To Investigate The Mechanism Of Qutan Huoxue Recipe In Treating Nonalcoholic Simple Fatty Liver Disease Based On Network Pharmacology And Experimental Verification

Objective:In this study,the potential targets and pathways of Qutan Huoxue Recipe in the treatment of NAFL were analyzed and predicted by network pharmacology,and the specific mechanism of Qutan Huoxue Recipe in the treatment of NAFL was verified by in vitro and in vivo experiments.Methods:1.Predicting the target of Qutan Huoxue Recipe in the treatment of NAFL through network pharmacology Network PharmacologyNetwork pharmacology analysis: Obtain the effective active components and related target genes of traditional Chinese medicines in Qutan Huoxue Recipe from TCMSP platform,extract NAFL related genes from five databases:genecards,OMIM,Pharm Gkb,TTD and Drug Bank;construct the network of active component-gene-target and PPI protein interactions with Cytoscape software and String platform;finally analyze GO and KEGG through Metascape platform,and carry out subsequent animal and cell experiments for verification according to the prediction results.2.To observe the effect of Qutan Huoxue Recipe on NAFL rats and verify the preliminary regulatory relationship of microRNA-27a/p38 MAPKα/AQP9In vivo experimental verification: To preliminarily verify the therapeutic effect of Qutan Huoxue Recipe on NAFL rats at the animal level and its regulatory relationship on microRNA-27a/p38 MAPKα/AQP9.Thirty-two8-week-old male SD rats of clean grade weighing 180 ～ 200 g were adaptively fed for 1 week and randomly divided into normal control group(n = 6)and high-fat diet feeding group(n = 26).The NAFL rat model was induced by high fat diet feeding in the high fat diet feeding group.After 6 weeks of feeding,two rats were randomly sacrificed from the high-fat diet-fed group and HE staining was performed to confirm that the modeling had been successful,and the remaining high-fat diet-fed rats were randomly divided into four groups,namely,the NAFL model group,and the low,medium,and high dose groups of Qutan Huoxue Recipe(5.4 g/kg,10.8 g/kg,and 21.6 g/kg,n = 6)and sacrificed after 4 weeks of continuous gavage.Liver function(serum ALT,AST content),blood lipid metabolism(serum LDL,HDL,TG,TC content),liver physiological and pathological changes(HE staining,SAF score),liver lipid accumulation(liver TC,TG content and oil red O staining)were assessed in each group.The expression of microRNA-27 a in serum/adipose tissue/liver tissue(q PCR),p38 MAPKα/AQP9 pathway m RNA in liver tissue(q PCR),and p38 MAPKα/p-p38 MAPKα/AQP9 pathway protein in liver tissue(Western blot,immunohistochemistry)were detected in each group.3.Mechanism of Qutan Huoxue Recipe Regulating Lipogenic Micro RNA-27 a Mediating p38 MAPKα/AQP9 Pathway on Hepatocyte Steatosis in NAFLIn vitro validation:(1)To verify the regulatory relationship of microRNA-27a/p38MAPK/AQP9 and its mechanism of action on steatosis in NAFL hepatocytes.A NAFL in vitro primary hepatocyte model(collagenase digestion)was established to verify the direct binding target of microRNA-27 a to p38 MAPKα(luciferase reporter assay α).Simulants of microRNA-27 a were transferred into NAFL rat primary hepatocytes and divided into three groups: control group(Control),transfection negative control group(NC),and microRNA-27 a overexpression group(mi R-27a(+)).The contents of microRNA-27a/p38MAPKα/AQP9 m RNA(q PCR),protein expression of p38 MAPKα/p-p38MAPKα/AQP9 pathway(Western blot,immunofluorescence)and the degree of lipid accumulation(TG content and Oil Red O staining)in hepatocytes of each group were detected.(2)To investigate the effect of microRNA-27 a secreted by mature adipocytes on hepatocyte steatosis in NAFL induced by obesity.A rat primary adipocyte-hepatocyte co-culture system was established in vitro and divided into four groups: normal co-culture group(CO),NAFL co-culture group(NO),transfection negative control group(NC),and microRNA-27 a silencing group(microRNA-27a(-)).The expression of microRNA-27 a in adipocytes/hepatocytes and p38 MAPKα/AQP9 pathway m RNA in hepatocytes(q PCR),p38 MAPKα/p-p38 MAPKα/AQP9 pathway protein in hepatocytes(Western blot),and the degree of hepatocyte lipid accumulation(TG content and Oil Red O staining)were detected in each group.(3)To investigate the specific mechanism of Qutan Huoxue Recipe in improving hepatocyte steatosis by affecting lipogenic microRNA-27a/p38MAPKα/AQP9.To screen the optimal serum concentration of Qutan Huoxue Recipe(CCK8).Six groups were established: control group(NC),microRNA-27 a silencing group(microRNA-27a(-)),microRNA-27 a silencing followed by drug addition group(microRNA-27a(-)+ qthx),and low,middle,and high dose groups of Qutan Huoxue Recipe.Qutan Huoxue Recipe intervened the rat primary adipocyte-hepatocyte co-culture model in vitro,and detected the expression of microRNA-27 a in adipocytes/hepatocytes and p38MAPKα/AQP9 pathway m RNA in hepatocytes in each group(q PCR),and the expression of p38 MAPKα/p-p38 MAPKα/AQP9 pathway protein in hepatocytes in each group(Western blot)to assess the degree of lipid accumulation in hepatocytes in each group(TG content and oil red O staining).Results:1.The results of network pharmacological gene screening showed that Qutan Huoxue Recipe had 130 effective active components and 1243 corresponding target genes,and 92 intersection genes of Qutan Huoxue Recipe and NAFL disease were obtained.PPI network,GO and KEGG enrichment analysis showed that Qutan Huoxue Recipe could treat NAFL by regulating microRNA transcription regulation,circulatory system processes,lipid metabolism and other signaling pathways.It is predicted that p38 MAPKα may be a key target gene of Qutan Huoxue Recipe in the treatment of NAFL.2.The results showed that Qutan Huoxue Recipe could significantly reduce the contents of TC,TG,LDL,ALT and AST in serum of NAFL rats,increased serum HDL content in NAFL rats,improve the liver function and relieve the hepatic steatosis of NAFL rats.And it could increase the serum/fat/liver microRNA-27 a content and inhibit the expression of p38 MAPKα/p-p38MAPKα/AQP9 pathway proteins(P < 0.05).3.The results showed that microRNA-27 a directly acted on the 3 ′UTR of p38 MAPKα m RNA.After overexpression of microRNA-27 a,its content was significantly increased in primary hepatocytes of NAFL rats,while the expression of p38 MAPKα/p-p38 MAPKα/AQP9 pathway m RNA was significantly decreased,and the degree of hepatocyte lipid accumulation was significantly improved.Micro RNA-27 a expression levels were significantly decreased in primary mature adipocytes and hepatocytes of rats in the NAFL model group.The results of co-culture showed that the content of microRNA-27 a in hepatocytes of NAFL co-culture group was significantly decreased,while the expression of p38 MAPKα/p-p38 MAPKα/AQP9 pathway was significantly increased,and the degree of lipid accumulation was more severe.Silencing microRNA-27 a in adipocytes significantly decreased microRNA-27 a content in hepatocytes,while the expression of p38MAPKα/p-p38 MAPKα/AQP9 pathway was significantly increased,and the degree of lipid accumulation in hepatocytes was further aggravated.The results of drug intervention showed that the optimal effective concentration of serum containing Qutan Huoxue Recipe was 5%,7.5% and 10%.Compared with the control group,microRNA-27 a levels were decreased,the expression of p38MAPKα/p-p38 MAPKα/AQP9 pathway was up-regulated,and the degree of lipid accumulation was aggravated in hepatocytes of NAFL rats in the microRNA-27 a silencing group.However,compared with the microRNA-27 a silencing group,there was no significant difference in the post-silencing plus group.Meanwhile,compared with the control group,the content of microRNA-27 a in hepatocytes significantly increased,the expression of p38MAPKα/p-p38 MAPKα/AQP9 pathway significantly decreased,and the degree of lipid accumulation in hepatocytes was significantly reduced in the Qutan Huoxue Recipe 5%,7.5%,and 10% medicated serum groups(P < 0.05).Conclusions:1.Network pharmacological analysis revealed that Qutan Huoxue Recipe mainly treats NAFL by regulating microRNA transcription,circulatory system processes,adipocytokines and other biological processes,and p38 MAPKα may be the core target gene of Qutan Huoxue Recipe in the treatment of NAFL.2.In vitro and in vivo experiments have demonstrated that Qutan Huoxue Recipe has a significant anti-NAFL effect.3.Qutan Huoxue Recipe may ameliorate hepatocyte steatosis by up-regulating lipogenic microRNA-27 a and inhibiting p38 MAPKα to down-regulate AQP9 expression.