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. 2019 Oct 18;12(10):1517-1523.
doi: 10.18240/ijo.2019.10.01. eCollection 2019.

Celastrol inhibits migration, proliferation and transforming growth factor-β2-induced epithelial-mesenchymal transition in lens epithelial cells

Li-Ping Wang  1 , Bao-Xin Chen  1 , Yan Sun  1 , Jie-Ping Chen  1 , Shan Huang  1 , Yi-Zhi Liu  1
Affiliations

Celastrol inhibits migration, proliferation and transforming growth factor-β2-induced epithelial-mesenchymal transition in lens epithelial cells

Li-Ping Wang et al. Int J Ophthalmol. .

Abstract

Aim: To investigate the mechanism of celastrol in inhibiting lens epithelial cells (LECs) fibrosis, which is the pathological basis of cataract.

Methods: Human LEC line SRA01/04 was treated with celastrol and transforming growth factor-β2 (TGF-β2). Wound-healing assay, proliferation assay, flow cytometry, real-time polymerase chain reaction (PCR), Western blot and immunocytochemical staining were used to detect the pathological changes of celastrol on LECs. Then, we cultured Sprague-Dawley rat lens in medium as a semi-in vivo model to find the function of celastrol further.

Results: We found that celastrol inhibited the migration of LECs, as well as proliferation (P<0.05). In addition, it induced the G2/M phase arrest by cell cycle-related proteins (P<0.01). Moreover, celastrol inhibited epithelial-mesenchymal transition (EMT) by the blockade of TGF-β/Smad and Jagged/Notch signaling pathways.

Conclusion: Our study demonstrates that celastrol could inhibit TGF-β2-induced lens fibrosis and raises the possibility that celastrol could be a potential novel drug in prevention and treatment of fibrotic cataract.

Keywords: cataract; celastrol; fibrosis; lens; transforming growth factor-β2.

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Figures

Figure 1
Figure 1. Celastrol inhibited the migration and proliferation of LECs
A: LECs were treated with or without celastrol (1 µmol/L) for 12h. Pictures were captured every 4h and the black lines represented the wound edges (scale bar 100 µm); B: The distance of LECs migration was measured at different time points; C: LECs were treated with 0, 0.5, 1 µmol/L celastrol for 0, 1, 2, 3, 4d. Data were the mean±SD from an experiment which was repeated three times. aP<0.05 vs the control group.
Figure 2
Figure 2. Celastrol induced G2/M phase arrest by cell cycle-regulated proteins
A, B: LECs were treated with 0, 1, 2 µmol/L celastrol for 24h. Cell cycle was detected by flow cytometry. The percentage of LECs in G2 phase was calculated; C: LECs were treated with 0, 0.5, 1, 1.5, 2 µmol/L celastrol for 48h. The protein expression levels of cdc2 and cyclin B1 were detected by Western blot analysis. D: Quantitative analysis of protein levels were detected three times. Data were derived by three independent experiments. bP<0.01 vs the control group.
Figure 3
Figure 3. Celastrol inhibited TGF-β2-induced EMT in LECs
LECs were cultured in the absence or presence of TGF-β2 (5 ng/mL) with celastrol (1 µmol/L) for 24-48h. A: The mRNA expression level of FN and Col IV in LECs was detected by real-time PCR; B, C: The protein expression level of FN was determined by Western blot analysis; D: Immunofluorescence analysis of FN (green), Col IV (green) and α-SMA (red) were observed using confocal microscopy (scale bar 50 µm). Data were derived by three independent experiments. aP<0.05, bP<0.01 vs the TGF-β2 group.
Figure 4
Figure 4. Celastrol inhibited TGF-β2 signaling pathway by suppressing the phosphorylation of Smad2/3 and downregulating the Jagged/Notch signaling pathway
LECs were cultured in the absence or presence of TGF-β2 (5 ng/mL) with celastrol (1 µmol/L) for 24-48h. A: The mRNA expression levels of Notch2, Notch3 and Jagged1 in LECs were detected by real-time PCR; B-D: The protein expression levels of p-Smad2/3, Smad2/3, Notch1, Notch3 and Jagged1 were determined by Western blot analysis. Data were derived by three independent experiments. bP<0.01 vs the TGF-β2 group.
Figure 5
Figure 5. Celastrol suppressed TGF-β2-induced ASC in the whole lens culture semi-in vivo mode
LECs were cultured in the absence or prescence of TGF-β2 (5 ng/mL) with celastrol (1 µmol/L) for a week. A: The morphology of lens was captured by dissecting microscope (scale bar 500 µm); B: The staining of parafin section of α-SMA (red) was captured by confocal microscopy (scale bar 100 µm).
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