[Effect of SB431542 on autophagy and epithelial mesenchymal transition in retinal pigment epithelial cells induced by high glucose].

Abstract

Objective: Exploring the effect of transforming growth factor β receptor inhibitor SB431542 on autophagy and the formation of retinal epithelial mesenchymal transition (EMT) in retinal pigment epithelial cells under high glucose conditions. Methods: This study was an experimental research. RPE cells were divided into normal group (N group), mannitol group (M group), high glucose group (HG group), high glucose combined with DMSO group (HG+DMSO group) and high glucose combined with SB431542 group (HG+SB431542 group). MTT assay was used to detect cell proliferation. Cell scratch assay was used to detect cell migration. Phalloidin staining was used to detect Fibrotic actin, real-time quantitative PCR (qPCR) and immunofluorescence were used to detect the expression of EMT related proteins Vimentin and E-cadherin. The cell autophagy staining kit detects the expression of autophagosomes. Western blotting (WB), qPCR, and immunofluorescence were used to detect the expression of Beclin1. One-way ANOVA and LSD-t test were used for statistical analysis. Results: The MTT assay showed that the cell optical density values of HG+DMSO group and HG+SB431542 group were 2.02±0.10 and 1.35±0.04, respectively (t=15.39, P<0.001); The results of cell scratch assay showed that the migration rates of cells in HG+DMSO group and HG+SB431542 group were 58.33%±2.07% and 28.17%±1.94%, respectively (t=26.07, P<0.001); Ghost pen cyclic peptide staining revealed spindle shaped changes in HG+DMSO group cells, while HG+SB431542 group cells were able to restore their normal polygonal structure; The qPCR and cell immunofluorescence results showed that the mRNA levels of vimentin in the HG+DMSO group and HG+SB431542 group were 1.03±0.04 and 0.93±0.05, respectively, with fluorescence intensities of (61 828±760) and 46 680±671 AU (t_qPCR=3.85, P=0.003; t_IF=36.62, P<0.001); The mRNA levels of epithelial calcium adhesion protein were 0.86±0.03 and 1.00±0.04, respectively, with fluorescence intensities of (38 637±880) and (54 988±1 264) AU (t_qPCR=8.89, P<0.001; t_IF=26.01, P<0.001); Autophagy staining showed that the fluorescence intensity of autophagosomes in HG+DMSO group and HG+SB431542 group was (22.75±1.39) and (33.21±1.95)AU, respectively (t=10.70, P<0.001); WB, qPCR, and cell immunofluorescence results showed that the protein levels of Beclin1 in the HG+DMSO group and HG+SB431542 group were 0.38±0.04 and 0.75±0.08, respectively, and the mRNA levels were 0.77±0.08 and 1.05±0.05, respectively. The fluorescence intensities were (42 639±1 713) and (49 027±1 024) AU, respectively (t_WB=9.51, P<0.001; t_qPCR=6.90, P<0.001; t_IF=7.84, P<0.001). Conclusion: SB431542 inhibits high glucose induced EMT by inducing autophagy in the RPE cells.