孕酮对缺氧缺血性新生大鼠海马神经元凋亡和自噬的影响

Effects of progesterone on hippocampus neuronal apoptosis and autophagy in neonatal Hypoxia-ischemia rats

目的:探讨孕酮对缺氧缺血性脑损伤新生大鼠海马神经元的自噬和凋亡的影响。方法:40只新生大鼠随机分成假手术组和手术组,其中手术组又分为高剂量孕酮处理组、低剂量孕酮处理组及溶剂对照组,每组10只。预先腹腔注射生理盐水或孕酮,通过左颈动脉结扎术建立缺血模型,然后在92%氮气和8%氧气混合气体的恒温容器装置中缺氧处理1 h。24 h后,取大鼠海马组织,免疫印迹检测组织AKT、凋亡相关蛋白Bcl-2,Bax,Capsase-3的水平,以及自噬相关蛋白LC3,Beclin-1和p62的表达水平;提取新生大鼠原代海马神经细胞培养,孕酮处理后免疫印迹检测细胞AKT及凋亡相关蛋白表达,流式细胞术检测细胞凋亡水平。结果:与溶剂对照组相比,缺氧缺血动物模型中海马神经元AKT蛋白表达降低,抗凋亡蛋白Bcl-2、活化型Caspase-3和自噬相关蛋白Beclin-1表达降低,凋亡蛋白Bax和自噬相关蛋白p62表达增加,差异均有统计学意义(P<0.05)。在海马原代神经细胞模型中,孕酮处理后海马原代神经细胞AKT表达降低,促凋亡相关蛋白Bax及活化型Caspase-3表达降低,流式细胞结果显示孕酮处理后海马原代神经细胞早晚期凋亡细胞数减少,差异均有统计学意义(P<0.05)。结论:孕酮可通过抑制AKT信号通路,降低由自噬和凋亡途径介导的神经元死亡。

Objective:To investigate the effect of progesterone on hippocampus neuronal apoptosis and autophagy in neonatal Hypoxia-ischemia rats.Methods:Forty neonatal rats were randomly divided into sham operation group and operation group.The operation group was divided into high-dose progesterone treatment group,low dose progesterone treatment group and solvent control group,ten rats in each group.Normal saline or progesterone were injected intraperitoneally in advance.An HIBD model in neonatal rat by ligation its left carotid artery was established and exposing in hypoxia condition for 1 hour.Then it was anoxic for 1 hour in a constant temperature container with 92%nitrogen and 8%oxygen.After 24 hours,the hippocampal tissue was obtained,with detection of AKT signal pathway in tissue cells by Western blotting,the level of Bcl-2,Bax,Capsase-3,the expression levels of autophagy related proteins LC3,Beclin-1 and p62.Meanwhile,the primary neuron from neonatal rats in hippocampus was isolated and cultured.Western blotting was performed to detect the expression and activity of AKT and apoptosis associated protein under the treatment of progesterone or not in the glucose and oxygen deprivation(GOD)assay,and flow cytometry was used to detect the number of apoptotic cells.Results:Compared with the solvent control group,progesterone can significantly reduce the activation of AKT signaling pathway in hippocampal neurons in hypoxic-ischemic animal model.The expression of Bcl-2、Caspase-3 and Belin-1 decreased,the expression of Bax and p62 increase,with statistical significant(P<0.05).In the primary hippocampal neuron model,the expression of AKT in the primary hippocampal neuron dreased after progesterone treatment,the expression of Bax and Caspase-3 decreased.Flow cytometry results showed that the number of apoptotic cells decreased in the early and late stage of primary hippocampal neurons after progesterone treatment,with statistical significant(P<0.05).Conclusion:Progesterone can reduce neuronal death mediated by autophagy and apoptosis by inhibiting AKT signaling pathway.