Function And Mechanism Of NLRP6 In NAFLD Induced By High-Fat Diet

BackgroundNon-alcoholic fatty liver disease（NAFLD）is one of the main causes of liver disease in the world,and its incidence in China is increasing year by year,showing a trend of younger age.At present,the pathogenesis of NAFLD is not completely clear,and the regulatory role of inflammasomes in NAFLD-related metabolic diseases has become increasingly prominent.NLRP6 is a member of the NLRs family of pattern recognition receptor,which can assemble into inflammasomes.In recent years,it has been reported that NLRP6 is related to the occurrence and development of NAFLD,but the specific mechanism is still unclear.In this study,NLRP6-knockout mice and cell lines were taken as the research objects to explore the role of NLRP6 in the pathogenesis of NAFLD,so as to provide a theoretical basis for the prevention and treatment of diseases related to lipid metabolism disorders.ObjectiveIn this study,we investigated the effect of NLRP6 expression on fatty metabolism of hepatocytes,and clarified the molecular mechanism of NLRP6 regulating the accumulation of lipid droplet in the liver.Methods1.Establishment of NAFLD model induced by high-fat diet in vivoWild-type mice and NLRP6^-/-male mice of C57BL/6 strain were randomly divided into a control group and a model group.The model group was given a high-fat diet to establish a NAFLD model,while the control group was fed a normal diet.The basic energy metabolism indexes of mice were detected with metabolism cage during modeling.Serum biochemical indicators were detected after modeling.The sections of liver tissue were stained with HE and Oil Red O to observe the histopathological changes and lipid droplet accumulation in the liver.The expression levels of liver lipid synthesis related factors and inflammatory cytokines were detected by q RT-PCR and Western blot.2.In vitro cell modelNCTC1469 cell line was cultured in vitro,and the NLRP6 overexpression and NLRP6 knockout models were constructed by plasmid transfection.We also isolate and cultured primary mouse hepatocytes in vitro.The cells were treated with fatty acid to establish a cell lipomatosis model,and the accumulation of intracellular lipid droplets was analyzed by Oil Red O staining and triglyceride detection.The expression levels of lipid synthesis related factors in hepatocytes were detected by q RT-PCR and Western blot.Results1.NLRP6 is highly expressed in the liver（hepatocytes）,the expression of NLRP6 in hepatocytes was significantly reduced under the high-fat condition.2.In the HFD-induced NAFLD model group,NLRP6 deletion did not affect the mouse basal metabolic indicators,but it increased the mouse lipid content and aggravated hepatocyte steatosis by up-regulating the expressions of ADRP and CIDEC and down-regulating the expression of Apo B100.Moreover,the mouse co-house experiment showed that the aggravation of HFD-induced fatty liver by NLRP6 deletion was not dependent on intestinal flora disorder.3.In NLRP6-knockout NCTC1469 cells and primary hepatocytes,after treatment with FA（0.5 m M）,the intracellular lipid content and the diameter of lipid droplets were increased.The expression of ADRP and CIDEC increased significantly,while the expression of Apo B100 decreased significantly.4.In hepatocytes overexpressing NLRP6,the intracellular lipid content and the diameter of lipid droplets were significantly reduced after treatment with FA（0.5 m M）.ADRP and CIDEC expression decreased significantly.5.In the HFD-induced NAFLD model group,NLRP6^-/-mice exhibited increased inflammatory cell infiltration and increased inflammatory responses in the liver compared with wild-type mice.Besides,the co-house experiment showed that the aggravation of HFD-induced hepatitis due to NLRP6 deletion was closely related to the intestinal flora disorder.6.In the MCD-induced NAFLD model group,NLRP6^-/-mouse showed more severe steatosis,inflammatory cell infiltration,and fibrosis than wild-type mice.ConclusionThis study indicated that NLRP6 can inhibit the occurrence and development of NAFLD.It can inhibit the accumulation of lipid droplets in hepatocytes by regulating the expression of ADRP,CIDEC and Apo B100,and thus alleviating the symptoms of NAFLD.