半胱氨酸天冬氨酸蛋白酶-1活化在胆红素神经毒性中的作用研究

目的 明确半胱氨酸天冬氨酸蛋白酶-1(caspase-1)活化是否参与胆红素神经毒性的发生机制;VX-765抑制caspase1活化是否能抑制胆红素神经毒性,发挥神经保护作用.方法 建立胆红素脑病动物模型,Western blot检测脑组织caspase-1蛋白的表达,ELISA法检测炎症因子IL-1β的水平,免疫荧光染色检测脑组织中GFAP蛋白的表达;VX-765干预后动态观察各组新生鼠的神经系统临床表现,记录新生鼠体质量变化,评估生活能力.原代培养大鼠皮层星型胶质细胞分为胆红素组、VX-765组、对照组:改良MTT法检测细胞存活率,Western blot检测细胞caspase-1蛋白的表达,ELISA法检测培养液上清IL-1β的水平.结果 胆红素脑病动物模型,建模后12 h,胆红素组较对照组,脑组织中活化型caspase-1表达增加(P＜0.05),IL-1β水平增高(P＜0.01),脑组织切片皮层区星型胶质细胞活化(P＜0.05).与胆红素组相比,VX-765组新生鼠建模后异常神经系统表现减少(P＜0.01),生活能力改善(P＜0.05).原代培养大鼠皮层星型胶质细胞,胆红素干预6h后,活化型caspase-1表达显著高于对照组(P＜0.05);与胆红素组相比,VX-765干预可抑制caspase-1活化(P＜0.05),提高细胞存活率(P＜0.05),减少培养液上清IL-1β的释放(P＜0.01).结论 胆红素可诱导活化型caspase-1表达增加;VX-765抑制caspase-1活化可减轻胆红素神经毒性,提高原代培养星型胶质细胞存活率,减少炎症因子释放.

Role of caspase-1 activation in bilirubin neurotoxicity in vivo and in vitro

Objective To investigate whether the activity of caspase-1 is involved in the pathogenesis of bilirubin neurotoxicity,and whether VX-765 (caspase1 specific inhibitor) exerts neuroprotective effect in the process.Methods Neonatal SD rats were randomly divided into Kernicterus group (n =42),control group (n =30) and VX-765 treatment group (n =12,50 mg/g,via intra peritoneal injection).Kernicterus model was established by injecting 1 μL/g bilirubin into posterior cistern in the rats.The expression of caspase-1 was assessed by Western blotting,the content of IL-1 β in the brain tissues was measured by ELISA,and the expression of GFAP protein was detected by immunofluorescence assay.Further,VX-765 was administered intraperitoneally at 24 h and 1 h before model establishment,typical neurological manifestations and body weight were dynamically recorded to evaluate their life activities.Primarily cultured rat cortical astrocytes were randomly divided into control group,bilirubin group and VX-765 intervention group.The survival rate of the astrocytes was assessed with modified MTT assay,expression of caspase-1 was detected by Western blotting,and content of IL-1β in the supernatant was measured by ELISA.Results At 12 h after bilirubin injection,the expression of caspase-1 (P ＜ 0.05) and content of IL-1β (P ＜ 0.01) were significantly higher in the kernicterus model group,and the activation of astrocytes in the cortex of brain tissue was observed (P ＜ 0.05),when compared with the control group.Meanwhile,VX-765 intervention resulted in decreased abnormal neural behaviors (P ＜ 0.01) and improved life activities (P ＜ 0.05).In primarily cultured rat cortical astrocytes,bilirubin exposure for 6 h enhanced the expression of activated caspase-1 when compaired with the control group (P ＜ 0.05),but VX-765 intervention suppressed the activation of caspase-1 (P ＜ 0.05),increased the cell survival (P ＜ 0.05),and reduced the release of IL-1β in the supernatant (P ＜ 0.01).Conclusion Bilirubin induces the expression of activated caspase-1.While VX-765 alleviates the bilirubin-induced neurotoxicity by inhibiting the activation of caspase-1,and then improves the survival rate of primarily cultured rat cortical astrocytes and reduces the release of inflammatory factors.