抑肺饮抑制肺癌转移的机制研究

Study on Mechanism of Yifei Yin Inhibiting Lung Cancer Metastasis

【目的】观察抑肺饮对肺癌细胞肿瘤坏死因子α(TNF-α)-核转录因子kappaB(NF-κB)信号通路以及细胞上皮间质转化(EMT)的影响,探讨抑肺饮对肺癌细胞生长和转移的抑制作用及机制。【方法】制备抑肺饮含药血清,通过四甲基偶氮唑盐(MTT)实验、侵袭实验和迁移实验观察抑肺饮对肺癌细胞A549增殖、侵袭和迁移能力的影响。以TNF-α诱导A549细胞建立细胞EMT模型,采用荧光定量聚合酶链反应(PCR)法观察抑肺饮对细胞EMT的影响,采用蛋白免疫印迹(Western Blot)法观察抑肺饮对A549细胞中EMT标志物蛋白表达水平及NF-κB p65磷酸化水平的影响,采用荧光素酶报告基因实验观察抑肺饮对NF-κB启动子活性的影响。建立Lewis肺癌移植瘤小鼠模型,称瘤体质量,观察肺脏转移灶情况,采用免疫组织化学法检测瘤组织磷酸化NF-κB p65的表达水平,荧光定量PCR法检测瘤组织B细胞淋巴瘤-xL(Bcl-xL)、细胞周期蛋白D1(CyclinD1)的mRNA表达水平。【结果】(1)抑肺饮含药血清能够抑制肺癌A549细胞的增殖、侵袭、迁移能力;(2)抑肺饮含药血清能够上调上皮细胞标志物E-钙黏蛋白(E-cadherin)的表达水平,下调间质细胞标志物波形蛋白(Vimentin)的表达水平,抑制NF-κB p65磷酸化水平和NF-κB启动子活性;(3)体内研究结果显示,抑肺饮高剂量组的瘤质量和肺转移率,瘤组织中NF-κB p65磷酸化表达水平,靶基因Bcl-xL、CyclinD1的mRNA表达水平显著低于模型组,且抑肺饮联合顺铂使用时抑制效果最佳。【结论】抑肺饮可能通过干预TNF-α-NF-κB信号通路,调节细胞EMT,从而发挥抑制肺癌细胞增殖和转移的作用。

Objective To observe the effects of Yifei Yin on tumor necrosis factorα(TNF-α)-nuclear transcription factor-kappaB(NF-κB)signaling pathway and epithelial mesenchymal transition(EMT)in lung cancer cells,and to explore the inhibitory effect of Yifei Yin on growth and metastasis of lung cancer cells and its mechanism.Methods The Yifei Yin drug-containing serum was prepared,and the effect of Yifei Yin on the proliferation,invasion and migration of lung cancer A549 cells was detected by methyl thiazolyl tetrazolium(MTT)assay,invasion assay and migration assay,respectively.A549 cells were induced by TNF-αinto EMT model.Fluorescence quantitative polymerase chain reaction(PCR)was used to observe the effect of Yifei Yin on cellular EMT,Western blotting assay was used to observe the effect of Yifei Yin on the expression levels of EMT marker proteins and phosphorylation level of NF-κB p65 in A549 cells,and the luciferase reporter assay was used to observe the effect of Yifei Yin on the activity of NF-κB promoter.The Lewis lung cancer tumor transplantation mouse model was established,and the tumor mass was measured,the lung metastasis was observed,the expression level of phosphorylated NF-κB p65 in tumor tissue was detected by immunohistochemistry,and the mRNA expression levels of B-cell lymphoma-xL(Bcl-xL)and cyclin D1(CyclinD1)in tumor tissue were detected by fluorescence quantitative PCR.Results(1)Yifei Yin serum inhibited the proliferation,invasion and migration of lung cancer A549 cells;(2)Yifei Yin-containing serum up-regulated the expression level of epithelial marker E-cadherin,down-regulated the expression level of mesenchymal marker Vimentin,and inhibited the phosphorylation level of NF-κB p65 and the activity of NF-κB promoter.(3)In vivo experimental results showed that the tumor body mass and lung metastasis rate,phosphorylation level of NF-κB p65 in tumor tissue,and mRNA expression levels of target gene Bcl-xL and CyclinD1 in high-dose Yifei Yin group were significantly lower than those in the model group,and the inhibition effect of Yifei Yin combined with cisplatin was the best.Conclusion Yifei Yin plays a role in inhibiting the proliferation and metastasis of lung cancer cells by intervening TNF-α-NF-κB signaling pathway thus regulating EMT.