Investigation Of Exploring The Material Basis Of Phlegm Syndrome In Nonalcoholic Steatohepatitis Based On The Lipid Overload-induced Mitochondrial Dysfunction

Objective: The aim of this study lies in: 1)To explore the mechanisms of phlegm syndrome in alcoholic steatohepatitis(NASH)based on the mitochondrial dysfunction caused by lipid overload;2)to investigate the regulatory role of mTORC1/SREBP1/CAV1 signaling pathway in the lipid overload-induced mitochondrial dysfunction;3)to enrich the essence and connotation of phlegm syndrome,and provides approaches and targets for the prevention and treatment of NASH.Methods:1.Collecting and screening the clinical research literatures of NASH,extracting the syndrome types and elements of NASH,and analyzing the etiology and pathogenesis of NASH and their association with phlegm syndrome.2.Collecting and screening the relevant literatures on animal models of phlegm syndrome induced by diet,sorting out and summarizing the model construction methods and model evaluation contents.3.In vitro,free fatty acid(FFA)was used to induce the lipid overloaded hepatocyte model,the lipid droplet deposition was evaluated by oil red O staining,TMRE fluorescence probe was used to assess mitochondrial membrane potential,seahorse XF energy metabolic meter was used to assess mitochondrial respiration.In vivo,high fat diet(HFD)was used to induce NASH phlegm syndrome mouse model,the general condition of mice was observed,the blood levels of lipid,glucose and insulin were measured,and the liver tissues were stained with H&E,oil red O,Masson,and nonalcoholic fatty liver disease activity score(NAS)were calculated.The contents of liver lipid and ATP were measured.Liver mitochondria were isolated,JC-1 fluorescence probe was used to assess mitochondrial membrane potential;LC-MS was used to detect lipids of mitochondrial.Erchen decoction(ECD),as a phlegm resolving prescription,was used for counter-evidence.4.In vitro,lipid overload hepatocytes induced by FFA were treated with mTORC1 inhibitor rapamycin(RAP)and Cav1-RNAi virus vector.Lipid deposition,mitochondrial membrane potential and mitochondrial respiration were also measured,m RNA and protein expression were evaluated using real-time PCR and western blot.In vivo,the NASH mice with phlegm syndrome induced by HFD were treated with mTORC1 activator MHY and ECD,then their NASH phenotypes were detected.Liver tissue lipid and ATP contents,mitochondrial membrane potential and expressions of m RNA and protein were evaluated.Results:1.A total of 6 articles were included.There were 11 NASH syndromes reported.The top three were liver depression and spleen deficiency syndrome,dampness-heat accumulation syndrome,and phlegm-dampness syndrome.In terms of the separation of syndrome elements,3 location elements were identified,namely,liver,spleen and kidney;there were 9 kinds of character elements,and the top three were: dampness,qi stagnation and phlegm.The pathogenesis of NASH is complex and multifold while phlegm runs throughout NASH.2.A total of 108 articles were included.There are several specific approaches using diet to induce phlegm syndrome animal model.High-fat diet with fat content more than 10% for2-8 weeks is the most commonly used modeling approach;while fatigue,weight gain,reduced activity,reduced diet and elevated blood lipids(TC,TG,LDL-C)are the basic assessments for phlegm syndrome animal model.3.In vitro,FFA induced obvious lipid deposition in hepatocytes,and successfully replicated the model of lipid overload.FFA induced the decrease of mitochondrial membrane potential,mitochondrial respiration and ATP production in hepatocytes.In vivo,HFD caused animals with decreased activity,fatigue,reduced diet,increased body weight along with abdominal circumference,increased TG,decreased FFA,appeared insulin resistance,and NAS>5,which proved that we have successfully established NASH mice with phlegm syndrome.Liver lipid levels was increased,the membrane potential along with ATP level were decreased,and the lipid distribution of the mitochondria isolated from the liver was chaotic in mice with NASH phlegm syndrome.Intervention of ECD achieved amelioration of the aforementioned indexes.4.In vitro,RAP decreased FFA-induced lipid droplet deposition,enhanced mitochondrial membrane potential,mitochondrial respiration and ATP production,down-regulated p-m TOR,p-S6 K,n-SREBP1 protein expression,along with up-regulated CAV1 m RNA and protein expression.Knockdown of CAV1 resulted in enhancement of lipid droplet deposition,decrease of membrane potential,mitochondrial respiration and ATP production.Knockdown of CAV1 with RAP treatment restored the aforementioned phenotypes.In vivo,MHY aggravated the phenotype of NASH phlegm syndrome mice,increased liver lipid level,decreased mitochondrial membrane potential,upregulated the expression of p-m TOR,p-S6 K and n-SREBP1 protein,along with down-regulated the expression of CAV1 m RNA and protein in NASH phlegm syndrome mice.The joint intervention of MHY and ECD could ameliorate the above indexes.Conclusion:1.The pathogenesis of NASH is complicated and multifold,"phlegm" is the pathological basis of NASH,phlegm syndrome almost runs through the process of occurrence and development of NASH,and therapy against phlegm is the basic treatment approach.2.Mitochondrial dysfunction induced by lipid overload is the material basis of NASH phlegm syndrome,which may be related to the imbalance of mitochondrial lipidome caused by excessive lipid deposition.3.mTORC1/SREBP1/CAV1 signaling pathway plays a regulatory role in mitochondrial dysfunction induced by lipid overload,which might be the internal regulatory mechanism of NASH phlegm syndrome.