The Protective Effects Of Salubrinal On Rat Liver Injury Induced By Brain Death

Liver transplantation was often the only treatment option at end-stage liver failure. The worldwide demanding for donor organs continued to exceed the number of organs available for transplantation. The main source of organ transplantation is from brain death donor. Brain death is the complete and irreversible loss of brain function. Brain death has definite effects on hemodynamic stability, immunologic changes, hormone regulation and others in donor organs. The risk of kidney and liver graft dysfunction is higher in grafts from BD donors than in grafts from living donors. Increased levels of apoptosis mediators in the donor are associated with a poor graft dysfunction. Thus, brain death is believed to be an important antigen-independent risk factor harming the organs before transplantation.The increasing in apoptosis was the main factor that leads to liver injury induced by brain death.Aparting from the mitochondrial pathway and extrinsic pathway leading to apoptosis, the endoplasmic reticulum stress pathway is the third way leading to apoptosis.The endoplasmic reticulum(ER) regulates the protein synthesis and also is the place where the folding and accumulation of the synthesized proteins occur. It also regulates cellular stress and intracellular calcium level. Generally, when an external stress occurs, the ER can protect the cells through the unfolded protein response(UPR); however, if such stress persists or is too strong, the UPR will ultimately activate the ER stress apoptosis signaling proteins and then cause cellular apoptosis. As shown in our previous study, the ER stress was activated in BD rats, and the expressions of the ER stress-related apoptosis proteins increased.PKR-like ER kinase(PERK) is a serine threonine kinase. After the initiation of the ER stress, it can mediate the cellular apoptosis by phosphorylating the eukaryotic translation initiation factor 2α(e IF2α). By selectively inducing the phosphorylation of e IF2-alpha and suppressing its dephosphorylation, salubrinal can fight against the ER stress-induced cellular apoptosis; meanwhile, it does not protect the apoptosis stimulations that are not related with ER.While research has demonstrated that salubrinal can alleviate the cell apoptosis, whether salubrinal can reduce the BD-induced injuries and its potential molecular biological mechanisms remain unclear. The research on the liver injury induced by brain death will provide theoretical basis for drug targerts with the protection of organ function. Objective:To observe the effect of salubrinal on rat liver in BD rats and explorve its relevant mechanisms Methods:In order to explore the mechanism, this research is divided into three parts: Firstly, establishing of modified brain death model: Brain death was induced by increasing intracranial pressure(ICP) with inflating an intracranial placed balloon-catheter.and it was confirmed by flat-line EEG, physical signs of apnea, and absence of brain stem reflexes. Donor management was performed after brain death. ICP and physiologic variables were continuously monitored during the entire 6h follow-up. Secondly, brain death is associated with endoplasmic reticulum stress and apoptosis in rat liver : on the basis of modified brain death model, SD rats were randomly divided into sham operation group(group A) and brain death group(group B). Activation of ER stress and apoptosis related protein expression were examined by the Western blot and immunohistochemical staining(IHC).In additional, apoptosis was assessed by terminal deoxynucleotide transferase-mediated d UTP nick-end labeling(TUNEL) assay and transmission electron microscopy(TEM). Changes in expression of proteins involved in pathways leading to initiation of ER stress and apoptosis were studied by the real-time PCR. Finally, protective effects of Salubrinal on liver Injury in rat models of brain death: The Sprague–Dawley(SD) rats were randomized into three groups: BD group; salubrinal group(administered with salubrinal before BD); and DMSO group(administered with dimethyl sulfoxide before BD). In the salubrinal groups, the salubrinal was administered one hour before the induction of BD. In the DMSO groups, the DMSO was administered one hour before the induction of BD. After the modeling was completed, the venous blood and liver samples were harvested. The m RNA expressions of Chop and Caspase-12 were detected by quantitative polymerase chain reaction(q PCR). The protein expressions of PERK, p-e IF2α, e IF2α, Chop, and Caspase-12 were detected by Western blotting. The distribution and expressions of Chop and Caspase-12 in liver tissues were determined by immunohistochemistry. The expressions of alanine aminotranferease(ALT) and aspartate aminotransferase(AST) were detected with an automatic biochemical analyzer. Apoptosis of hepatic cells were detected using the TUNEL method. Results:Firstly, Rat model of brain death could be performed in a standardized, reproducible and successful way: after brain death is confirmed, it can maintain the state of brain death 6 hour with the effective respiratory and circulatory support and mean arterial pressure is above 80 mm Hg. Secondly, ER stress and response is in mediating brain death-induced apoptosis and liver injury Brain death induces a variety of signature ER stress markers including ER stress-specific X box-binding protein-1(Xbp-1) and up-regulation of glucose-regulated protein 78(Grp78). Furthermore, brain death causes up-regulation of C/EBP homologous protein(Chop) and Caspase12. Consistent with this, TUNEL and TEM were observed confirming apoptosis in the liver following brain death. Finally, Salubrinal can remarkably alleviate the apoptosis of hepatic cells in BD rats :The m RNA and protein expressions of Chop and Caspase-12 as well as the protein expressions of PERK, e IF2α, and p-e IF2α showed no significant difference between the BD group and the DMSO group. Compared with the BD group, the salubrinal group had significantly higher p-e IF2α level and lower P-PERK level 2h and 6h after the induction of BD(P<0.05). However, the expression level of e IF2α showed no such significant difference(P>0.05). As shown by q PCR, after the salubrinal treatment, the m RNA expression of Chop significantly dropped 4h after BD, and so did the m RNA expression of Caspase-12(P<0.05). Western blotting and immunohistochemistry indicated that the protein expressions of Chop and Caspase-12 also significantly decreased after salubrinal treatment. In addition, salubrinal treatment was also associated with the improved liver functions and decreased apoptosis of hepatic cells. Conclusions:The rat model of brain death could be performed in a standardized, reproducible and successful way.The initiation of ER stress and an important role of the ER stress response is proved to mediate brain death-induced apoptosis and liver injury.Salubrinal can remarkably alleviate the apoptosis of hepatic cells in BD rats, and such a protective effect may be achieved via the PERK-e IF2α signaling pathway.