Rho-associated Protein Kinase Inhibitor(Y-27632) Treatment Promotes Proliferation And Phagocytosis In Trabecular Meshwork Cells

Purpose:Reductions in trabecular meshwork?TM?cellularity inhibit aqueous humor?AH?outflow,which is the main cause of primary open-angle glaucoma.In addition,the dysfunction of TM cells which can clear obstructive debris will reduce the outflow of AH.Previous studies have shown that the new intraocular pressure?IOP?-reducing drug Rho-associated protein kinase inhibitor?ROCKi?can promote the proliferation of human corneal endothelial cells,however,it is still unknown whether it can also promote proliferation of TM cells.Here,we aimed to investigate the effect of a ROCKi?Y-27632?on TM cell proliferation and phagocytosis.Methods:Morphology of pTM,iHTM and GTM3 cells was observed by inverted phase contrast microscopy,expression of cell biomarkers:TIMP3,MMP3 and COL IV proteins was detected by immunofluorescence and expression of dexamethasone-induced myocilin protein by western blot detection to identify these cells.The effects of various concentrations of Y-27632?0,10,30,100 and 200?M?on F-actin were assessed using immunofluorescence.pTM,iHTM and GTM3 cells were used to perform effects of Y-27632 on proliferation of TM cells.CCK-8 was used to detect the effects of different concentrations of Y-27632?0,10,30,100 and 200?M?on the proliferation of iHTM and GTM₃ cells.The effect of 100?M Y-27632 on the proliferation of pTM cells was evaluated by cell counting and Ki67immunofluorescence staining.Cell phagocytosis was evaluated in i HTM and GTM₃ cells.The effects of 100?M Y-27632 on phagocytosis of iHTM and GTM3 cells were assessed by immunofluorescence and flow cytometry.C57BL/6J and Tg-MYOC^Y437H437H mice were used to investigate the proliferative effects of Y-27632 on TM cells in vivo.Mice were injected with100?M of Y-27632 under the bulbar conjunctiva for 4 consecutive days,and the IOP was measured for 2 weeks to observe the effect of Y-27632 on IOP.Then the AH outflow facility was detected 2 weeks after IOP measurement.The number of TM cells in mice TM tissue was counted using immunofluorescence.Results:pTM,iHTM and GTM₃ cells all have the characteristics of TM cells.Immunofluorescence showed that different concentrations of Y-27632?10,30,100 and 200?M?all affected the cytoskeleton of iHTM and GTM₃ cells.CCK-8 analysis showed that 100?M of Y-27632 significantly promoted the proliferation of i HTM cells and 10,30,100 and200?M of Y-27632 significantly promoted the proliferation of GTM3 cells.Cell counting and Ki67 immunofluorescence staining showed that 100?M of Y-27632 promoted pTM cell proliferation.In GTM3 cells,phagocytosis was significantly greater in the Y-27632 group than in the control group,nearly reaching the level of phagocytosis in i HTM,as determined using immunofluorescence and flow cytometry.In Tg-MYOC^Y437H437H mice,treatment with 100?M of Y-27632 significantly decreased IOP,and after injection it can still reduce IOP and increase AH outflow facility in the medium-and long-term.The number of TM cells in Tg-MYOC^Y437H437H mice was significantly improved after Y-27632 administration.Conclusions:ROCKi?Y-27632?can promote the proliferation and phagocytosis of TM cells,then reduce IOP and increase AH outflow in the medium-and long-term.These results suggest that Y-27632 may increase the number of TM cells and promote the phagocytic function of TM cells,thereby the improving function of TM cells can achieve the effect of lowering IOP in the medium-and long-term,which may be a new mechanism of ROCKi.This study provides the new theoretical supports for the application of ROCKi in the field of glaucoma treatment.