Study Of The Role And Mechanism Of Cardiolipin Synthetase 1 In Nonalcoholic Steatohepatitis

Nonalcoholic fatty liver disease(NAFLD)is a common chronic liver disease,with its prevalence rate of 25% in the world population,can be divided into nonalcoholic fatty liver(NAFL)and nonalcoholic steatohepatitis(NASH)according to whether there is specific inflammation,and NAFL will develop into NASH if effective measures are not taken.NASH,as an increasingly serious and concerned health problem,the prevalence is about 2-5%,characterized by lipid accumulation,insulin resistance,severe inflammation and fibrosis in the liver.Patients with NASH have the increased risk of cirrhosis,hepatocellular carcinoma(HCC),liver failure and the potentially fatal possibility.As a multifactor disease,NASH has complex pathogenesis and a lot of influencing factors,although extensive clinical and basic researches have been conducted in this field,there are currently no effective drugs.Therefore,studying the complicated molecular mechanisms and pathological processes in the development of NASH and developing key therapeutic targets and fruitful s trategies for NASH treatment are of great importance.Cardiolipin(CL)-a mitochondria specific phospholipid that is necessary to maintain the structure and function of mitochondrial.The final step in the de novo mitochondrial synthesis of CL is catalyzed by cardiolipin synthase 1(CRLS1).CRLS1 belongs to CDP-alcohol phosphatidyl transferase class-I family,which is localized in the mitochondria and has been found to be highly expressed in the liver,is involved in apoptosis and HCC.Recently,Elahu G et al.reported that the depletion of CL in brown and beige fat abolished glucose uptake and the adipose CRLS1 levels positively correlated with insulin sensitivity.Thus,adipose CL is a key control point for organismal metabolic fitness and glucose homeostasis.Considering the close involvement of insulin sensitivity in NASH progression,we hypothesized that CRLS1 may be essential during the progression of hepatic steatosis.However,the specific roles of CRLS1 and the related underlying mechanisms associated with NASH remain unexplored.Considering that hepatocytes account for 60% of the total number of cells in liver,that are the main unit of liver function and play central roles in the process of liver steatosis,this paper mainly studies the role and the related mechanisms of CRLS1 in hepatocytes.We first examined the protein content of CRLS1 in the liver of wild-type mice under the high-fat and high-cholesterol(HFHC)diet-induced NASH model,and found that the expression of CRLS1 reduced significantly,compared with the normal chow diet-fed control mice,and then observed the same phenomenon in palmitic acid-stimulated L-02 liver cells.Based on these results,we speculated that CRLS1 may play a role in NASH pathological processes in hepatocytes.Studies on CRLS1 overexpressing L-02 cells showed that CRLS1 ameliorates lipid accumulation and inflammation.Conversely,the lack of CRLS1 aggravates these symptoms.On the account of the results in vitro,we fed hepatocyte-specific CRLS1-knockout(CRLS1-HKO)mice and their corresponding control mice on a 16-week of HFHC diet.What we observed were that the body weight,blood glucose and the liver weight of CRLS1-HKO mice were prominently elevated compared with the floxed control mice.In addition,the contents of triglyceride and total cholesterol in blood were also raised substantially.Pathological section staining,Western blot and qRT-PCR analysis revealed that lipid deposition,lipid metabolism disorder,inflammatory reaction and fibrosis in the liver cells of CRLS1-deficient mice aggravated markedly.After verifying the function of CRLS1 in the pathological process of NASH,by means of RNA sequencing analysis,we further picked out ten genes that may be related to CRLS1 under the HFHC diet induced NASH conditions,through digital gene expression profiling technology.The expression of these ten genes were detected in wild-type mice treated with normal chow diet and HFHC diet for 16 weeks and activating transcription factor 3(ATF3)was speculated may be involved in the regulation of NASH by CRLS1 with review of the literature.The depth research confirmed that CRLS1 suppresses the expression of ATF3,while overexpression of ATF3 partially reverses lipid metabolism disorders and inflammatory responses alleviated by CRLS1 overexpression,clarified the role of ATF3 in the development of NASH regulated by CRLS1.The prevalence of NASH is increasing year by year,that is one of the major causes of liver dysfunction.Previous research has indicated that adipose CRLS1 levels are associated with insulin sensitivity;however,the precise roles of CRLS1 and underlying mechanisms involving CRLS1 in the pathological process of NASH have not been elucidated.Here,we discovered that the protein level CRLS1 was significantly downregulated in HFHC diet-induced mice model.In vitro studies demonstrated that overexpressing of CRLS1 markedly attenuated palmitic acid induced hepatic steatosis and inflammation in hepatocytes,whereas shRNA mediate CRLS1 knockdown aggravated these abnormalities.Moreover,HFHC diet induced fatty degeneration of liver,inflammatory response and fibrosis significantly aggravated in CRLS1-HKO mice during NASH development.ATF3 was identified as the key differentially expressed gene after CRLS1 hepatocyte specific knockout through transcriptomic analysis,and our investigation further showed that CRLS1 suppressed ATF3 expression in palmitic acid-stimulated hepatocytes,while ATF3 partially reverses lipid accumulation and inflammation inhibited by CRLS1 overexpression under metabolic stress.In conclusion,CRLS1 ameliorates liver steatosis,inflammation and fibrosis during the pathological process of NASH by inhibiting the expression of ATF3.