摘要
目的研究淫羊藿女贞子含药血清合用地塞米松对自噬激活剂雷帕霉素(Rap)诱导的气道平滑肌细胞(ASMCs)自噬、凋亡及增殖活性的影响。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)、Rap组(正常大鼠血清+Rap)、激素组(正常大鼠血清+地塞米松+Rap)、中药组(Rap+淫羊藿、女贞子含药血清)、合用组(Rap+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Rap刺激ASMCs,48 h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蛋白表达并评价细胞增殖活性;Annexin V-FITC/PI法检测细胞凋亡活性;Western blotting检测LC3蛋白相对表达;细胞免疫荧光法检测Bax、Bcl-2、Beclin-1、P53、Caspase-3、mTOR、p-mTOR蛋白表达。结果激素可协同Rap诱导ASMCs自噬活性增加,使LC3Ⅱ/Ⅰ蛋白比值进一步增加,下调p-mTOR表达,但对Beclin-1及m TOR表达无影响,抑制ASMCs细胞活力及增殖活性,可增加凋亡水平,上调Bax、Caspase-3及P53表达,下调Bcl-2表达。中药组及合用组均能显著抑制Rap诱导的ASMCs自噬,下调LC3Ⅱ/Ⅰ蛋白比值及Beclin-1蛋白表达,并改善Rap导致的细胞活力、增殖活性降低。此外中药能抑制凋亡活性升高,上调Caspase-3蛋白表达,下调P53蛋白表达;中西药合用能下调p-mTOR蛋白表达,但对凋亡活性无显著影响。与激素单用比较,合用组下调LC3Ⅱ/LC3Ⅰ比值,调节ASMCs细胞活力、增殖活性及凋亡活性均有统计学差异(P<0.05或P<0.01)。结论中药联合糖皮质激素可抑制激素协同Rap诱导的ASMCs过度自噬状态,并改善被抑制的细胞活性,使ASMCs趋于正常。
Objective:To investigate 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 activator rapamycin(Rap).Methods:The primary ASMCs of rats were cultured and randomly divided into 5 groups:normal control(Nc)group(normal rat serum),Rap group(normal rat serum+Rap),DEX group(normal rat serum+Rap+Dex),TCM group(Rap+HE&FLL rat serum)and combination group(Rap+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 Rap to stimulate ASMCs,which were examined after 48 h.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 to evaluate cell proliferation activity;Annexin V-FITC/PI assay to estimate apoptosis activity;Western blotting to detect LC3,protein relative expression;cell immunofluorescence to measure the protein expressions of Bax,Bcl-2,Beclin-1,P53,Caspase-3 m TOR and p-mTOR.Results:DEX was synergistic with Rap to induce the increase of autophagy activity of ASMCs,further increasing the ratio of LC3Ⅱ/Ⅰ protein,and down-regulating the expression of p-mTOR,but had no effect on the expression of Beclin-1 and mTOR;it could inhibit the activity and proliferation of ASMCs cells,increase the level of apoptosis,up-regulate the expression of Bax,Caspase-3 and p53,and down-regulate the expression of Bcl-2.The TCM group and combination group could significantly inhibit the autophagy of ASMCs induced by Rap,down-regulate LC3Ⅱ/Ⅰ protein ratio and Beclin-1 protein,and im-prove the decrease of cell viability and proliferation activity caused by Rap.In addition,the TCM group could in-hibit the increase of apoptosis activity,up-regulate Caspase-3 protein and down-regulate P53 protein;the combi-nation group coul
作者
王瀚
龙玉婷
马紫童
李玉曼
马再娜
于萍
刘仁慧
Wang Han;Long Yuting;Ma Zitong;Li Yuman;Ma Zaina;Yu Ping;Liu Renhui(Capital Medical University,Beijing 100069,China)
出处
《中国中医急症》
2022年第9期1354-1357,1371,共5页
Journal of Emergency in Traditional Chinese Medicine
基金
国家自然科学基金项目(81873115)
北京市自然科学基金资助项目(7202014)。
关键词
气道平滑肌细胞
自噬
凋亡
淫羊藿
女贞子
糖皮质激素
雷帕霉素
Airway smooth muscle cells
Autophagy
Apoptosis
Herba Epimedii
Fructus Ligustri Lucidi
Glucocorticoid
Rapamycin
