Catalpol represses the migration, proliferation and epithelial-mesenchymal transition of TGF-β2-stimulated lens epithelial cells via TGF-β/Smad and Notch1 signaling pathways

Purpose: To investigate the role of catalpol in posterior capsule opacification (PCO). Methods: Human lens epithelial cells (SRA01/04) were treated with TGF-β2 or co-treated with TGF-β2 and different concentrations of catalpol. Cell migration and viability were assessed via wound healing and CCK8, respectively. Epithelial-mesenchymal transition and the underlying mechanism of action were evaluated using western blot. Results: Treatment with TGF-β2 significantly increased cell viability and promoted the migration of SRA01/04 (p < 0.001). However, catalpol significantly reduced cell viability and repressed the migration of TGF-β2-stimulated SRA01/04 (p < 0.001). Moreover, TGF-β2-stimulated increases in fibronectin, α-smooth muscle actin (α-SMA), snail and vimentin. Decreases of E-cadherin and connexin-43 in SRA01/04 were reversed by catalpol. Moreover, TGF-β2-stimulated the up-regulation of p-smad2/3, while SRA01/04 was down-regulated by catalpol, but attenuated TGF-β2-stimulated increases in Notch1 and Jagged1 in SRA01/04. Conclusion: Catalpol inhibits TGF-β2-stimulated migration, proliferation and epithelial-mesenchymal transition of SRA01/04 through the inactivation of TGF-β/Smad and Notch1 signaling. Catalpol might be a novel preventive agent for PCO. However, the effect of catalpol on animal models of PCO should be investigated further.


INTRODUCTION
Posterior capsular opacification (PCO) is caused by the migration and proliferation of residual lens epithelial cells (LECs) in equatorial lens capsules after cataract surgery [1]. About 30 % of adults and 100 % of children suffer vision loss due to posterior cataract [2]. Although a variety of pharmacological and surgical strategies, such as anterior vitrectomy, posterior continuous curvilinear capsulorhexis, laser capsulotomy, and lens capsule polishing have been assessed for the prevention of PCO, implantation of intraocular lens is the most effective method for now [2]. However, design and materials of the intraocular lens are known as risk factors for PCO [3]. Therefore, effective strategies for the prevention of PCO are still urgently needed.
Previous studies have shown that transforming growth factor-β2 (TGF-β2) stimulated capsular fibrosis, cell migration, differentiation, proliferation, and epithelial-mesenchymal transition (EMT) of LECs, also contribute to the development of PCO [4]. The EMT of LECs, characterized by gain of mesenchymal cell features and loss of epithelial cell features, contributes to the loss of cell adhesion and gives rise to myofibroblasts and fibroblasts, leading to PCO [5]. Blockage of TGF-β2-stimulated abnormal proliferation and EMT is considered to be an effective strategy for PCO [5].
Catalpol inhibited cell proliferation and migration, and blocked the EMT of osteosarcoma [6]. Moreover, the application of catalpol reduced the cell migration of human umbilical vein endothelial cells, and exerted anti-inflammatory as well as antiangiogenic effects against corneal neovascularization [7]. Catalpol reduced secretion of lactate dehydrogenase, and suppressed oxidative stress and inflammation in retinal ganglion cells, which attenuated diabetic retinopathy [8]. Catalpol suppressed the activation of TGF-β/smad2/3 pathway so as to exhibit a nephroprotective effect against diabetes mellitus-associated complications [9]. Therefore, catalpol was hypothesized to suppress TGF-β2stimulated proliferation and EMT of LECs through the inactivation of TGF-β/smad2/3 pathway.

Cell viability and migration assays
The SRA01/04 was seeded into 96-well plates, and treated with TGF-β2 or catalpol. A total of 10 μL CCK8 (Beyotime, Beijing, China) was added into each well for 2 h. Absorbance at 450 nm was measured using microplate reader (Bio-Rad, Hercules, CA, USA). For wound healing assay, SRA01/04 was seeded into 6-well plates, and treated with TGF-β2 or catalpol. A p20 pipette tip was used to make a straight line scratch in the middle of the well. The suspended cells were removed, and the scratch gap was photographed under a light microscope (Olympus, Tokyo, Japan) 24 h later. The wound width was analyzed with Image J v.1.46 (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis
All the data were obtained from at least three independent experiments and expressed as mean ± SEM, and analyzed via student's t test or one-way analysis of variance using SPSS software. P < 0.05 was considered statistically significant.

DISCUSSION
Posterior capsule opacification (PCO) is related to the pathological progression in residual LECs [4]. Catalpol has been shown to suppress cell proliferation and metastasis of various tumors [6]. In this study, catalpol suppressed cell proliferation, migration and EMT of TGF-β2stimulated SRA01/04, suggesting the possibility that catalpol might be a potential agent for PCO. The TGF-β pathway is an inducer of EMT, and TGF-β2-stimulated LECs have been widely used as model of PCO [10,11]. In this study, TGF-β2 was used to induce an increase of cell proliferation and migration in SRA01/04. This TGF-β2 induced the activation of Smad2/3 signaling during the development of PCO [12]. Protein expressions of mesenchymal biomarkers, including fibronectin, α-SMA, Snail and vimentin, was up-regulated in SRA01/04 followed by TGF-β2. However, the protein expression of epithelial biomarkers, E-cadherin and connexin-43, were down-regulated. Moreover, phosphorylation of smad2/3 was increased in TGF-β2-stimulated SRA01/04, suggesting that TGF-β2 induced EMT of LECs through the activation of smad2/3 signaling.

CONCLUSION
Catalpol inhibits TGF-β2-stimulated proliferation, migration and EMT of LECs through inactivation of TGF-β/Smad and Notch1 signaling. Therefore, catalpol might be a novel preventive agent for PCO. However, the effect of catalpol in animal models of PCO should be investigated in further research.

Acknowledgements
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Funding
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Ethical approval
None provided.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflict of Interest
No conflict of interest associated with this work.

Contribution of Authors
We declare that this work was done by the authors named in this article, and all liabilities pertaining to claims relating to the content of this article will be borne by the authors. Xiaoyu Li and Honglei Ma designed the study and carried them out, supervised the data collection, analyzed the data, interpreted the data, prepared the manuscript for publication and reviewed the draft of the manuscript. All authors have read and approved the manuscript.

Open Access
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