Cigarette Smoke Extract Induce Apoptosis Of RLE-6TN Cells Via TGF-B1/SMAD2 Pathway

Objective:To investigate the effect of Cigarette Smoke Extract (CSE) on the apoptosis of rat alveolar Type Ⅱ cell line (RLE-6TN) and explore the possible mechanism in it. Methods:Using the different concentration of CSE (0.5%-2.0%) co-cultured with rat alveolar epithelial cell line (RLE-6TN) for 48 hours. Morphological changes of apoptotic cells were observed by fluorescence microscope after Hochest staining; Rate of cell apoptosis was measured by flow cytometry; Expression levels of TGF-β1, p-Smad2 and activity of apoptotic related protein Cleaved caspase-3 were examined by Western blotting. Then inhibitor of the transforming growth factor-β1 (TGF-β1) type I receptor (SB431542) was administered to RLE-6TN prior to CSE exposure. Expression of p-Smad2 and activity of Cleaved caspase-3 were examined by Western blotting. Results:Hoechst staining showed that the cells in each group which treated with CSE had chromatin condensation gathered, however, Nuclei in control group exhibited uniform light blue fluorescence. Apoptotic cell percentages of control group and each CSE treatment group were (3.671±1.04)%, (16.606±6.645)%, (18.626±3.596)% and (31.035±4.373)%, respectively. Apoptotic cell percentage of each CSE treatment group were significant higher than the control group (P<0.05). The early apoptosis rate of each CSE treatment group was (5.270±0.526)%, (22.563±1.048)%, (27.467±0.735)% and (32.580±0.413)%, respectively. Which was significant higher than the control group (P<0.05). Western Blot results showed that protein expression of TGF-β1, smad2, phosphorylation smad2 (p-smad2) and activity of caspase-3 in each CSE treatment group were significantly increased, when compared with the control group (P<0.05). After TGF-β1 receptor inhibitor (SB431542) co-cultured with RLE-6TN for 48 h, expression of p-Smad2 and activity of Cleaved caspase-3 in SB431542 treament group were significantly decreased when compared with the control group (P<0.05); Expression of p-Smad2 protein and activity of caspase-3 in SB431542+2.0% CSE-treated group was significantly lower than 2.0% CSE treatment group (P<0.05). Conclusion: 1. CSE could active the TGF-β1/Smad2 pathway and induce the apoptosis of RLE-6TN.2. Blocking of TGF-β1/Smad2 signal pathway by SB431542 could abrogate the CSE induced apoptosis of RLE-6TN. TGF-β1/Smad2 signal pathway may be involved in the CSE induced apoptosis of rat alveolar cells.