Estradiol’s Anti-apoptotic Effect Via SIRT₁/P⁵³ Pathway In Human Lens Epithelial Cells

Objective:Human lens epithelial cell line HLE-B3 was cultured in vitro to induce apoptosis with a certain concentration of hydrogen peroxide（H₂O₂）,which mimic the mechanism of age-related cataract（ARC）in vivo.To observe the proliferation and apoptosis of human lens epithelial cells（HLECs）dealt with estradiol（E₂）at different concentrations and expressions of silent information regulator 1（SIRT₁）,P⁵³,acetylated P⁵³(Ac-P⁵³)in each group.We want to further know about the protective mechanism of E₂ on H₂O₂-induced HLE-B₃ cell oxidative damage and the role of SIRT₁/P⁵33 pathway of human lens epithelial cells（HLECs）.Methods:1.Different concentrations of H₂O₂(0μmol.L^-1,50μmol.L^-1,100μmol.L^-1,200μmol.L^-1,400μmol.L^-1)were used to dealt with human lens epithelial cells HLE-B₃ in vitro and with different times（0 h,1 h,6 h,12 h,24h）.According to the cell counting kit-8（CCK-8）and flow cytometry,the optimal concentration and appropriate time are selected.2.HLE-B₃ were cultured in vitro with different concentrations of nicotinamide（NAM）(0μmol.L^-1,25μmol.L^-1,50μmol.L^-1,100μmol.L^-1,200μmol.L^-1).The optimal concentration of NAM was selected based on the results of CCK-8.3.HLE-B₃ was randomly divided into 5 groups:control group,model group(100μmol.L^-1H₂O₂),low concentration estradiol group(100μmol.L^-1H₂O₂+0.01μmol.L^-1E₂),medium concentration estradiol group(100μmol.L^-1H₂O₂+0.1μmol.L^-11 E₂),high concentration estradiol group（100μmol.L^-11 H₂O₂+1μmol.L^-11 E₂）.The cell proliferation rate was detected by CCK-8,and apoptosis rate was detected by flow cytometry to observe the effects of different concentrations of E₂ on the activity and apoptosis rate of LECs.We observed the cell growth status with a light microscope.We detected the expression of SIRT₁ mRNA and P⁵33 mRNA by real time fluorescence quantitative PCR（RT-qPCR）.Western-blot was used to detect SIRT₁,P⁵³,Ac-P⁵33 protein expression,and confocal immunofluorescence was used to detect SIRT₁distribution and fluorescence intensity.4.HLE-B₃ was randomly divided into 3 groups,control group,model group(100μmol.L^-1H₂O₂),and NAM group(100μmol.L^-1H₂O₂+50μmol.L^-1NAM).The morphological changes of HLECs in each group were observed.CCK-8 and Flow cytometry was used to detect the proliferation and apoptosis rates of HLECs in each group,and Western-blot was used to detect the expression of Ac-P⁵33 protein.Statistical Analysis:SPSS19.0 statistical software was used to perform statistical analysis on the data of each group of experiments.The comparison between the mean of multiple groups of samples was by one-way analysis of variance（ANOVA）,and the comparison between two groups was by LSD-t test.Data were presented as the mean±standard error of the mean.Differences were considered statistically significant when the P was less than 0.05.Results:1.The optimal concentration of H₂O₂ and NAM:according to the results of CCK-8 and flow cytometry,select 100μmol.L^-11 H₂O₂ as the appropriate concentration to induce oxidative damage in HLE-B₃ cells,with an action time of 12 h;50μmol.L^-11 NAM is the best concentration for HLE-B₃,the action time is 12h.2.CCK-8 results showed that the proliferation rate of the low,medium and high concentration estradiol group was higher than that of the model group（P<0.05）.The proliferation rate of cells in the NAM group was lower than that of the control group and model group（P<0.05）.3.Flow cytometry showed that the apoptosis rate of the model group was higher than that of other groups（P<0.05）.Compared with the apoptosis rate between the groups,control group<high concentration estradiol group<low concentration estradiol group<model group（P<0.05）.There was no statistical difference between the low and medium E₂ groups（P>0.05）.And the apoptosis rate of LECs in the NAM group was higher than that in the model group（P<0.05）.4.RT-qPCR results showed that the expression of SIRT₁ mRNA increased with the increase of estradiol concentration.The expression of SIRT₁mRNA:High concentration estradiol group>medium estradiol group>low concentration estradiol group>model group>control group（P<0.05）;the expression of P⁵33 in control group was lower than that of other groups（P<0.05）;and there was no significant difference between the other groups（P<0.05）.5.Western blot results showed that the expression of SIRT₁ protein increased with the increase of estradiol concentration（P<0.05）;the expression of Ac-P⁵33 protein in model group was higher than that in control group and other estradiol group（P<0.05）,the expression of Ac-P⁵33 protein:low concentration estradiol group>medium concentration estradiol group>high concentration estradiol group（P<0.05）.However,the expression of P⁵33 protein in control group was lower than that in any other groups（P<0.05）;the overexpression of Ac-P⁵33 protein in the NAM group was significantly higher than that in model group and control group（P<0.05）.6.The results of confocal immunofluorescence showed that the fluorescence intensity of SIRT₁ was the strongest in the high-concentration E₂group,and the comparison between each two groups was showed that high concentration estradiol group>medium concentration estradiol group>model group>control group（P<0.05）.There was no statistically significant difference between the low concentration estradiol group and model group or medium concentration estradiol group（P>0.05）.It was found to be mainly localized in the nucleus.Conclusion:1.E₂ can reduce the apoptosis rate of HLE-B₃ and has a protective effect on HLECs at physiological concentration.2.SIRT₁/P⁵33 pathway is involved in the apoptosis process of HLECs after oxidative damage and it plays an important role in it.3.One of the protective mechanisms of E₂ on HLE-B₃ may be through the SIRT₁/P⁵33 pathway in HLECs.As the estradiol concentration increases,SIRT₁expression increases,the level of Ac-P⁵33 decreases,which reduces HLE-B₃apoptosis.