[The role of transforming growth factor-β1/connective tissue growth factor signaling pathway in paraquat-induced pulmonary fibrosis]

H H Li, Q Cai, Y P Wang, H R Liu, M Huang
Chinese Journal of Industrial Hygiene and Occupational Diseases 2016 July 20, 34 (7): 484-488
Objective: To investigate the effects of Paraquat on human embryonic lung fibroblasts (MRC5) and explore the role of transforming growth factor-β1/connective tissue growth factor signaling pathway in paraquat-induced pulmonary fibrosis. Methods: MRC5 cells were cultured with different concentration of PQ (0, 12.5, 25, 50, 100, 200, 400 μmol/L) for 24 h. The viability of cells was measured by MTT. The protein level of TGF-β1 were analyzed by ELISA after PQ treatment (0, 25, 50, 100 μmol/L) . To examine whether TGF-β1/CTGF signaling pathway was involved in paraquat-induced cytotoxicity, cells was divided into 6 groups: (1) control; (2) 25 μmol/L PQ group; (3) 50 μmol/L PQ group; (4) 100 μmol/L PQ group; (5) TGF-β1 positive control group (50 μmol/L rhTGF-β1) ; (6) stimulate group (100 μmol/L PQ+50 μmol/L TGF-β1) . The protein levels of p-Smad2, p-Smad3 and CTGF were assayed by western blot. The mRNA level of CTGF was assayed by real time RT-PCR. Results: MTT showed that cell viability decreased with increasing PQ concentration (P<0.05) . The protein expression of TGF-β1 treated with PQ (25, 50, 100 μmol/L) significantly increased compared with control in a dose-independent manner (P<0.05) . Exposure to PQ (25, 50, 100 μmol/L) induced increase of protein levels of p-Smad2 and p-Smad3. Noteworthy, the expression of p-Smad2 and p-Smad3 were dramatically increased following PQ plus TGF-β1 stimulation (P<0.05) . Exposure to PQ (50, 100μmol/L) induced increase of CTGF protein expression and similar greatly increase following PQ plus TGF-β1 stimulation (P<0.05) . Real time RT-PCR showed CTGF mRNA in all groups also significantly up-regulated compared with control (P<0.05) . Conclusion: TGF-β1 regulates the expression of target gene CTGF to exhibit its pro-fibrogenic effects by activating TGF-β1/Smad signaling pathway in PQ-induced pulmonary fibrosis.

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