Conbercept reverses TGF‑β2-induced epithelial-mesenchymal transition in human lens epithelial cells by regulating the TGF-β/Smad signaling pathway

doi:10.12122/j.issn.1673-4254.2024.08.04

Abstract

Abstract:

Objective To investigate the mechanism by which conbercept reverses transforming growth factor-β₂ (TGF‑β₂)-induced epithelial-mesenchymal transition (EMT) in human lens epithelial cells (HLECs). Methods Cultured HLEC SRA01/04 cells were treated with TGF‑β₂, conbercept, or both, and the changes in cell proliferation, apoptosis, and migration were observed using MTT assay, flow cytometry, scratch assay, and Transwell assay. Western blotting and qRT-PCR were used to detect the changes in the expression of EMT-related epithelial cell markers (E-Cadherin, α‑SMA, and Snail), extracellular matrix components, and genes related to the TGF-β/Smad signaling pathway. Results Conbercept significantly reduced TGF-β₂-induced EMT of SRA01/04 cells, decreased the expression levels of mesenchymal and extracellular matrix markers α-SMA, Snail, collagen I, collagen IV, and FN1, and upregulated the protein and mRNA expressions of E-cadherin (P<0.05). Transwell assay showed significantly lower cell migration ability in TGF-β₂+conbercept group than in TGF-β₂ group (P<0.05). Conbercept also inhibited the increase in Smad2/3 phosphorylation levels in HLEC-SRA01/04 cells with TGF-β₂-induced EMT (P<0.01). Conclusion Conbercept inhibits TGF‑β₂ induced EMT by downregulating the expression of pSmad2/3 in TGF‑β/Smad signaling pathway, indicating a potential therapeutic strategy against visual loss induced by posterior capsule opacification.

Key words: posterior capsule opacification, conbercept, epithelial-to-mesenchymal transition, transforming growth factor-β₂

Mengyun ZHU, Jianfeng WANG. Conbercept reverses TGF‑β₂-induced epithelial-mesenchymal transition in human lens epithelial cells by regulating the TGF-β/Smad signaling pathway[J]. Journal of Southern Medical University, 2024, 44(8): 1459-1466.

Figures/Tables 11

Tab.1 The sequences of primer

Gene	Forward primer	Reverse primer
E-cadherin	GAAGTGTCCGAGGACTTTGG	CAGTGTCTCTCCAAATCCGATA
Snail	CGGAAGCCTAACTACAGCGA	GGACAGAGTCCCAGATGAGC
GAPDH	CAGCCTCAAGATCATCAGCA	TGTGGTCATGAGTCCTTCCA
α-SMA	ATAGACATGCCGCCCTTCTT	GGCATCAAGGTACCCACAGA
FN1	AAGACCAGCAGAGGCATAAGG	TGTAGGGGTCAAAGCACGAG
Collagen Ⅰ	TTGAGTTGTATCGTGTGGTG	AGAAGATGAAAATGAGACTG
Collagen Ⅳ	TAGAGAGGAGCGAGATGTTC	GTGACATTAGCTGAGTCAGG

Fig.1 Effects of conbercept on survival rate of lens epithelial cell line HLEC-SRA01/04. *P<0.05 vs 0 mg/mL at the same time point; #P<0.05 vs same concentration for 24 h; &P<0.05 vs the same concentration for 48 h.

Fig.3 Effects of conbercept on migration ability of lens epithelial cells (Scale bar=200 μm). **P<0.01 vs control.

Fig.4 Inverted microscopy of morphological changes of human lens epithelial cells SRA01/04 induced by TGF-β2 (Scale bar=200 μm).

Fig.5 TGF-β2 at 10 ng/mL for 24 h induces EMT of human lens epithelial cells SRA01/04 cells. A: Expression level of E-cadherin after TGF-β2 treatment detected by qRT-PCR. B: Expression level of α-SMA after TGF-β2 treatment detected by qRT-PCR. C: Expression level of Snail after TGF-β2 treatment detected by qRT-PCR. *P<0.05, **P<0.01 vs control.

Fig.6 Western blotting for detecting TGF-β2-induced EMT in HLEC-SRA01/04 cells treated with TGF-β2 for 24 h by detecting epithelial cell markers E-cadherin and mesenchymal cell markers α‑SMA and Snail. *P<0.05, **P<0.01 vs control.

Fig.7 Western blotting for detecting expressiona of E-cadherin, α-SMA and Snail in TGF-β2-induced HLEC-SRA01/04 cells after conbercept treatment. At least 3 replicates were performed in each experiment. **P<0.01, ***P<0.001 vs control; #P<0.05, ##P<0.01, ###P<0.001 vs TGF-β2.

Fig.9 Effect of conbercept on TGF-β2-induced expression of ECM genes in HLEC-SRA01/04 cells. A: Collagen I mRNA levels in cells treated with TGF-β2 and conbercept for 24 h detected by real-time PCR. B: FN1 mRNA levels in cells treated with TGF-β2 and conbercept for 24 h The mRNA levels. C: Collagen IV mRNA levels in cells treated with TGF-β2 and conbercept for 24 h. At least 3 replicates were performed in each experiment. **P<0.01, ***P<0.001 vs control; #P<0.05, ##P<0.01 vs TGF-β2.

Fig.10 Effects of conbercept on migration of SRA01/04 cells induced by TGF-β2. At least 3 replicates were performed in each experiment. Giemsa staining, Scale bar: 50 μm. ***P<0.001 vs control; ##P<0.01 vs TGF-β2.

Fig.11 Conbercept inhibits TGF-β/Smad signaling pathway in SRA01/04 cells induced by TGF-β2. The expressions of Smad2/3 and p-Smad2/3 were detected by Western blotting. At least 3 replicates were performed in each experiment. **P<0.01 vs control; ##P<0.01 vs TGF-β2.

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Conbercept reverses TGF‑β₂-induced epithelial-mesenchymal transition in human lens epithelial cells by regulating the TGF-β/Smad signaling pathway

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