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  

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

作　　者：冯洁[1] 李梦文[1] 韦倩 李胜君[1] 华子瑜[1] 

机构地区：[1]重庆医科大学附属儿童医院新生儿科、儿童发育疾病研究教育部重点实验室、儿童发育重大疾病国家国际科技合作基地、认知发育与学习记忆障碍转化医学重庆市重点实验室,重庆400014

出　　处：《第三军医大学学报》2017年第18期1796-1802,共7页Journal of Third Military Medical University

基　　金：国家自然科学基金青年科学基金(81200459);重庆市基础科学与前沿技术研究一般项目(CSTC2013jcyj A0020)~~

摘　　要：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。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-1β 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 supernat

关 键 词：胆红素 神经毒性 星型胶质细胞 半胱氨酸天冬氨酸蛋白酶-1 炎症 

分 类 号：R345[医药卫生—基础医学] R349.29