Y27632对哮喘气道重塑幼年大鼠气管平滑肌细胞骨架的影响

Effect of Rho kinase inhibitor Y27632 on the cytoskeleton of airway smooth muscle in young asthmatic rats with airway remodeling

目的 探讨幼年哮喘气道重塑大鼠中气管平滑肌细胞骨架的变化及RhoA/ROCK信号通路的影响.方法 应用抗原致敏液对幼年SD大鼠激发8周,原代培养纯化气管平滑肌细胞（ASMC）,并给予Rho激酶抑制剂Y27632干预,通过免疫荧光、实时定量PCR、Western blot等方法,检测气管平滑肌细胞骨架F-actin、α-tubulin的变化.结果 （1）哮喘气道重塑组ASMC中F-actin平均灰度值高于对照组（P＜0.01）,加入Y27632预处理后,F-actin平均灰度值明显降低（P＜0.01）;（2）哮喘气道重塑组α-tubulin的荧光信号强度、密度较对照组增高,而Y27632干预组微管α-tubulin的荧光信号明显减弱（P＜0.05）;（3）哮喘气道重塑组α-tubulin蛋白的表达较对照组显著增加（P＜0.01）,Y27632预处理组α-tubulin蛋白的表达明显下降（P＜0.05）.结论 幼年哮喘气道重塑大鼠中存在着气管平滑肌细胞骨架的变化,RhoA/ROCK信号通路可能起着重要作用.

Objective To investigate the alteration of the cytoskeleton of airway smooth muscle cells in young asthmatic rats with airway remodeling and the effect of RhoA/ROCK signal pathway.Methods Airway smooth muscle cells （ASMCs） were primary cultured and purified from Sprague-Dawley（SD） rats that were induced by ovalbumin （OVA） inhalation for 8w,then incubated by Pho kinase inhibitor Y27632.Real time quantitative polymerase chain reaction （RT-PCR）,Western blot,and immunohistochemistry were used to measure the alteration of F-actin,and α-tubulin in the cytoskeleton of airway smooth muscle.Results （1） The asthma group showed a high average gray value of F-actin in ASMC than control groups,especially 8 weeks;and were significantly down in the group after adding Y27632（P 〈0.01）.（2） The intension and intensity of fluorescence signal of α-tubulin in asthma groups in 8 weeks were higher than control greup（P 〈0.01）,and were significantly decreased in Y27632 group.（3） A higher expression of α-tubulin protein was shown in the asthma group in 8 weeks relative to control group（P 〈0.01）,and was significantly down-regulated in Y27632 group（P 〈0.05）.Conclusions Alteration of the cytoskeleton of airway smooth muscle exists in young asthmatic rats and the RhoA/ROCK signal pathway possibly plays a significant role.