Protective Effect And Mechanism Of G-protein-coupled Receptor GPR116 On Acute Drug-induced Liver Injury Induced By Acetaminophen

BackgroundAcetaminophen（APAP）is a kind of acetanilide antipyretic and analgesic,which is widely used.Although relatively safe,APAP is a dose-dependent hepatotoxin that can cause acute drug-induced liver injury.The use of APAP at therapeutic doses of more than a few days has shown elevation of serum transaminases in one-third of patients,and APAP overdose,which can lead to severe liver injury or even death,is a major cause of drug-induced acute liver failure.However,most patients have missed the best time to treat when they are hospitalized.Therefore,in-depth investigation of the occurrence and development mechanism of acute drug-induced liver injury caused by excessive APAP will provide experimental and theoretical basis for the development of new and more effective intervention targets and treatment measures.APAP is metabolized within hepatocytes and converted by cytochrome P 450enzymes 2E1（CYP 2E1）to n-acetyl-p-benzoquinone imine（NAPQI）,which can rapidly consume glutathione（GSH）,covalently bind to mitochondrial proteins,endoplasmic reticulum proteins and cause massive hepatocellular necrosis.In hepatocytes,the endoplasmic reticulum is a major site for protein synthesis,folding and maturation,and drug metabolism.In APAP treated hepatocytes,when the amount of unfolded or misfolded proteins exceeds the folding capacity of the endoplasmic reticulum,endoplasmic reticulum stress can be initiated and,if excessive severe or sustained,trigger hepatocyte necrosis.G-protein-coupled receptor 116（GPR116）is one of the G-protein-coupled receptor family members.This family is evolutionarily conserved adhesion receptors.GPR116plays important roles in stabilizing pulmonary surfactant,insulin resistance,vascular remodeling,and activation of pulmonary macrophages,but roles in acute liver injury are rarely reported,and the relationship between GPR116 and ER stress has not been reported.In this study,we first set out from the clinical samples of patients with drug-induced liver injury,and then through the establishment of APAP induced acute drug-induced liver injury animal model,to explore the actual role and possible mechanism of GPR116in APAP induced acute drug-induced liver injury in mice from the in vivo level,and further verified it at the cell level in vitro.Methods1、The research of changes of GPR116 expression in drug-induced liver injuryExperiment 1:Immunohistochemical staining was performed to detect the expression of GPR116 in human liver tissue after drug-induced liver injury.Experiment 2:8-12 weeks male C57BL/6 mice（20-25 g）were randomly divided into control group and APAP group.After 16 hours of fasting（consumption the original GSH in the body）,APAP（250 mg/kg）was intraperitoneally injected to establish the model of acute drug-induced liver injury.The control group was intraperitoneally injected with the same volume of PBS.The liver tissues of mice were collected 2,4,6,12and 24 hours after APAP administration,and the changes of GPR116 expression in liver tissues of mice were detected by real-time quantitative PCR（Q-PCR）,Western blot assay and immunohistochemical staining.2、The research of protective effect of GPR116 on APAP-induced acute drug-induced liver injury in miceWe constructed GPR116 hepatocyte-specific knockout mice(GPR116^fl/flAlb^cre-/-mice).8～12 weeks male GPR116^fl/flAlb^cre+/+mice and GPR116^fl/flAlb^cre-/-mice（20～25g）were randomly divided into GPR116^fl/flAlb^cre+/++PBS group,GPR116^fl/flAlb^cre-/-+PBS group,GPR116^fl/flAlb^cre+/++APAP group,and GPR116^fl/flAlb^cre-/-+APAP group.After 16 hours of fasting（consuming the original GSH in the body）,APAP（250 mg/kg）was intraperitoneally injected to establish the model of acute drug-induced liver injury.The control group was intraperitoneally injected with the same volume of PBS.Enzyme linked immunosorbent assay（ELISA）was used to detect alanine aminotransferase（ALT）and alanine aminotransferase（AST）in serum of mice when APAP was highly hepatotoxic（24 h）.The degree of liver injury was detected by Hematoxylin and Eosin（H&E）staining,and the apoptosis of hepatocytes was detected by Td T-mediated d UTP Nick-End Labeling（TUNEL）.The 72-hour survival rate of the two kinds of mice was observed by intraperitoneally injected with lethal dose of APAP（500 mg/kg）.3、The research of mechanism of GPR116 protecting APAP-induced acute drug-induced liver injury in mice8～12 weeks male GPR116^fl/flAlb^cre+/+mice and GPR116^fl/flAlb^cre-/-mice（20～25 g）were randomly divided into GPR116^fl/flAlb^cre+/++PBS group,GPR116^fl/flAlb^cre-/-+PBS group,GPR116^fl/flAlb^cre+/++APAP group,and GPR116^fl/flAlb^cre-/-+APAP group.After16 hours of fasting（consuming the original GSH in the body）,APAP（250 mg/kg）was intraperitoneally injected to establish the model of acute drug-induced liver injury.The control group was intraperitoneally injected with the same volume of PBS.At the time of the highest expression of GPR116（4 h）,the liver tissues of mice were taken,and the endoplasmic reticulum stress（ER stress）markers and c-Jun N-terminal kinase（JNK）activation were detected by Q-PCR,Western blot and immunohistochemical staining.4、The research of protective effect of GPR116 on APAP induced hepatocyte injury in vitroExperiment 1:The normal mouse liver cell lines（AML-12 cells）were cultured in vitro and divided into control group and APAP group after platting.The control group was added with the same amount of medium,and the APAP group was added with APAP（5 m M）to induce liver cell injury.The expression of GPR116 in AML-12 cells was detected by Q-PCR and Western blot.Experiment 2:GPR116 si RNA was transfected to interfere with the expression of GPR116 in AML-12 cells（NC as negative control）.After interference,the cells were divided into NC control group,si GPR116 control group,NC+APAP group and si GPR116+APAP group.The control group was added with the same amount of medium,and the APAP group was added with APAP（5 m M）to induce liver cell injury.The cell viability of AML-12,namely the cytotoxicity of APAP,was determined by CCK-8 kit.Results1、GPR116 was up-regulated in liver during drug-induced liver injuryCompared with normal human liver tissue,the expression of GPR116 protein in liver tissue of patients with drug-induced liver injury was significantly up-regulated.The m RNA and protein expression of GPR116 in liver tissue of normal C57 mice after APAP-induced liver injury increased significantly and reached the peak value at 4 h after APAP-induced liver injury,and then decreased.2、Successful construction and identification of hepatocellular specific deletion GPR116 miceThe expression of GPR116 protein in the liver tissue of GPR116^fl/flAlb^cre-/-mice decreased significantly compared with that of GPR116^fl/flAlb^cre+/+mice,but there was no difference in the expression of GPR116 in other organs,which proved the knockout efficiency of hepatocyte specific knockout mice.3、Hepatocyte specific deletion of GPR116 aggravated APAP-induced liver injuryGPR116^fl/flAlb^cre-/-mice showed stronger hepatotoxicity and higher mortality after excessive APAP treatment compared with GPR116^fl/flAlb^cre+/+mice.Specific knockdown GPR116 hepatocytes exacerbated APAP-induced hepatocyte apoptosis.4、GPR116 protected APAP-induced liver injury by inhibiting endoplasmic reticulum stress and JNK activationThe expression of endoplasmic reticulum stress markers in liver tissue of GPR116^fl/flAlb^cre-/-mice was significantly increased in m RNA level and protein level,and the level of JNK phosphorylation was significantly increased in the early stage after excessive APAP treatment.It showed that GPR116 could protect liver injury by inhibiting endoplasmic reticulum stress and JNK phosphorylation.5、TUDCA reduced the aggravation of liver injury caused by GPR116deficiencyTreatment of APAP-treated mice with endoplasmic reticulum stress specific inhibitor TUDCA reduced liver injury in GPR116^fl/flAlb^cre+/+mice and GPR116^fl/flAlb^cre-/-mice,and reversed hepatic toxicity induced by GPR116 gene deletion,further demonstrating that GPR116 could protect acute drug-induced liver injury induced by excessive APAP-induced by inhibiting endoplasmic reticulum stress.6、GPR116 was up-regulated and protective in APAP-induced AML-12 cellsThe expression of GPR116 m RNA and protein increased after APAP stimulation of AML-12 cells in vitro.GPR116 si RNA was used to interfere with the expression of GPR116 in AML-12 cells,which showed enhanced sensitivity to APAP and significantly increased cell mortality,and the results of in vivo experiments were confirmed.ConclusionGPR116 expression was up-regulated in liver cells during drug-induced liver injury,reducing hepatocyte death by inhibiting endoplasmic reticulum stress and JNK phosphorylation,thus playing a protective role in drug-induced liver injury.