The Rat Liver Ischemia Reperfusion Mir - 146 - A Study

Part ⅠDown-regulation of miR-146a in the early stage of liverIschemia-Reperfusion InjuryIschemia reperfusion (IR) injury often occurs clinically during liver transplantation,hepatectomy, hypovolemic shock and disseminated intravascular coagulation. Liver ishighly sensitive to IR injury, however, the mechanism of this injury remainsincompletely defined. Toll-like receptor4(TLR4), an innate immunity receptor,several studies discovered TLR4and innate immunity play an important role in IRinjury. Aside from recognizing PAMPs, TLR4also recognize endogenous ligands thatreleased from damaged cells or necrotic cells and subsequently induce aninflammatory response, as well as induce activation and maturation of endritic cells(DCs). In addition to secretion of cytokines and chemokines, DCs also canup-regulation T cells. So interfere in the TLR4signal pathway may represent afascinating future tool for the development of new therapeutic strategies in regard toalleviate IR injury. TLR4has attracted significant attention in the study of IR injuryin recent years due to its key role in immune responses and down-regulation of theexpression of TLR4by vasoactive intestinal peptide (VIP) can attenuate liver IRinjury.MicroRNAs (miRNAs) are a class of21-23-nucleotide noncoding RNAs that actas regulators of gene expression transcriptionally. miRNA in C. elegans, Drosophila,plants, mammals, and even wides present in virus. miRNA genes are single copy,multiple copies gene clusters and other forms exist in the genome, the vast majoritylocated in the intergenic region, and its transcription is independent of other genes.Type of small RNA expression on the organization and time-specific gene expression of important regulatory molecules regulate other functions, plays an important rolein a variety of biological processes of organisms such as development, differentiation,growth, proliferation, apoptosis, metabolism, and homeostasis. miRNAs act on targetmRNAs by binding to miRNAs recognition elements, typically within3′-UTRs,leading to translational inhibition and/or induction of mRNAs cleavage, therebydown-regulating expression of protein products. One of the most important miRNAs,miR-146a, has been demonstrated to be one of the key molecules in oncogenesis andthe inflammatory response. There are no data available regarding the expression ofmiR-146a in liver ischemia reperfusion (IR) injury.Aim: The present study aimed to explore the expression of miR-146a and therelationship between miR-146a and TLR4signaling pathways in a rat model ofwarm IR injury.Methods: A partial warm hepatic IR injury model was used. The expression ofmiR-146a was detected by real-time RT-PCR. The expression of TLR4, TRAF6, andIRAK1protein was assessed by Western blotting. The signaling pathways inducedby TLR4were also assessed.Results: The expression of hepatic miR-146a was down-regulated in IR injuryduring the24hours after reperfusion and reached a plateau6hours after reperfusion.In this study we also found that increases in TLR4, TRAF6and IRAK1wereaccompanied by decreased miR-146a during the24hours after reperfusion, andpeaked at6hours. Immunohistochemistry showed the cytoplasmic expression ofcells positive for TLR4, and the nuclear expression of cells positive for NF-κB p65and c-jun was increased in the IR groups after reperfusion.Conclusion: Down-regulation of miR-146a in the early stage of liver ischemiareperfusion injury and possibly through the TLR4-mediated signaling pathwayregulating hepatic ischemia reperfusion injury. Part ⅡProtective effect of recombinant human erythropoietin onliver ischemia-reperfusion injury in ratsErythropoietin EPO(erthrpoietin EPO), a peptide hormone, in the mammaliankidney and liver of a molecular mass of46kDa glycoprotein cytokines. It canstimulate immature red blood cells hyperplasia, hemoglobin and red blood cellsmature, mainly used to treat a variety of reasons due to anemia. EPO promoteerythropoiesis biological effect is completed by specific EPO receptor (EPOR) in thebone marrow erythroid progenitor cell surface. EPO combined with EPOR, EPOR inthe formation of dimers, through signal transduction pathways regulating theproliferation and differentiation of erythroid. EPO is not only the classic endocrinepathway can also play a tissue-specific role of hormones and cytokines with a varietyof functions from the autocrine and paracrine manner.Recent years non-hematopoietic biological effect gradually aroused the concern,the EPO has a number of organs and tissue protection and anti-inflammatory effects.The inflammatory response involved in a variety of injury, but EPO can reduce therelease of many proinflammatory cytokines, reducing the infiltration ofinflammatory cells, can also activate the endogenous protective mechanisms, whichplay an important anti-inflammatory effects. Protective effect of EPO may increasethe anti-apoptotic gene expression, inhibit inflammation, reduce nitric oxide(NO)-mediated damage and direct antioxidant effects.1985recombinant human erythropoietin (EPO)(rHuEPO) successful and widely used in clinical research hasgreatly accelerated the basic and applied research on EPO. Reported in the studies ofrHuEPO can reduce a variety of organs and tissues of ischemia and reperfusion (IR)injury, but rHuEPO little studied in the liver IR injury.Aim: To explore the effect of recombinant human erythropoietin (rHuEPO) onliver ischemia-reperfusion injury in rats and discuss the possible mechanism of it.Method: An ischemia reperfusion(IR) process of1h ischemia and then6hreperfusion was established in male Sprague-Dawley rat.The30rats were randomlydivided into three groups of A(sham-operated),B(model) and C(treatment).The levelsof serum ALT, AST were detected at reperfusion for6hours.The expression ofinduced nitricoxide synthase(iNOS) and endothelial nitric oxide synthase (eNOS)were examined by Western blot and RT-PCR.Result: Compared with the B group of C,rHuEPO significantly reduced serumALT,AST(P＜0.01).The expression of iNOS in C group were significantly lowerthan those in B group(P＜0.05). Compared with the B group, the eNOS in the Cgroup was no significant difference(P＞0.05).Conclusion: These results indicated that rHuEPO down-regulating the expressionof iNOS and decreased the liver ischemic-reperfusion injury.