[Protection of liver sinusoidal endothelial cells from hypoxia-reoxygenation induced apoptosis by alpha-1 antitrypsin in vitro].
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; 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.


CONCLUSIONS
alpha-1 antitrypsin protects LSEC from apoptosis during hypothermia and hypoxia-reoxygenation injury by decreasing NO production and inhibiting MMP activity.
Yang
Hong
H
Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing 100029, China.
Zhang
Wen-Jian
WJ
Wu
Lian-Qiu
LQ
Gu
Feng
F
Ye
Li-Ya
LY
Li
Jing
J
Xu
Shi-Qing
SQ
Xu
Ya-Ping
YP
Lou
Jin-Ning
JN
D017209:Apoptosis / Q000187:drug effects*; D015687:Cell Hypoxia; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology*; D006801:Humans; D007035:Hypothermia; D008099:Liver / Q000166:cytology*; D015427:Reperfusion Injury / Q000517:prevention & control*; D000515:alpha 1-Antitrypsin / Q000494:pharmacology*
D000515:alpha 1-Antitrypsin