Effects Of Vitamin D On Nonalcoholic Fatty Liver Disease Through The PPARα/CPT1A Pathway

Objective:This study conducted to detect the relationship between serum25（OH）D₃and severity of non-alcoholic fatty liver disease（NAFLD）,and to explore the potential mechanism of vitamin D₃（VD₃）in treatment of NAFLD by regulating liver lipid metabolism,so as to provide scientific basis for revealing the potential improvement effect of VD₃on NAFLD.Methods:1.Case-control study:381 NAFLD patients and 350 controls were recruited from the Health Management Center of Affiliated Hospital of Southwest Medical University.The participants’height and weight were measured and recorded by a professional physician.Heart blood was collected and centrifuged to obtain serum for following detection.The levels of AST,ALT,TG,TC,HDL-C,LDL-C,GGT,ALP,ALB,FPG,INS,PLT were detected by automatic biochemical analyzer.Serum 25（OH）D₃was detected by CLIA.NFS,FIB-4 and FLI were calculated as noninvasive assessment of NAFLD.Multivariate logistic regression models were used to assess the strength of association between 25（OH）D₃and the risk of NAFLD.2.In vivo study:this study was divided into two stages.In the first stage,5-week-old male rats（n=48）were randomly divided into two groups:chow diet group（CON,n=16）and high fat diet group（HFD,n=32）.After feeding for 7weeks,8 rats were sacrificed to determine model established,respectively.The remaining rats were divided into CON group（n=8）,HFD group（n=8）,HFD+VD group（n=8）,and HFD+Oil group（n=8）to receive weekly intervention for 7 weeks.In the second stage,5-week-old male rats（n=24）were randomly divided into HFD+DMSO group（n=8）,HFD+MK group（n=8）and HFD+MK+VD group（n=8）to receive weekly intervention for 7 weeks after being fed high fat diet for 7 weeks.During the period,general information such as weight and food intake were measured.At the 14^thweek,serum was collected,and liver was separated and weighed,and serum AST,ALT,TG,TC,LDL-C,HDL-C and GLU levels were detected by automatic biochemical analyzer.IL-6,MCP-1,TNF-αand 25（OH）D₃were detected by ELISA.HE staining and oil red O staining were used to evaluate the pathological changes of liver.Hepatic TG and FFA were measured.The expression of lipid metabolic protein,such as PPARα,CPT1A,CD36,FABP1 in liver were detected by western blot.3.In vitro study:the steatosis model of Hep G2 cells was induced by intervention of 0.5m M oleic acid（OA）for 24h,and then treated with calcitriol at 25,50,100 and 200n M for 24h.The optimal intervention concentration of calcitriol was detected by CCK8,oil red O staining,intracellular TG and TC detection.Hep G2 was treated with different doses of MK886（0.1μM,1μM,10μM and 100μM）for 0.5h,1h and 2h,respectively.CCK8 and western blot were used to determine the best time and dose of MK886.Western blot was used to detect the expression of lipid metabolic related protein including PPARα,CPT1A,CD36,FABP1.Results:1.Case-control study:（1）Compared with control group,BMI,WC,SBP,DBP,ALT,AST,GGT,ALP,INS,GLU,HOMA-IR,TC,TG,LDL were increased and HDL,25（OH）D₃were decreased in case group;The rates of vitamin D insufficiency,hypertension,diabetes,overweight and obesity in the NAFLD group were higher than those in the controls,and this trend exists when stratified by sex.（2）25（OH）D₃was negatively correlated with ALT,INS,HOMA-IR and positively correlated with HDL-C in the case group.（3）Insufficient vitamin D was a risk factor for NAFLD when FLI<60（OR 1.423,95%CI 1.021-1.982）,FLI≥60（OR 1.920,95%CI 1.057-3.489）,NFS<-1.455（OR 1.591,95%CI 1.148-2.203）and FIB-4<1.3（OR 1.774,95%CI1.260-2.497）.（4）Serum vitamin D level was not associated with NAFLD in univariate model.After adjusting for age,sex,INS,GLU,AST,ALT and ALP（Model 3）,high serum vitamin D level（25（OH）D₃>20ng/m L）was a protective factor for NAFLD(OR 0.019,95%CI 0.001-0.619,P_trend<0.001).2.In vivo study:（1）At the 7^thweek,HFD significantly increased serum ALT,TG,TC,LDL-C,decreased AST/ALT and HDL-C,and hepatic steatosis was observed by staining,which means NAFLD model was estabolished.（2）At the 14^thweek after HFD,the body weight,energy intake,liver weight,liver index,NAS score,oil red O mean density,hepatic TG and FFA were significantly increased,and the liver tissue structure was damaged with a large number of red-stained lipid droplet.（3）Hepatic TG and FFA were significantly reduced after the application of Vitamin D₃with amelioration of hepatic steatosis.（4）Vitamin D₃treatment significantly decreased serum HDL-C,AST,GLU,MCP-1 and increased serum 25（OH）D₃.（5）Vitamin D₃treatment significantly decreased the protein expression of PPARαand CTP1A,but had no effect on the expression of CD36 and CPT1A in liver.（6）In the presence of MK886 intervention,the improvement effects of VD3 on PPARα,CPT1A,CD36,FABP1 in the liver of NAFLD rats were inhibited.3.In vitro study:（1）Difference concentration of 1,25（OH）₂D₃had no toxic effect on Hep G2 cells.（2）50n M 1,25（OH）₂D₃improved OA induced steatosis in Hep G2 cells and 25,100,200n M 1,25（OH）₂D₃aggravated OA-induced steatosis in Hep G2 cells.（3）The expression of PPARαwas lowest when treated with 10μM MK886 for 1h.（4）MK886 and 1,25（OH）₂D₃inhibited PPARαexpression in Hep G2 cells.Conclusion:（1）The serum level of 25（OH）D₃in NAFLD patients was lower than that in control group.（2）Normal level of VD₃status（>20ng/m L）has protective effect on NAFLD.（3）VD₃supplementary can improve liver function,hyperlipidemia,inflammatory factors and hepatic steatosis in NAFLD,which is related to PPARα/CPT1A pathway.