Therapeutic Effect And Preliminary Mechanism Of Qushi Huayu Decoction On Nonalcoholic Steatohepatitis(NASH)Complicated With Liver Fibrosis In Mice

Objective:1.Establish a model of non-alcoholic fatty liver hepatitis with liver fibrosis in mice;2.Using this model to evaluate the efficacy of traditional Chinese medicine compound Qushi Huayu Recipe on non-alcoholic steatohepatitis(NASH)combined with liver fibrosis;3.Based on the significant effect of phlegm and phlegm and blood stasis on NASH combined with liver fibrosis,transcriptomics was further used to analyze the possible mechanism of action.Method:1.Using high-trans fatty acid feed diet combined with high-sugar drinking water(HFHC)to induce C57BL/6J mice with NASH and liver fibrosis animal models,and biochemical or pathological dynamic evaluation of the model at 16 weeks,20 weeks and 24 weeks respectively.To determine the appropriate modeling period for the preparation of the NASH combined liver fibrosis model.2.Using the above-mentioned suitable modeling period of mouse nonalcoholic steatohepatitis combined with fibrosis model,the experimental mice were randomly divided into:normal group,model group,phlegm-dampness phlegm treatment group,Fuzheng Huayu prescription group,oleic acid control group.The drug was administered four weeks after administration.The main observations were:liver tissue triglyceride(TG)content,liver tissue hydroxyproline(HYP)content,fasting blood glucose(FBG),fasting insulin(FINS)and insulin resistance index(HOMA).-IR),liver function(serum ALT,AST activity),liver histopathology(HE,oil red O staining,Sirius red staining),liver fibrosis related indicators(Col-1,Col-4,?-SMA and TGF-The expression of ?protein and mRNA levels),evaluated the efficacy of phlegm and blood stasis for the treatment of experimental nonalcoholic steatohepatitis with liver fibrosis.3.The transcriptome gene expression microarray technique was used to detect the transcriptome expression profiles of the liver tissues of the above groups.The differentially expressed genes in the normal group,the model group and the phlegm-dampness phlegm group were analyzed and analyzed,and the functions of the differential genes were analyzed.Signal path,etc.Result:1.High-trans fatty acid diet diet combined with high-sugar drinking water for 16 weeks,compared with normal mice,the liver TG and HYP levels in the model group were significantly increased;serum ALT and AST activities were significantly increased;FBG,FSI And HOMA-IR was significantly increased;HE and oil red O staining showed that hepatic steatosis was obvious,inflammatory cell infiltration occurred,and some hepatocytes showed balloon-like changes;Sirius red staining showed that some mice(60%)began to appear.Collagen fiber deposition in the three regions of the liver acinus.At the end of the 20th week,HE and oil red O staining showed that the liver tissue of the model group showed obvious macrofollicular fat-like changes and vesicular fatty-like changes,and inflammatory cell infiltration was more common;Sirius red staining showed that the liver tissue appeared star-like The distribution of sinusoidal fibrosis and fibrosis stage are all F1-2.HE staining at 24 weeks showed that the liver tissue of the model mice had severe macrofollicular and vesicular fatty-like changes(range 66%-99%),with significant hepatocyte ballooning and lobular inflammatory lesions.The wolf red staining showed that the sinus periplasmic fibrosis of all the liver tissues of mice was obvious,and the cage-like fibrosis was changed.Some of them had tight bridging formation,and the fibrosis stage was mostly F2-F3 stage.2.After the model was established for 24 weeks,the mice were given phlegm and blood stasis for 4 weeks.The pharmacodynamic results showed that compared with the model group,the liver TG of the phlegm-dampness phlegm group HYP content was significantly decreased;serum ALT and AST activities were significantly decreased;FBG,FSI and HOMA-IR were significantly decreased.HE staining showed that the fat vacuoles in the liver tissue of the phlegm-dampness and phlegm-removing group were significantly reduced,the degree of inflammatory cell infiltration was significantly reduced,and the number of inflammatory cells was significantly reduced;the red staining of Sirius showed a significant decrease in collagen fibers,and the collagen fibers were loose and had signs of disappearance of breakage.More than half of the fibrosis stages are F0-F1.The levels of Col-1,Col-4,?-SMA and TGF-? protein and mRNA in the liver tissues of Danghu Huayu Recipe were significantly lower than those in the model group.3.Transcriptome microarray results of liver tissue:There were 135 differential genes with different logarithmic value of Log2FC in the difference gene between the two groups in the phlegm-dampness group and the model group,and the number of differential genes was 135,and 84 genes were up-regulated(including Dusp9,Cyp2b13 and Gdf3,etc.,down-regulated 51 genes(including Hspala,Timd4,Cxcl13,etc.),these 135 differential genes involved 21 signaling pathways(including extracellular matrix receptor interaction pathway,cell adhesion molecule pathway,retinol metabolism)Pathway,bile secretion pathway and MAPK signaling pathway,etc.).The difference between the model group and the normal group,the phlegm-dampness phlegm group and the model group was opposite.There were 33 genes with an absolute value of Log2FC greater than 2 and statistically significant(P<0.05)(including Rgs4,Ighgl,Elmo1,Gcnt1).,Prune2,Msx1,Cdkn2b,and Scandl,etc.,these 33 differential genes involved 49 KEGG pathways,among which there were statistically significant differences(P<0.05)(including FoxO signaling pathway,TGF? signaling pathway,NF-?B signaling pathway).And 18 of the PI3K-Akt signaling pathways,etc.)Conclusion:1.High-trans fatty acid diet diet combined with high-sugar drinking water for 24 weeks,can successfully induce a mouse model of non-alcoholic steatohepatitis with liver fibrosis in combination with obesity and hyperinsulinemia.2.Qushi Huayu Recipe can significantly alleviate liver steatosis and inflammation,improve liver fibrosis and insulin resistance in NASH mice with liver fibrosis.3.phlegm-dampness and phlegm may treat NASH with liver fibrosis through immune,metabolic,cancer signaling pathway-related genes and signaling pathways(extracellular matrix receptor interaction,MAPK signaling pathway,PI3K-Akt signaling pathway,etc.).