Vitamin D Deficiency Exacerbates Hepatic Oxidative Stress During Acetaminophen-induced Acute Liver Injury In Mice

Background and objective acetaminophen(APAP)can cause extensive liver injury and hepatic oxidative stress,while vitamin D has anti-inflammatory and antioxidant stress effects.Recent experiments confirmed that vitamin D protected against oxidative stress,inflammation and hepatorenal damage during APAP-induced acute liver injury(ALI)in rat.It is unclear whether vitamin D deficiency aggravates APAP-induced acute liver injury and hepatic oxidative stress.The purpose of this study was to establish a model of vitamin D deficiency aggravating APAP-induced liver injury,to research the effect of vitamin D deficiency on liver oxidative stress and its possible mechanism.Methods 96 male ICR mice were divided into vitamin D deficiency(VDD)group and control(Ctrl)group at random,with 48 mice in each group.In VDD group,mice were fed with vitamin D deficient fodder.In Ctrl group,mice were fed with standard fodder.Six weeks later,mice were divided into four groups at random: Ctrl,APAP,VDD,VDD+APAP.In APAP and VDD+APAP groups,mice were intraperitoneally injected with sublethal dose of APAP(150 mg/kg).In Ctrl and VDD groups,mice were treated with equal volume of saline in the same way.Mice were given standardized fasting for12 hours before administration of APAP.Mice were sacrificed at different time points(2,6 or 24 h)after APAP injection.We recorded body weight and liver weight.We collected Serum samples to detect serum 25(OH)D,alanine aminotransferase(ALT)and aspartate aminotransferase(AST).We kept liver samples for following studies.Some of the liver samples were fixed in paraformaldehyde for hematoxylin and eosin(H&E)staining,some were frozen in-80℃ degree refrigerator for subsequent detection of GSH,MDA and WB related proteins oxygenase-1(Ho-1),NOX-2,3-nitrotyrosine(3NT),and some were frozen for RT-PCR detection of glutathione peroxidase(Gshpx),superoxide dismutase 1(Sod1),catalase enzymes(Cat)and Ho-1.In order to study the effect of vitamin D deficiency on the survival rate of mice induced by APAP,20 mice were divided into APAP group and VDD+APAP group.All mice were intraperitoneally injected with lethal dose of APAP(300mg/kg).We observed the survival of mice for 7days.Results The levels of serum 25(OH)D of vitamin D deficient mice were obviously decreased compared with the mice fed with normal diet for 6 weeks.There was no difference in body weight among different groups.The liver weight and liver weight/body weight in APAP group were higher than those in Ctrl group at 24 h after APAP.The liver weight and liver weight/body weight in APAP+VDD group were higher than those in APAP group.Serum ALT and AST were increased slightly in APAP group.VDD aggravated the increase of serum ALT and AST induced by APAP.Mild structural disorder and liver necrosis were observed at each time point after APAP.VDD significantly increased the area of liver necrosis induced by APAP.Further experiments showed that APAP-induced liver GSH depletion and lipid peroxidation were increased in VDD-fed mice.In addition,APAP induced up-regulation of liver antioxidant genes such as heme oxygenase-1(Ho-1),glutathione peroxidase(Gshpx),superoxide dismutase 1(Sod1)and catalase(Cat).Ho-1,NOX-2 and 3-NT protein levels only increased slightly or had no change in APAP group.VDD aggravated the increase induced by APAP.We analyzed the effects of VDD on the survival rate.No obvious difference on the survival rate was observed between two groupsConclusion The aim of this study was to explore the role of vitamin D deficiency in APAP-induced acute liver injury.The results indicated that sublethal dose of APAP only caused mild liver injury while vitamin D deficiency aggravated APAP-induced liver injury.Vitamin D deficiency aggravated hepatic oxidative stress and vitamin D deficiency may aggravate APAP-induced oxidative stress by increasing Ho-1,NOX-2and 3-NT.