雌二醇通过激活SIRT1/P53通路对人晶状体上皮细胞抗凋亡作用研究

Anti-apoptotic effect of estradiol on human lens epithelial cells via SIRT1/P53 pathway

目的探究雌二醇(estradiol,E2)对H2O2诱导的人晶状体上皮细胞(HLE-B3细胞)氧化损伤的保护作用及抗凋亡机制。方法不同浓度H2O2作用于HLE-B3细胞,探索最佳浓度。将HLE-B3细胞随机分成5组空白对照组,模型组(100μmol·L^-1 H2O2),低、中、高浓度E2组(100μmol·L^-1 H2O2+0.01μmol·L^-1、0.10μmol·L^-1、1.00μmol·L^-1 E2),观察各组细胞形态变化。采用CCK-8和流式细胞学检测细胞的增殖与凋亡,观察不同浓度的E2对HLE-B3细胞的影响。RT-qPCR检测沉默信息调节因子1(silent information regulator 1,SIRT1)mRNA、P53 mRNA表达。Western blot检测SIRT1、肿瘤抑制蛋白P53、乙酰化P53(acetylate P53,Ac-P53)蛋白表达,免疫荧光化学检测SIRT1定位及荧光强度。结果100μmol·L^-1H2O2为诱导HLE-B3细胞氧化应激的最佳浓度。CCK-8检测结果显示低、中、高浓度E2组的细胞增殖率均高于模型组(均为P<0.05)。流式细胞学检测结果显示模型组细胞凋亡率均高于其他各组,两两比较空白对照组<高浓度E2组<中浓度E2组<低浓度E2组<模型组(均为P<0.05)。RT-qPCR及Western blot检测结果表明SIRT1表达随着E2浓度的升高而增加,高浓度E2组>中浓度E2组>低浓度E2组>模型组>空白对照组(均为P<0.05)。Ac-P53在空白对照组表达与高浓度E2组差异无统计学意义(P>0.05),其余组间Ac-P53表达比较模型组>低浓度E2组>中浓度E2组>高浓度E2组(均为P<0.05)。空白对照组中P53表达均低于其他各组(均为P<0.05),其余各组之间两两比较,差异均无统计学意义(均为P>0.05)。共聚焦免疫荧光化学检测结果示SIRT1荧光强度随着E2浓度升高而增强。结论E2对HLE-B3细胞的保护作用与SIRT1/P53通路相关,在生理浓度范围化学检测内,随着E2浓度的增加,SIRT1表达增强,抑制Ac-P53,减少HLE-B3细胞凋亡。

Objective To investigate the protective and anti-apoptotic mechanisms of estradiol(E2)on H2O2-induced oxidative damage in human lens epithelial cells(HLE-B3 cells).Methods HLE-B3 cells were treated with different concentrations of H2O2,and the optimal concentration was selected.HLE-B3 cells were randomly divided into 5 groups normal control group,model group(100μmol·L^-1 H2O2),low,medium and high concentration of E2 group(100μmol·L^-1 H2O2+0.01,0.10 and 1.00μmol·L^-1 E2),to observe the morphological changes of cells in each group.The cell proliferation and apoptosis were detected by cell counting kit-8(CCK-8)and flow cytometry,to observe the effect of different concentrations of E2 on HLE-B3 cells.The expression of silent information regulator 1(SIRT1)mRNA and P53 mRNA were detected by RT-qPCR.Protein expression of SIRT1,tumor suppressor protein P53 and acetylate P53(Ac-P53)were detected by Western blot.The distribution and fluorescence intensity of SIRT1 were detected by immunofluorescence chemistry.Results 100μmol·L^-1 H2O2was the optimal concentration to induce oxidative stress in HLE-B3 cells.CCK-8 detection results showed The proliferation rates of low,medium and high concentrations of estradiol groups were higher than that in model group(all P<0.05).Flow cytometry test showed that the apoptosis rate in the model group was higher than that in other group normal control group<high concentration of E2 group<medium concentration of E2 group<low concentration of E2 group<model group(all P<0.05).RT-qPCR and Western blot test showed that the expression of SIRT1 increased with the rising of E2 concentration high concentration of E2 group>medium concentration of E2 group>low concentration of E2 group>model group>normal control group(all P<0.05).The expression of Ac-P53 in normal control group was higher than that in high concentration of E2 group,and no statistical difference was found(P>0.05)model group>low concentration of E2 group>medium concentration of E2 group>high concentration of E2 group(all P<0.05).The expression of P53 in normal control group was lower than that in other group(all P<0.05),and no statistical difference was found by pairwise comparison among other groups(all P>0.05).Confocal immunofluorescence chemistry showed that the fluorescence intensity of SIRT1 increased with rising of estradiol concentration.Conclusion E2 plays protective effect on HLE-B3 cells via SIRT1/P53 pathway,to inhibit Ac-P53 and decrease HLE-B3 cell apoptosis;the expression of SIRT1 increases with rising of E2 concentration in the physiological concentration range.