磷酸酶PTEN在新生大鼠缺氧缺血性脑损伤神经元凋亡中的作用

Changes of Phosphatase PTEN in Neuronal Apoptosis in Neonate Rats with Hypoxic-Ischemic Brain Damage

目的观察新生大鼠缺氧缺血性脑损伤(hypoxic-ischemic brain damage,HIBD)时,大脑皮层第10号染色体上磷酸酶及张力蛋白同源缺失的基因(PTEN)蛋白、磷酸化(p)-PTEN蛋白、促凋亡蛋白Bim mRNA及蛋白表达变化,探讨抑制PTEN活性对新生大鼠缺氧缺血(HI)神经元凋亡的保护作用机制。方法将128只10日龄SD大鼠随机分为4组:缺氧缺血组、假手术组、PTEN脂质磷酸酶抑制剂双过氧化钒(bisperoxovanadium,bpv)干预组和生理盐水溶剂组。缺氧缺血组在乙醚麻醉下行右侧颈总动脉结扎,氮氧混合气(92%N2、8%O2)中缺氧2.5h;假手术组不结扎颈总动脉,不作缺氧处理。bpv干预组和溶剂对照组予腹腔内注射bpv和生理盐水,30min后做缺氧缺血处理。各组分别于建模后0.5h及24h处死动物取大脑皮层,应用Western blot检测PTEN、p-PTEN及Bim的蛋白含量。实时荧光定量PCR(Real-Time PCR)定量检测Bim mRNA表达水平,应用TUNEL染色法检测凋亡细胞。结果缺氧缺血组于HI后0.5h及24h,PTEN蛋白无明显变化,p-PTEN蛋白表达降低。HI后0.5h,Bim mRNA及蛋白表达增加,与假手术组比较差异有统计学意义(P<0.05),HI后24h,Bim mRNA及蛋白恢复至基线水平。bpv干预组PTEN蛋白无明显变化,p-PTEN蛋白表达增加,Bim mRNA及蛋白表达降低,与生理盐水组和缺氧缺血组相比,差异有统计学意义(P<0.05)。bpv干预组于HI后24h,TUNEL染色阳性细胞数较生理盐水组和缺氧缺血组减少,凋亡指数降低,差异有统计学意义(P<0.05)。结论新生大鼠缺氧缺血性脑损伤时,PTEN发生去磷酸化,活性增高,抑制PTEN活性可下调前凋亡蛋白Bim mRNA及蛋白表达,减少神经细胞凋亡。

Objective To examine the changes in expression of phosphatase and tensin homolog deleted on chromosome ten（PTEN）protein,p-PTEN protein and Bim（Bcl-2interacting mediator of cell death）mRNA in the cortex of neonate rat brains with hypoxic-ischemic brain damage（HIBD）and to explore the mechanisms of neuroprotective effects of PTEN inhibition.Methods One hundred and twenty-eight neonate（10days）SD rats were divided into four groups：hypoxia-ischemia（HI）,sham control（Sham）,bisperoxovanadium（bpv）,and normal saline（NS）group.Rats in the HI group had their right common carotid arteries（CCA）exposed and ligated,and were then exposed to hypoxia in a chamber filled with 8%oxygen（balanced with nitrogen）for 2.5h.Rats in the sham control group had their right CCA surgically exposed without ligation and exposure to hypoxia.Rats in the bpv treated group received intraperitoneal injections of bpv,30 min before HI was induced.Instead of bpv,rats in the NS-treated group received intraperitoneal injections of NS.Cerebral cortex samples of the rats were collected 0.5hand 24 hafter hypoxia.Western blot was used to detect the protein expression of PTEN,p-PTEN and Bim.Real-Time PCR was used to detect the level of BimmRNA.TUNEL staining was used to detect apoptotic cells.Results No significant changes of PTEN protein were observed in the rats exposed to HI.However,pPTEN protein decreased in the rats exposed to HI（0.5hand 24h）compared with those exposed to sham surgery（P〈0.01）.Compared with the sham controls,Bim mRNA and protein increased in the rats exposed to HI（0.5h,P〈0.01）and then returned to the baseline level 24 hafter HI.No significant changes of PTEN protein were observed in the bpv-treated rats.However,p-PTEN protein increased and Bim mRNA and protein decreased in the bpv-treated rats（0.5hand 24 h,P〈0.01）compared with those in the HI group and NS-treated group.TUNEL positive cells also reduced in the bpv-treated rats（24h,P〈0.01）compared with those in the HI group and NStreated group.Conclusion PTEN activities increase in the brains of neonate rats with hypoxic-ischemic brain damage.PTEN activity inhibition can decrease the level of pro-apoptotic protein BimmRNA,leading to reduction of neuronal apoptosis.