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目的探讨α1抗胰蛋白酶对人肝窦内皮细胞(LSEC)冷缺氧复氧损伤的保护作用。方法人LSEC在体外培养,通过低温、缺氧复氧建立缺氧复氧损伤实验模型,观察α1抗胰蛋白酶对LSEC缺氧复氧损伤的影响。细胞凋亡应用原位免疫组织化学和DNA梯状条带方法检测;产生基质金属蛋白酶(MMP)的活性应用酶谱方法分析;一氧化氮的产生通过检测亚硝酸盐含量进行分析;一氧化氮合酶的表达采用细胞免疫组织化学方法进行分析。结果低温、缺氧复氧引起LSEC凋亡,而α1抗胰蛋白酶抑制LSEC的凋亡。应用一氧化氮的抑制剂Nω硝基左旋精氨酸(LNAME)或MMP的抑制剂BB3103明显减少缺氧复氧诱导的LSEC的凋亡,而应用外源性一氧化氮的供体S亚硝基N乙酰青霉氨(SNAP)则明显增加LSEC的凋亡,表明一氧化氮和MMP在LSEC凋亡过程中是重要的介导物。在缺氧复氧过程中,一氧化氮合酶表达增强,一氧化氮产生增多,产生MMP的活性增强。α1抗胰蛋白酶明显抑制缺氧复氧过程中MMP的释放,并通过抑制一氧化氮合酶的表达减少一氧化氮的产生。结论α1抗胰蛋白酶通过抑制一氧化氮和MMP的产生保护LSEC的冷缺氧复氧损伤。
Objective To investigate the protective effect of α1 antitrypsin on cold anoxia-reoxygenation injury in human hepatic sinusoidal endothelial cells (LSECs). METHODS: Human LSECs were cultured in vitro and hypoxic-reoxygenation injury was induced by hypoxia and reoxygenation. The effects of α1 antitrypsin on hypoxic-reoxygenation injury were observed. Apoptosis was detected by in situ immunohistochemistry and DNA ladder method. The activity of matrix metalloproteinases (MMPs) was analyzed by zymography. Nitric oxide production was analyzed by detecting nitrite content. Nitric oxide Synthase expression was analyzed by cellular immunohistochemistry. Results Low temperature hypoxia and reoxygenation induced apoptosis of LSEC, while α1 antitrypsin inhibited the apoptosis of LSEC. Nitric oxide inhibitor Nω-nitro-L-arginine (LNAME) or MMP inhibitor BB3103 significantly reduced hypoxia-reoxygenation-induced apoptosis of LSEC, while application of exogenous nitric oxide donor S nitrosamine N-acetyl penicillamine (SNAP) significantly increased the apoptosis of LSEC, suggesting that nitric oxide and MMPs are important mediators in LSEC apoptosis. During hypoxia and reoxygenation, the expression of nitric oxide synthase increased, the production of nitric oxide increased, and the activity of producing MMP increased. α1 antitrypsin significantly inhibits MMP release during hypoxia-reoxygenation and decreases nitric oxide production by inhibiting nitric oxide synthase expression. Conclusion α1 antitrypsin protects cold-hypoxia-reoxygenation injury of LSEC by inhibiting the production of nitric oxide and MMPs.