Study On Prevention And Treatment Mechanism Effect Of Scutellaria Against Alcoholic Fatty Liver Disease Based On The Gut Microbiota And The Intestinal Mucosal Barrier

Objective:Non-alcoholic fatty liver disease(NAFLD)is a chronic disease that manifests as abnormal proliferation of connective tissue in the liver.The high incidence rate in the world has attracted great attention,poor diet is one of the main causes of NAFLD,a high-fat diet causes liver lipid accumulation,lipid metabolism disorders,damage to the mucous barrier,and damage to the parenthetic axis,and leads to liver damage.Scutellaria baicalensis Georgi.is a commonly used clinical Chinese medicine for the treatment of various inflammatory injury diseases,but its potential anti-inflammatory and anti-liver injury mechanism of action is unknown.Given the important role of gut microbiota in pathogenesis and development,this study aimed to investigate the molecular biological mechanism of anti-liver injury of Scutellaria baicalensis based on the"Intestinal bacterial flora-Mucosal barrier".Non-targeted metabolomics was used to study mouse feces,combined with 16Sr RNA high-throughput sequencing to explore the microbial diversity of microbiota,and explain the mechanism of action of Scutellaria baicalensis in the prevention and treatment of fatty liver disease,which provides a theoretical basis for the prevention and treatment of fatty liver disease in the later stage.Methods:1.Establish the analysis method of active ingredients of skullcap extract:the active ingredients in Scutellaria baicalensis extract were qualitatively and quantitatively analyzed by ultra-high performance liquid chromatography-quadrupole rod-electrostatic field orbital well tandem mass spectrometry(UPLC-Q-Orbitrap MS/MS)and ultra-performance liquid chromatography-triple quadrupole mass spectrometry(UPLC-QQQ MS).2.Pharmacodynamic study of Scutellaria baicalensis extract on NAFLD model mice:a model of NAFLD disease in C57BL/6J mice was established by high-fat diet induction method,normal saline was given to the blank group and the model group,and high,medium,and low doses of Scutellaria baicalensis extract and the positive drug silybin capsules were given to the treatment group for a total of 10 weeks.By investigating the weight gain,liver index,liver lipid metabolism,liver injury,serum inflammatory level,and liver pathological changes of mice,the pharmacodynamic effect of Scutellaria baicalensis extract on NAFLD was determined.3.Metabolomics study of Scutellaria baicalensis extract on NAFLD model mice:Mass Hunter software collected the obtained mass spectrometry data by UPLC-Q-Orbitrap-MS detection,and after preprocessing the original mass spectrometry data by QA software,the retention time,mass-charge ratio and peak area of metabolites were obtained,and the SIMCA14.0 software was used for unified analysis.Scutellaria baicalensis extract was screened for the prevention and treatment of potential biomarkers of NAFLD based on VIP>1and p<0.05,and Metabo Analyst 5.0 was used to analyze the differential metabolite enrichment pathway.4.Effects of Scutellaria baicalensis extract on intestinal microbial diversity in NAFLD model mice:16Sr RNA high-throughput sequencing technology was used to analyze fecal samples of NAFLD mice,analyze alpha diversity,beta diversity,species composition of intestinal flora,community structure differences in mice based on sequence similarity clustering OTU,and predict metabolic pathways for KEGG functional genes,and screen for different strains that play a key role in intestinal flora changes before and after the intervention of Scutellaria baicalensis extract.5.Based on the"Intestinal bacterial flora-Mucosal barrier",the effect of Scutellaria baicalensis extract on the prevention and control of NAFLD model mice was investigated:the histopathological changes,inflammatory factor levels,expression of tight junction proteins and mucosal permeability changes of NAFLD mice were investigated,and combined with 16Sr RNA flora.To investigate the molecular biological mechanism of anti-liver injury of Scutellaria baicalensis extract.Results:1.In this study,a total of 6 flavonoids were identified by the qualitative analysis of Scutellaria baicalensis extract by UPLC-Q-Orbitrap-MS,namely baicalein,baicalein,baicalein,baicalein I,baicalein,and Melaleucin A.The quantitative analysis of UPLC-QQQ-MS contained baicalin 94.12%,wogonoside 3.31%,baicalein 1.2%and wogonin0.65%.The average dosing recovery rate of the four flavonoids was 106.28%～109.84%,and the linear relationship(r~2≥0.999)was good in the respective linear concentration range,and the RSD of precision,repeatability,and stability were less than 2%.In this study,the analysis method of active ingredients of Scutellaria baicalensis extract was established to be simple to operate,stable,and reliable.2.In this study,the liver of NAFLD mice was analyzed by HE staining,and a large amount of steatosis was observed in liver cells,and inflammatory cell infiltration and slight fibrosis signals appeared in liver lobules.Oil red O adipose staining showed the formation of a large number of red lipid droplets,increased lipid deposition,and vacuolar denaturation;There were no obvious fibrotic cords in the liver tissue during Masson staining,indicating that NAFLD mice were successfully modeled.After the intervention of Scutellaria baicalensis extract,it could inhibit weight gain(p<0.05)and liver hypertrophy(p<0.05)in mice with NAFLD,significantly reduce TC(p<0.05),TG(p<0.05)and LDL-C(p<0.05)levels,AST(p<0.05)and ALT(p<0.05)levels,and significantly increase HDL-C(p<0.05)level,which in turn reduces lipid accumulation in the liver.At the same time,the intervention of Scutellaria baicalensis extract significantly reduced the levels of inflammatory factors IL-6(p<0.05),TNF(p<0.05),and IL-1β(p<0.05),thereby alleviating liver inflammation.The dose of Scutellaria baicalensis extract improved liver inflammatory damage and had the best ability to remove serum lipids,indicating that Scutellaria baicalensis extract had a protective effect on the liver of NAFLD mice.3.In this study,32 potential differential metabolites in the feces of NAFLD mice were screened for ESI~+/ESI~-mode based on VIP>1 and p<0.05.A total of 24 compounds were recovered after the intervention of Scutellaria baicalensis extract,among which chenodeoxycholic acid sulfate(p<0.001),chenodeoxycholic acid(p<0.001),cholic acid(p<0.001),ursodeoxycholic acid(p<0.001),taurocholic acid(p<0.001),deoxycholic acid(p<0.001),the related pathways are mainly Primary bile acid biosynthesis and Secondary bile acid biosynthesis.Metabo Analyst5.0 combined with the KEGG enrichment differential metabolite pathway,it was found that Scutellaria baicalensis extract mainly affected Primary bile acid biosynthesis and Taurine and hypotaurine metabolism.4.In this study,through 16Sr RNA high-throughput sequencing of mouse feces of each group,it was found throughαdiversity andβdiversity that Scutellaria baicalensis extract significantly reduced the bacterial amount,reduced the Chao1 index(p<0.05),Simpson index(p<0.05),significantly reduced the ACE index(p<0.01),and changed the diversity and richness of the intestinal flora of NAFLD mice.Based on the phylum and genus levels,Scutellaria baicalensis extract inhibited the propagation of harmful bacteria Firmicutes(p<0.05),Desulfobacterota(p<0.05),Proteobacteria(p<0.05),unclassified＿Muribaculaceae(p<0.05)and Helicobacter(p<0.05),and increased the propagation of beneficial bacteria Bacteroidota(p<0.05)and Bacteroides(p<0.05)and Lachnospiraceae＿NK4A136＿group(p<0.05)genera to intervene in the structural composition and diversity of gut microbes in NAFLD mice.Through LEf Se analysis of community structure differences,statistically different colony species were sought between groups,and the final attribution was consistent with the species composition analysis.The composition and difference analysis of the KEGG metabolic pathway showed significant differences in 41 intestinal microbiota metabolism categories in NAFLD mice and 47intestinal flora metabolism categories after Scutellaria baicalensis extract intervention.5.In this study,the colon of NAFLD mice was analyzed pathologically by HE staining,and it was observed that the mucosal epithelial structure was destroyed and missing,the crypt structure was severely lost,the glands were partially missing and arranged in disorder,the folds became shorter and the gap widened,and a large number of inflammatory cells were infiltrated by the mucosa.After the intervention,the levels of TNF-α(p<0.01),IL-1β(p<0.01),IL-6(p<0.01),LPS(p<0.01),and DAO level(p<0.05)were significantly reduced,thereby reducing the level of colitis and repairing intestinal damage.Immunofluorescence staining showed that the fluorescence intensity and fluorescence area of tight junction protein expression increased after the intervention of Scutellaria baicalensis extract,and the expression of tight junction protein was determined by western blotting,and Scutellaria baicalensis extract could increase ZO-1(p<0.01),Occludin(p<0.05)and Claudin1(p<0.05)protein expression to improve intestinal mucosal barrier function."Intestinal flora and mucosal barrier"correlation analysis The levels of inflammatory factors,intestinal mucosal injury factors and tight junction protein expression levels in colonic tissue of NAFLD mice were significantly positively correlated with Firmicutes,Desulfobacterota and Proteobacteria,Helicobacter and unclassified＿Muribaculaceae bacteria.The phylum Bacteroidota and the genera Lachnospiraceae＿NK4A136＿group and Bacteroides were significantly negatively correlated.Conclusion:Scutellaria baicalensis extract can reduce liver lipid accumulation,relieve liver inflammation,and effectively improve metabolic levels in the body.By regulating the homeostasis of intestinal microbiota,inhibiting the reproduction of harmful bacteria,increasing the number of beneficial bacteria,enhancing the expression of intestinal tight junction protein,and repairing the intestinal mucosal barrier function,it can prevent and treat non-alcoholic fatty liver disease.