Effects Of Resveratrol On Proliferation Of Human Tenon’s Capsule Fibroblasts And Possible Mechanism

Part I Effects of resveratrol on proliferation of human Tenon’s capsule fibroblastsObjective:To investigate the effects of resveratrol on proliferation and apoptosis of human Tenon’s capsule fibroblasts in Vitro,and compare the changes at the different concentrations of resveratrol and different action duration.Methods:The Tenon’s capsule tissues,derived from patients with glaucoma filtration surgery (conditions needed),were cultivated in vitro by means of tissue-sticking method,and the third to sixth generations of HTFs were used.MTT method was applied to evaluate the proliferation and the biologic activities of cultivated human Tenon’s capsule fibroblasts(HTFs),which was exposured to the Resveratrol with different concentrations (10,20,40,80μg/ml) for12,24,36and48hours respectively.2. The cultivated cells were incubated with Res in various concentrations (10,20u,40,80μg/ml) for24hours. The change of cells apoptosis was assayed by AO-EB,Hoechst33258staining and Annexion V-propyl iodide ingot double staining flow cytometry respectively.Results:1.The results showed by MTT assay Compared with the control, resveratrol could inhibit the proliferation of human Tenon’s capsule fibroblasts significantly at the concentrations of10,20,40,80μg/ml, within12-48hours(p<0.05),and it was dependent on the concentrations and the duration of treatment. That is, as the extension of intervention time and increase of drug concentration, the inhibition rate of proliferation of HTFs was also enhanced.when cells were treated with resveratrol at the concentrations of10,20,40,80μg/ml for12h,an average of9.62%,18.86%,29.05%and51.05%decrease in cells proliferation was observed;when cells with the same treatment for24h,the proliferation inhibition rate of HTFs were13.86%,23.53%,34.76%and54.14%respectively,and those of16.86%,29.17%,40.88%and62.72%for36h while23.18%, 32.35%,59.20%,and69.05%for48h respectively.2The results of AO/EB and Hochest33258staining showed that cell nuclear manifested with shrinkage or a fragmental thick dense in vitro HTFs treated with resveratrol at different concentration for24h. The apoptosis phenomenon was highlighted with the increase of drug concentration.3The results of Annexin V-FITC/PI staining showed that in vitro HTFs apoptosis was promoted after treated with resveratrol at concentration of10ug/ml,20ug/ml,40ug/ml,80ug/ml for24h. It also showed that apoptosis rate increased in a dose-dependent manner.Conclusions:The inhibitory effect of Resveratrol on the proliferation of human Tenon’s capsule fibroblasts in vitro was significant, and it was time-dependent and dose-dependent in a certain range. Resveratrol could also promote the apoptosis of cells,the effect was in a dose-dependent manner. Part Ⅱ Effect of resveratrol on TGF-β gene expression of human Tenon’s capsule fibroblastsObjective:To explore the effect of resveratrol on TGF-β gene expression of human Tenon’s capsule fibroblast (HTFs).Methods:HTFs cultured with tissue adherence were divided into control group and drug intervention group, which were cultured for24h in normal condition and in resveratrol intervention at concentration of10μg/ml,20μg/ml,40μg/ml,80μg/ml respectively. Then, the protein and mRNA levels of TGF-β1and TGF-β2in HTFs were detected by western blot and RT-PCR.Results:There was significant difference in protein and mRNA levels of TGF-β1and TGF-β2in two groups. Compared to control group, the protein and mRNA levels of TGF-β1were decreased by33.51%-62.21%and40.12%-63.15%respectively, and the protein and mRNA levels of TGF-β2were decreased by38.53%-72.13%and42.33%-68.16%respectively after resveratrol intervention with various concentration.Conclusion:Resveratrol can significantly down-regulate the expression of the protein and mRNA levels of TGF-β1and TGF-β2in HTFs.