The Roles And Mechanisms Of Growth Arrest And DNA-Damage Gene 45a (GADD45a) In APAP-Induced Liver Injury

Drug-induced liver injury(DILI)has been one of the most important diseases induced by drugs,it also has been a major problem that currently threatens human health and drug development.DILI has been a major cause of acute liver failure in the U.S.and some western countries.Acetaminophen(N-acetyl-p-aminophenol,APAP)has been a typical drug causing drug induced liver injury,it also been the first cause which can induce acute liver failure even death in some western countries.Although there has been some studies about the hepatotoxicity of DILI,our understanding about the exact occurrence and progress mechanism of DILI just like the tip of the iceberg.We have already realized that the final clinical prognosis of DILI patients depends on the interaction between the injury factors and the adaptive protection mechanism.However,it is regrettable that an amount of studies focused the mechanism of APAPinduced liver injury exclusively on the former,there are very few studies focused on the adaptive protection mechanism of DILI.GADD45(growth arrest and DNA-damage gene 45,GADD45)participated in the activities of cell cycle regulation,DNA repair,cell survival,inflammation and stress response.There have been some studies reported that the upregulated expression of GADD45 a in hepatocytes can enhance the cell proliferation,and inhibit the activation of p38 and JNK.However,the exact function and mechanism of GADD45 a in AILI is not clear until now.In our present study,we constructed APAP-induced hepatotoxicity cell model and APAP-induced liver injury mouse model.We used a variety of molecular biological and morphological methods,such as RNA-seq,real-time quantitative PCR,western blot,LC-MS,siRNA transfection,transfecting adenovirus,H&E,oil red O staining,immunohistochemistry and immunofluorescence staining and so on,to illuminate the roles and mechanisms of GADD45 a in APAP-induced liver injury by in vivo and in vitro experiments,and our study will provide the theoretical and experimental bases for GADD45 a as targets for treating APAP induced liver injury.The present study is composed of three parts as following: Part I: Transcriptional analysis of drug-induced liver injury and the discovery of GADD45 a.Firstly,we constructed APAP-induced hepatotoxicity cell model and APAP-induced liver injury mouse model,then observed the global transcriptome profiles of APAP induced liver injury both in mouse and cells model by RNA sequencing.Importantly,we found that the mRNA expression level of GADD45 family displayed marked change in response to APAP treatment both in vivo and vitro,particularly,GADD45 a presented the most obviously increase.Part Ⅱ: The role of GADD45 a in APAP induced liver injury.1.We examined the expression level of GADD45 a in DILI using human examples and some exprements in vivo and vitro.Firstly,we detected the expression level of GADD45 a in DILI patients and healthy volunteers by immunohistochemistry staining,we found that the expression of GADD45 a was obviously increased in livers from DILI patients.Secondly,we established the drug induced liver injury model in vivo and vitro to further test GADD45 a expression.We established the mice model by APAP intraperitoneally(i.p.)injection with acetaminophen at a dosage of 300 mg/kg(body weight).We chose AML-12 cells,established the cell model by APAP treatment at a concentration of 20 uM.We measured the mRNA and protein expression levels of GADD45 a using RT-PCR and Western blot,the results showed that the expression of GADD45 a up regulated obviously.2.We overexpressed GADD45 a to test the effect of GADD45 a in APAP induced liver injury.Firstly,we enhanced the expression of GADD45 a both in AML-12 cells and mice by injecting the Ad-GADD45 a adenovirus.Then,we tested the mRNA levels of the genes involving in hepatic lipid/glucose metabolism,tested DNA fragment by TUNEL staining and the accumulation of small lipid droplets.We found that the DNA fragment and the accumulation of small lipid droplets induced by APAP were alleviated obviously after we overexpressed GADD45 a.Moreover,the genes involving in the fatty acid beta-oxidation pathway and TCA cycle were significantly increased after GADD45 a overexpression,decreased the accumulation of lipids in hepatocytes.We further analyzed the global transcriptome profiles of APAP induced liver injury,the findings indicated that the genes relating to lipid metabolism increased after overdose APAP treatment.We applied Liquid chromatography and mass spectrometry(LC-MS)to analyze the levels of several metabolites in APAP-treated AML-12 cell samples.Our data showed that the overexpression of GADD45α can increase the levels of TCA cycle metabolites after APAP treatment.Finally,we isolated primary mouse hepatocytes,then transfected primary mouse hepatocytes and AML-12 cells with GADD45a-siRNA,the DNA fragment and accumulation of small lipid droplets were more aggravated compared to the control group.These data suggested that GADD45 a play an important protective role against APAP induced liver injury.Part Ⅱ: The underlying mechanisms of GADD45 a protecting against APAP induced acute liver injury.1.The activation and role of AMPK pathway during the process of APAP induced liver injury.Firstly,we found that AMPK pathway was obviously activated in APAP induced liver injury by the analysis of RNA-seq,the genes expression involving in the AMPK pathway also upregulated in line with the activation of AMPK pathway.We then tested the activation of AMPK in vivo and vitro after APAP treatment using Western blot,the results showed an obviously activation of AMPK pathway in response to APAP exposure.When we knockdown the expression of AMPKa in primary mouse hepatocytes,DNA fragmentation was more aggravated and the number of small lipid droplets was increased using TUNEL staining and oil red O staining.These data suggested that AMPK pathway play an important role in APAP induced liver injury.2.The effect of GADD45 a on hepatic AMPK pathway.We used Ad-GADD45 a infection to induce the overexpression of GADD45 a in AML-12 cells,then the cells were exposed to APAP to induce the hepatotoxicity.We found that P-AMPK was increased significantly in the AML-12 cells overexpressing GADD45 a and the levels of P-S6 K and P-S6,which are downstream proteins of the AMPK pathway,were consistent with the levels of P-AMPK using Western blot.Furthermore,we reduced the expression of GADD45α using siRNA in primary mouse hepatocytes.We found that when GADD45α was knocked down,the protein levels of P-AMPK was decreased compared with the levels in the control-siRNA group.Collectively,these results indicate that GADD45α can promote AMPK activation during the process of APAPinduced hepatotoxicity.3.The underlying mechanisms of GADD45 a promoting the activation of AMPK pathway.We have already found that GADD45 a was increased in APAP induced acute liver injury,and the AMPK pathway was activated at the same time.We further study the underlying relationship between GADD45 a and AMPK.Firstly,we found that GADD45α interacted directly with Ppp2 cb using co-immunoprecipitation.We further confirmed the direct interaction between Ppp2 cb and AMPKα.Then,we knocked down the expression of Ppp2 cb and overexpressed GADD45α in AML-12 cells.Then,the levels of P-AMPK after APAP treatment were detected by Western blot.We found that GADD45α enhanced the activation of P-AMPK after APAP treatment;however,the enhancement was offset by the knockdown of Ppp2 cb expression.We also examined the expression levels of genes that were downstream of P-AMPK in primary mouse hepatocytes with knocking-down Ppp2 cb followed with the overexpression of GADD45 a,we found that the increase in mRNA expression mediated by GADD45α overexpression was dramatically reversed after Ppp2 cb was knocked down.Taken together,these results demonstrate that GADD45α promote the activation of AMPK by interacting directly with Ppp2 cb.In conclusion,GADD45 a expression was up regulated after APAP treatment,then the up-regulated GADD45 a offset the inhibition of Ppp2 cb protein on AMPK activation by binding directly to Ppp2 cb protein,promoted the phosphorylation of AMPK,promoted the expression of genes involving in lipid/glucose metabolism,regulated hepatic lipid/glucose metabolism homeostasis,promoted cell survival,ultimately alleviated the liver injury caused by APAP.