虾青素调控AMPK-SIRT1通路对七氟醚诱导HT22神经细胞损伤的影响

Astaxanthin regulates AMPK-SIRT1 pathway on the effect of sevoflurane-induced HT22 nerve cell injury

目的研究虾青素对七氟醚诱导的海马神经元HT22细胞活力和细胞凋亡的作用与机制。方法体外培养HT22细胞分为对照组、七氟醚组、七氟醚+虾青素(1.25、2.50、5.00μmol/L)组;七氟醚组给予4%七氟醚刺激细胞6 h;七氟醚+虾青素组七氟醚刺激细胞前给予不同浓度的虾青素处理2 h。采用MTT实验检测各组HT22细胞存活率,DCFH-DA荧光探针和硫代巴比妥酸法分别检测各组细胞内的活性氧(ROS)水平和丙二醛(MDA)含量,流式细胞术和TUNEL检测各组细胞凋亡,Western blotting检测各组细胞中CyclinD1、Cleaved Caspase-3、Bax和Bcl-2、AMPK、p-AMPK、SIRT1蛋白的表达。结果与对照组比较,七氟醚组HT22细胞存活率显著下降和细胞凋亡率显著增加(P<0.05),CyclinD1、Bcl-2、p-AMPK和SIRT1蛋白表达减少(P<0.05),Cleaved Caspase-3、Bax蛋白表达增加(P<0.05),ROS活性和MDA含量降低(P<0.05)。与模型组比较,七氟醚+虾青素HT22细胞Cleaved Caspase-3、Bax蛋白表达下调(P<0.05),CyclinD1、Bcl-2、p-AMPK和SIRT1蛋白表达上调(P<0.05),ROS活性和MDA含量降低(P<0.05),且呈剂量相关性。结论虾青素对七氟醚诱导的HT22细胞凋亡和氧化应激损伤具有保护作用,其机制可能与调控AMPK-SIRT1通路有关。

Objective To study the effect and mechanism of astaxanthin on the viability and apoptosis of hippocampal neuron HT22 cells induced by sevoflurane.Methods HT22 cells cultured in vitro were divided into control group,sevoflurane group,sevoflurane+astaxanthin(1.25,2.50,5.00μmol/L)group.The cells of sevoflurane group was given 4%sevoflurane stimulation and treated for 6 h;The cells of sevoflurane+astaxanthin group was treated with different concentrations of astaxanthin for 2 h before 4%sevoflurane stimulation.The MTT experiment was used to detect the survival rate of HT22 cells in each group,and the DCFH-DA fluorescent probe and thiobarbituric acid method were performed to determine the levels of ROS and MDA content.Flow cytometry and TUNEL were applied to detect cell apoptosis in each group,and Western blotting was used to detecte expression of CyclinD1,Cleaved caspase-3,Bax and Bcl-2,AMPK,p-AMPK and SIRT1 proteins.Results Compared with the control group,the survival rate of HT22 cells in the sevoflurane group was significantly decreased and the apoptosis rate was significantly increased.CyclinD1,Bcl-2,p-AMPK and SIRT1 protein expression were decreased,Cleaved Caspase-3,Bax protein expression were increased,and ROS activity and MDA content were decreased.Compared with the model group,the expression of Cleaved Caspase-3 and Bax protein were down-regulated in the astaxanthin group,and the expression of CyclinD1,Bcl-2,p-AMPK and SIRT1 protein were up-regulated,the activity of ROS and the content of MDA were decreased,which were dose-dependent.Conclusion Astaxanthin has a protective effect on sevoflurane induced HT22 cells apoptosis and oxidative stress injury,and its mechanism may be correlated to the regulation of AMPK-SIRT1 pathway.