PTE inhibits TGF-β1-induced cell proliferation, EMT and accumulation of ECM
The chemical structure of PTE was shown in Fig. 1A. As exhibited in Fig. 1B, PTE at a dose of 30–100 µmol/L significantly reduced the cell viability of 10 ng/ml TGF-β1 exposed A549 and AEC cells (Fig. 1B). Furthermore, our preliminary experiments showed that 10 ng/ml TGF-β1 caused EMT after 48 h exposure in A549 and AEC cells (Fig. 1C and D). Here, the N-cadherin and Vimentin expression in cells were significantly increased after incubation with 10 ng/ml TGF-β1 (Fig. 1C and D), while E-cadherin protein levels were decreased (Fig. 1C and D). 30 µmol/L PTE suppressed N-cadherin and Vimentin protein levels, and rescued E-cadherin expression in TGF-β1-exposed cells (Fig. 1C and D). For accumulation of ECM, the results from western blot analysis confirmed the increased protein levels of α-SMA and fibronectin, as well as collagen 1, which induced by TGF-β1 in A549 and AEC cells (Fig. 1E and F). PTE significantly reduced the α-SMA, fibronectin, and collagen 1 expression (Fig. 1E and F). In addition, incubation with PTE alone, without TGIF1, seemed to have no effect on the EMT and accumulation of ECM compared into normally cultured A549 and AEC cells (Fig. 1C-F).
PTE promotes apoptosis and autophagy in TGF-β1-induced cells
Some studies have reported that PTE induces apoptosis in ovarian[26] and pancreatic cancer cells[27]. In this study, we found that the treatment of TGF-β1 significantly decreased the proportion of apoptosis cell (Fig. 2A and B), and inhibited the expression of Bax and caspase-3, and induced the expression of Bcl2 (Fig. 2C and D). The treatment of PTE rescued the apoptosis which inhibited by TGF-β1 (Fig. 2A and B), elevated Bax and caspase 3 protein levels, and degraded Bcl2 in A549 and AEC cells (Fig. 2C and D). We also focused on PTE in cell autophagy. As shown in Fig. 2E and F, TGF-β1significantly inhibited LC3-II, Beclin-1 and p21 protein expression, and enhanced LC3-I and Beclin-1 protein expression in A549 and AEC cells, which all were reversed by PTE. Moreover, incubation with PTE alone, without TGIF1, had no effect on the apoptosis and autophagy compared into normally cultured A549 and AEC cells (Fig. 2).
Identification of the differentially expressed genes in PTE-treated cells
We further performed transcriptome high-throughput sequencing on A549 cells incubated with TGF-β1 alone (TGF-β1), or TGF-β1 and PTE (TGF-β1 + PTE) for 24 h to identify differentially expressed genes caused by PTE. The cross-linked RNA fragments were isolated, converted into cDNA libraries, and performed to high-throughput sequencing with Illumina HisEq. TGF-β1 samples produced 8.4430 Gb clean bases, and 95.74% reads were compared to the reference genome, and measured 14848 genes. TGF-β1 + PTE samples produced 8.4045 Gb clean bases, and 95.54% reads were compared to the reference genome, and measured 15451 genes. Compared with PTE samples, 2898 differentially expressed genes were detected in TGF-β1 + PTE samples (Fig. 3). Among them, 2037 genes were differentially up-regulated and 861 genes were differentially down-regulated.
PTE inhibits pulmonary fibrosis by down-regulating ASIC2
Among the 861 differentially down-regulated genes, we found that PTE inhibited pulmonary fibrosis by down-regulating ASIC2. As shown in Fig. 4A and B, PTE significantly reduced the protein levels of ASIC2 compared with TGF-β1 group. To explore the role of ASIC2, the ASIC2 overexpression plasmid was used to rescue the protein levels of ASIC2 compared with TGF-β1 + PTE group. Furthermore, the restoration of ASIC2 protein levels stimulated the EMT (Fig. 4C and D) and accumulation of ECM (Fig. 4E and F), and inhibited apoptosis and autophagy (Fig. 5) compared with TGF-β1 + PTE group.