| Background:Liver failure is a group of clinical syndrome characterised by coagulopathy,jaundice,hepatic encephalopathy and ascites with varous precipitating factors.Acute liver failure(ALF)is a subtype known as rapid aggravation which presenting hepatic encephalopaghy in no more than two weeks and high mortality.ALF can be caused by virus,alcohol,toxins,metabolic and genetic diseases,but irrespective of etiology,ALF results from rapid and extensive hepatic apoptosis and necrosis.Although the nature of ALF has been extensively studied,the pathogenesis is still unclear.The endoplasmic reticulum(ER)is an organelle that has essential roles in multiple cellular processes that are required for cell survival and normal cellular functions.Endoplasmic reticulum stress(ER stress)is induced by physiological and/or pathological stress signals,leading to the accumulation of unfolded or misfolded proteins in the ER.If protein aggregation is persistent and the stress cannot be resolved,the signaling switches from a pro-survival to a pro-apoptotic ER stress response.Sustained or massive ER stress leads to apoptosis.As reported previously,severe ER stress promotes liver injury by inducing hepatocyte apoptosis in D-galactosamine(D-GalN)and lipopolysaccharide(LPS)treated mice.Peroxisome proliferator-activated receptors(PPARs)are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors.To date,three subtypes of PPARs(a,(3,y)have been identified in many species,including humans.PPARa has been reported to regulate lipid metabolism,inflammation,cell differentiation and apoptosis.Studies have demonstrated that PPARα plays a different role in cancer cells.PPARa activation is widely employed in hepatocarcinogenesis protocols for rodents in which its anti-apoptotic action is assumed to play an important role;however,activation of PPARa by exogenous agonists causes inhibition of tumor cell growth in cell lines derived from colorectal cancer.Recent study has reported that activation of PPARα ameliorates hepatic insulin resistance to increased ER stress,but others have inverse results.Although our studies have demonstrated that PPARa activation protects the liver from acute injury by promoting the autophagy pathway in.the D-GalN/LPS-induced ALF mouse model,the underlying mechanisms of the PPARa and ER stress in ALF required further elucidation.Objection:In this study,the mice model of ALF was established through intraperitoneal injection with D-GalN/LPS.We use Wy-14643 to activate PPARa,and siRNA to inhibit.Meanwhile,4-phenylbutyric acid(4PBA)is used to relieve ER stress.To determine the mutual action of PPARa and ER stress,we detected the levels of serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),and the expresssions of glucose-regulated protein 78(Grp78)、Grp94、C/EBP homologous protein(CHOP)、cysteinnyl aspartate specific proteinase-3(Caspase-3)、Cleaved caspase-3(markers for ER stress and apoptosis)in liver tissue.The pathological changes were observed via HE staining.Moreover,to study the effects of PPARa regulation on hepatocyte apoptosis induced by ER stress,the primary mouse hepatocytes were treated with tunicamycin(TM)or thapsigargin(TG),which increases ER stress,and the indicated conditions including Wy-14643 or PPARα siRNA.The changes in the qualitative index of cell proliferation,levels of lactate dehydrogenase(LDH)in the cell supernatants,and the expressions of PPARα、CHOP、Cleaved caspase-3 in cells were measured.The results provided a novel and experimental basis for the prevention and treatment of ALF with PPARa.Methods:1 The effects of PPARα on ER stress-induced hepatocyte apoptosis in mice model of ALFMale C57BL/6 mice at the age of 8-12 weeks were purchased from the Capital Medical University(Beijing,China)and fed freely with a standard chow diet and water;they were housed under specific pathogen-free conditions for 1 week before the experiments.All animals received humane care according to the Capital Medical University Animal Care Committee guidelines.Male C57BL/6 mice were divided into three groups randomly.The mice were injected intraperitoneally with Wy-14643(6mg/kg)or vehicle(DMSO)2 hours prior to D-GalN(700mg/kg)and LPS(10μg/kg)exposure(n=22/group).The control mice were pretreated with vehicle(DMSO)2 hours before PBS injection(n=10).The survival rate was analyzed in D-GaIN/LPS-treated mice and Wy+D-GaIN/LPS-treated mice up to 24 hours after D-GalN/LPS injection(n=10/group).The remaining mice were euthanized with chloral hydrate(1.0g/kg)6 hours after D-GalN/LPS treatment,and the liver and serum samples were collected for analysis.Blood biochemical indicators were measured by using a multi-parametric analyzer,according to an automated procedure.Liver tissue was fixed with 10%neutral formaldehyde and then embedded in paraffin.The specimens were cut into 5μm sections,which were then stained with hematoxylin and eosin(H&E)and observed under light microscopy.Apoptosis in liver sections was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)using the InSitu Cell Death Detection Kit.The expressions of markers for ER stress such as Grp78、Grp94、CHOP were detected via real-time quantitative polymerase chain reaction(qRT-PCR)and western blot,respectively.The expressions of Caspase-3 and Cleaved caspase-3 in the liver were measured via western blot.2 The regulatory role of inhibition of ER stress in D-GaIN/LPS-induced ALFThe experiment consisted of two parts.First,the male C57BL/6 mice were divided into three groups randomly.A chemical chaperone that relieves ER stress,4-PBA(100mg/kg;Sigma,St Luis,MO),was dissolved in PBS and administered intraperitoneally 6 hours prior to D-GalN/LPS exposure.The mice were sacrificed at 6 hours after D-GalN/LPS treatment.The expression and distribution of PPARa in mice liver were measured via qRT-PCR、western blot and immunofluorescence staining.Then the male C57BL/6 mice were divided into five groups randomly.Mice were pretreated with control siRNA(50μM/kg)or PPARα siRNA(50μM/kg)via tail vein injection 24 hours prior to D-GalN/LPS treatment and then injected with 4-PBA or PBS 6 hours prior to D-GalN and LPS exposure(n=24/group).The control mice were injected with only PBS(n=10).The survival rate was analyzed in the D-GalN/LPS-treated mice,the 4-PBA+D-GalN/LPS-treated mice and the PPARa siRNA+4-PBA+D-GalN/LPS-treated mice up to 24 hours after D-GalN/LPS injection(n=10/group).The remaining mice were euthanized with chloral hydrate 6 hours after D-GalN/LPS treatment,and the serum and liver samples were collected for further analysis.Liver injury was estimated by biochemical serum markers such as ALT,AST and by pathological examination.The expressions of markers for ER stress such as Grp78、Grp94、CHOP were detected via qRT-PCR and western blot,respectively.3 The effect of PPARa regulation on ER stress-induced hepatocyte apoptosis in vitro3.1 The expression profile of PPARa in the progression of ER stress-induced hepatocyte apoptosis in vitroThe livers of 7-week old mice were perfused with collagenase-containing Hank’s solution,and viable hepatocytes were isolated by Percoll isodensity centrifugation.The primary hepatocytes were cultured in DMEM medium with high glucose containing 10%fetal bovine serum and incubated with known ER stress inducers TM and TG for various times or at increasing doses.The primary hepatocytes were treated with only DMSO as a control or 40μg/ml TM or 1μg/ml TG for 3,6,12 or 24hours.Moreover,the primary hepatocytes were treated with increasing concentrations of TM(2.5,5,10,25,or 50μg/ml)or TG(0.25,0.5,1,2.5,or 5μg/ml)for 12 hours.Relative PPARα mRNA expression was measured by qRT-PCR.The protein levels of PPARa,CHOP and Cleaved caspase-3 were measured by western blot.3.2 The role of PPARα in the intrinsic potential of hepatocyte apoptosis triggered by ER stressThe primary hepatocytes were cultured in DMEM medium with high glucose containing 10%fetal bovine serum and we used specific siRNA to knock down the expression of PPARα and Wy-14643 to activate PPARa.The experiment is composed of two parts.First,the hepatocytes were transfected with PPARα siRNA(5nM)for 24 hours,followed by TM(40μg/ml)or TG(1μg/ml)for 6 hours.Then the hepatocytes were incubated with Wy-14643(50μM)or DMSO for 2 hours and then stimulated with TM(40μg/ml)or TG(1μg/ml)for 24 hours.The primary hepatocytes were treated with only DMSO as a control.Cell viability or apoptosis was measured by MTT assay or LDH activity assay,respectively,in different groups.The expressions of PPARα,CHOP and Cleaved caspase-3 were measured by western blot from the different groups.The results were expressed as the means±standard deviation(SD).Normality and homogeneity of variance were tested before single-factor analysis of variance.All data were compared between the two groups through Least-Significant-Difference method.Two-tailed P<0.05 was considered statistically significant.SPSS 19.0 version was used to compute the collected data.Results:1 The effects of PPARa on ER stress-induced hepatocyte apoptosis in mice model of ALF1.1 The levels of PPARa were gradually reduced and apoptotic cells were increased in D-GaIN/LPS-treated ALF miceOur studies have demonstrated that the gene and protein of PPARa were gradually reduced throughout the procession of ALF.In the survival analysis the mice in the D-GalN/LPS group began to die 6 hours after D-GalN/LPS administration,and the survival rate stabilized at 60%(6 of 10 mice)at 24h.The levels of serum ALT(1465.15±559.81U/L),AST(4037.83± 1774.55U/L)in the D-GalN/LPS-treated group were higher than those in the control group(36.85±6.91U/L,41.90±10.05U/L)(both P<0.01).Histopathological examinations showed that the structure of liver tissues was destroyed,the hepatic cord was disarranged and large necrosis area was present.Meanwhile,compared with the control group,a large number of TUNEL-positive cells were observed and the levels of Cleaved caspase-3(17 and 19 kDa)increased in the D-GalN/LPS-treated group(both P<0.01).1.2 PPARa activation decreases hepatocyte apoptosis,thus protecting against ALFWe first evaluated whether PPARa activation could rescue liver injury by applying Wy-14643,a PPARa ligand activator.In the survival analysis the mice pretreatment with Wy-14643 before D-GalN/LPS administration reduced the mortality,and the survival rate was 90%(9 of 10 mice).With respect to liver damage,liver function showed significantly lower ALT(524.53±380.83U/L)and AST(1227.151362.84U/L)levels in the Wy-14643 pretreatment group compared with the D-GalN/LPS administration group.The gross morphology of the liver after Wy-14643 pretreatment appeared to be substantially better,and the liver architecture was well preserved.To explore the potential protective mechanism of PPARa against ALF induced by D-GalN/LPS,we measured apoptotic cells in the three groups.The Wy-14643 pretreatment group displayed significantly fewer apoptotic hepatocytes.Moreover,consistently with the TUNEL data,the increase of Cleaved caspase-3 was attenuated by Wy-14643 pretreatment(all P<0.05).Thus,these results suggested that PPARα activation significantly reduced apoptotic cells and thereby protected mice from ALF induced by D-GalN/LPS.1.3 PPARa activation relieves ER stress in D-GalN/LPS-induced ALFTo examine the effects of PPARα on D-GalN/LPS-induced ER stress in mice,we measured the levels of mRNA and protein for ER stress mediators.The expression of Grp78,Grp94 and CHOP,which are the classical ER stress markers,was increased significantly after D-GalN/LPS administration but was significantly attenuated by pretreatment with Wy-14643.These alterations were confirmed by western blot analyses(all P<0.05).The results showed that PPARα activation suppressed ER stress during D-GalN/LPS-induced ALF.2 The regulatory role of inhibition of ER stress in D-GaIN/LPS-induced ALF2.1 Inhibition of ER stress increases the expression of PPARα in D-GalN/LPS-induced ALFOur studies have demonstrated that PPARa were gradually reduced throughout the procession of ALF.A small chemical chaperone,4-PBA,has been shown to reduce ER stress both in vivo and in vitro.We evaluated whether ER stress inhibition could promote the expression of PPARα in the context of ALF.The qRT-PCR and western blotting results showed that,compared with D-GalN/LPS treatment alone,pretreatment with 4-PBA promoted the expression of PPARa.Similar results were obtained by immunofluorescence staining of liver tissue.Moreover,our results also showed that the expression of PPARa was cytoplasmic rather than nuclear.These results indicated that the expression of PPARa is promoted by 4-PBA pretreatment in D-GalN/LPS-induced ALF(all P<0.05).2.2 Inhibition of ER stress protects mice from ALF through PPARαmechanismsWe used siRNA to knock down the expression of PPARa in mice.The specific inhibition of PPARα in the liver by siRNA in vivo was confirmed by the reduced levels of PPARa in the mice.The results showed that the hepatic protection by 4-PBA in the ALF was abolished by knockdown of PPARa,which was evidenced by the decreased survival rate,significantly higher levels of ALT and AST and abnormal gross morphology and less preserved liver architecture as observed from histology.Meanwhile,the knockdown of PPARα reversed the expression levels of Grp78,Grp94 and CHOP in 4-PBA-pretreatment ALF mice.Thus,these results demonstrated that the mechanism of hepatoprotection by ER stress inhibition depends on PPARa activity(all P<0.05).3 The effect of PPARα regulation on ER stress-induced hepatocyte apoptosis in vitro3.1 The expression profile of PPARa in the progression of ER stress-induced hepatocyte apoptosis in vitroThe qRT-PCR and western blot results showed that the expression of PPARa was significantly up-regulated in the early stage of TM or TG-induced ER stress and was significantly down-regulated in the later time points of TM or TG treatment compared with the control group.Moreover,there was a difference in responses at different doses of TM or TG,compared with the control group.The low dose of TM or TG markedly up-regulated PPARa expression,whereas the high dose of TM or TG reduced the expression of PPARa.Moreover,for the longer time and higher dose of TM or TG treatment,the CHOP and Cleavage of caspase-3 were increased.Therefore,these results indicated that mild ER stress promotes the expression of PPARα,and severe ER stress reduces the expression of PPARα(all P<0.05).3.2 The role of PPARα in the intrinsic potential of hepatocyte apoptosis triggered by ER stressUnder the conditions of mild ER stress,we used specific siRNA to knock down the expression of PPARa.TM or TG treatment for 6 hours increased the release of LDH from the hepatocytes and decreased hepatocyte viability;down-regulation of PPARa by siRNA further increased the LDH levels from the hepatocytes and further decreased hepatocyte viability.Western blot analysis showed that PPARα siRNA increased the levels of CHOP and Cleaved caspase-3 compared with TM or TG-treated cells.Under conditions of severe ER stress,we used Wy-14643 to activate PPARa.Compared with 24 hour treatment of hepatocytes with TM or TG,activation of PPARa by Wy-14643 significantly decreased the hepatocyte levels of LDH and increased hepatocyte viability.Western blot analysis also showed that Wy-14643 decreased the levels of CHOP and Cleaved caspase-3,as compared with TM or TG-treated cells(all P<0.05).Therefore,the activation or expression of PPARα was a key point of balance between hepatocyte survival promoted by mild ER stress and hepatocyte apoptosis induced by severe ER stress.Conclusions:1 PPARα activation decreased the expression of ER stress markers(Grp78Grp94 and CHOP)and inhibited hepatocyte apoptosis,thereby protecting against ALF.2 The liver protection by 4-PBA was due to the induction of PPARαexpression,because 4-PBA pretreatment promoted up-regulation of PPARa,and inhibition of PPARα by siRNA treatment reversed liver protection and increased hepatocyte apoptosis.3 Thes mild ER stress promoted the expression of PPARα,and severe ER stress reduced the expression of PPARα.4 The activation or expression of PPARα was a key point of balance between hepatocyte survival promoted by mild ER stress and hepatocyte apoptosis induced by severe ER stress.5 PPARα may be useful as a potential therapeutic strategy to ameliorate ALF. |
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