Effect And Mechanism Of Ire1-mediated Endoplasmic Reticulum Stress On Hepatocellula Apoptosis In Experimental Fulminant Hepatic Failure

Objective: Fulminant hepatic failure(FHF) is a syndrome with the characteristic of severe liver function damage caused by a great quantity of hepatocellular necrosis resulted from many reasons. The characteristics of FHF are acute onset, rapid progression, many complications, high mortality and poor prognosis. It is currently the hot and tough issue at home and abroad. Its pathogenesis is complex and has not been fully clarified so far. The current studies suggest that FHF is closely related to hepatocellular apoptosis. But the mechanism has not been known well. Broadly speaking, there are three pathways in apoptosis: death receptor pathway, mitochondrion pathway and endoplasmic reticulum pathway. Of which, endoplasmic reticulum stress(ERS) mediated apoptosis pathway recently discovered is related to many liver diseases, and it is a hot point of domestic and international research. Endoplasmic reticulum(ER) is the biggest membrane network structure in cell. Its functions are the synthesis and processing of proteins so as to be involved in metabolism and cell signal processing. There are many reasons for physiological dysfunction , the imbalance of Ca2+ and the accumulation of misfolded and unfolded proteins in the ER. Such subcellular pathological phenomena are called ERS. It is an important mechanism by which the misfolded proteins return to the correct conformation and to be further processed. ERS is a self-protection mechanism for cells, but too much and continuous ERS will harm cells even cause apoptosis. The deposition of a large number of membrane proteins induces ER overload response(EOR),and nuclear factorκB (NF-κB) is activated during ERS. Inositol requiring 1( IRE1) play a central role in the EOR pathway.IRE1 is invovled in all the ERS stages such as adpatation , alert, apoptosis as an ER transmembrane protein .In ERS,activated IRE1s are able to recruit tumor necrosis factor receptor associated factor 2(TRAF2)to be IRE1-TRAF2 complex , by which NF-κB is activated.NF-κB as a multiway regulation nuclear transcription factor exists widely in cells. It is closely related to apoptosis .It is involved in transcriptional regulation of apoptosis-related genes and plays a dual role in apoptosis inhibition and apoptosis promotion. Activation of NF-κB is reported to be mediated via TRAF2,another study demonstrated that activation of NF-κB was also reported to be mediated via NIK and activation of NF-κB inducing kinase(NIK) was reported to be mediated via TRAF2,so activation of NF-κB is supposed to be through the pathway IRE1/TRAF2/ NIK/ NF-κB. The family of cysteine aspirate proteases,the so called caspases,are critical mediators of programmed cell death. Caspase-12 as a member of the family of cysteine aspirate proteases is localized to the ER and activated via ERS, but not via membrane- or mitochondrial-targeted apoptotic signals. Recently, it has been demonstrated that apoptosis mediated via ERS is related to many kinds of diseases such as hepatitis B virus ,nonalcoholic fatty liver disease, Alzheimer's disease ,diabetes and so on. But there is still little research on how hepatocellular apoptosis is mediated via ERS in FHF.In this research, Wistar rats were administered with D-GalN and LPS to be FHF animal model, and the control group were administered with the same volumetric saline simultaneously. Immunohistochemistry,reverse transcriptase polymerase chain reaction (RT-PCR) methods were used to detect the expressions of Caspase-12, IRE1, NF-κB, NIK in the two groups. Hematoxylin-eosin (HE) staining, flow cytometry (FCM) were used to evaluate hepatocellular apoptosis in the two groups. The relationships between IRE1 expression and hepatocellular apoptosis, Caspase-12 were analized respectively,and the relationship between Caspase-12 expression and hepatocellular apoptosis was analized, the effect and mechanism of ERS on FHF were eventually discussed .The relationships between NF-κB activation expression and hepatocellular apoptosis,IRE1,NIK were analized respectively,the effect and mechanism of NF-κB activated via IRE1-mediated ERS pathway, were eventually discussed . These discussions may be good for clarifying the pathogenesis of FHF.Methods:1 study objects: 60 masculinity depuratory Wistar rats weighted 180-200g were randomly divided into model group 30 and PDTC group 30. Fulminant hepatic failure model was manufactured by peritoneal injection of D-galactosamine 800 mg/kg,afterward intradermally injection of LPS 10μg/kg in the model group. The control group were administered with the same volumetric saline simultaneously.2 Sample collection and conservation: 6 rats in model group and control group respectively were sacrificed at 2,4,8,12,24 hours after injection by femoral vein exanguinate. Adequate liver tissue was respectively fixed by 10% neutral formaldehyde solution,70% ethanol. Some of the liver tissue was freezed in liquid nitrogen immediately.3 Routine HE staining of hepatic tissue: Liver histopathology change was observed under the light microscope.4.Immunohistochemistry staining of hepatic tissue: The expressions of Caspase-12, IRE1, NF-κBp65 and NIK proteins were detected by immunohistochemistry PV method.5 The expression of Caspase-12, IRE1, NF-κB and NIK mRNA were detected by RT-PCR in the two groups.6 The apoptotic rate was detected by flow cytometry method.7 Statistically analysis: SPSS 13.0 statistical software was used for statistical analysis,data were analyzed by ANOVA, grade and rank and Spearman's rank correlation test.Results:1 Observation of rat general state of health: General state of health was aggravated gradually with administration time in model group. Rats became hydroposia decrease,horripilation,downcast,reaction dullness and drowsiness with the time. But in control group, general state of health was good. 2 Liver tissues morphology general observation: (1) Liver tissues showed hyperaemia,bleeding point,partly congestion and necrosis in model group. The fragility of hepatic tissue increased and there was massive necrosis in the model group with the time. Liver tissue in control group was normal, bright red and soft. (2) Hepatic tissue HE staining: The pathological change was aggravated gradually with the time in model group. Hepatocyte edema and acidophilia,apoptotic bodies,inflammatory cell infiltration,liver tissue hemorrhage,hepatocellular necrosis and hepatic lobules disorganization were discovered in liver tissue under light microscope. In control group, hepatic tissue was normal.3 Changing of hepatocyte apoptosis: The apoptotic rate detected by FCM exhibited an upward trend of apoptotic hypatocytes with the time in model group(P<0.05). In control group, hepatocellular apoptotic rate was low(P>0.05), and less than the model group at 4,8,12,24 hours respectively(P <0.05).4 Caspase-12 protein and mRNA expression in liver tissue in the two groups: (1) The expression of Caspase-12 protein: The positive staining cell is mainly hepatocyte in model group,and most cytoplasm staining,increased with time. And the difference had statistical significance (P<0.01). The expression of Caspase-12 protein was not discovered in liver tissue in control group.(2) The expression of Caspase-12 mRNA: In model group, it increased with time(P<0.01). There was a peak at 8 hour after admistration and then decreased gradually.In control group , there was weak expression of Caspase-12 mRNA and the expressin of Caspase-12 mRNA were less than the model group at 4,8,12,24 hours respectively(P <0.05).5 IRE1αprotein and mRNA expression of hepatic tissue in the two groups: (1) The expression of IRE1αprotein: The positive staining cell is mainly hepatocyte in model group,and most cytoplasm staining,increased with time (P<0.01). There was no expression of IRE1αprotein of liver tissue in control group.(2) The expression of IRE1αmRNA: In model group, it increased with time gradually(P<0.01). In control group , there was a little expressin of IRE1αmRNA . The expressin of IRE1αmRNA were less than the model group(P <0.05).6 NF-κB p65 protein and mRNA expression in liver tissue in the two groups: (1) The expression of NF-κB p65 protein: The positive staining cell was mainly hepatocyte in model group,and most nuclear staining,increased with time,and was strongly positive at 12 hour, then decreased at 24 hour. The expression of NF-κB p65 protein in control group was not discovered and less than the control group (P<0.05). (2)The expression of NF-κB mRNA: In model group, it increased after administration and peaked at 12 hour,then gradually decresed(P<0.01). In control group, liver tissue showed less NF-κB mRNA expression compared with the model group at 2, 4,8,12,24 hours respectively (P<0.05).7 NIK protein and mRNA expression of liver tissue in the two groups: (1) The expression of NIK protein: The positive staining cell was mainly hepatocyte in model group,and most cytoplasm staining,And the difference had no statistical significance (P>0.05). The expression of NIK protein was not discovered in liver tissue in control group. (2) The expression of NIK mRNA: In model group liver tissue showed more NIK mRNA expression compared with the model group (P<0.05).But there were no difference at different time points in model group or in control group (P>0.05).8 The relationships of IRE1αand hepatocellular apoptotic rate ,Caspase-12,and the relationship of Caspase-12 and hepatocellular apoptotic rate in experimental fulminant hepatic failure:There were positive correlations between IRE1αand Caspase-12,hepatocellular apoptotic rate(r= 0.733, P=0.000;r= 0.715, P=0.000). There was positive correlation between Caspase-12 and hepatocellular apoptotic rate(r= 0.586, P=0.001).9 The relationship of NF-κB and hepatocellular apoptotic rate ,IRE1α,NIK in experimental fulminant hepatic failure:There were positive correlations between NF-κB and hepatocellular apoptotic rate,IRE1α(r=0.515 , P=0.004; r= 0.763, P=0.000).There was no correlation between NF-κB and NIK(P>0.05). Conclusions:1 The rat model induced by D-GalN+LPS can reflect the pathological changes of fulminant hepatic failure. Hepatocellular apoptosis was observed morphologically,cytologically and from gene level. It's confirmed that hepatocyte apoptosis plays an important role in the progression of FHF.2 In experimental FHF, there were increased expressions of IRE1α, Caspase-12 with time and there were positive correlations between IRE1αand Caspase-12,hepatocellular apoptotic rate. There was positive correlation between Caspase-12 and hepatocellular apoptotic rate.So it is confirmed that ERS play an important rol in FHF and the IRE1α/ TRAF2/ Caspase-12 signal transduction pathway may be an important mechanism for ERS inducing hepatocellular apoptosis.3 In experimental FHF,there was increased expression of NF-κB with time and there was positive correlation between NF-κB and hepatocellular apoptotic rate. So it is confirmed that NF-κB may play an important role in FHF hepatocellular apoptosis.4 In experimental FHF,there was positive correlation between NF-κB p65 and IRE1α,there was no correlation between NF-κB p65 and NIK. So it is confirmed that NF-κB may play an important role in ERS , but NIK may be not a connection molecule between TRAF2 and NF-κB p65 in the IRE1α/ TRAF2/ NF-κB signal transduction pathway.