摘要
目的探讨吉祥草中纤细薯蓣皂苷(gracillin)诱导人非小细胞肺癌A549细胞自噬的作用及机制。方法以A549细胞为对象,采用CCK-8法检测不同浓度(0.25、0.5、1、2、4μmol/L)gracillin作用不同时间(12、24、48 h)对细胞增殖的影响。与不加药物的对照组进行比较,采用生物透射电子显微镜观察gracillin(2μmol/L)作用24 h对细胞自噬小体形成的影响;通过GFP-LC3质粒转染实验检测gracillin(0.25、0.5、1、2μmol/L)作用24 h后GFP-LC3在细胞自噬小体膜上的聚集情况;采用实时定量聚合酶链式反应法和Western blot法检测gracillin(0.25、0.5、1、2μmol/L)作用24 h后A549细胞中序列相似性家族102成员A(FAM102A)mRNA和蛋白的表达水平,以及自噬相关蛋白[p62、Beclin-1、微管相关蛋白1轻链3B(LC3B)]和磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(又名Akt)信号通路相关蛋白的表达水平。结果gracillin对A549细胞具有一定的增殖抑制作用,且呈浓度和时间依赖趋势;其作用24 h的半数抑制浓度为2.55μmol/L。gracillin作用24 h后,细胞胞浆内可见含双层膜结构的自噬小体;自噬小体膜上可见明显的GFP-LC3绿色荧光斑点,且有随药物浓度升高而增多的趋势。与对照组比较,gracillin不同浓度组细胞中FAM102A mRNA和蛋白的表达水平(0.5、1、2μmol/L组)、Beclin-1蛋白的表达水平(1、2μmol/L组)和LC3B-Ⅱ/LC3B-Ⅰ比值(2μmol/L组)均显著升高,p62蛋白的表达水平(1、2μmol/L组)和Akt蛋白(1、2μmol/L组)、PI3K蛋白(2μmol/L组)的磷酸化水平均显著降低(P<0.05或P<0.01)。结论gracillin可能通过上调FAM102AmRNA和蛋白的表达、抑制PI3K/Akt信号通路来促进A549细胞发生自噬,从而发挥抑制细胞增殖的作用。
OBJECTIVE To investigate the effect and mechanism of gracillin from Reineckia carnea on autophagy in non-small cell lung cancer A549 cells.METHODS Using A549 cells as subjects,the effects of different concentrations of gracillin(0.25,0.5,1,2,4μmol/L)on the proliferation of cells were detected by CCK-8 after being treated for different time(12,24,48 h).Compared with the control group without medication,the effect of gracillin(2μmol/L)on the formation of autophagosomes in cells was observed by transmission electron microscope after 24 h of exposure.The aggregation of GFP-LC3 on autophagosome membrane was detected by GFP-LC3 plasmid transfection after being treated with gracillin(0.25,0.5,1,2μmol/L)for 24 h.Quantitative real-time PCR and Western blot assay were used to detect the mRNA and protein expressions of family with sequence similarity 102 member A(FAM102A),the expressions of autophagy-related proteins[p62,Beclin-1,microtubule-associated protein 1 light chain 3B(LC3B)],and the expressions of phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway-related proteins in A549 cells after being treated with gracillin(0.25,0.5,1 and 2μmol/L)for 24 h.RESULTS Gracillin significantly inhibited the proliferation of A549 cells in a concentration-and time-dependent manner.The IC50 was 2.55μmol/L at 24 h.After 24 h of gracillin treatment,autophagosomes with bilayer membrane structure were found in the cell cytoplasm,and GFP-LC3 green fluorescent spots on autophagosome membrane were obvious,representing an increasing trend as drug concentration.Compared with the control group,mRNA and protein expressions of FAM102A(0.5,1,2μmol/L groups),protein expression of Beclin-1(1,2μmol/L groups)and LC3B-Ⅱ/LC3B-Ⅰratio(2μmol/L group)were significantly increased in different concentrations of gracillin groups,while the protein expression of p62(1,2μmol/L groups),and the protein phosphorylations of Akt(1,2μmol/L groups)and PI3K(2μmol/L group)were all decreased significantly(P<0.05 or P<0.01).CONCLUSIONS Gracillin can promote excessive autophagy in A549 cells by up-regulating mRNA and protein expressions of FAM102A and inhibiting PI3K/Akt signaling pathway,thus inhibiting cell proliferation.
作者
李燕
李亚梅
雷歌燕
康佳兰
刘明轩
张敏鸿
杨建琼
LI Yan;LI Yamei;LEI Geyan;KANG Jialan;LIU Mingxuan;ZHANG Minhong;YANG Jianqiong(Dept.of Pharmacy,the First Affiliated Hospital of Gannan Medical University,Jiangxi Ganzhou 341000,China;Clinical Medicine Research Center,the First Affiliated Hospital of Gannan Medical University,Jiangxi Ganzhou 341000,China;Dept.of Pharmacy,Taihe People’s Hospital,Jiangxi Ji’an 343700,China)
出处
《中国药房》
CAS
北大核心
2024年第8期912-917,共6页
China Pharmacy
基金
国家自然科学基金项目(No.82260985)
江西省卫生健康委科技计划项目(No.202210939)
江西省教育厅科学技术研究项目(No.GJJ2201459)
赣州市科技计划重点实验室项目(No.2022DSYS9969)。
