益气养精方对肺癌细胞Apelin/APJ信号通路的影响

摘要

Objective To explore the influence of Yiqi Yangjing prescription on cell proliferation,invasion and angiogenesis of A549 and H1975 lung cancer cells via Apelin /APJ signaling pathway. Methods Human lung cancer cell line A549 and H1975 were divided into blank control group,half maximal inhibitory concentration (IC50) of Yiqi Yangjing prescription group,Apelin-13 group and IC50 of Yiqi Yangjing prescription + Apelin-13 group. Cell invasion was tested by counting kit-8 (CCK-8) and Transwell assay. Expressions of Apelin,APJ, vascular endothelial growth factor-A(VEGF-A) ,hypoxia inducible factor-1α(HIF-1α) and Smad3 mRNA and protein were tested by real-time fluorescence quantitative polymerase chain reaction and western blotting. Results According to the test results of CKK-8,the experimental dose of Apelin-13 was 0.1 μmol /L. IC50 of Yiqi Yangjing prescription in A549 and H1975 cells was 1 g /L and 0.5 g /L,respectively. Transwell assay showed that A549 and H1975 cell amounts of lower chamber in Apelin-13 group were significantly more than those in blank control group; the cell amounts in Yiqi Yangjing group were significantly less than those in blank control group; the cell amounts in Yiqi Yangjing + Apelin-13 group were slightly more than those in blank control group. Polymerase chain reaction showed that Smad3 expression of A549 cells in Yiqi Yangjing group was significantly higher than that in blank control group; expressions of Apelin,APJ,HIF-1α,Smad3 and VEGF-A mRNA in Yiqi Yangjing group and Yiqi Yangjing + Apelin-13 group were significantly lower than those in blank control group(all P < 0.05). Western blotting showed that expressions of Apelin,APJ,phosphatidylinositol-3-kinase(PI3K) ,protein kinase B (Akt) and VEGF-A of A549 cells were significantly down-regulated and Smad3 expressed more in Yiqi Yangjing group; expressions of PI3K and Smad3 in Yiqi Yangjing + Apelin-13 group were significantly lower and APJ and VEGF-A were significantly higher than those in blank control group(all P < 0.05). Expressions of Apelin,PI3K, Akt,Smad3 and VEGF-A of H1975 cells in Yiqi Yangjing group and Yiqi Yangjing +Apelin-13 group were significantly lower and APJ was significantly higher than those in blank control group (all P < 0.05 ). Conclusion Yiqi Yangjing prescription can inhibit the proliferation and invasion of lung cancer cells, antagonize Apelin-13 promoting cell proliferation and invasion; it can also down-regulate the expression of angiogenesis factors such as VEGF,HIF-1α and PI3K through Apelin /APJ signaling pathway.%目的 探讨益气养精方对人肺癌细胞株A549和H1975中可促进细胞增殖侵袭及血管形成的Apelin /APJ信号通路的影响.方法 将人肺癌细胞株A549和H1975分为空白对照组、半数抑制浓度(IC50) 益气养精方组、Apelin-13组、IC50益气养精方+ Apelin-13组.通过细胞侵袭实验计数Transwell小室下室面的细胞数,实时荧光定量聚合酶链反应法检测Apelin、APJ、血管内皮生长因子A(VEGF-A) 、缺氧诱导因子1α(HIF-1α) 及Smad3 mRNA的表达,蛋白质印迹法检测各组相关蛋白的表达.结果 经细胞增殖-毒性检测法检测,本研究选择0.1 μmol /L的Apelin-13处理2种肺癌细胞,A549细胞和H1975细胞益气养精方IC50浓度分别为1 g /L和0.5 g /L.乏氧微环境下A549细胞与H1975细胞的Transwell细胞侵袭实验结果显示,Apelin-13组下室面的细胞数明显多于空白对照组,益气养精方组下室面的细胞数明显少于空白对照组,益气养精方+ Apelin-13组下室面的细胞数略多于空白对照组.实时荧光定量聚合酶链反应法检测结果显示,除益气养精方组A549细胞株Smad3的表达高于空白对照组外,益气养精方组和益气养精方+ Apelin-13组Apelin、APJ、HIF-1α、Smad3及VEGF-A mRNA的表达均低于空白对照组,差异均有统计学意义(均P < 0.05) .蛋白质印迹法检测结果显示,益气养精方组A549细胞株Apelin、APJ、磷脂酰肌醇3-激酶(PI3K) 、蛋白激酶B(Akt) 及VEGF-A蛋白的表达量均明显低于空白对照组,Smad3的表达明显高于空白对照组; 益气养精方+ Apelin-13组PI3K、Smad3表达低于空白对照组,APJ、VEGF-A蛋白的表达量高于空白对照组,差异均有统计学意义(均P < 0.05) .益气养精方组和益气养精方+ Apelin-13组H1975细胞株Apelin、PI3K、Akt、Smad3及VEGF-A蛋白的表达均明显低于空白对照组,而APJ表达均高于空白对照组,差异均有统计学意义(均P < 0.05) .结论 益气养精方能抑制肺癌细胞的增殖侵袭能力,并能拮抗Apelin-13对肺癌细胞增殖侵袭的促进作用; 同时能通过Apelin /APJ通路对血管生成相关因子VEGF、HIF-1α、PI3K等起到一定的下调作用.