허혈성 뇌질환시 소량의 sodium nitroprusside (SNP) 전처리가 LPS와 phorbol 12-myristate 13-acetate (PMA)에 대한 세포손상을 방어하는지를 조사하기 위하여 MTT assay, LDH 분비 측정, Nitrite formation 측정, Electrophoretic mobility shift assay (EMSA), 광학현미경적 관찰, Hoechst staining으로 조사하여 다음과 같은 결과를 얻었다. 1. C6 glial 세포에서 LPS와 PMA에 의하여 생성되어지는 iNOS의 발현과 NF-kB의 활성은 LPS와 PMA을 처리하기 24시간 전에 100uM의 SNP를 전처리 함으로써 억제되었다. 2. SNP 전처리는 LPS와 PMA에 의해서 유도되어지는 세포막독성과 사립체독성을 현저히 억제하였다. 3. SNP 전처리는 iNOS의 활성을 억제함으로써 염색실의 응축이나 DNA의 분절 등을 억제하였고 caspase 3의 활성을 억제하였다. 이상의 결과로써, 허혈성 뇌질환에 대한 SNP 전처리는 NF-kB의 활성을 저해하여 일산화질소의 생성을 억제함으로써 세포막독성 및 사립체의 손상을 방어할 뿐만 아니라 caspase의 활성을 억제하여 아폽토시스를 방지하는 것으로 생각한다.
Nitric oxide (NO) is mainly involved in brain ischemic damage to elucidate the protective mechanism of NO pretreatment on ischemic-induced cytotoxicity. This study was investigated whether NO pretreatment inhibits the increase of iNOS expression by lipopolysaccharide (LPS) combined phorbol 12-myristate 13-acetate (PMA) via regulating NF-kB activation in C6 glial cells. C6 glial cells with LPS and PMA for 72 hours markedly induced NO, but sodium nitroprusside (SNP) (100 nM) pretreatment before exposure of LPS and PMA significantly supressed NO production, iNOS expression and NF-kB activation by LPS and PMA. In addition, LPS and PMA treatment for 72 hours induced severely cell death and LDH release from cell into media in C6 glial cells. However SNP pretreatment before treatment of LPS and PMA significantly protected LPS and PMA induced cytotoxicity. Treatment with LPS and PMA induced caspase 3 activation follewed by chromosomal condensation, and fragmentation of nuclei in C6 glial cells. SNP pretreatment before exposure to LPS and PMA supressed caspase 3 activation and inhibited chromosomal condensation and fragmentation of nuclei. From these above results, it is suggest that the protective effects of SNP pretreatment against LPS and PMA induced cytotoxicity may be mediated by inhibiting the expression of iNOS via regulating NF-kB activation.
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