Effects Of Heat Shock Protein 70 On The Inflammation After Local Ischemia-reperfusion In The Rats

AIMThe injury incurred by ischemia reperfusion is the concerned widely problem in the field of liver surgery. Now many studies indicate that the injury incurred by hepatic ischemia reperfusion suffers usually two distinct phases. The hepatocellular injury and its metabolizable unbalance during the first phase mostly induced by the enhanced lipid peroxidant stress, the increased production of radical oxygen species (ROS) and the activated Kupffer cells. The recruited neutrophils during the second phase induced by proinflammatory cytokines , including adhesion molecules and chemokines caused the inflammatory reaction. These damages have direct relation with the success in operation and the prognosis of illness, so seeking the available method for protecting hepatocyes from ischemia reperfusion injury will have the important and clinic signification.The tissue and cell disposed in the thermal environment usually occur the heat shock stress and produce a kind of heat shock proteins ( HSPs). HSPs are a kind of high conservative proteins. Among them the HSP70 family is an endo-genic and protective protein, which helps the tissue and cell to enhance the endurance to the bad stimulations. It has confirmed that HSP70 protects the liver from ischemia reperfusion injury, but the protective mechanism is unclear so far. In this study, we aim to discuss the effects of HSP70 on the inflammation after the hepatic partial ischemia reperfusion in the rats.MATERIAL AND METHOD1. Animal grouping and model preparation 1.1 Animal groupingThe rats were randomly into three groups: A group: hepatic local ischemia reperfusion (IR) group B group: heat stress pretreatment + hepatic local ischemia reperfusion (H + IR) groupC group: injecting quercetin + heat stress pretreatment + hepatic local ischemia reperfusion ( Q + H + IR) group1. 2 model preparationInjecting the inhibitor of HSP70 expression: to inject quercetin (i. p. ) Heat stress pretreatment: The rats were anesthetized and fitted with a digital thermal sensor inserted in their returns 6cm beyond the anus. Then the rats were placed in 45 T! ± 0. 5t oven and heated until their ractal temperature reached 42^. Their temperature at 421 for 15min, and then they were taken out.Hepatic local ischemia reperfusion model: All rats were anesthetized with pentobarbital sodium (40mg/kg. BW , i. p. ). The abdomen was opened in a median laparatomy, and the ligment of hepatic left and middle lobars was detached and champed for 40min using an atraumatic microvascular clip. Reperfusion was accomplished by removing the clip.2. Measured items2.1 The expression of HSP70 in liver: Western blot analysis2.2 The intercellular adhesion molecule-1 ( ICAM-1 ) expression: Using strept avidin-biotin-enzyme complex (SABC) assay. Trie negative control was performed with PBS that insteaded of anti-HSP70 antibody.2. 3 The extent of neutrophilic infiltration in liver; myeloperoxidase (MPO) activity assay2.4 Hepatic function assay: The serum levels of aspartate amintransferase (AST) and alaninetransaminase (ALT) were measured by auto-biochemical an-alyzer.2.5 Hepatic histology study: liver samples were fixed in 2.5% glutaralde-hyde to study the histopathologically hepatic changes with the transmission electron microscope. The un-ischemic tissue was taken to observed contrastively.3. Statistical analysisThe data were expressed an mean SEM. The significance of differences was determined by ANOVA. A p value <0.05 was considered to be significant.RESULTS1. After heat stress pretreatment, the HSP70 expression in liver gradually increased and peaked at 16h, and then decreased as time went.2. The findings of Western blot analysis indicated that quercetin inhibited the HSP70 expression in a dose-dependent way. Namely the dose of injecting quercetin increased, While the the HSP70 expression decreased. Quercetin obviously inhibited the HSP70 expression in the dose of 7mg/kg. BW.3. The HSP70 expression in liver in each group: In IR group without heat stress pretreatment , the HSP70 expression was at the low level in 24h, and gradually decreased as time went. In H + IR group, the HSP70 expression significantly higher compared with IR group. In Q + H + IR group, the HSP70 expression was inhibited obviously.4. In the liver tissue, the positive product of ICAM-1 expression presented in many brown-yellow pellets with immunohistochemical assay. These pellets exited in cellular membrane of endothelial cells of central veins. The results of image quantitative analysis indicated the ICAM-1 expression increased , then decreased in each group, and peaked at 6h. The ICAM-1 expression was lower at each time point in H + IR group (p <0.01}. There was no significant difference between IR group and Q + H + IR group at each time point (p >0.05}.5. In each group the MPO activity in the liver tissue increased earlier and then decreased at each time point. The MPO activity in H + IR group was lower than that in IR group and Q + H + IR group (p<0.01) at the same time point, and there was no significant difference between IR group and Q + H + IR group...