K-115 Inhibits The Activation Of Human Tenon's Fibroblasts By Regulating Autophagy

Objective:Human Tenon's fibroblasts(HTFs)were cultured by tissue block adherence method.Activation of HTFs induced by transforming growth factor-?1(TGF-?1).And then,to study the regulation of ROCK inhibitor K-115 on the activation of HTFs,and to explore its related mechanism.To provide theoretical support at the cellular level for K-115 to inhibit scar formation after glaucoma filtering Surgery(GFS).Methods:1 Tenon's sac tissues was taken from glaucoma patients during operation and primary HTFs were cultured by tissue block adherence method.2 Establish a cell model of HTFs activation induced by TGF-?1.CCK-8 method was used to determine the optimal concentration and time of TGF-?1.3 To study the effect of K-115 on the activation of HTFs,HTFs were pretreated with K-115 at different concentrations for 2 hours,and then HTFs were induced with the optimum concentration of TGF-?1,CCK-8 method was used to determine the effect of K-115 pretreatment on the proliferation of HTFs activated by TGF-?1,and the optimal concentration of K-115 was determined.Transwell method was used to study the effect of K-115 pretreatment on the migration ability of HTFs activated by TGF-?1.Immunofluorescence method was used to study the effect of K-115 pretreatment on the expression of Smooth Muscle Actin(a-SMA)in HTFs activated by TGF-?1.4 To study the mechanism of K-115 regulating HTFs activation,Cyto-ID staining,flow cytometry was used to study the effects of K-115 pretreatment at different concentrations on the levels of autophagy and apoptosis of HTFs activated by TGF-?1.At the same time,qRT-PCR and Western blot were used to study the effects of different concentrations of k-115 pretreatment on the expression of autophagy-related proteins Beclin-1 and LC3 after activation of HTFs induced by TGF-?1.Result:1 Human Tenon's fibroblasts were successfully cultured by tissue block adherence method.2 The cell model of HTFs activation induced by TGF-?1 was established.CCK8 method showed that the optimum concentration of TGF-?1 was 5 ng/ml.24 hours may be the best time to observe the effect of TGF-?1 on proliferation.3 CCK8 method confirmed that the cell proliferation ability of K-115 group was significantly lower than that of TGF-?1 group,and the optimum concentration of K-115 was 10umol/L.Transwell migration experiment showed that K-115 coula inhibit the cell migration ability of activated cells induced by TGF-?1.Cell immunofluorescence assay confirmed that the expression of ?-SMA in HTFs induced by TGF-?1 increased significantly,while the positive expression of filamentous fluorescent substance in cytoplasm after K-115 pretreatment decreased significantly compared with that induced by TGF-?1 alone.4 Cyto-ID staining showed that the autophagy level of HTFs activated by TGF?1 increased,while K-115 pretreated at different concentrations(5umol/L,10umol/L)decreased the autophagy level of HTFs activated cells in a concentration-dependent manner.Flow cytometry showed that K-115 pretreatment had no significant effect on the apoptotic level of cells.The results of qRT-PCR and Western blot showed that different concentrations of K-115 pretreatment could inhibit the up-regulation of autophagy-related proteins Beclin-1 and LC-3 induced by TGF-?1 in a concentration-dependent manner.Conclusion: ROCK inhibitor K-115 has no obvious toxic effect on HTFs cultured in vitro.It can reduce the activation of TGF?1 on human Tenon's fibroblasts to a certain extent.Its mechanism may be to regulate the autophagic function of HTFs.