芒果苷通过PI3K/Akt/mTOR通路对蛛网膜下腔出血后神经细胞的保护作用

Protective effect of mangiferin on nerve cells after subarachnoid hemorrhage via PI3K/Akt/mTOR pathway

目的探讨芒果苷(MF)在蛛网膜下腔出血(SAH)后早期阶段对神经细胞的保护作用及机制。方法采用小鼠来源神经瘤母细胞(Neuro-2a细胞)和氧合血红蛋白(OxyHb)建立体外SAH模型,分为对照组、蛛网膜下腔出血组(SAH)及芒果苷干预组(MF+SAH)3组,利用免疫荧光检测受损线粒体与溶酶体的结合;利用JC-1和DCFH-DA分别检测线粒体膜电位(Δψm)和活性氧(ROS);Western blot分别检测细胞内磷脂酰肌醇-3-激酶(PI3K)、蛋白激酶B(Akt)和雷帕霉素靶蛋白(mTOR)的表达,胞浆、线粒体中凋亡相关蛋白淋巴细胞瘤-2(Bcl-2)、Bcl2关联X蛋白(Bax)、细胞色素C(Cyt-C)和Caspase-3的表达。结果与对照组相比,SAH组线粒体膜电位(Δψm)明显去极化(P<0.05),细胞内ROS水平增多(P<0.01),p-PI3K、p-Akt、p-mTOR的表达水平显著减少(P<0.05),胞浆内Cyt-C表达水平显著增加(P<0.05),线粒体内Cyt-C表达水平显著减少(P<0.05),Bax和Caspase-3表达水平显著增加(P<0.05),Bcl-2表达水平显著减少(P<0.05);与SAH组相比较,MF+SAH组Δψm和ROS明显恢复(P<0.05),p-PI3K、p-Akt、p-mTOR的表达水平明显增加(P<0.05),胞浆内Cyt-C表达水平显著下降(P<0.05),线粒体内Cyt-C表达水平显著增加(P<0.05),Bax和Caspase-3表达显著减少(P<0.05),Bcl-2表达显著增加(P<0.05)。结论芒果苷可以通过稳定线粒体膜电位,减少ROS的产生,改善线粒体功能障碍,从而抑制SAH后早期阶段神经细胞的凋亡。

Objective To investigate the protective effect and mechanism of mangiferin(MF)on nerve cells in the early stage after subarachnoid hemorrhage(SAH).Methods The SAH model in vitro was established by using the Neuro-2a cells originated from the mouse neuroblastoma cells and OxyHb.The SAH models were divided into the control group(CON),SAH group and mangiferin intervention group(MF+SAH).The binding of damaged mitochondria to lysosome was detected by using the immunofluorescence;the mitochondrial membrane potential(Δψm)and reactive oxygen species(ROS)were measured by JC-1 and DCFH-DA,respectively;the expressions of phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt),PKB)/rapamycin target protein(mTOR)were detected by Western blot;the expressions of apoptosis related proteins lymphoma-2(Bcl-2),BCL2 associated X protein(Bax),cytochrome C(Cyt-C)and cleaved-caspase-3(Caspase-3)in cytoplasm and mitochondria were detected.Results Compared with the control group,theΔψm in the SAH group was significantly depolarized(P<0.05),the intracellular ROS content was increased,the expression levels of p-PI3K,p-Akt and p-mTOR were significantly decreased(P<0.05),the expression of Cyt-C in cytoplasm was significantly increased(P<0.05),the expression of Cyt-C in mitochondria was significantly decreased(P<0.05),the expression of Bax and Caspase-3 was significantly increased(P<0.05),and the expression of Bcl-2 was significantly decreased(P<0.05);compared with SAH group,Δψm and ROS in the MF+SAH group were significantly recovered compared with before(P<0.05),the p-pi3k,p-Akt,p-mTOR expression levels were significantly increased(P<0.05),Cyt-C expression in cytoplasm was significantly decreased(P<0.05),Cyt-C expression in mitochondria was significantly increased(P<0.05),the Bax and Caspase-3 expression levels were significantly decreased(P<0.05),and the Bcl-2 expression level was significantly increased(P<0.05).Conclusion Mangiferin can inhibit the nerve cells apoptosis in the early stage after SAH by stabilizing the mitochondrial membrane potential,reducing the ROS production and improving the mitochondrial dysfunction.