The Protective Effects Of Vitamin D₃ On Liver Injury Induced By Isoniazid And Rifampicin In Rats And Its Mechanism

Objective: Vitamin D(VD)not only regulates the metabolism of calcium and phosphorus in vivo,but also participates in the regulation of endocrine system and immune system.In recent years,domestic and foreign studies showed that VD could improve liver injury induced by hexachlorobenzene or D-galactosamine through inhibiting oxidative stress and down-regulating expression of inflammatory factors.However,studies on the effects of VD on anti-tuberculosis drug induced liver injury(ATLI)have been rarely reported so for.Therefore,isoniazid(INH)and rifampicin(RFP)were used to establish rat liver injury model in this study.To observe whether different doses of VD3 intervention could improve ATLI and explore whether the mechanisms are related to the improvement of oxidative stress,inflammatory reaction and bile acid metabolism in the liver.Methods: After one week of adaptive feeding,50 SPF male Sprague-Dawley(SD)rats were randomly divided into 5 groups according to their body weight: normal control(control)group,model group,VD3 low dose(VD-L)group,VD3 medium dose(VD-M)group,VD3 high dose(VD-H)group.All rats were given intragastric administration twice a day with an interval of 2 hours.Firstly,control group was given 0.5% sodium carboxymethyl cellulose(CMC-Na);model,VD-L,VD-M and VD-H groups were given INH(100 mg/kg/d)+ RFP(150 mg/kg/d).Secondly,control and model groups were fed with soybean oil,VD-L,VD-M and VD-H groups were fed with 1 ug/kg/d,5 ug/kg/d and 10 ug/kg/d VD respectively.The intervention lasted for 21 days.Fasting for 12 hours after last intervention,and then weighing,anaesthesia.Blood was extracted from abdominal aorta and separation for serum and plasma.Viscera were acquired after dissected and stored in the refrigerator at-80 ?.Serum markers of liver injury were detected by automatic biochemical analyzer.Pathological changes of liver tissues were observed by HE staining.The activities of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)and the concentration of malondialdehyde(MDA)were detected in rat liver homogenate.Enzyme-linked immunosorbent assay(Elisa)was used to determine the concentration of tumor necrosis factor-?(TNF-?),interleukin 1?(IL-1?),interleukin-10(IL-10)in rat liver tissue homogenate and high transfer rate group B1(HMGB1)in serum.The expression levels of bile acid transporters Bsep and Ntcp m RNA were detected by real-time quantitative PCR.Liver bile acid receptor FXR and transporter Ntcp protein levels were detected by Western-blot.Results: 1.Compared with control group,the weight gain of rats in model group and VD3 intervention groups were slower.After 7 days of intervention,the body weight of rats in control group were higher than that in other groups(P<0.05).There was no significant difference in body weight of rats between VD3 intervention groups and model group.2.Serum biomarkers(AST,ALP,TBA,TBIL,DBIL)of liver injury in model group were higher than that in control group(P<0.05).VD3 intervention significantly inhibited the increase of serum AST,ALP and DBIL levels.Pathological examination showed inflammatory cell infiltration and mild degeneration and necrosis of liver cells in model group.And VD3 intervention improved the pathological phenomenon of inflammatory cell infiltration and necrosis of liver cells.3.The activities of SOD and GSH-PX were lower and the concentration of MDA was higher in model group than those in control group(P<0.05).The activity of GSH-Px was higher and the concentration of MDA was lower in VD-H group than those in model group(P<0.05).4.The concentrations of TNF-?,IL-1? in liver tissue and HMGB1 in serum in model group were higher than those in control group(P<0.05).High dose VD3 intervention significantly inhibited the increase of TNF-?,IL-1? and HMGB1 concentrations induced by INH+RFP(P<0.05).5.Compared with control group,the expression of Bsep m RNA and Ntcp m RNA decreased in model group(P<0.05).The expression of Bsep m RNA and Ntcp m RNA in VD-H group were higher than those in model group.And the expression of Ntcp m RNA in VD-M group was also higher than that in model group(P<0.05).6.INH and RFP significantly reduced the expression of bile acid receptor FXR and transporter Ntcp protein(P<0.05).High dose VD3 intervention effectively improved the reduction of FXR and Ntcp protein levels induced by drugs.The expression of FXR protein in VD-M group was also higher than that in model group(P<0.05).Conclusion: VD3 could reduce the level of serum markers of liver injury induced by INH and RFP and improve the pathological changes of liver tissue,which suggests that VD3 supplementation has an improvement to ATLI.The related mechanisms for improving ATLI by VD include:VD3 could inhibit oxidative stress and reduce the damage of lipid peroxidation to liver cells by improving antioxidant enzymes activities.VD3 could be used as a HMGB1 inhibitor to reduce the expression of TNF-? and IL-1?,thus inhibiting the inflammatory response of liver.VD3 could promote the expression of Bsep and Ntcp m RNA,increase the level of FXR and Ntcp protein,promote the circulation of bile acid and improve the cholestasis.