SB431542 partially inhibits high glucose-induced EMT by restoring mitochondrial homeostasis in RPE cells

Abstract

Background: The epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells participated in the development of retinal fibrosis. SB431542 is a small molecule inhibitor with inhibitory effects on the ALK4, ALK5 and ALK7. Our study aimed to explore the effect of SB431542 on the EMT of RPE cells and to provide new ideas for the treatment of retinal fibrosis.

Methods: We performed fundus fluorescein angiography, optical coherence tomography and hematoxylin-eosin staining in vivo to observe the effect of SB431542 on choroidal neovascularization (CNV)-induced retinopathy. The proliferation, migration, cytoskeleton, adhesion, reactive oxygen species (ROS), mitochondrial morphology and membrane potential of RPE cells were observed in vitro through fluorescein diacetate staining, Cell Counting Kit-8 experiment, wound healing assay, phalloidin staining, immunofluorescence, MitoSOX, DCFH-DA, MitoTracker and JC-10 staining. Western blot, reverse transcription quantitative and immunofluorescence were used to detect the expression of EMT-related markers, pERK1/2, pGSK3β and β-catenin.

Results: SB431542 significantly alleviated retinopathy in the CNV model. The proliferation, migration and adhesion in RPE cells decreased to a certain extent in SB431542 treatment. SB431542 partially normalized the structure of RPE cells. The expression levels of E-cadherin increased, while the expression levels of laminin and N-cadherin decreased with SB431542 treatment. SB431542 reduced the production of total ROS, mitochondrial SOX and recovered the mitochondrial membrane potential to a certain degree. In addition, our study showed that SB431542 downregulated the phosphorylation of ERK1/2, GSK3β and the expression of β-catenin.

Conclusion: SB431542 improved EMT in RPE cells by maintaining mitochondrial homeostasis via the ERK1/2 and GSK3β/β-catenin pathways. Video Abstract SB431542 inhibits EMT in RPE cells under high glucose conditions.

Keywords: Epithelial mesenchymal transformation; Mitochondria; Proliferative diabetes retinopathy; RPE; SB431542.

Fig. 1

Effects of SB431542 on retinopathy in CNV models. A, D FFA observation fluorescein leakage and quantitative analysis. B, E Observation of retinopathy area via OCT and quantitative analysis. C, F Observation of retinopathy area via HE staining and quantitative analysis. ****P<0.0001, ***P<0.001

Fig. 2

Effects of SB431542 on the proliferation and migration of RPE cells. A-B FDA staining detected cell density and quantitative analysis. (C)CCK-8 detected cell viability. D-E Wound healing assay detected the cell migration rate and quantitative analysis. ****P<0.0001, ***P<0.001

Fig. 3

Effects of SB431542 on the cytoskeleton and adhesion of RPE cells. A Phalloidin stained cytoskeleton protein F-actin for evaluating the cytoskeleton. B Expression of adhesive protein vinculin was evaluated via immunofluorescence. C Fluorescence intensity of vinculin was quantitatively detected. D Enlarged images of the cytoskeleton protein F-actin and adhesive protein vinculin. ***P<0.001

Fig. 4

Effects of SB431542 on the expression of EMT-related markers. A WB detected the expression levels of laminin, N-cadherin and E-cadherin. B Quantitative protein expression of laminin. C Quantitative protein levels of N-cadherin. D Quantitative protein levels of E-cadherin. E The mRNA level of laminin was detected via RT-qPCR. F The mRNA level of N-cadherin was detected via RT-qPCR. G The mRNA level of E-cadherin was detected via RT-qPCR. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05

Fig. 5

Effect of SB431542 on mitochondrial dysfunction induced by high glucose. A Comparing the levels of mitochondrial ROS and total ROS in each group by using MitoSOX and DCFH-DA staining. B Quantitative detection of mitochondrial ROS levels. C Quantitative detection of total ROS levels. D Mito-Tracker Red CMXRos was used to evaluate mitochondrial morphological changes. E Quantitative detection of total area. F Quantitative detection of mean form factor. G Quantitative detection of mean branch length. ***P<0.001

Fig. 6

Effect of SB431542 on the mitochondrial dysfunction induced by high glucose. A Detection of mitochondrial membrane potential via JC-10 staining. B Enlarged images of JC-10 staining in high glucose and high glucose+SB431542 groups. C Quantitative detection of monomer production. D Quantitative detection of polymer production. ***P<0.001

Fig. 7

Effect of SB431542 on the expression of ERK1/2 activity. A Quantitative protein levels of pERK1/2. B WB detection of the expression of ERK1/2 activity. C Quantitative fluorescence intensity of pERK1/2. D Immunofluorescence detection of the expression of pERK1/2. ****P<0.0001, ***P<0.001

Fig. 8

Effects of SB431542 on the expression of pGSK3β and β-catenin induced by high glucose. A WB detection of the expression of pGSK3β and β-catenin. B Quantitative analysis of the protein levels of pGSK3. C Quantitative detection of the protein levels of β-catenin. D Quantitative fluorescence intensity of pGSK3β. E Immunofluorescence detection of the expression of pGSK3β. F Quantitative fluorescence intensity of β-catenin. G Immunofluorescence detection of the expression of β-catenin. ****P<0.0001, ***P<0.001