AMPK/mTOR通路对慢性支气管哮喘大鼠气道平滑肌细胞增殖的影响及机制  

Effects and mechanism of AMPK/mTOR pathway on proliferation of airway smooth muscle cells in rats with chronic bronchial asthma

作　　者：徐灵彬 韩新鹏[3] 吴昌归 Lingbin Xu;Xinpeng Han;Changgui Wu(Department of Pulmonary and Critical Care Medicine,the First Affiliated Hospital,Air Force Medical University of PLA,Xi′an 710032,China;Second Department of Pulmonary and Critical Care Medicine,Shaanxi Provincial People′s Hospital,Xi′an 710068,China;Department of Pulmonary and Critical Care Medicine,Xi′an International Medical Center Hospital,Xi′an 710100,China)

机构地区：[1]空军军医大学第一附属医院呼吸与危重症医学科,西安710032 [2]陕西省人民医院呼吸与危重症医学二科,西安710068 [3]西安国际医学中心医院胸科医院呼吸与危重症医学科,710100

出　　处：《国际呼吸杂志》2021年第22期1712-1719,共8页International Journal of Respiration

基　　金：国家自然科学基金(81470223)。

摘　　要：目的观察二甲双胍对慢性支气管哮喘(哮喘)动物模型的气道平滑肌细胞(ASMCs)增殖的影响及机制。方法使用OVA致敏法制备慢性哮喘模型, 末次激发48 h后处死大鼠, 使用酶消化法进行ASMCs的原代分离培养。对原代培养的ASMCs的一磷酸腺苷活化的蛋白激酶α1(AMPK-α1)、S期激酶相关蛋白(SKP-2)的表达使用shRNA进行干扰, 之后采用Western blot检测AMPK/mTOR通路相关蛋白的表达。结果在原代培养的ASMCs中, 二甲双胍可激活AMPK通路, 能明显抑制ASMCs的增殖;雷帕霉素亦可明显抑制细胞的增殖, 两者均存在明显的量效关系(P值均<0.05)。沉默AMPK基因表达后, 可使ASMCs增殖增加(P<0.01), 进一步分析发现mTOR及下游的p70s6k磷酸化增加(P<0.05), 从而上调SKP-2基因表达(P<0.05)、下调P21基因表达(P<0.01);沉默SKP-2基因表达, 再干扰AMPK基因的表达, 可逆转其促进细胞增殖的作用(P<0.01)。结论 AMPK/mTOR通路对ASMCs增殖的调节是通过调节SKP2表达, 进而影响P21水平实现的。二甲双胍作为AMPK的激动剂, 有潜在的抑制哮喘患者气道平滑肌增生、进而改善气道重塑的作用。Objective To observe the effects and mechanism of metformin on the proliferation of airway smooth muscle cells(ASMCs)in chronic bronchial asthma(asthma)model.Methods The rat model of chronic asthma was established,and then the rats were killed 48 hours after the last challenge,with ASMCs isolated and cultured by enzyme digestion method.shRNA was used to interfere the expression of mTOR,AMPK-α1 and SKP-2 in primary cultured ASMCs.Western blot was used to detect the expression of AMPK/mTOR pathway related proteins.Results In the primary culture of ASMCs,metformin activated AMPK pathway and significantly inhibited the proliferation of ASMCs in a dose-dependent manner,and rapamycin also significantly inhibited the proliferation of ASMCs,both of which have obvious dose-response relationship(both P<0.05).ASMCs proliferation was increased by silencing AMPK gene expression(P<0.01).Further analysis showed increased phosphorylation of mTOR and downstream p70s6k(P<0.05),which up-regulated SKP-2 gene expression(P<0.05)and down-regulated P21 gene expression(P<0.01).Silencing SKP-2 gene expression and interfering AMPK gene expression reversed the effect of SKP-2 gene on cell proliferation(P<0.01).Conclusions AMPK/mTOR pathway regulates the proliferation of ASMCs by regulating the expression of SKP-2 and thus affecting the level of P21.As an agonist of AMPK,metformin may have potential effects on inhibiting airway smooth muscle proliferation and improving airway remodeling.

关 键 词：哮喘 模型 动物 气道平滑肌 增殖 AMPK/mTOR通路 SHRNA干扰 

分 类 号：R562.25[医药卫生—呼吸系统]