Cigarette smoke extract promotes proliferation of airway smooth muscle cells in asthmatic rats via regulating cyclin D1 expression

Abstract

Background Increased proliferation of airway smooth muscle cells (ASMCs) are observed in asthmatic patients and smoking can accelerate proliferation of ASMCs in asthma. To elucidate the molecular mechanisms leading to these changes, we studied in vitro the effect of cigarette smoke extract (CSE) on the proliferation of ASMCs and the expression of cyclin D1, an important regulatory protein implicated in cell cycle. Methods ASMCs cultured from 8 asthmatic Brown Norway rats were studied. Cells between passage 3 and 6 were used in the study and were divided into control group, pcDNA3.1 group, pcDNA3.1-antisense cyclin D1 (ascyclin D1) group, CSE group, CSE+pcDNA3.1 group and CSE+pcDNA3.1-ascyclin D1 group based on the conditions for intervention. The proliferation of ASMCs was examined with cell cycle analysis, MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. The expression of cyclin D1 was detected by reverse transcriptase-PCR (RT-PCR) and Western blotting. Results (1) The percentage of S+G2M phase, absorbance value at 490 nm wavelength (A490) and the expression rate of PCNA protein in CSE group were (31.22±1.17)%, 0.782±0.221, (90.2±7.0)% respectively, which were significantly increased compared with those of control group ((18.36±1.02)%, 0.521±0.109, and (54.1±3.5)%, respectively) (P <0.01). After the transfection with antisense cyclin D1 plasmid for 30 hours, the percentage of S+G2M phase, A490 and the expression rate of PCNA protein in ASMCs were much lower than in untreated cells (P <0.01). (2) The ratios of A490 of cyclin D1 mRNA in CSE group was 0.288±0.034, which was significantly increased compared with that of control group (0.158±0.006) (P <0.01). After the transfection with antisense cyclin D1 plasmid for 30 hours, the ratios of A490 of cyclin D1 mRNA in ASMCs was much lower than in untreated cells (P <0.01). (3) The ratios of A490 of cyclin D1 protein expression in CSE group was 0.375±0.008, which was significantly increased compared with that of control group (0.268±0.004) (P <0.01). After the transfection with antisense cyclin D1 plasmid for 30 hours, the ratios of A490 of cyclin D1 protein expression in ASMCs was much lower than in untreated cells (P <0.01). Conclusion CSE may increase the proliferation of ASMCs in asthmatic rats via regulating cyclin D1 expression.