[Effect of extracellular signal-regulated kinase antisense oligodeoxynucleotides on the proliferation of passively sensitized human airway smooth muscle cells].
OBJECTIVE
Airway smooth muscle (ASM) cell proliferation is a key feature of airway remodeling in asthma. The extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway is one of the most important transduction pathways involved in the proliferation of ASM of asthmatic rats. However, its role in the human ASMCs proliferation remains unclear.


METHODS
HASMCs were cultured in vitro and passively sensitized with 10% serum from asthmatic patients, and non-asthmatic human serum treated HASMCs served as the control. Then, HASMCs were transfected with ERK sense, antisense and mismatched oligodeoxynucleotides (ODN). The proliferations of HASMCs were detected by flow cytometry analysis, MTT colorimetric assay, [³H] thymidine incorporation and proliferating cell nuclear antigen (PCNA) in immunofluorescence staining respectively. The apoptosis of HASMCs were detected by in situ end labeling and Annexin-V FITC PI double staining. The expressions of ERK mRNA, ERK protein and phosphorylation of ERK1/2(p-ERK1/2) protein were measured by RT-PCR and Western blotting, respectively.


RESULTS
The percentage of S + G₂/M phase, absorbance (A(490)) value, DNA synthesis value and the expression of PCNA protein in HASMCs passively sensitized with 10% serum from asthmatic patients were (22.48 ± 2.04)%, (0.507 ± 0.090), (3869 ± 396) cpm/10⁶ cells, (11.25 ± 1.21), respectively. After treated with ERK oligodeoxynucleotides, these measurements were decreased to (14.21 ± 1.21)%, (0.271 ± 0.021), (2811 ± 182) cpm/10⁶ cells and (5.25 ± 0.60), respectively (F = 65.594, 39.676, 61.111, 120.321, respectively, P < 0.05). The apoptotic index (13.96 ± 1.72) and the percentage of the early apoptotic cells (9.17 ± 0.47)% in HASMCs from group AODNs were significantly increased compared to those of chronic asthma group, which were (5.37 ± 0.05), (3.26 ± 0.04)%, respectively (F = 98.181, 65.444, respectively, P < 0.05). The expression of ERK mRNA (0.43 ± 0.06) and the activation ratio of ERK (63 ± 6)% in HASMCs from group AODNs were significantly decreased compared to those of chronic asthma group, which were (0.89 ± 0.09), (87 ± 8)%, respectively (F = 78.043, 87.288, respectively, P < 0.05). ERK antisense ODN inhibited the proliferation of HASMCs and induced the apoptosis of HASMCs, but the sense and the mismatched ones did not have these effects.


CONCLUSIONS
ERK antisense ODN inhibited the proliferation and increased the apoptosis in cultured HASMCs passively sensitized with 10% serum from asthmatic patients. The result suggests that ERK signaling pathway may contribute to the proliferation and apoptosis of HASMCs in asthmatic patients.
Bai
Jing
J
Department of Respiratory Medicine, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Liu
Xian-sheng
XS
Xu
Yong-jian
YJ
Zhang
Zhen-xiang
ZX
Xie
Min
M
Ni
Wang
W
D000328:Adult; D017209:Apoptosis / Q000187:drug effects; D001249:Asthma / Q000097:blood / Q000276:immunology*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics; D005260:Female; D006801:Humans; D007106:Immune Sera / Q000276:immunology / Q000494:pharmacology*; D008297:Male; D008875:Middle Aged; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology*; D012137:Respiratory System / Q000378:metabolism*; D015398:Signal Transduction
D007106:Immune Sera; D020319:Oligodeoxyribonucleotides, Antisense; D048049:Extracellular Signal-Regulated MAP Kinases