α-1抗胰蛋白酶对肝窦内皮细胞缺氧复氧损伤保护作用的实验研究

Protection of liver sinusoidal endothelial cells from hypoxia-reoxygenation induced apoptosis by α-1 antitrypsin in vitro

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 effect of α-1 antitrypsin on ischemia-reperfusion injury of human liver sinusoidal endothelial cells (LSECs). Methods LSECs were cultured and put into 4℃ refrigerator for 12 hours and then into 37℃ culture box with 95% O_2 and 5% CO_2 for 2-6 hours to establish an experimental hypoxia-reoxygenation injury model. The LSECs were inoculated in 24-pit culture plate and L-NAME, NO inhibitor, SNAP,a NO supplier, BB3103,a matrix metalloproteinase (MMP) inhibitor, or α-1 antitrypsin of different concentrations were added. The LSECs were put into 4℃ refrigerator for 12 hours and then into 37℃ culture box with 5%CO_2 for 2-6 hours. The supernatant was collected to detect the production of nitric oxide (NO) and activity of MMPs. LSECs were cultured under conditions of presence or absence of α-1 antitrypsin and normal temperature and oxygen concentration, low-temperature and hypoxia, and low temperature and hypoxia-reoxygenation respectively. Then the supernatant was collected to detect the activities of MMP-2 and MMP-9 with zymography and the production of NO with determination of nitrite concentration and expression of eNOS和iNOS by immunohistochemistry. Results Immunohistochemistry showed that LSECs were α-1 trypsin positive, RT-PCR showed LSECs did not express α-1 trypsin mRNA. TUNEL staining showed that hypothermia for 12 hours caused apoptosis of LSECs, apoptosis of LSECs was more obvious after hypothermia and hypoxia-reoxygenation; however, with the presence of α-1 trypsin apoptosis of LSECs was significantly decreased after hypothermia and hypoxia-reoxygenation. Hypoxia-reoxygenation induced apoptosis was significantly decreased by L-NAME and BB3103 and increased by SNAP. significantly decreased the apoptosis Zymography showed a significant increase of MMP production, in the forms of proMMP-2和proMMP-9, in LSECs, paralleling with the number of apoptotic LSECs; and ~α-1 trypsin significantly inhibited the MMP activity during hypothermia and hypoxia-reoxygenation dose-dependently. Paralleling with the number of apoptotic LSECs, the expression of iNOS and eNOS, especially the former, was significantly increased after hypothermia and hypoxia-reoxygenation. L-NAME significantly decreased and SNAP significantly increased the production of NO during hypothermia and hypoxia-reoxygenation. The NOS expression and NO production of LSECs during hypothermia and hypoxia-reoxygenation were inhibit by α-1 trypsin dose-dependently.Conclusion α-1 antitrypsin protects LSEC from apoptosis during hypothermia and hypoxia-reoxygenation injury by decreasing NO production and inhibiting MMP activity.