[Protection of liver sinusoidal endothelial cells from hypoxia-reoxygenation induced apoptosis by alpha-1 antitrypsin in vitro].
摘要
OBJECTIVE: To investigate the effect of alpha-1 antitrypsin on ischemia-reperfusion injury of human liver sinusoidal endothelial cells (LSECs). METHODS: LSECs were cultured and put into 4 degrees C refrigerator for 12 hours and then into 37 degrees C 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 alpha-1 antitrypsin of different concentrations were added. The LSECs were put into 4 degrees C refrigerator for 12 hours and then into 37 degrees C 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 alpha-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 and iNOS by immunohistochemistry. RESULTS: Immunohistochemistry showed that LSECs were alpha-1 trypsin positive, RT-PCR showed LSECs did not express alpha-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 alpha-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 and proMMP-9, in LSECs, paralleling with the number of apoptotic LSECs; an