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; and alpha-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 alpha-1
trypsin dose-dependently.
CONCLUSION: