外源性H_(2)S对血管性痴呆大鼠缺血再灌注后神经元线粒体损伤及其相关信号通路的影响

Effects of exogenous H_(2)S on mitochondrial damage of neurons and related signaling pathways after ischemia reperfusion in vascular dementia rats

目的以二血管(2-VO)法制作血管性痴呆(VaD)大鼠模型,探讨神经元自噬及相关信号通路的变化及外源性硫化氢(H_(2)S)的神经保护作用。方法改良2-VO结扎SD大鼠双侧颈总动脉制作VaD模型,按缺血进程急性缺血期、缺血损伤期、损伤恢复期共设置1、7、30 d 3个时间段,每个时间段各有假手术(Sham)组、模型(VaD)组、阳性对照[尼莫地平(Nimodipine)组、硫氢化钠(NaSH)低剂量(Low-NaSH)组、NaSH高剂量(High-NaSH)组。为保持药物处理时间一致,1 d及7 d,各组术前灌胃给药30 d,30 d各组术后灌胃给药30 d,VaD组和Sham组生理盐水灌胃,Nimodipine组Nimodipine灌胃给药,Low-NaSH组、High-NaSH组NaSH灌胃给药。利用水迷宫检测30 d各组学习记忆能力。Western印迹检测海马神经元线粒体蛋白中Beclin1、P62、蛋白激酶B(Akt)、P-Akt、哺乳动物雷帕霉素靶蛋白(mTOR)、P-mTOR表达。结果术后1 d,与Sham组比较,VaD组Beclin1、P-Akt、P-mTOR表达显著降低;与VaD组比较,不同剂量NaSH组Beclin1表达显著降低,Nimodipine组及不同剂量NaSH组P-Akt表达显著降低(P<0.01,P<0.05)。术后7 d,与Sham组比较,VaD组P62、P-Akt、P-mTOR表达显著降低,Beclin1表达显著升高;与VaD组比较,Nimodipine组及不同剂量NaSH组P62表达显著升高、Beclin1表达显著降低,不同剂量NaSH组P-Akt表达显著升高,High-NaSH组P-mTOR表达显著升高(P<0.05,P<0.01)。术后30 d,与Sham组比较,VaD组Beclin1、P62表达显著升高,P-mTOR、P-Akt表达显著降低;与VaD组比较,Nimodipine组及不同剂量NaSH组Beclin1、P62表达显著降低,P-mTOR、P-Akt表达显著升高;与Low-NaSH组比较,High-NaSH组Beclin1、P62表达显著降低,P-mTOR、P-Akt表达显著升高(P<0.05,P<0.01)。结论缺血再灌注会导致神经细胞线粒体功能受损,且引发线粒体自噬,外源性H_(2)S可减轻VaD大鼠海马神经元线粒体自噬程度并保护线粒体功能,从而改善VaD大鼠学习记忆功能,推测其保护作用是通过上调磷脂酰肌醇3蛋白激酶(PI3K)/Akt/mTOR信号通路实现的。

Objective Vascular dementia(VaD)rats model by 2-vessel occlusion(2-VO)methed was made to investigate the changes of autophagy and related signaling pathways in neurons and the neuroprotective effect of exogenous hydrogen sulfide(H_(2)S).Methods The VaD model was made by 2-VO ligation of bilateral common carotid arteries in SD rats,according to the ischemic process,acute ischemia stage,ischemic injury stage and injury recovery stage were set 1,7,30 d three time periods,each time period had sham operation(Sham)group,model(VaD)group,positive control[Nimodipine group,NaSH low dose(Low-NaSH)group,NaSH high dose(High-NaSH)group].In order to maintain consistent drug treatment time,1 d and 7 d groups were given intragastric administration for 30 d before operation,30 d groups were given intragastric administration for 30 d after operation,VaD group and Sham group was given intragastric administration of normal saline,Nimodipine group was given intragastric administration of nimodipine,Low-NaSH group and High-NaSH group were given intragastric administration of NaSH.The water maze was used to assess the learning and memory ability of each 30 d group.The expressions of Beclin1,P62,protein kinase B(Akt),P-Akt,mammalian target of rapamycin(mTOR),P-mTOR in the mitochondria protein of hippocampal neurons were detected by Western blotting.Results At 1 d after operation,compared with Sham group,the expressions of Beclin1,P-Akt and P-mTOR were significantly decreased in VaD group;compared with the VaD group,the expressions of Beclin1 in different doses of NaSH group were significantly decreased,and the expression of P-Akt in Nimodipine group and different doses of NaSH group was significantly decreased(P<0.01,P<0.05).At 7 d after operation,compared with the Sham group,the expressions of P62,P-Akt and P-mTOR in the VaD group were significantly decreased,and the expressions of Beclin1 was significantly increased;compared with the VaD group,the expression of P62 was significantly increased in the Nimodipine group and different doses of NaSH group,and the expressions of Beclin1 was significantly decreased,the expression of P-Akt in different doses of NaSH group was significantly increased,and the expression of P-mTOR in High-NaSH group was significantly increased(P<0.05,P<0.01).At 30 d after operation,compared with Sham group,the expressions of Beclin1 and P62 were significantly increased,the expressions of P-mTOR and P-Akt were significantly decreased in VaD group;compared with VaD group,the expressions of Beclin1 and P62 in Nimodipine group and different doses of NaSH group were significantly decreased,the expressions of P-mTOR and P-Akt were significantly increased;compared with the Low-NaSH group,the expressions of Beclin1 and P62 in the High-NaSH group were significantly decreased,and the expressions of P-mTOR and P-Akt were significantly increased(P<0.05,P<0.01).Conclusions Ischemia-reperfusion could cause damage to mitochondrial function of nerve cells and induce mitochondrial autophagy.Exogenous H_(2)S could attenuate the degree of mitochondrial autophagy in hippocampal neurons of VaD rats and protect mitochondrial function,thereby improving learning and memory abilities in VaD rats.It's speculated that its potective effect might be achieved by up-regulating the phosphatidylinositol 3-kinase(PI3K)/Akt/mTOR signaling pathway.