竹节参总皂苷通过肾素-血管紧张素系统抑制肾癌细胞增殖的研究

Inhibitory effect of total Panax japonicus saponins on renal carcinoma cells through renin-angiotensin system

目的探讨竹节参总皂苷(TPJS)抑制肾癌细胞增殖的作用机制。方法培养人肾癌ACHN和A498细胞,采用不同浓度的TPJS处理ACHN和A498细胞,观察细胞形态学变化,分析肾癌细胞生长情况。采用菌落形成试验测定形成的菌落数量;采用发光仪测定Caspase Glo 3/7活性;采用乳酸脱氢酶细胞毒性检测试剂盒评估TPJS的细胞毒性;采用细胞计数试剂盒检测TPJS对肾癌细胞增殖的影响;采用异硫氰酸荧光素Annexin V凋亡检测试剂盒进行凋亡检测;采用Western Blot检测凋亡相关蛋白Caspase 9、B细胞淋巴瘤2(Bcl-2)和Bcl-2样蛋白4(Bax)在肾癌细胞中的表达;采用酶联免疫吸附试验(ELISA)测定总蛋白提取液中血管紧张素Ⅱ(AngⅡ)、AngⅡ1型受体(AT1R)、血管内皮生长因子(VEGF)和环氧化酶2(COX-2)的浓度,分析AngⅡ、AT1R、VEGF和COX-2对TPJS诱导的细胞生长抑制和凋亡的影响。结果细胞形态学结果显示,未经处理的肾癌细胞生长良好,经TPJS处理的细胞形态扭曲、变圆并发生凋亡。菌落形成试验结果显示,与0μmol/L TPJS处理的细胞相比,TPJS治疗6 h后菌落形成显著减少,且TPJS处理后显著增强了细胞毒性和Caspase 3/7活性。流式细胞术检测结果显示,TPJS治疗24 h后可诱导肾癌细胞凋亡,并且TPJS可以上调Caspase 9和Bax的水平,下调Bcl-2的水平。与采用0μmol/L TPJS处理的细胞相比,ACHN和A498细胞系经TPJS治疗后呈剂量依赖性的方式显著降低了AngⅡ、AT1R、VEGF和COX-2的水平。AT1R和VEGF可逆转TPJS对细胞生长的抑制作用,而且细胞凋亡试验结果与细胞生长结果相似。结论TPJS可能通过AT1R/VEGF途径抑制肾癌细胞增殖并诱导凋亡。

Objective To explore the mechanism of total Panax japonicus saponins(TPJS) in inhibiting the proliferation of renal carcinoma cells. Methods ACHN and A498 cells were cultured and treated with different concentrations of TPJS, the growth of ACHN and A498 cells was analyzed by cell morphology. The number of colonies formed was determined by colony formation test;the activity of Caspase Glo 3/7 was determined by luminescence meter;the cytotoxicity of TPJS was evaluated by the lactate dehydrogenase cytotoxicity test kit;cell counting kit was used to detect the effect of TPJS on the proliferation of renal cell carcinoma cells;apoptosis was detected with fluorescein isothiocyanate annexin V apoptosis detection kit;the western blot was used to detect the expression of apoptosis related protein Caspase 9, B cell lymphoma 2(Bcl-2) and Bcl-2 like protein 4(Bax) in renal cell carcinoma cells;the concentrations of angiotensin Ⅱ(AngⅡ), AngⅡ type 1 receptor(AT1 R), vascular endothelial growth factor(VEGF) and cyclooxygenase-2(COX-2) in total protein extract were measured by enzyme-linked immunosorbent assay(ELISA), and the effects of AngⅡ, AT1 R, VEGF and COX-2 on TPJS-induced cell growth inhibition and apoptosis were analyzed. Results The result of morphological analysis showed that the untreated renal carcinoma cells grew well, while the cells treated with TPJS were distorted, rounded and apoptotic;the results of colony formation test showed that compared with the cells treated with 0 μmol/L TPJS,the colony formation decreased significantly after 6 hours of TPJS treatment,and TPJS treatment significantly increased cytotoxicity and Caspase 3/7 activity. The results of flow cytometry showed that TPJS was able to induce apoptosis of renal cell carcinoma cells after 24 hours of treatment,in addition,TPJS was able to up-regulate the levels of Caspase 9 and Bax,and down-regulate the level of Bcl-2. Compared with the cells treated with 0 μmol/L TPJS,the levels of AngII,AT1R,VEGF and COX-2 in both ACHN and A498 cell lines were significantly reduced in a dose-dependent manner after TPJS treatment. AT1R and VEGF were able to reverse the inhibitory effect of TPJS on cell growth,and the results of apoptosis test were similar to those of cell growth. Conclusion TPJS may inhibit the proliferation and induce apoptosis of renal cell carcinoma cells through AT1R/VEGF pathway.