ABSTRACT
Objective To investigate the expression of microRNA-34a (miR-34a) and silent information regulator 1 (SIRT1) in human lens epithelial cells under H2O2-induced oxidative stress.Methods Different concentrations of H2O2 (0 μmol · L-1,100 μ mol· L-1,200 μmol · L-1,300 μmol · L-1,and 400 μmol · L-1) were used to stimulate SRA01/04 cells for 24 hours.Cell viability was measured using cell counting kit-8 (CCK-8) assay.Cell apoptosis was detected by flow cytometry.Expression levels of miR-34a/SIRT1 were measured by RT-PCR.Results CCK-8 assay showed that a certain concentration range of H2O2 had a proliferation inhibition on SRA01/04 cells.There was a dose response relationship between 100 μmol · L-1 and 400 μmol · L-1.Compared with 0 μmol · L-1 H2O2 group,the difference was statistically significant (all P ＜ 0.01).According to flow cytometry results,apoptotic rate of SRA01/04 cells in control group and H2O2(100-300 μmol · L-1) groups were (6.1 ± 1.2)％,(26.3 ± 1.8)％,(32.5 ± 2.2) ％,and (64.7 ± 5.3) ％.Compared with 0 μmol · L-1 H2 O2 group,the differences were statistically significant (all P ＜ 0.01).RT-PCR test results showed that the expression of miR-34a increased significantly in a dose-dependent manner after the SRA01/04 cells treated with different concentrations of H2O2,while SIRT1 expression level was decreased,there were significant differences compared with control group (all P ＜ 0.001).Conclusion There is a significantly increase of miR-34a and decrease of SIRT1 in human lens epithelial cells under the oxidative stress of a certain concentration of H2O2.Down-regulated expression of miR-34a can increase the survival rate of human lens epithelial cells under H2O2-induced oxidative stress.
ABSTRACT
Background JAK/ signal transducer and activator of transcription 3 (STAT3) signal pathway plays a critical role during the sclera remodeling of experimental myopia.As a tyrosine kinase inhibitor,AG490 can inhibit the activation of this pathway.But whether AG490 plays a role in delaying the development of myopia is not completely clear.Objective This study was to investigate the inhibition of AG490 to activation of STAT3 signaling pathway and the sequential arresting effect on the sclera remodeling in form-deprived myopia (FDM) models.Methods Forty guinea pigs were randomly divided into the normal control group,model control group,PBS control group and AG490 treatment group.FDM models were established by the occlusion of the right eyes of guinea pigs for consecutive 4 weeks using translucent goggles in the model control group,PBS control group and AG490 treated group,and 25 μl PBS or AG490 were respectively injected into vitreous since the first day of modeling in two-day interval till the fourth week in the PBS control group and AG490 treated group.Refractive state and axial length were examined with retinoscopy and A-scan ultrasonography before and 4 weeks after experiment.The experimental eyes were extracted in the fourth week,and the expressions of scleral STAT3,p-STAT3,metal matrix proteinase-2 (MMP-2) proteins and STAT3 mRNA,MMP-2 mRNA were detected by immunocytochemstry and semi-quantitative reverse transcription PCR (RT-PCR) respectively.The use and care of experimental animals followed ARVO.Results Compared to the normal control group,the negative refraction power and axial length were significantly increased in the model control group,PBS control group and AG490 treated group,and the axial length in the AG490 treated group was smaller than those in the model control group and PBS control group,showing significant differences among the 4 groups (refraction:F =89.063,P =0.000;axial length:F =96.145,P =0.000).The expressions of STAT3,MMP-2 and p-STAT3 in scleral tissue were weaker in the normal control group.The expressional values (A values) of STAT3,p-STAT3 and M MP-2 were 0.064 ± 0.016,0.019 ± 0.002 and 0.155 ± 0.052 in the AG490 treated group,which were lower than 0.129±0.008,0.071 ±0.021,0.425 ±0.004 of the model control group and 0.130±0.004,0.069±0.002,0.421 ±0.042 of the PBS control group (STAT3:t =4.641,9.364,both at P＜0.01;p-STAT3:t =4.638,4.488,both at P＜ 0.05;MMP-2:t =9.123,9.029,both at P ＜ 0.05),however,these expressions were still higher than those of the normal control group (t =2.674,2.251,2.682,all at P ＜0.05).The expressional levels (A values) of STAT3 mRNA and MMP-2 mRNA in the AG490 treated group were 0.295±0.032 and 0.569±0.019,which were significantly lower than 0.547±0.015 and 0.782±0.051 in the model group as well as 0.544±0.015 and 0.779±0.048 in the PBS control group (STAT3 mRNA:t =10.115,11.703,both at P＜0.01;MMP-2 mRNA:t =9.218,9.494,both at P＜0.01).The expressional levels (A values) of STAT3 mRNA and MMP-2 mRNA in the AG490 treated group were still higher than those in the normal control group (t=2.576,3.565,both at P＜0.05).Conclusions AG490 can ultimately inhibit the development of axial myopia by arresting the activation of STAT3 signaling pathway in the FDM eyes and further regulating the expression of MMP-2 in sclera and delaying the remodeling of sclera.