after gentian fraction administration.5.LC-MS serum non-targeted metabolomics and 16 S r DNA high-throughput sequencing were used to study the effects of gentian key pharmacodynamic components on NAFLD.Metabolomic analysis revealed that the addition of gentian key pharmacodynamic components significantly altered the levels of amino acids,Gentiopicroside,and Cinnoline,Galabiosylceramide,and Tryptophyl-Tyrosine and other metabolites were involved in the regulation of bile secretion,tryptophan metabolism and lipid metabolism pathways.The analysis of intestinal bacteria revealed that the key pharmacodynamic components of gentian changed the community composition structure of intestinal bacteria,characterized by an increase in beneficial bacteria and a decrease in harmful bacteria.In addition,correlation analysis showed that the effect of gentian fractions on metabolites was positively correlated with the intestinal flora Bacteroides,Lactobacillus,Muribaculum,and Prevotellaceae＿UCG＿001.Finally,joint analysis revealed that metabolites were correlated with the regulation of Firmicutes and Actinobacteriota spp and positively correlated with lipid levels.Conclusion: The key medicinal components of Long Dan were defined based on the yield of Long Dan,the content of the quality control component gentiopicroside and the intensity of hepatoprotective and anti-inflammatory activity.The cellular level activity and mechanism of action studies showed that the key potent fraction of Long Dan has good hepatoprotective effects,through inhibiting lipid accumulation,anti-inflammation,and fatty acid oxidation,etc.The specific mechanism is related to the activation of TLR4/NF-κB,SREBP-1c/ACC1/FAS,PPARα/CPT-1 and AMPK signaling pathways.Meanwhile,the key pharmacodynamic components of Long Dan also had good cholestatic effects,which were associated with the m RNA and protein expression levels of FXR,FGFR4,and CYP7A1,key factors regulating the cholesterol-bile acid metabolic pathway.The combined metabolomics and intestinal flora analysis showed that the key pharmacodynamic components of Long Dan could improve intestinal flora,lipid metabolism and bile acid metabolism in model animals,constructing a map of the correlation between intestinal microbiota and blood metabolism,revealing key intestinal microbial metabolite combinations,and laying a foundation for further research on the disease mechanism of NAFLD.This study provides a theoretical and experimental basis for the prevention and treatment of hepatobiliary injury disease and provides new ideas for the practical development and application of key pharmacological components of Long Dan and the diversification of Long Dan quality evaluation indexes.