乏氧环境下益气养精方对肺癌细胞apelin分子表达的影响

Effects of Enhancing Qi and Nourishing Essence Formula on the Expression of Apelin in Lung Adenocarcinoma Cells under Hypoxic Condition

目的观察益气养精方对乏氧微环境下肺癌细胞中具有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 CoCl2加入培养基模拟乏氧微环境,实验分组为乏氧组、常氧益气养精方组、乏氧益气养精方组及空白对照组,分别作用于人肺癌细胞株A549和H1975。采用CCK-8法和Transwell实验观察各组细胞的增殖、侵袭能力,RT-qPCR、Western Blot方法观察apelin/APJ信号通路相关因子VEGF、HIF-1α、PI3K等在各组干预细胞中的mRNA和蛋白表达。结果 CCK-8实验测得,缺氧环境能促进肺癌细胞增殖,益气养精方作用于A549、H1975的IC50分别为为1 mg·mL^-1和0.5 mg·mL^-1,且在常氧和缺氧微环境中均能抑制肺癌细胞增殖。Transwell实验结果显示,益气养精方组下室面细胞数量明显少于对照组(P <0.01),乏氧微环境中下室面细胞数明显多于对照组(P <0.01),加入益气养精方后亦可见减少。RT-qPCR结果显示,益气养精方在乏氧微环境中能不同程度下调血管生成相关因子HIF-1α、VEGF、APJ及Apelin的表达(P <0.05)。Western Blot检测发现,益气养精方在乏氧环境中能使相关因子(VEGF、PI3K、APJ)的表达量明显下调,具有统计学意义。结论益气养精方在乏氧微环境中能抑制肺癌细胞的增殖和侵袭,同时能通过Apelin/APJ通路对血管生成相关因子VEGF、HIF-1a、PI3K等起到一定的下调作用。

Objective To observe the effect of enhancing qi and nourishing essence formula on cell proliferation, invasion and angiogenesis of lung adenocarcinoma cells under hypoxic condition via Apelin/APJ signaling pathway. Methods CoCl2 working fluid was used to produce hypoxic microenvironment. Human lung cancer cell lines A549 and H1975 were treated with hypoxic, enhancing qi and nourishing essence formula, hypoxia enhancing qi and nourishing essence formula and control group respectively. The effect of each group on cell proliferation and invasion was observed by CCK-8 assay and Transwell assay. The expression of angiogenesis-related factors, such as VEGF, HIF-1α and PI3 K, was observed by RT-qPCR experiment and Western Blotting assay. Results CCK-8 assay showed that hypoxic environment could promote the proliferation of lung cancer cells. The IC50 of Enhance Qi Nourish Jing formula was determined to be about 1 mg/ml for A549 cells, and 0.5 mg/ml for H1975 cells. The formula can inhibit the proliferation of lung cancer cells in no matter normal or hypoxic microenvironment. Transwell results showed that the number of cells in the lower chamber of the enhancing qi and nourishing essence formula group was significantly lower than that in the control group(P < 0.01), and the number of cells in the hypoxic microenvironment was significantly higher than that in the control group(P < 0.01), the reduction can also been observed after treated with the formula. The results of RT-qPCR showed that enhancing qi and nourishing essence formula could down-regulate the expression of angiogenesis-related factors HIF-1α, VEGF, APJ and Apelin in the hypoxic microenvironment(P < 0.05). Western Blot analysis showed that enhancing qi and nourishing essence formula can significantly down-regulate the expression of related factors(VEGF,PI3 K, APJ) in a hypoxic environment, with statistically significant. Conclusion Enhancing qi and nourishing essence formula can inhibit the proliferation and invasion of lung cancer cells in hypoxic microenvironment. At the same time, it can down-regulate the angiogenesis-related factors VEGF, HIF-1 a and PI3 K through Apelin/APJ pathway.