用乌司他丁抑制炎症反应减轻大鼠移植肝再灌注损伤的实验研究

Alleviation of reperfusion injury for liver graft via inhibiting inflammatory response by ulinastatin

目的 研究乌司他丁（ulinastatin,UTI）对移植肝再灌注损伤的保护作用及相关机制.方法 建立Sprague-Daw-ley同系大鼠肝移植（OLT）模型,受体分为3组,每组8只大鼠：假手术组、对照组和乌司他丁组.假手术组大鼠仅接受麻醉、开腹、肝周韧带游离及关腹操作；乌司他丁组及对照组大鼠建立OLT模型,门静脉开放后,乌司他丁组大鼠经尾静脉注射乌司他丁1×104 U/kg,对照组大鼠注射等量生理盐水.再灌注后6及24 h每组分别处死4只大鼠,获取下腔静脉血标本及肝脏标本.检测各组大鼠血清ALT水平及肝组织形态学变化、肝细胞凋亡情况；用免疫组化方法检测肝脏Kupffer细胞浸润,用荧光定量RT-PCR检测肝脏肿瘤坏死因子（tumor necrosis factor,TNF）-α、白细胞介素（interleukin,IL）-6、IL-1β及单核细胞趋化蛋白（monocyte chemoattractant protein,MCP）-1mRNA表达水平,用酶联免疫吸附试验检测受体血清内上述炎症介质含量；用蛋白质印迹技术检测肝脏内核因子（nuclear factor,NF）-κB亚单位P65磷酸化水平.结果 与对照组相比,在同一时点检测乌司他丁组大鼠血清ALT水平明显降低（P＜0.01）,肝脏组织学损伤情况明显改善（P＜0.01）,肝细胞凋亡明显减少（P＜0.01）；肝脏内TNF-α、IL-1β、IL-6、MCP-1的mRNA表达降低（P＜0.01）,血清内上述炎症介质含量明显降低（P＜0.01）；P65磷酸化水平明显下降.结论 乌司他丁能够减轻大鼠移植肝再灌注损伤,其机制可能与其抑制再灌注后炎症反应有关.

Objective To evaluate the protective effect of ulinastatin on reperfusion injury of liver graft in rats and the related mechanisms.Methods The orthotopic liver transplantation （OLT） model between syngeneic Sprague-Dawley rats was used,and recipient rats were allocated in three groups：sham operation （SO） group,control group,and ulinastatin （UTI） group.Just after OLT procedure,rats in UTI group received ulinastatin injection at a dose of 1 × 104 IU/kg by tail vein,and rats in control group received saline injection with equal dose.Four rats at 6 hour and the others at 24 hour after OLT were sacrificed; the sera and liver were collected.The ALT levels in serum were detected and hepatic histological changes and hepatocellular apoptosis were examined.The infiltration of Kupffer cells was detected by immunochemistry analysis,and the relative mRNA levels of tumor necrosis factor （TNF）-oα,interleukin （IL）-6,IL-1β and monocyte chemoattractant protein （MCP）-1 in the liver tissue and the content of these cytokines in the sera were measured with real time PCR and ELISA.The activation of transcription factor NF-κB was measured with Western blotting analysis.Results Compared with the rats in control group,at the same time-point rats in UTI group exhibited lower levels of ALT （P 〈0.01）,better hepatic histological changes （P 〈 0.01）,less hepatocellular apoptosis （P 〈 0.01）,lower mRNA expression of TNF-α,IL-6,IL-1β and MCP-1 in the liver tissue and lower content of these cytokines in the sera （P 〈 0.01）.Moreover,the activation of NF-κB in UTI group was significantly down-regulated in comparison with that in control group.Conclusion Ulinastatin could reduce reperfusion injury for liver graft via inhibiting inflammatory response and deserve deep research as novel protective agent.