Necrostatin －1对创伤失血性休克大鼠肝脏巨噬细胞炎性蛋白－1α表达的影响

摘要

目的：探讨程序性坏死特异性抑制剂－1（necrostation－1， Nec－1）对创伤失血性休克大鼠肝脏巨噬细胞炎性蛋白－1α（ MIP－1α）的影响及其机制。方法采用左下肢股骨、胫骨骨折及腹部软组织损伤并失血、再灌注的方法制备大鼠创伤失血性休克模型，将40只雄性SD大鼠按随机数字表法随机分为模型组、DMSO组、Nec－1组、假手术组，每组10只。假手术组仅进行麻醉、分离血管、结扎血管，并不进行创伤失血和再灌注。模型组为创伤失血性休克大鼠模型，不进行任何干预。 DMSO组建立创伤失血性休克大鼠模型，再灌注前5 min前股静脉给予DMSO溶剂。 Nec－1组于再灌注5 min股静脉给予Nec－11 mg／kg。于再灌注后24 h取动物血清及肝脏组织。检测血清丙氨酸氨基转移酶（ ALT）、天门冬氨酸氨基转移酶（ AST）水平；HE染色观察肝脏病理变化；酶联免疫分析法（ ELISA）分析血清MIP－1α含量；RT－PCR技术检测肝脏组织MIP－1αmRNA含量；蛋白质免疫印迹法（ Western blotting）检测肝脏组织MIP－1α含量。结果模型组血清ALT、AST及血清MIP－1α含量与DMSO组比较差异无统计学意义（P＞0．05）， Nec－1组24 h血清ALT、AST及血清MIP－1α含量较模型组及DMSO组有明显下降（P＜0.05）。光镜下模型组及DMSO组可见肝小叶结构破坏、淤血、炎性细胞浸润，Nec－1组肝组织损伤明显减轻。 Nec－1组MIP－1αmRNA及MIP－1α蛋白含量低于模型组及DMSO组（P＜0.05）。结论Nec－1可以有效降低创伤失血性休克对肝脏的损伤，减少MIP－1α蛋白的表达，减少免疫细胞对组织的浸润及破坏。%Objective To investigate the effect and mechanism of necrostatin-1 (Nec-1) on the expression of hepatic macrophage inflammatory protein-1α( MIP-1α) in hemorrhagic-traumatic shock rats.Methods The model was Sprague-Dawley ( SD) rats suffered hemorrhagic -traumatic shock.A number of 40 male SD rats were divided into model group, DMSO group, Nec-1 group and sham group with 10 rats in each group by randomized digital number method.Rats in sham group were only received anesthesia, separating and ligating blood vessels, without trauma induced hemorrhagic and reperfusion. Rats in model group were received hemorrhagic -traumatic shock without other intervention.Rats in Nec-1 group were received 1mg/kg Nec-1 through femoral vein 5 minutes before reperfusion, while the rats in DMSO group were received the same amount of solvent.The serum and liver tissues of each group were collected 24 hours after reperfusion.Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected by automatic biochemistry analyzer.The pathology changes in liver were observed by hematoxylin -eosin ( HE ) staining.The expression of MIP-1αin serum was detected by ELISA.The MIP-1αmRNA in the liver was determined by reverse transcription-polymerase chain reaction ( RT -PCR ) .The protein expressions of MIP -1αwere assessed by Western blotting analysis.Results Compared with model group and DMSO group, there was no different between the expressions of ALT, ATS and MIP-1αin serum (P>0.05).Levels of serum ALT, AST and MIP-1α24 hours after reperfusion in Nec-1 group were significantly decreased compared with model group and DMSO group (P<0.05).Under light microscopy, it was noted that hepatic lobule destroyed, the blood extravasated, and the immunocyte infiltrated.Compared with DMSO group and model group, the expression levels of MIP-1αprotein and MIP-1αmRNA in Nec-1 group were significantly decreased (P<0.05).Conclusion Nec-1 can remarkable protect the liver of rats with hemorrhagic-traumatic shock, decrease the expression of MIP-1αprotein, reduce the infiltration of immunocyte.