Effects of tacrolimus on the TGF-β1/SMAD signaling pathway in paraquat-exposed rat alveolar type II epithelial cells

Paraquat is a highly toxic pesticide, which often causes pulmonary interstitial fibrosis after poisoning, and there is no specific antidote. At present, limited studies have reported that tacrolimus, as an immunosuppressant, can inhibit pulmonary fibrosis, but the specific mechanism remains unknown. The aim of the present study was to demonstrate the effect of tacrolimus on the TGF-β1 pathway associated with pulmonary fibrosis in paraquat exposed alveolar type II epithelial cells, and to identify the antipulmonary fibrosis mechanism of tacrolimus The rat alveolar epithelial type II RLE-6TN cell line was exposed to paraquat and treated with or without tacrolimus for 24 h, or with a TGF-β1 receptor type I/II inhibitor (LY2109761) for 1, 4, 8 or 16 h. MTT assays were used to detect the viability of rat alveolar type II epithelial cells under these different treatment conditions, while the concentrations of TGF-β1, SMAD3, SMAD7 and connective tissue growth factor (CTGF) in the cell culture supernatant were determined using ELISAs. Additionally, reverse transcription-quantitative PCR and immunofluorescence were used to analyze the mRNA and protein expression levels of TGF-β1, SMAD3, CTGF and SMAD7. The results demonstrated that the inhibition of the proliferation of RLE-6TN cells exposed to 200 nmol/l paraquat was 26.05±2.99%. The inhibition rate of 10 ng/ml tacrolimus on paraquat-exposed alveolar type II epithelial cells was 18.40±3.49%. The inhibition rate caused by 5 µmol/l LY2109761 was 26.56±4.49%. The expression levels of TGF-β1, SMAD3 and CTGF, as well as their concentrations in the culture supernatant, were significantly downregulated in the tacrolimus group compared with the paraquat group. However, both the concentration and expression levels of SMAD7 were significantly upregulated in the tacrolimus group compared with the paraquat group. In conclusion, tacrolimus can reduce the levels of TGF-β1, SMAD3 and CTGF, increase the level of SMAD7 in TGF-β1 signaling pathway and protect the development of pulmonary fibrosis in paraquat exposed alveolar epithelial cells.