Bai Jing, Liu Xian-sheng, Xu Yong-jian, Zhang Zhen-xiang, Xie Min, Ni Wang
Department of Respiratory Medicine, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Zhonghua Jie He He Hu Xi Za Zhi. 2010 Jul;33(7):524-9.
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.
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.
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.
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.
气道平滑肌（ASM）细胞增殖是哮喘气道重塑的关键特征。细胞外信号调节激酶1/2（ERK1/2）信号通路是参与哮喘大鼠ASM增殖的最重要转导通路之一。然而，其在人ASM细胞增殖中的作用仍不清楚。
体外培养人ASMC（HASMC），并用哮喘患者的10%血清进行被动致敏，以非哮喘患者血清处理的HASMC作为对照。然后，用ERK正义、反义及错配寡脱氧核苷酸（ODN）转染HASMC。分别采用流式细胞术分析、MTT比色法、[³H]胸腺嘧啶核苷掺入法及免疫荧光染色检测增殖细胞核抗原（PCNA）来检测HASMC的增殖。通过原位末端标记法和膜联蛋白V FITC PI双染法检测HASMC的凋亡。分别用逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测ERK mRNA、ERK蛋白的表达及ERK1/2（p-ERK1/2）蛋白的磷酸化水平。
用哮喘患者10%血清被动致敏的HASMC中，S + G₂/M期百分比、吸光度（A(490)）值、DNA合成值及PCNA蛋白表达分别为(22.48 ± 2.04)%、(0.507 ± 0.090)、(3869 ± 396) cpm/10⁶细胞、(11.25 ± 1.21)。用ERK寡脱氧核苷酸处理后，这些指标分别降至(14.21 ± 1.21)%、(0.271 ± 0.021)、(2811 ± 182) cpm/10⁶细胞和(5.25 ± 0.60)（F分别为65.594、39.676、61.111、120.321，P < 0.05）。AODNs组HASMC的凋亡指数（13.96 ± 1.72）和早期凋亡细胞百分比（9.17 ± 0.47）%与慢性哮喘组相比显著增加，慢性哮喘组分别为(5.37 ± 0.05)、(3.26 ± 0.04)%（F分别为98.181、65.444，P < 0.05）。AODNs组HASMC中ERK mRNA表达（0.43 ± 0.06）和ERK激活率（63 ± 6）%与慢性哮喘组相比显著降低，慢性哮喘组分别为(0.89 ± 0.09)、(87 ± 8)%（F分别为78.043、87.288，P < 0.05）。ERK反义ODN抑制HASMC增殖并诱导其凋亡，但正义及错配ODN无此作用。
ERK反义ODN抑制用哮喘患者10%血清被动致敏的培养HASMC的增殖并增加其凋亡。结果提示ERK信号通路可能参与哮喘患者HASMC的增殖和凋亡。
