Objective To investigate the mechanism of connective tissue growth factor (CTGF) and PI3K/Akt, RhoA/ROCK signaling pathways in alveolar epithelial-mesenchymal transition (EMT) caused by paraquat (PQ) poisoning.
Methods RLE-6TN cells were divided into four groups, namely the control group, PQ group, irrelevant plasmid group, and interfering plasmid group. Cells in the interfering plasmid group were transfected with CTGF-shRNA plasmid, while those in the irrelevant plasmid group were transfected with plasmid that had no interfering effect on CTGF. The expression of the mRNAs and proteins of the PI3K/Akt and RhoA/ROCK signaling pathways and the molecules related to EMT were detected and analyzed. In order to further elucidate the relationship between the roles of the two signaling pathways in EMT in RLE-6TN cells caused by PQ poisoning, RLE-6TN cells were divided into five groups, namely the control group, PQ group, PI3K inhibition group, RhoA inhibition group, and co-inhibition group. The expression of EMT-related molecules, mRNAs, and proteins in the cells in each group was detected and analyzed.
Results After specific inhibition of CTGF expression, the expression of CTGF, PI3K, Akt, RhoA, and ROCK mRNAs and proteins in the cells of the interfering plasmid group was reduced compared with those of the PQ group and the irrelevant plasmid group (P < 0.05/6). The examination of the expression of EMT-related molecules in RLE-6TN cells showed that E-cadherin mRNA and protein expression were elevated and α-SMA mRNA and protein expression were decreased in cells in the interfering plasmid group, compared with the PQ group and the irrelevant plasmid group (P < 0.05/6). After blocking the signaling pathway activity using specific inhibitors, the cellular α-SMA mRNA and protein expression were decreased in the PI3K-inhibited, RhoA-inhibited, and combined-inhibited groups compared with the PQ group (P < 0.05/10), whereas the E-cadherin mRNA and protein expression were elevated (P < 0.05/10). Compared with the PI3K-inhibited and RhoA-inhibited groups, cellular E-cadherin protein expression was elevated (P < 0.05/10) and α-SMA protein expression was decreased (P < 0.05/10) in the combined treatment group.
Conclusions CTGF can promote the process of PQ-induced EMT in alveolar epithelial cells by activating the PI3K/Akt and RhoA/ROCK signaling pathways, and the PI3K/Akt pathway and the RhoA/ROCK pathway have synergistic roles in PQ-induced EMT in alveolar epithelial cells.
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