title: [Protection of liver sinusoidal endothelial cells from hypoxia-reoxygenation induced apoptosis by alpha-1 antitrypsin in vitro].
abstract: 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.
authors.affiliation: Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing 100029, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Yang
authors.forename: Wen-Jian
authors.initials: WJ
authors.lastname: Zhang
authors.forename: Lian-Qiu
authors.initials: LQ
authors.lastname: Wu
authors.forename: Feng
authors.initials: F
authors.lastname: Gu
authors.forename: Li-Ya
authors.initials: LY
authors.lastname: Ye
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Shi-Qing
authors.initials: SQ
authors.lastname: Xu
authors.forename: Ya-Ping
authors.initials: YP
authors.lastname: Xu
authors.forename: Jin-Ning
authors.initials: JN
authors.lastname: Lou
mesh_terms: 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*
chemical_list: D000515:alpha 1-Antitrypsin