iNOS抑制剂阻断由细菌脂多糖诱导活性小胶质细胞对少突胶质细胞前体毒性作用的研究

目的 探讨由细菌脂多糖(lipopolysaccharide,LPS)诱导的活性小胶质细胞(MG)对少突胶质细胞(OL)前体的毒性作用及选择性iNOS抑制剂1400W对毒性作用的阻断效果.方法 取2日龄SD大鼠脑内MG和OL前体共培养,分为共培养对照组,共培养LPS组以及共培养LPS+1400W组.对共培养细胞经LPS 100 ng/ml诱导后48 h,分别应用硝酸还原比色法检测一氧化氮(NO)含量,免疫细胞染色法检测过氧亚硝酸盐(ONOO-)含量,Westem blot法检测诱生型一氧化氮合酶(Inducible Nitric Oxide Synthase,iNOS)蛋白合成量,Hochest 33342/PI荧光染色观察细胞凋亡形态学改变,以及流式细胞仪检测细胞凋亡率.结果 与共培养对照组比LPS可诱导培养细胞内NO含量(82.27±3.41)tunol/L vs(167.86±9.87)μmol/L,t=8.593,P<0.01]、ONOO-含量(6.14±1.27)×107/L vs(34.38±7.75)×107/L,t=5.892,P<0.01]以及iNOS蛋白合成量相对值(0.18 4-0.027)vs.(0.79±0.068),t=9.26,P<0.01]明显增高,OL前体的凋亡率亦显著增加(6.73 4±1.39)%vs.(24.77 4±2.05)%,t=12.619,P<0.01].应用1400W 10 μmol/L则可显著抑制因LPS诱导而增高的NO含量(69.55±5.07)μmol/L,t=8.896,P<0.01]、ONOO-含量(10.33±3.47)×107/L,t=14.96,P<0.01]以及iNOS蛋白的合成量(0.35±0.042,t=5.506,P<0.01),并显著降低了OL前体的细胞凋亡率(11.8±2.06)%,t=7.715,P<0.01].结论 NO、iNOS以及ONOO-等物质在LPS诱导OL前体的死亡通路中扮演了重要角色,1400W通过选择性抑制iNOS,减少了NO以及ONOO-的生成,从而有效阻断了由LPS诱导活性MG对OL前体的毒性作用,提高了OL前体的存活率.

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 micmglia 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-cultttre LPS plus 1400W group.After cultured ceils were induced by LPS (100 ng/ml)for 48 hours,the concentration of nitric oxide(NO) was measured by nitric acid-deoxidize-colorimetry,the level of peroxynitrite(ONOO-)was determined by immunocytochemistry,and the synthetic level of iNOS was deleted 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)μmol/L vs(167.86±9.87)μmol/L,t=8.593,P<0.01],ONOO-(6.14±1-1.27)x 107/L vs.(34.38±7.75)×107/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)μmol/L,t=8.896,P