丙泊酚对H_(2)O_(2)诱导的HT22细胞凋亡的保护作用

Protective effect of propofol against H_(2)O_(2)-induced apoptosis in HT22 cells

目的探讨丙泊酚对过氧化氢(H_(2)O_(2))诱导的HT22细胞凋亡的影响及其对沉默信息调节因子2相关酶1(SIRT1)-叉头框蛋白O1(FoxO1)信号通路的影响。方法首先利用H_(2)O_(2)建立HT22细胞损伤模型,丙泊酚干预后,利用细胞计数试剂盒(CCK8)检测细胞存活率和筛选H_(2)O_(2)合适作用浓度,活性氧荧光探针(DCFH-DA)检测细胞内活性氧(ROS)水平,烟酰胺腺嘌呤二核苷酸(NAD)/还原型烟酰胺腺嘌呤二核苷酸(NADH)检测试剂盒检测细胞内NAD含量,Westernblot检测SIRT1、FoxO1及细胞凋亡相关蛋白Cleaved-Caspase3、Bcl2关联X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)、细胞色素C(Cyt C)的表达水平。结果与对照组比较,在一定浓度范围内,随着H_(2)O_(2)浓度增加,HT22细胞存活率逐渐下降,差异具有统计学意义(P<0.05)。H_(2)O_(2)诱导细胞损伤的IC50为234.13μM。与对照组比较,100μM H_(2)O_(2)处理HT22细胞24 h后,HT22细胞存活率为75.62%。本研究通过实验摸索和调整结合参考文献最终确定100μM作为本次实验的处理浓度。H_(2)O_(2)组细胞内ROS及凋亡相关蛋白Cleaved-Caspase3、Bax、Cyt C蛋白的表达水平显著高于对照组,丙泊酚组低于对照组,差异均具有统计学意义(P<0.05)。H_(2)O_(2)和丙泊酚联合处理组细胞内ROS及凋亡相关蛋白Cleaved-Caspase3、Bax、Cyt C蛋白的表达水平低于H_(2)O_(2)组,高于丙泊酚组,差异具有统计学意义(P<0.05)。H_(2)O_(2)组细胞内SIRT1和FoxO1蛋白的表达水平、NAD水平明显低于对照组,丙泊酚组明显高于对照组,差异均具有统计学意义(P<0.05)。H_(2)O_(2)和丙泊酚联合处理组细胞内SIRT1和FoxO1的表达水平、NAD水平高于H_(2)O_(2)组,低于丙泊酚组,差异均具有统计学意义(P<0.05)。结论丙泊酚对H_(2)O_(2)诱导HT22细胞损伤具有的保护作用,其作用机制可能与激活SIRT1-FoxO1信号通路来减轻氧化应激水平,降低细胞凋亡有关。

Objective To investigate the effect of propofol on hydrogen peroxide(H_(2)O_(2))-induced apoptosis in HT22 cells and its effect on the silent information regulator 2-related enzyme 1(SIRT1)-forkhead box protein O1(FoxO1)signaling pathway.Methods Firstly,HT22 cell injury model was established using H_(2)O_(2).After propofol intervention,cell viability and screening of the appropriate action concentration of H_(2)O_(2)were detected using a cell counting kit-8(CCK8),intracellular reactive oxygen species(ROS)levels were detected by 2,7-dichlorofluorescein diacetate(DCFH-DA),and intracellular NAD levels were detected by nicotinamide adenine dinucleotide(NAD)/reduced nicotinamide adenine dinucleotide(NADH)assay kit,the expression levels of SIRT1,FoxO1 and apoptosis-related proteins Cleaved-Caspase 3,Bcl2-associated X protein(Bax),B lymphocytoma-2(Bcl-2)and cytochrome C(Cyt C)were detected by Westernblot.Results Compared with the control group,the survival rate of HT22 cells gradually decreased with increasing H_(2)O_(2)concentration in a certain concentration range,and the difference was statistically significant(P<0.05).The IC50 of H_(2)O_(2)-induced cell injury was 234.13μM.The survival rate of HT22 cells was 75.62%after 24 h treatment with 100μM H_(2)O_(2)compared with the control group.In this study,100μM was finally determined as the treatment concentration for this experiment through experimental exploration and adjustment and combined with references.The expression levels of intracellular ROS and apoptosis-related proteins Cleaved-Caspase 3,Bax,and Cyt C proteins in the H_(2)O_(2)group were significantly higher than those in the control group;thoes in the propofol group were lower than those in the control group;the differences were all statistically significant(P<0.05).The expression levels of intracellular ROS and apoptosisrelated proteins Cleaved-Caspase 3,Bax,Cyt C protein in H_(2)O_(2)and propofol combined treatment group were lower than those in H_(2)O_(2)group,but higher than those in propofol group,and the differences were all statistically significant(P<0.05).The expression levels of intracellular SIRT1 and FoxO1 proteins and NAD of the H_(2)O_(2)group were significantly lower than those of the control group,and the propofol group was significantly higher than the control group,the differences were all statistically significant(P<0.05).The expression levels of intracellular SIRT1,FoxO1 and NAD in H_(2)O_(2)and propofol combined treatment group were higher than those in H_(2)O_(2)group,but lower than those in propofol group,and the differences were all statistically significant(P<0.05).Conclusion Propofol has a protective effect on H_(2)O_(2)-induced apoptosis in HT22 cells,and its mechanism may be related to the activation of SIRT1-FoxO1 signaling pathway to reduce the level of oxidative stress and reduce apoptosis.