Effects Of Vitamin D Deficiency On The Pathogenesis Of Alcoholic Fatty Liver In Mice

Objective The AFL model of mouse was established by using Lieber-Decarli alcoholic liquid diet to observe the effects of vitamin D deficiency on the incidence of alcoholic fatty liver in mice.Methods Six-week-old C57BL/6J male mice were randomly divided into four groups.They were control group(Control),vitamin D deficiency group(VDD),alcohol group(EOH)and alcohol+vitamin D deficiency group(EOH+VDD).Alcohol group mice were fed modified Lieber-De Carli liquid diets(containing 4%(w/v)alcohol).The normal control group were fed normal liquid diets without ethanol.The vitamin D deficiency group were fed diets which deficient in vitamins D and without ethanol,mice in alcohol+vitamin D deficiency group were fed diets with ethanol and deficient in vitamins D.During the feeding period,the daily feed intake and weekly body weight of mice in each group were recorded and fed continuously for 6 weeks.After the experiment,the mice were sacrificed and blood samples were collected for the detection of subsequent lipids,ALT and AST activities,25(OH)D and the level of inflammatory cytokines.The liver was collected and weighed,partially stored rapidly at-80°C for detection of GSH、MDA and triglyceride content,real-time quantitative PCR and western blot analysis;the other part was fixed with 4% paraformaldehyde and embedded in OCT to prepare paraffin sections and frozen sections for liver injury and hepatic steatosis evaluation.Results After 6 weeks of modeling,serum 25(OH)D levels in VDD group and EOH+VDD group were 13.32 ± 0.81ng/ml and 12.78±2.1ng/ml respectively,which were significantly lower than those in Control group(55.34±2.59ng/ml)and EOH group(51.68±3.53ng/ml),while alcohol caused significant hepatic injury and hepatic steatosis.Compared with the normal control group,the liver weight,liver coefficient and serum ALT and AST activity of the mice in the alcohol group were significantly increased;the expression of Ki67 and the number of TUNEL apoptotic cells in the liver tissue were significantly increased,and the pathological sections of the liver tissue were visible a large number of lipid deposition,varying degrees of steatosis and inflammatory cell infiltration;TG levels in serum and liver tissue and m RNA levels of some lipid transport related genes were significantly increased;GSH activity decreased significantly while MDA content elevated in liver homogenate and the m RNA levels of proinflammatory cytokines TNFα and chemokines MCP1 and KC were significantly increased.Compared with the alcohol group,vitamin D deficiency further increased the mouse liver coefficient and serum ALT,AST levels,but no effect on hepatocyte proliferation and hepatocyte apoptosis and fat lesions in liver tissue;the positive area ratio and integral optical density value of Oil Red O staining tended to increase,but the difference was not statistically significant.Vitamin D deficiency had no effect on the content of TG in serum and liver,and the expression of lipid synthesis and transport related genes.But further upregulated the liver lipid metabolism-related gene m RNA levels;vitamin D deficiency had no effect on liver GSH levels,but further increased MDA content aggravate alcohol-induced lipid peroxidation,while further enhanced nitric oxide synthase expression and significantly decreased the expression of GSH-Px,SOD1 and catalase antioxidant enzyme genes;vitamin D deficiency further significantly upregulated proinflammatory cytokines TNFα,IL1β and TGFβ and chemotaxis factors MCP1 and KC m RNA levels.Conclusions Vitamin D deficiency is not a direct risk factor for fatty liver disease,but vitamin D deficiency aggravates alcohol-induced liver injury.The mechanism may be related to vitamin D deficiency to promote oxidative stress,to release inflammatory cytokines and chemokine-induced liver inflammation.