基于p53/AMPK信号通路观察益气扶正解毒汤对A549细胞自噬水平和生长的影响  

Effect of Yiqi Fuzheng Jiedu Decoction on Autophagy and Growth of A549 Cells:An Exploration Based on p53/AMPK Signaling Pathway

作　　者：吴俏兰 宋婷[2] 陈泽涛[2] 陈修保[2] 张宜晨 陈维达[2] WU Qiao-lan;SONG Ting;CHEN Ze-tao;CHEN Xiu-bao;ZHANG Yi-chen;CHEN Wei-da(First Clinical Medical College,Shandong University of Traditional Chinese Medicine(TCM),Jinan 250014,China;Affiliated Hospital of Shandong University of TCM,Jinan 250014,China)

机构地区：[1]山东中医药大学第一临床医学院,济南250014 [2]山东中医药大学附属医院,济南250014

出　　处：《中国实验方剂学杂志》2021年第22期65-75,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(82004281)。

摘　　要：目的:探讨益气扶正解毒汤对A549细胞自噬水平和生长的作用及机制。方法:制备益气扶正解毒汤含药血清干预A549肺癌细胞。蛋白免疫印迹法(Western blot)检测A549细胞中自噬蛋白微管相关蛋白1轻链3(LC3),自噬关键分子酵母Atg6同系物1(Beclin1),p62,p53蛋白和腺苷酸活化蛋白激酶(AMPK),磷酸化AMPK(p-AMPK),哺乳动物雷帕霉素靶蛋白(mTOR)和磷酸化mTOR(p-mTOR)蛋白的水平,免疫荧光(IF)检测微管相关蛋白1A/1B轻链3B(MAP1LC3B)蛋白水平,5-乙炔基-2′脱氧尿嘧啶核苷(EDU)染色、侵袭实验(Transwell)和β-半乳糖苷酶染色分别检测含药血清对细胞增殖、侵袭、衰老情况的影响;设置自噬抑制剂甲基腺嘌呤(3-MA,5 mmol·L^(-1))干预组,即分为10%胎牛血清组(空白组),10%正常血清组(正常组),10%含药血清低、中、高剂量组,10%高剂量含药血清加3-MA(高剂量+3-MA)组,检测各组细胞增殖、侵袭、衰老水平;并设置p53抑制剂(PFT-α,10μmol·L^(-1))组,即分为正常组,PFT-α组,高剂量组,高剂量+PFT-α组,检测自噬相关蛋白LC3-Ⅱ,Beclin1表达水平和MAP1LC3B免疫荧光强度及各组细胞增殖、侵袭、衰老的情况。结果:与空白组、正常组比较,干预A549细胞48 h,益气扶正解毒汤中、高剂量组LC3-Ⅱ和Beclin1蛋白水平呈剂量依赖性升高(P<0.01);益气扶正解毒汤低、中、高剂量组p62,p-mTOR/mTOR蛋白下降(P<0.05,P<0.01),p53,p-AMPK/AMPK蛋白表达升高(P<0.01);益气扶正解毒汤低、中、高剂量组A549细胞增殖、侵袭数量明显降低,细胞衰老数量显著增多(P<0.01),同时MAP1LC3B免疫荧光强度增强,且益气扶正解毒汤高剂量组效果最佳;与益气扶正解毒汤高剂量组比较,益气扶正解毒汤高剂量+3-MA组、益气扶正解毒汤高剂量+PFT-α组发生增殖、侵袭的细胞数量均增加,而发生衰老的细胞数量明显减少(P<0.05,P<0.01),同时,高剂量+PFT-α组的MAP1LC3B免疫荧光强度减弱及自噬相关蛋白LC3-ⅡObjective: To investigate the effect and mechanism of Yiqi Fuzheng Jiedu decoction(YQFZJDD)on autophagy and growth of A549 cells. Method:A549 cells were intervened with YQFZJDDcontaining serum prepared in advance. The levels of microtubule-associated protein 1 light chain 3(LC3),homologue of yeast autophagy-related gene 6(Beclin1),p62,p53,adenosine 5’-monophosphate-activated protein kinase(AMPK),phosphorylated AMPK(p-AMPK),mammalian target of rapamycin(mTOR),and phosphorylated mTOR(p-mTOR)were detected by Western blot assay,and microtubule-associated protein 1 A/1 B light chain 3 B(MAP1 LC3 B) by immunofluorescence(IF) assay. The proliferation, invasion, and senescence of A549 cells were separately measured by 5-ethynyl-2’-deoxyuridine(EDU)staining,Transwell assay, and β-galactosidase staining. In the presence of autophagy inhibitor 3-methyladenine(3-MA,5 mmol·L^(-1)),the cells were divided into the 10% fetal bovine serum(blank)group,10% control serum(control)group,low-,medium-,and high-dose 10% YQFZJDD-containing serum groups,and high-dose 10%YQFZJDD-containing serum + 3-MA group,followed by the measurement of A549 cell proliferation,invasion,and senescence. In the adoption of p53 inhibitor Pifithrin-α(PFT-α,10 μmol·L^(-1)),the cells were divided into the control group,PFT-α group,high-dose YQFZJDD-containing serum group,and high-dose YQFZJDDcontaining serum + PFT-α group. Then the LC3-Ⅱ and Beclin1 expression,MAP1 LC3 B fluorescence intensity,as well as A549 cell proliferation,invasion and senescence were determined. Result: Compared with the blank group and control group,YQFZJDD-containing serum at the medium and high doses up-regulated the protein expression levels of LC3-Ⅱ and Beclin1 in A549 cells after 48-h intervention in a dose-dependent manner(P<0.01). Besides,YQFZJDD-containing serum at the low-,medium-,and high-doses down-regulated p62 and p-mTOR/mTOR expression(P<0.05,P<0.01),elevated p53 and p-AMPK/AMPK(P<0.01),decreased the number of proliferative and invasive cells,increased the nu

关 键 词：益气扶正解毒汤 A549细胞 自噬 p53/腺苷酸活化蛋白激酶(AMPK)通路 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学]