1400W对脂多糖诱导少突胶质细胞前体死亡通路的阻断研究

1400W blocks death pathway of LPS-induced activated-microglia to preOLs

目的建立细菌脂多糖(LPS)诱导的2日龄感染型脑室周围白质软化(PVL)新生大鼠动物模型,探讨iNOS抑制剂1400W对LPS诱导脑白质少突胶质细胞(OL)前体死亡的体内阻断效果。方法 2日龄新生大鼠随机分为假手术组、PVL组、1400W-即刻组、1400W-8h组、1400W-16h组以及1400W-24h组,分别在LPS注射后即刻、8 h、16 h以及24 h经皮下注射1400W 20 mg/kg。于造模后第5天处死取脑,分别进行光镜下脑白质病理评估、硝酸还原法检测一氧化氮(NO)含量、Western blot法检测诱生型一氧化氮合酶(iNOS)表达量、免疫组织化学染色检测过氧亚硝酸盐(ONOO-)含量以及OL前体数量。结果 LPS诱导可引起新生大鼠脑白质明显损伤,脑内iNOS表达明显上调,NO和ONOO-含量显著增加,脑白质内少突胶质细胞前体标记物(O4)标记的OL前体显著减少。与PVL组比较,1400W-即刻组、1400W-8h组以及1400W-16h组新生大鼠的脑白质病理均获明显改善,iNOS蛋白合成量、NO及ONOO-生成量均显著降低,OL前体数量明显增加(P<0.05)。1400W-24h组的各项检测结果与PVL组相似,均无明显改善。结论体内研究确认1400W通过抑制iNOS的表达上调、减少脑内NO及ONOO-的生成量,从而阻断OL前体的死亡通路,发挥对脑白质的保护效果。在LPS诱导后16 h内应用1400W,均有良好的脑保护作用。

Objective To explore the efficacy of inductible nitric oxide synthase（iNOS） inhibitor 1400W in vivo in blocking the death pathway of lipopolysaccharide（LPS）-induced activated-microglia to preoligodendrocytes（preOLs） in neonatal rats with infective-type periventricular leukomalacia（PVL） induced by LPS.Methods Two-day-old neonatal rats were randomly divided into： a sham-operated group,an untreated PVL group,and four 1400W-treated PVL groups that were subcutaneously administrated with 20 mg/kg of 1400W at 0 h,8 hrs,16 hrs,and 24 hrs after LPS induction,respectively.The brain specimens were obtained 5 days after LPS induction.The pathological assessment of cerebral white matter was performed under a light microscope.Concentrations of nitric oxide（NO） were measured by nitric acid-deoxidize colorimetry.Synthesis of iNOS was determined by Western blot analysis.Peroxynitrite（ONOO-） level and the amount of preOLs were determined by immunocytochemistry.Results The obvious injuries of periventricular white matter,massive loss of positive O4-labelled preOLs,and increased levels of NO,ONOO-and iNOS were observed in neonatal rats with PVL.Compared to the untreated PVL group,the use of 1400W at 0 h,8 hrs and 16 hrs after LPS induction significantly improved white matter injuries,reduced the levels of NO,ONOO-and iNOS,and increased the amount of O4-labelled preOLs.However,the use of 1400W at 24 hrs after LPS induction did not result in the improvements.Conclusions iNOS inhibitor 1400W can effectively block the toxicity of LPS-activated microglia to preOLs and protect cerebral white matter through inhibiting iNOS and reducing the production of NO and ONOO-.The use of 1400W within 16 hrs after LPS induction may provide cerebral protections in neonatal rats with PVL.