Caspase-1活化在胆红素诱导的大鼠海马神经元损伤中的作用

摘要

Objective To investigate the role of caspase-1 activation in bilirubin-induced neuronal injury and the protective effect of VX-765 against bilirubin-induced neurotoxicity in cultured primary rat hippocampal neurons. Methods Cultured primary rat hippocampal neurons were exposed to DMSO(control group),50 μmol/L bilirubin,or 50 μmol/L bilirubin 1 h after 50 μmol/L VX-765 treatment.The expressions of NLRP3 and caspase-1 in the neurons were detected by Western blotting,and the relative cell survival and death rates were assessed with a modified MTT assay, lactate dehydrogenase assay and Typan blue staining. Interleukin-18 (IL-18) concentration in the culture supernatant was measured using enzyme-linked immunosorbent assay(ELISA).Results In cultured primary rat hippocampal neurons,bilirubin exposure for 3 and 6 h caused significant increases in the expressions of NLRP3 and activated caspase-1 compared with those in the control group(P<0.05). Pretreatment of the cells with VX-765 obviously suppressed bilirubin-induced activation of caspase-1 (P<0.05). The relative survival rate of the neurons was(84.02±2.31)% in VX-765 intervention group,significantly higher than that in bilirubin group (P<0.05)but lower than that in the control group(P<0.05);LDH release rate in VX-765 intervention group was(10.78±1.58)%, significantly lower than that in bilirubin group(P<0.05)but higher than that in the control group(P<0.05).The cell death rate in VX-765 intervention group was(5.58±1.23)%,significantly lower than that in bilirubin group(P<0.05)but higher than that in the control group (P<0.05). Conclusion In cultured primary rat hippocampal neurons, caspase-1 activation plays a role in bilirubin-induced neurotoxicity, and VX-765 treatment provides protection against bilirubin-induced neuronal injury by inhibiting caspase-1 activation.%目的 研究半胱氨酸天冬氨酸蛋白酶-1(caspase-1)活化及VX-765在胆红素诱导大鼠海马神经元损伤中的作用.方法 原代培养大鼠海马神经元分为胆红素组、对照组、VX-765组;胆红素组给予胆红素(50 μmol/L),对照组给予同体积药物溶剂二甲基亚砜(DMSO),VX-765组在胆红素干预前1 h给予VX-765(50 μmol/L);Western blot检测NLRP3、活化caspase-1表达,改良MTT、乳酸脱氢酶(LDH)释放率及台盼蓝染色检测细胞相对存活率及死亡率,ELISA法检测原代培养上清IL-18水平.结果胆红素诱导原代培养大鼠海马神经元3、6 h,NLRP3、活化caspase-1蛋白表达明显高于对照组(P<0.05).VX-765干预后,活化caspase-1表达与胆红素组相比明显降低(P<0.05).分组干预细胞24 h,VX-765组的相对存活率为(84.020±2.311)%,明显高于胆红素组(P<0.05),低于对照组(P<0.05);VX-765组LDH释放率为(10.780±1.577)%,明显低于胆红素组(P<0.05),高于对照组(P<0.05);VX-765组台盼蓝染色阳性率为(5.580±1.234)%,明显低于胆红素组(P<0.05),高于对照组(P<0.05).结论 在原代培养的大鼠海马神经元中,caspase-1活化参与胆红素神经毒性的发生,VX-765抑制其活化发挥神经保护作用.