Protective Effect Of Rosiglitazone On Acetaminophen Induced Acute Liver Injury And Its Mechanism

Background and Objective Drug-induced liver injury(DILI)has become a fatal threat to human health.Acetaminophen is one of the most common non-steroidal anti-inflammatory drugs(NSAID).Overdoses of Acetaminophen can potentially cause liver injury,and even acute liver failure.Current studies have demonstrated that APAP-induced liver injury is provoked probably via multiple mechanisms,such as inflammatory reaction,oxidative stress,mitochondrial autophagy and endoplasmic reticulum stress,etc.Peroxisome proliferators-activated receptor γ(PPARγ)is a member of the nuclear hormone receptor superfamily.Accumulated evidence has demonstrated that besides the physiological functions such as regulating lipid metabolism,regulating immunity,anti-tumor and so on,PPAR γ probably exerts protective effect upon liver injury induced by varying factors.Thiazolinedione drugs(TZDs)are capable of binding with PPAR γto activate relevant functions of PPAR γ.Several studies showed that RSG had potent anti-inflammatoryand anti-oxidative effects.Nevertheless,whether RSG alleviates hepatic sterile inflammation and oxidative stress in the progression of APAP-induced liver injury needs to be determined.In this study,mouse models with APAP-induced acute liver injury were established.The protective effect of rosiglitazone(RSG)upon APAP-induced acute liver injury was investigated,aiming to preliminarily explore the underlying mechanism on the perspectives of inflammatory reaction and oxidative stress.Methods1.To deliver dynamic observation of APAP-induced acute liver injury and evaluate the protective effect of RSG upon APAP-induced acute liver injury,60 healthy male CD-1mice were randomly divided into 10 groups,6 animals in each group.In the control group,intraperitoneal injection of physiological saline alone was administered.In the RSG alone group,intragastric administration of RSG at a dose of 20mg/kg was given at48,24 and 1 h before medicine administration in the APAP group.Meantime,an equivalent quantity of physiological saline was delivered via intraperitoneal injection.In the APAP group(four groups,means APAP 0h group,APAP 1h group,APAP 4h group,APAP 24 h group),single intraperitoneal injection of APAP at a dose of 300 mg/kg was delivered.The mice were sacrificed at 0,1,4,and 24 h after APAP administration.In the RSG plus APAP group(four subgroups,means APAP+RSG 0h group,APAP+RSG1h group,APAP+RSG 4h group,APAP+RSG 24 h group),intragastric administration of RSG was given at 48,24 and 1 h prior to intraperitoneal injection of APAP.The animals were sacrificed at 0,1,4,and 24 h after administration of APAP.All mice were subject to fasting at 12 h before medicine administration.Serum sample was collected for detection of ALT,AST and total bilirubin(TBIL)levels.Partial liver tissues were prepared for HE staining and cellular apoptosis detection.2.To assess the effect of RSG upon the survival rate of mice with APAP-induced acute liver injury,healthy male CD-1 mice were randomly divided into two groups,10 in each group.In the APAP group,single intraperitoneal injection of APAP at a dose of 300mg/kg was delivered.In the RSG+APAP group,intragastric administration of 20 g/kg RSG was given at 48,24 and 1 h before intraperitoneal injection of APAP.Meantime,APAP at a dose of 300 mg/kg was given via intraperitoneal injection simutaneously.The survival rate was calculated and the survival curve was delineated between two groups.3.To observe the effect of RSG upon the metabolism of phase I metabolic enzyme CYP2E1of APAP,healthy male CD-1 mice was randomly separated into the control,RSG alone,APAP alone and RSG+APAP groups,6 in each group.The processing methods were described above.At 0.5 h after APAP treatment,the animals were sacrificed and the liver tissue was prepared for detection of CYP2E1.4.In order to observe effect of RSG on the activity of GSH metabolic enzyme in acute APAP-induced liver injury,twenty four healthy male CD-1 mice were randomly assigned into the control,RSG alone,APAP alone and RSG plus APAP groups,6 in each group.The treatment procedures were described in section 1.All mice were sacrificed at 1 h after APAP treatment.Fresh liver tissue homogenate was prepared for the detection of activity of GST,GSH-Rd and GSH-Px enzymes.Results1.Protective effect of RSG upon APAP-induced acute liver injury ALT was slightly increased at 1 h after APAP treatment,which was continuously ascended along with the time of APAP intervention and peaked at 24 h after APAP treatment.The changing pattern of AST was parallel to that of ALT.The serum level of TBIL began to elevate at 4 h after APAP treatment and peaked at 24 h.While in RSG pretreatment groups,the serum levels of ALT,AST and TBIL were significantly lower than those during the same time interval in the APAP alone group,suggesting that RSG can significantly mitigate the severity of APAP-induced liver injury.HE staining demonstrated that liver congestion and edema was observed at 1 h following APAP administration,evident spotty necrosis,bridging necrosis and submassive necrosis of the hepatocytes were noted at 4 h,and significantly massive necrosis was seen at 24 h after medicine administration.TUNEL assay revealed that a slight quantity of liver cell apoptosis was noted at 1 h after APAP treatment,and the quantity of apoptotic cells was significantly increased at 4 h and peaked at 24 h following APAP intervention.In the RSG+APAP group,hepatocellular degeneration and the severity and area of necrosis were slighter,the quantity of apoptotic cells was significantly less compared with those in the APAP alone group at the same time interval.2.Effect of RSG on the survival rate of mice with APAP-induced acute liver injury The experimental results prompted that the 7-day survival rate was calculated as 50% in the APAP alone group,significantly lower than 80% in the RSG plus APAP group.3.Effect of RSG on the metabolism of phase I metabolic enzyme CYP2E1 The results demonstrated that overdose of APAP did not affect the expression of CYP2E1.RSG exerted no effect upon the expression of CYP2E1 in mice with acute APAP-induced liver injury.4.Effect of RSG on inflammatory response caused by acute APAP-induced liver injury4.1 Effect of RSG on serum CRP in mice with acute APAP-induced liver injury Detection of serum CRP indicated that compared with the control group,the level of CRP began to slightly increase at 4 h after APAP treatment,and significantly ascended at 24 h.Pretreatment with RSG could reduce the increasing level of CRP mediated by APAP.4.2 Effect of RSG on the MAPK signaling pathway during APAP-induced liver injury To evaluate the effect of RSG upon the MAPK signaling pathway during APAP-induced liver injury,the phosphorylated levels of JNK,ERK1/2,P38 MAPK and AKT as the members of MAPK superfamily were quantitatively measured by Western blot.The results prompted that the phosphorylation of these four molecules were activated during APAP-induced liver injury.Pretreatment with RSG could significantly down-regulate the APAP-induced activation of phosphorylated levels of four molecules.4.3 Effect of RSG on the NF-κB signaling pathway in acute APAP-induced liver injury The nucleus expression of NF-κB in APAP-induced liver injury tissue was detected by Western blot and demonstrated that compared with the control group,the expression levels of P65 and P50 in the liver nucleus were up-regulated within 1 h after APAP intervention,suggesting that the NF-κB was activated early after medicine administration.Pretreatment with RSG could significantly down-regulate the nucleus expression of NF-κB.4.4 Effect of RSG on inflammation-related cytokines in acute APAP-induced liver injury.The effect of RSG upon the expression of inflammation-related cytokines including TNF-α,KC,COX-2 and IL-1 at the presence of APAP-induced liver injury was evaluated by RT-PCR.Results demonstrated that compared with the control group,the expression levels of pro-inflammation cytokines including TNF-α,KC and were significantly up-regulated during the liver injury in the APAP group.The degree of up-regulated expression of these cytokines was consistent with the degree of the up-regulated expression of liver enzymes.In the RSG plus APAP group,the expression levels of TNF-α and KC were significantly down-regulated compared with those in the APAP group during the same time interval.No statistical significance was noted in the transcription status of IL-1,COX-2 among all groups.5.Effect of RSG on oxidative stress pathway in APAP-induced liver injury5.1 Overdose of APAP may cause the exhaustion of glutathione To observe the dynamic variation of glutathione(GSH)consumption and the effect of RSG upon the GSH content in APAP-induced liver injury,liver tissue homogenate was prepared for detection of GSH.The results demonstrated that the GSH in the liver tissue was exhausted at 1 h after APAP administration,which was gradually increased at 4 h and almost restored to the level in the control group at 24 h following APAP treatment.Pretreatment with RSG may significantly ease the consumption of GSH caused by APAP.5.2 Effect of RSG on the activity of GSH metabolic enzyme in acute APAP-induced liver injury.Twenty four healthy male CD-1 mice were randomly assigned into the control,RSG alone,APAP alone and RSG plus APAP groups,6 in each group.The treatment procedures were described in section 1.3.All mice were sacrificed at 1 h after APAP treatment.Fresh liver tissue homogenate was prepared for the detection of activity of GST,GSH-Rd and GSH-Px enzymes.Activities of hepatic GSH-Px,GSH-Rd and GST,were markedly reduced at 1 h after APAP injection.Of interest,RSG pretreatment significantly attenuated reduction of hepatic GSH-Rd and GST activities during APAP-induced acute liver injury.5.3 Effect of RSG on expression of lipid peroxide products in acute APAP-induced liver injury The liver tissue homogenate was prepared for detection of MDA content.The results demonstrated that the MDA content was significantly increased during the early stage(1h)of APAP-induced liver injury.No significant changes in the MDA content were observed at 4 h and those in the control group.No significant elevation was noted in the MDA content during each time interval after pretreatment with RSG.5.4 Effect of RSG on 3-nitrotyrosine(3NT)in acute APAP-induced liver injury The expression of 3NT in APAP-induced injured liver tissue was detected by Western blotting and the results prompted that the expression of 3NT was up-regulated at 1 h after administration of overdose APAP.The expression level of 3NT was gradually up-regulated over time.Pretreatment with RSG could significantly down-regulate the expression of 3NT after APAP administration.5.5 Effect of RSG on the expression of NOXs in acute APAP-induced liver injury The expression levels of NOX-2 and NOX-4 in APAP-induced injured liver tissue was detected by Western blotting and the results found that the expression of NOX-2 and NOX-4 was up-regulated at 1 h after administration of overdose APAP.The expression levels of NOX-2 and NOX-4 were steadily up-regulated over time.Pretreatment with RSG could significantly down-regulate the expression of NOX-2 and NOX-4 after APAP administration.Conclusion Taken together,our study suggests that pretreatment with RSG can significantly mitigate the acute liver injury induced by overdose of APAP.RSG plays a protective role in APAP-induced liver injury via inhibiting inflammatory response.In addition,it can protect and alleviate APAP-induced liver injury probably through suppressing the oxidative stress.