The Preliminary Study Of Protective Effects Of Active Form Of Vitamin D For Nonalcoholic Fatty Liver Disease

Objective:Nonalcoholic Fatty Liver Disease（NAFLD）has an increasing prevalence in the world as high as 58%-74%in obese population.The hepatic excessive lipid accumulation was accompanied by insulin resistance and characterized by over 5%of hepatic steatosis showed by liver biopsy.And the term“nonalcoholic”was defined as the amount of alcohol consumption less than 30grams one day in the male（20grams in the female）.NAFLD has a disease spectrum ranging from nonalcoholic fatty liver（NAFL）to nonalcoholic steatohepatitis（NASH）and hepatic carcinoma.The progression from NAFL to NASH is a key step in the development of NAFLD,involving many mechanisms.And oxidative stress is a major determinant of the pathogenesis and progression of NASH.The suppression of oxidative stress is a key therapeutic goal in the prevention of NASH progression.Nuclear factor-erythroid-2-related factor 2（Nrf2）is a transcription factor that activates antioxidant response elements（AREs）to modulate antioxidant capacity against oxidative stress.Vitamin D is a well-documented suppressor of oxidative stress and was proved to play an important role in the protection against inflammation in chronic liver diseases such as hepatitis.Therefore,we hypothysize that vitamin D can protect against NAFLD by alleviating the oxidative stress in liver and investigate whether vitamin D can activate Nrf2 pathway to enhance the antioxidative capacity.Methods:Part one.Male Sprague–Dawley rats were divided into three groups and treated with standard chow,HFD,or HFD plus intraperitoneal injection of1,25（OH）₂D₃（5μg/kg body weight,twice per week）,respectively,for 20 weeks.Rat’s general condition and body weight were recorded.Hyperinsulinemic-euglycemic clamp experiment was used to assess insulin sensitivity.Serum lipid profiles,hepatic function,intrahepatic lipid,and calcium levels were determined.Hepatic histology was examined using hematoxylin/eosin,Masson’s trichrome,and Oil Red O staining.Oxidative stress was assessed by measuring hepatic malondialdehyde（MDA）and F2α-isoprostane content.Expression of nuclear factor-erythroid-2-related factor 2（Nrf2）and downstream target genes was analyzed using quantitative RT-PCR.Part two.HepG2 cells were divided into three groups:group C,group HF（palmitic acid:oleic acid=1:2,0.1mmol/l）and group HF+VD(1,25（OH）₂D₃,10^-7mol/L).Oil O staining was used to assess lipid accumulation.MDA and antioxidant enzymes activity were measured.Expression of Nrf2 and downstream target genes was analyzed using quantitative RT-PCR.And expression of NRF2 in nucleus was analyzed using western-blot analysis.Analysis of variance（ANOVA）was used for multiple group comparisons.Results:The model of NAFLD rats with insulin resistance was successfully established.The rats in HFD group exhibited hyperlipidemia,hepatic injury and histopathologic characteristics of NAFLD.1,25（OH）₂D₃ treatment improved insulin resistance and serum lipid profile,reduced intrahepatic lipid levels,and attenuated hepatic steatosis and inflammation in HFD rats.Furthermore,MDA and F2α-isoprostane levels in liver tissue were reduced by 1,25（OH）₂D₃ administration.Although 1,25（OH）₂D₃ did not regulate the expression of Nrf2 mRNA,it did induce Nrf2 nuclear translocation.The expression of Nrf2 target genes was up-regulated by1,25（OH）₂D₃.1,25（OH）₂D₃ treatment reduced lipid accumulation and MDA content,enhanced antioxidant activity in high-fat cultured HepG2 cells.Expression of Nrf2mRNA was no change in three groups.And expression of Nrf2 target genes were up-regulated at 24-hour in both HF and HF+VD group and got up-regulation at48-hour in HF+VD group compared with gourp HF.Expression of NRF2 in nucleus of HepG2 cells were increased at 24-hour and 48-hour.Conclusions:We conclude that 1,25（OH）₂D₃ protects against HFD-induced NAFLD by attenuating oxidative stress,inducing NRF2 nuclear translocation,and up-regulating the expression of genes encoding antioxidant enzymes.