神经营养因子受体同源物2通过上调proNGF、sortilin、p75NTR表达诱导脑出血后血肿周围脑组织细胞凋亡

NRH2 induces cell apoptosis of cerebral tissues around hematomas after intracerebral hemorrhage through up-regulating pro NGF,sortilin and p75NTR expressions

目的探讨脑出血后不同时期血肿周围脑组织中神经营养因子受体同源物2(NRH2)、神经生长因子前体(pro NGF)、sortilin、神经营养因子受体p75(p75NTR)表达及与细胞凋亡的关系。方法收集临床脑出血后实施血肿清除术的周围组织标本,根据脑出血距离标本取出时间,分为6h以内(包括6h)、6h以上至24h(包括24h)、24h以上至72h(包括72h)、72h以上4组,同时取10例手术过程中血肿远隔部位掉落的组织块作为对照组,通过末端脱氧核糖核酸转移酶介导的脱氧尿苷三磷酸缺口末端标记法(TUNEL)测定脑细胞的凋亡指数(AI),采用实时荧光定量PCR、Western blot法分别检测脑组织中NRH2、pro NGF、sortilin、p75NTR mRNA和蛋白表达,Western blot法检测脑组织中Bcl-2、Bax的表达。体外培养大鼠皮层星形胶质细胞,经NRH2 siRNA或scramble siRNA转染后,Western blot法检测pro NGF、sortilin、p75NTR蛋白表达。结果与对照组以及6h以内的脑出血组相比,脑出血后6h以上各组AI升高,以24h以上至72h内组为最高,但6h以内的脑出血组AI与对照组无区别。随着脑出血时间的延长,pro NGF、p75NTR mRNA和蛋白表达水平逐渐升高,24h以上至72h达到高峰,72h后仍处于较高水平。与对照组和6h以内的脑出血组比较,脑出血后6h以上至24h内(包括24h),脑组织NRH2、sortilin的mRNA与蛋白表达水平及Bax表达水平即增加,24h以上至72h内达高峰,72h后仍维持在较高水平。但与对照组比较,6h以内的脑出血组上述指标无显著性差异。与对照组相比,6h以内的脑出血组Bcl-2表达水平无明显变化,脑出血后6h以上的脑出血各组脑组织Bcl-2表达水平降低,以24h以上至72h内处于最低值。NRH2 siRNA组pro NGF、sortilin、p75NTR蛋白表达水平明显低于空白对照组、scramble siRNA组。结论 NRH2在脑出血后血肿周围脑组织中表达增加,通过促进pro NGF、sortilin、p75NTR表达增加Bax/Bcl-2比率,从而诱导脑细胞凋亡。

Objective To observe the expressions of neurotrophin receptor homolog 2（NRH2）,nerve growth factor precursor（pro NGF）,sortilin and neurotrophin receptor p75（p75NTR） in cerebral tissues around hematomas in the different periods after intracerebral hemorrhage,and explore their relationships to cell apoptosis.Methods The specimens of cerebral tissues around hematomas were collected from the patients undergoing hematoma removal operation after intracerebral hemorrhage.These specimens were divided into four groups,namely ≤6 hours,6-24 hours（including 24 hours）,24-72hours（including 72 hours） and over 72 hours according to the time from intracerebral hemorrhage to specimen col ection.At the same time,10 brain tissues distant to hemorrhage that dropped in the operative process were col ected as a control group.Apoptosis index（AI） was examined in brain cel s by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling（TUNEL）.The expressions of NRH2,pro NGF,sortilin and p75 NTR mRNAs and proteins in brain tissues were detected through real-time quantitative PCR and Western blotting,respectively.Also,the expressions of Bcl-2 and Bax in brain tissues were analyzed using Western blotting.In vitro cultured astrocytes of rat cortex were transfected by NRH2 siRNA or scramble siRNA.The expressions of pro NGF,sortilin and p75 NTR proteins were detected using Western blotting.Results AI was higher in all groups of hemorrhage for 6 hours or longer than that in control and ≤6 hours groups,and AI in the group of24-72 hours after intrac erebral hemorrhage was the highest.However,there was no significant difference in AI between≤6 hours group and control group.With the extension of intracerebral hemorrhage time,the expression levels of pro NGF and p75 NTR mRNAs and proteins were gradually elevated,reached the peak in 24-72 hours,and maintained a higher level after72 hours,whereas there were no significant differences in the above indicators between ≤6 hours group and control group.In comparison with control group and ≤6 hours group,the expression levels of NRH2 and sortilin mRNAs and proteins and Bax expression started to increase in 6-24 hours,reached the peak in 24-72 hours,and then stayed a higher level after 72 hours,whereas there were no significant differences in the above indicators between ≤6 hours group and control group.There was no obvious change in Bcl-2 expression level between ≤6 hours group and control group.The level of Bcl-2 decreased in all groups of intracerebral hemorrhage for over 6 hours,and reached the nadir in 24-72 hours.Astrocytes transfected with NRH2 siRNA displayed a significant decrease in pro NGF,sortilin and p75 NTR protein levels as compared with scramble siRNA or blank control groups.Conclusion The expression of NRH2 would increase in the cerebral tissues around hematomas after intracerebral hemorrhage.NRH2 might enhance the ratio of Bax / Bcl-2 by promoting the expressions of pro NGF,sortilin and p75 NTR,thereby inducing brain cell apoptosis.