[Effect of 1400W, an inhibitor of inducible nitric oxide synthetase, on blocking the toxicity of lipopolysaccharide-induced activated microglia to preoligodendrocytes].
OBJECTIVE
To explore the toxicity of LPS-induced activated microglia to preoligodendrocytes (preOLs) and the effect of 1400W, a selective inhibitor of inducible nitric oxide synthetase (iNOS), on the blockage of the toxicity.


METHODS
Co-cultured microglia and preOLs obtained from two-day-old Sprague-Dawley (SD) rats were divided into three groups: co-culture control group, co-culture LPS group and co-culture LPS plus 1400W group. After cultured cells were induced by LPS (100 ng/ml) for 48 hours, the concentration of nitric oxide (NO) was measured by nitric acid-oeoxidize-colorimetry, the level of peroxynitrite (ONOO(-)) was determined by immunocytochemistry, and the synthetic level of iNOS was detected by Western blotting, respectively. The morphologic observation of apoptotic preOLs stained with Hoechst 33342/PI and the apoptotic rate of preOLs detected by flow cytometry were processed simultaneously. Data were analyzed with SPSS 11.0 software.


RESULTS
Compared to co-culture control group, there was significant increase in levels of NO [(82.27+/-3.41) micromol/L vs. (167.86+/-9.87) micromol/L, t=8.593, P<0.01], ONOO(-)[(6.14+/-1.27) x 10(7)/L vs. (34.38+/-7.75) x 10(7)/L, t=5.892, P<0.01], and iNOS [(0.18+/-0.027) vs. (0.79+/-0.068), t=9.26, P<0.01] induced by LPS in co-culture LPS group, and with a higher apoptotic rate of preOLs [(6.73+/-1.39)% vs. (24.77+/-2.05)%, t=12.619, P<0.01]. However, all levels of NO [(69.55+/-5.07) micromol/L, t=8.896, P<0.01], ONOO(-) [(10.33+/-3.47) x 10(7)/L, t=14.96, P<0.01] and iNOS (0.35+/-0.042, t=5.506, P<0.01) decreased significantly with the use of 1400W at a dose of 10 micromol/L in co-culture LPS plus 1400W group, and the apoptotic rate of preOLs [(11.8+/-2.06)%, t=7.715, P<0.01] was also reduced evidently.


CONCLUSIONS
NO, ONOO(-) and iNOS, etc. play important roles in the death pathway of preOLs induced by LPS. 1400W can block effectively the toxicity of LPS-activated microglia toxicity to preOLs through inhibiting iNOS selectively and reducing the production of NO and ONOO(-), and improve the survival rate of preOLs.
He
Ya-Fang
YF
Shanghai Institute for Pediatric Research, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China.
Chen
Hui-Jin
HJ
Qian
Long-Hua
LH
Chen
Guan-Yi
GY
D000578:Amidines / Q000494:pharmacology*; D000818:Animals; D001596:Benzylamines / Q000494:pharmacology*; D002478:Cells, Cultured; D008070:Lipopolysaccharides / Q000633:toxicity; D017628:Microglia / Q000187:drug effects* / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009836:Oligodendroglia / Q000187:drug effects* / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D000578:Amidines; D001596:Benzylamines; D008070:Lipopolysaccharides; C104820:N-(3-(aminomethyl)benzyl)acetamidine; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase