Pharmacological Interpretation Of Qutan Huoxue Decoction And Experimental Validation In The Treatment Of Nonalcoholic Steatohepatitis (NASH)

Objective:Qutan Huoxue Recipe is a prescription summed up by Professor Sun Tongjiao,a well-known traditional Chinese medicine practitioner for many years.Through preliminary clinical trials,it was found that it can treat nonalcoholic steatohepatitis(NASH),but its specific mechanism of action is still unclear.Network pharmacology predicts the action pathway of Qutan Huoxue Recipe in the treatment of NASH,and later cell experiments verify the specific action mechanism of Qutan Huoxue Recipe in the treatment of NASH.Methods:The active ingredients and target genes of Qutan Huoxue Recipe were obtained from the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform(TCMSP),and NASH disease genes were extracted from five databases: genecards,OMIM,Pharm Gkb,TTD and Drug Bank.These data were visualized and analyzed by Cytoscape software,and the active ingredient-gene-target network map,PPI-protein interaction map,and enrichment analysis of GO and KEGG were constructed,and cell experiments were performed later for verification.By isolating and co-culturing primary hepatocytes and primary kupffer cells of NASH model rats,an in vitro NASH rat cell model was established.NASH model group(N),5% Qutan Huoxue prescription group(L),7.5% Qutan Huoxue prescription group(M)and 10% Qutan Huoxue prescription group(H)five groups.The secretion of alanine aminotransferase(ALT),aspartate aminotransferase(AST),triglyceride(TG),total cholesterol(TC)and cell oil in primary hepatocytes after serum exposure Red O was used to detect cell steatosis;Western Blotting and q-PCR were used to detect the expressions of SOCS1,TLR4,Myd88 and NF-k B in primary rat hepatocytes and primary Kupffer cells;SOCS1 si RNA transfection reagent was designed to silence the primary cells The SOCS1 gene in Kupffer cells was verified by immunofluorescence,WB and q-PCR,and the expression of SOCS1/TLR4 cell signaling pathway-related factors was detected to explore the specific mechanism of Qutan Huoxue Recipe in the treatment of NASH.Results:(1)Using network pharmacology to analyze Qutan Huoxue Recipe,130 active ingredients of Qutan Huoxue Recipe and 1243 Chinese medicine target genes of Qutan Huoxue Recipe were obtained,from five genecards,OMIM,Pharm Gkb,TTD and Drug Bank.8579 NASH disease genes were extracted from the database,and 147 intersecting genes of Qutan Huoxue Recipe and NASH diseases were obtained.Through PPI network analysis,GO and KEGG enrichment analysis,it was shown that there is a complex relationship between the Chinese medicine target genes of Qutan Huoxue Recipe and NASH disease genes.Qutan Huoxue Recipe can treat NASH by improving multiple targets such as inflammatory pathways and lipid metabolism.Therefore,this study combined the results of previous experiments with the results of network pharmacology analysis and the prediction of the online JASPAR database to explore the specific mechanism of Qutan Huoxue Recipe in the treatment of NASH through the SOCS1/TLR4/NF-k B cell signaling pathway.(2)The results of in vitro cell experiments showed that Qutan Huoxue Recipe could significantly reduce ALT,AST,TG,TC of primary hepatocytes in rats,improve lipid deposition in hepatocytes,and relieve hepatocyte injury(P<0.05).In the model,it was found that the expressions of TLR4,Myd88 and NF-k B were significantly increased;when the serum containing Qutan Huoxue prescription was added,the expressions of TLR4,Myd88 and NF-k B decreased(P<0.05),and showed a certain dose-dependent tolerance;The online JASPAR database found that SOCS1 is related to the TLR4/NF-κB signaling pathway.At the same time,the expression of SOCS1 was not statistically significant after 6h,24 h,and 48 h of primary hepatocyte extraction(P>0.05),while in primary Kupffer cells,NASH The expression of SOCS1 in the group was significantly lower than that in the control group(P<0.05);after the SOCS1 protein expression in primary Kupffer cells was silenced by SOCS1 si RNA transfection reagent,the expressions of TLR4,Myd88 and NF-k B were significantly increased.After treatment(10%),the expression of SOCS1 was increased,and the expression of TLR4,Myd88 and NF-k B was decreased(P<0.05).Conclusion:The network pharmacology analysis has certain accuracy.The mechanism of Qutan Huoxue Recipe in the treatment of NASH mainly acts on cellular insulin resistance,lipid deposition,inflammation and other cell signaling pathways.The negative feedback regulation of SOCS1/TLR4/NF-k B signaling pathway in Kupffer cells can improve liver lipid deposition and inflammatory response,and play a role in the treatment of NASH.