胆红素对幼年大鼠海马细胞凋亡及水通道蛋白4表达的影响

The effects of bilirubin on cell apoptosis and the expression of aquaporin4 in hippocampus of young rats

目的:研究幼年胆红素脑病(BE)模型大鼠海马中水通道蛋白4(AQP4)的表达变化,并且探究其与海马细胞凋亡及认知功能障碍的关系。方法:采用7 d龄SD幼年大鼠经小脑延髓池注射未结合胆红素(UCB)建立BE模型,根据UCB作用时间的不同将其分为12 h、24 h、48 h、72 h和7 d组。采用HE和Nissl染色观察各组海马病理变化;应用TUNEL染色法检测海马细胞凋亡;应用Western Blot检测海马AQP4、LAMP1、caspase-3和胶质纤维酸性蛋白(GFAP)的表达变化;应用免疫荧光染色检测海马AQP4、LAMP1和GFAP的表达变化及AQP4^(+)/GFAP^(+)细胞数量的变化。结果:HE及Nissl染色显示,随着UCB作用时间的延长,海马神经细胞间隙不断变大,空泡逐渐增多,Nissl小体数量减少。TUNEL染色法显示海马凋亡细胞数量在UCB作用24 h组最多(P<0.05)。Western Blot结果显示,随着UCB作用时间的延长,海马细胞中caspase-3和LAMP1均上升至24 h达峰值(P<0.05)。免疫荧光结果显示,AQP4和GFAP均增加至48 h表达最高(P<0.05),AQP4^(+)/GFAP^(+)细胞数量在48 h组达最多(P<0.05)。结论:UCB的神经毒性作用可导致海马细胞凋亡增加至24 h达峰值,继发星型胶质细胞代偿性增生,并伴随AQP4的表达增加至48 h达峰值,以上变化可能与BE的认知功能障碍具有密切联系。

Objective:To study the changes of expression of aquaporin4(AQP4)in the hippocampus of the bilirubin encephalopathy(BE)model of young rats,and to explore its relationship with cells apoptosis and cognitive function in hippocampus.Method:7 d old SD young rats were injected with unconjugated bilirubin(UCB)solution into the cisterna magna to establish BE model.According to the different duration of UCB treatment,they were divided into 12 h,24 h,48 h,72 h,and 7 d groups.HE and Nissl stainings were used to observe the pathological changes of hippocampus;TUNEL staining was used to detect cells apoptosis in hippocampus;Western Blot was used to detect the expression of AQP4,lysosomal associated membrane protein-1(LAMP1),caspase-3,and glial fibrillary acidic protein(GFAP);Immunofluorescence staining was used to detect the expression of hippocampal AQP4,LAMP1,GFAP and the number of AQP4^(+)/GFAP^(+)cells.Result:HE and Nissl staining showed that with the prolongation of UCB treatment,the gap between nerve cells in hippocampus became larger,vacuoles increased,and the number of Nissl bodies decreased.TUNEL staining showed that the number of hippocampal TUNEL;cells were the most in UCB-treated 24 h group(P<0.05).Western Blot showed that with the prolongation of UCB treatment,both caspase-3 and LAMP1 in hippocampus rose to a peak at 24 h(P<0.05).Immunofluorescence results showed that both AQP4 and GFAP increased to the highest expression at 48 h(P<0.05)and the number of AQP4^(+)/GFAP^(+)cells increased to the most in UCB-treated 48 h group(P<0.05).Conclusion:The neurotoxic effect of UCB can cause an increase of cells apoptosis to a peak at 24 h in hippocampus,leading to compensatory proliferation of astrocytes,and accompanied by an increase of AQP4 with a peak at 48 h.Above-mentioned changes may be closely related to the cognitive dysfunction of BE.