基于自噬/凋亡研究淫羊藿女贞子合用糖皮质激素对3-MA诱导的ASMCs自噬抑制状态的作用

Effects of the Combination of Glucocorticoid and Herba Epimedi/Fructus Ligustri Lucidi on ASMCs in Autophagy Inhibition State Induced by 3-MA Based on Apoptosis/Autophagy

目的研究淫羊藿女贞子合用糖皮质激素对自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)抑制气道平滑肌细胞(airway smooth muscle cells,ASMCs)自噬的影响。方法培养大鼠原代ASMCs,随机分为5组,即正常组(正常大鼠血清)、3-MA组(正常大鼠血清+3-MA)、激素组(正常大鼠血清+3-MA+地塞米松)、中药组(3-MA+淫羊藿女贞子含药血清)、合用组(3-MA+淫羊藿女贞子含药血清+地塞米松)。根据分组分别加入正常大鼠血清或含药大鼠血清;除正常组外,其余各组同时加入3-MA刺激ASMCs,48h后进行检测。电镜观察细胞超微结构;MTT法检测细胞活力:免疫细胞化学法检测增殖蛋白Ki-67蛋白表达;Annexin V-FITC/PI法检测凋亡活性;Western Blot或免疫荧光法检测LC3、Bax、Bcl-2、P53、Caspase-3、Beclin-1、mTOR、p-mTO R蛋白表达。结果与3-MA组比较,各给药组可不同程度地增加3-MA抑制的自噬活性,上调LC3 II/I比值、Beclin-1表达,下调mTOR及p-mTOR表达下调;继续下调ASMCs细胞活力并上调凋亡活性,其中激素单用或与中药合用可显著下调Ki-67蛋白表达而抑制细胞增殖,各给药组Bax、Caspase-3、P53表达显著上调;此外,与激素组相比,合用组Beclin-1、mTOR、Bax、Bcl-2、Bax/Bcl-2、Caspase-3、P53均有统计学差异。结论激素、中药或二者合用均可重新激活3-MA引发的自噬阻断,进一步抑制ASMCs增殖、诱导细胞凋亡,且合用后在一定程度上发挥协同增效作用。

Objective To study the effect of Herba Epimedii and Fructus Ligustri Lucidi(HE&FLL)containing serum combined with dexamethasone(Dex)on autophagy,apoptosis,and proliferation activity of airway smooth muscle cells(ASMCs)induced by the autophagy inhibitor 3-methyladenine(3-MA).Methods The primary ASMCs of rats were cultured and randomly divided into 5 groups:normal control(Nc)group(normal rat serum),3-MA group(normal rat serum+3-MA),Dex group(normal rat serum+3-MA+Dex),TCM group(3-MA+HE&FLL rat serum)and combination group(3-MA+HE&FLL rat serum+Dex).Normal rat serum or medicated rat serum was added separately according to grouping;Except for Nc group,all other groups were simultaneously added with 3-MA to stimulate ASMCs,which were detected after 48h.Transmission electron microscope was used to observe cell microstructure;MTT assay to detect cell viability;immunocytochemistry to detect protein expression of proliferation protein Ki-67;Annexin V-FITC/PI assay to estimate apoptosis activity;Western blot or immunofluorescence to detect LC3,Bax,Bcl-2,P53,Caspase-3,Beclin-1,mTOR and p-mTOR.Results Compared with 3-MA group,each administration group could increase the autophagy activity inhibited by 3-MA in different degrees,up-regulate LC3 II/I and Beclin-1,down-regulate mTOR and p-mTOR;Continue to down-regulate cell viability and up-regulate apoptosis activity in ASMCs,in which using Dex alone or in combination with HE&FLL significantly down-regulated Ki-67 protein and inhibited cell proliferation,with significant up-regulation of Bax,Caspase-3,and p53 protein in each administration group;In addition,Beclin-1,mTOR,Bax,Bcl-2,Bax/Bcl-2,Caspase-3,and p53 were statistically increased in combination group compared with the Dex group.Conclusion The blockage of autophagy induced by 3-MA could be reactivated by Dex,HE&FLL or the combination of both,which further inhibited ASMCs proliferation and induced cell apoptosis,and the combination exerted synergistic effects to some extent.