摘要
目的探讨淫羊藿苷激活Ca2+/钙调蛋白依赖性的蛋白激酶Ⅱ(CaMKⅡ)-c-Jun氨基末端激酶(JNK)通路抑制人非小细胞肺癌A549细胞存活和转移特性的作用及其机制。方法采用不同浓度(0、0.1、0.25、0.5、1、2.5、5、10、20、50、100、200、300和400μmol)淫羊藿苷作用于人非小细胞肺癌A549细胞24 h后,应用CCK8法检测细胞存活率,Transwell检测细胞侵袭能力,划痕实验检测细胞迁移能力,免疫荧光法检测vimentin表达情况,Western blot法检测血管内皮生长因子(VEGF)、E-cadherin和N-cadherin的表达情况以及不同浓度(10、20、50μmol)淫羊藿苷处理后Ca2+/钙调蛋白依赖性的蛋白激酶Ⅱ(CaMKⅡ)和c-Jun氨基末端激酶(JNK)的磷酸化情况和单独或联合作用以10μmol JNK抑制剂SP600125对JNK磷酸化的影响。结果与0μmol淫羊藿苷组比较,100、200、300和400μmol淫羊藿苷组A549细胞存活率[分别为(72±8)%、(57±7)%、(48±6)%、(38±5)%]均降低(均P<0.01);与对照组比较,10、20、50μmol淫羊藿组侵袭细胞数量[分别为(84±12)、(30±8)、(10±5)个]均减少(均P<0.01),划痕愈合率[分别为(84±7)%、(70±6)%、(45±6)%]均降低(均P<0.01),VEGF[分别为(0.40±0.07)、(0.16±0.04)、(0.05±0.03)]、N-cadherin[分别为(0.70±0.08)、(0.26±0.05)、(0.08±0.04)]和vimentin[分别为(25.5±2.9)、(12.1±2.6)、(5.3±2.0)]表达均降低(均P<0.05),E-cadherin[分别为(0.13±0.05)、(0.44±0.06)、(0.95±0.08)]、p-CaMKⅡ/CaMKⅡ[分别为(0.16±0.05)、(0.29±0.07)、(0.42±0.07)]和p-JNK/JNK[分别为(0.30±0.06)、(0.46±0.08)、(0.84±0.09)]表达均增加(均P<0.05);且10μmol淫羊藿苷能减弱10μmol SP600125对JNK磷酸化的抑制作用。结论淫羊藿苷能上调CaMKⅡ、JNK磷酸化水平激活该通路来抑制人非小细胞肺癌A549细胞的存活和转移。
Objective To investigate inhibitive effect of icariin on survival and metastasis of human non-small cell lung cancer A549 cells via activating Ca2+/calmodulin-dependent protein kinaseⅡ(CaMKⅡ)-c-Jun amino terminal kinase(JNK)pathway and the mechanisms of the effect.Methods The A549 cells were administrated with icariin at different doses of 0,0.10,0.25,0.50,1.0,2.5,5.0,10,20,50,100,200,300 and 400μmol for 24 hours.Then,the survival of the A549 cells was detected with Cell Counting Kit-8(CCK8)and the invasion and migration capability the A549 cells were determined with Transwell assay and scratch assay.Immunofluorescence assay was used to measure vimentin expression.Western blot was adopted to detect expressions of vascular endothelial growth factor(VEGF),E-cadherin,N-cadherin,and the phosphorylation of Ca2+/calmodulin-dependent protein kinaseⅡ(CaMKⅡ)and c-Jun amino terminal kinase(JNK)in the A549 cells treated with different concentrations(10,20,50μmol)of icariin alone or combined with 10μmol JNK inhibitor SP600125.Results Significantly lower survival rates of 72±8%,57±7%,48±6%,and 38±5%were detected for the A549 cells treated with 100,200,300,and 400μmol icariin compared to that for the cells without incariin treatment(all P<0.01).Significantly decreased number of invasive cells(84±12,30±8,10±5;all P<0.01),rate of scratch healing(84±7%,70±6%,45±6%;all P<0.01),and expressions of VEGF(0.40±0.07,0.16±0.04,0.05±0.03),N-cadherin(0.70±0.08,0.26±0.05,0.08±0.04)and vimentin(25.5±2.9,12.1±2.6,5.3±2.0)(P<0.05 for all)but significantly increased expression of E-cadherin(0.13±0.05,0.44±0.06,0.95±0.08)and ratios of phosphorylated CaMKⅡ/CaMKⅡ(0.16±0.05,0.29±0.07,0.42±0.07)and phosphorylated JNK/JNK(0.30±0.06,0.46±0.08,0.84±0.09)(P<0.05 for all)were detected in the A549 cells treated with 10,20 and 50μmol icariin in comparison with those detected in the cells without incariin treatment.In addition,10μmol icariin alleviated the inhibition of 10μmol SP600125 on JNK phosphorylation in A549 cells.Conclusion Icariin can up-regulate the phosphorylation of CaMKⅡand JNK and activate the pathway to inhibit the survival and metastasis of human non-small cell lung cancer A549 cells.
作者
杨全中
张煜
王亚娟
程远芳
YANG Quan-zhong;ZHANG Yu;WANG Ya-juan(School of Basic Medical Science,Sanquan College of Xinxiang Medical University,Xinxiang,Henan Province 453003,China;不详)
出处
《中国公共卫生》
CAS
CSCD
北大核心
2020年第7期1014-1019,共6页
Chinese Journal of Public Health
基金
河南省教育厅2018年度人文社会科学研究一般项目(2018–ZZJH–430)
新乡医学院三全学院2017年度科技创新。
