[Effect of thrombin on cultured rat cerebral astrocyte injured by hypoxia/reoxygenation and its relationship with iNOS].
OBJECTIVE
To observe the effect of thrombin on the cytotoxicity of astrocytes injured by hypoxia/reoxygenation(H/R) and to explore its relationship with inducible nitric oxide synthase (iNOS).


METHODS
Primary astrocytes were cultured in DMEM with 10% approximately 15% calf serum and divided into 6 groups: a control group, a Tm control group, an H/R group, a Tm+H/R group, a hirudin (HR) control group, and a Tm+HR+ H/R group. The cell damage and viability were detected by the 3-(4, 5-di-methyl-thazol-2-yl)-2, 5 diphenyl-tetrazol-iumbromide (MTT) conversion method. The NO level in the cultured cell supernatant was assayed by Griess reagent. The flow cytometry was performed to evaluate the apoptosis rate of astrocytes. The iNOS mRNA was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Immunocytochemistry was used to observe the expression of iNOS protein.


RESULTS
The cell viability injured by H/R was lower than that of the control group, the NO production and apoptosis rate in the cell of H/R group were higher than those of the control group. Incubation of H/R cell with 10kU/L Tm enhanced the cytotoxicity of H/R stimulation compared with the cells injured by H/R. Hirudin can reverse the effect of thrombin. RT-PCR and immunocytochemistry analysis demonstrated that the levels of iNOS mRNA and iNOS protein increased in the cells treated by H/R. Tm enhanced the expression of iNOS mRNA and iNOS protein in the cells treated by H/R. Hirudin blocked the effect of Tm.


CONCLUSIONS
Increasing the level of iNOS and enhancing the production of NO may be the mechanism of thrombin cytotoxicity in astrocytes injured by H/R.
Yang
Li-juan
LJ
Department of Physiology, Xiangya School of Medicine, Central South University, Changsha 410078.
Wu
Li-xiang
LX
Liu
Fa-yi
FY
Zhou
Xuan
X
He
Fang
F
Zhang
Yan
Y
Huang
Bo-sheng
BS
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects*; D015687:Cell Hypoxia; D002478:Cells, Cultured; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013917:Thrombin / Q000494:pharmacology*
D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D013917:Thrombin