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

Background and objective Acetaminophen(APAP)is an antipyretic and analgesic drug widely used in clinic.Normal therapeutic dose of acetaminophen is relatively safe,and overdose often results in acute liver injury,even liver failure in severe cases.Many studies have confirmed that the mechanisms of acute liver injury induced by APAP include sterile inflammation,oxidative stress,mitochondrial dysfunction and so on.It is well known that vitamin D has anti-inflammatory activity.Recent study has shown that vitamin D has a protective effect on acute liver injury induced by acetaminophen through anti-inflammation.However,it is not clear whether Vitamin D deficiency aggravates hepatic inflammation during APAP-induced acute liver injury.The purpose of this study was to establish a mouse model of vitamin D deficiency and to observe the effect of vitamin D deficiency on acute liver injury induced by APAP.Methods Six-week-old male CD-1 mice were randomly divided into two groups:vitamin D deficiency group(n = 48)and control group(n = 48).The mice in vitamin D deficiency group were given vitamin D deficient diet,and the mice in control group were given standard fodder.Six weeks later,the mice were divided into four subgroups:Ctrl,VDD,APAP,VDD + APAP.In the APAP and VDD+APAP groups,mice were intraperitoneally injected with a sublethal dose of APAP(150 mg/kg).In the Ctrl and VDD groups,mice were treated with equal volume of saline in the same way.Mice were sacrificed at different time points(2,6 or 24 h)after APAP injection.Serum and liver were collected for further study.Results Compared with the control and APAP groups,the serum 25-(OH)D level of the mice fed with vitamin D deficient diet was significantly lower,indicating that the vitamin D deficient mouse model was successfully established.In the APAP group,the absolute liver weight and liver coefficient were increased 24 h after APAP,which were aggravated by vitamin D deficiency.The serum ALT levels in APAP group were only slightly increased after APAP,while vitamin D deficiency upregulated the ALT level.The change of serum AST was consistent with that of ALT.Then we evaluated the pathological changes of liver.In the APAP group,mild structural disorder and hepatic necrosis were observed at all time points after APAP,in which necrotic areas were only from 4% to 7%.However,vitamin D deficiency aggravated APAP-induced hepatic injury.We detected the hepatocyte apoptosis induced by APAP.The results showed that few TUNEL positive cells were observed in the liver of mice treated with a sublethal dose of APAP,while vitamin D deficiency significantly increased the number of TUNEL positive cells at 6 or 24 h after APAP.The expression of C-Caspase3 protein was consistent with the result of TUNEL staining.Then we evaluated the effect of APAP on pro-inflammatory cytokines in hepatic.In the APAP group,proinflammatory cytokines such as Tnf-α,chemokines such as Mcp-1,Kc and Mip2 were not significantly elevated,while vitamin D deficiency upregulated the expression of these genes.Western blotting showed that the level of p-JNK in the APAP group was increased significantly only 2 h after APAP,while the VDD + APAP group began to increase 6 h after APAP,and 24 h after APAP,VDD + APAP group was significantly higher than that of APAP group.These results suggest that vitamin D deficiency may aggravate hepatic inflammation during APAP-induced acute liver injury by up-regulating the level of JNK phosphorylation.Conclusion The above results suggest that:(1)vitamin D deficiency aggravates APAP-induced acute liver injury;(2)vitamin D deficiency exacerbates hepatic inflammation in APAP-induced acute liver injury;(3)vitamin D deficiency may aggravate hepatic inflammation during APAP-induced acute liver injury by up-regulating the level of JNK phosphorylation.