人参皂苷Rd抑制人非小细胞肺癌A549细胞的生长及作用机制

Inhibitory effect of ginsenoside Rd on the growth of non-small cell lung cancer A549 cells and its mechanism

目的探讨人参皂苷Rd(GS-Rd)对人非小细胞肺癌(NSCLC)细胞株A549细胞增殖、凋亡、侵袭和周期的影响及可能机制。方法采用MTT法,观察不同浓度(12.5、25、50、100μmol/L)GS-Rd体外作用NSCLC细胞系A549不同时间后对其细胞活力的影响;利用高内涵分析软件,观察不同浓度、不同时间作用于A549细胞系的荧光强度以评估其对细胞凋亡的影响;采用Transwell法观察不同浓度GS-Rd对A549细胞侵袭能力的影响;利用流式细胞仪检测A549细胞周期的阻滞效应;Western blotting检测经GS-Rd处理后A549细胞周期相关蛋白的表达情况。结果MTT法结果显示,12.5、25、50、100μmol/L GS-Rd处理A549细胞后的24 h增殖抑制率分别为3.54%、18.25%、26.58%和32.38%,48 h增殖抑制率分别为21.91%、27.15%、40.37%和46.44%。随着药物浓度的增加,其增殖抑制率也随之增加,呈剂量-时间依赖性(P<0.05)。高内涵分析显示,GS-Rd对细胞亦具有明显的促凋亡作用,且呈剂量-时间依赖性。Transwell小室结果显示,12.5、25、50μmol/LGS-Rd作用A549细胞后的穿膜细胞数分别为(78.2±16.32)个、(57.0±10.37)个和(42.8±15.98)个,空白组为(89.8±12.87)个。与空白组比较,GS-Rd组的细胞侵袭数目明显减少,并呈浓度依赖性(P<0.05)。流式细胞仪检测结果显示,与空白组比较,GS-Rd作用A549细胞24 h后,出现G_(0)/G_(1)期细胞比例逐渐上升,而S期细胞比例逐渐下降,细胞周期阻滞于G_(0)/G_(1)期。Western blotting检测结果显示,磷酸化Akt表达下降,且p21、p27表达上升。结论GS-Rd可直接抑制NSCLC细胞系A549的增殖,亦可通过阻滞细胞周期于G_(1)期抑制细胞增殖,且降低了A549细胞系的侵袭能力。

Objective To explore the cell proliferation,apoptosis,invasion and the cell cycle of human non-small cell lung cancer(NSCLC)cell line A549 which was treated by ginsenoside Rd(GS-Rd)in vitro.Methods MTT method was used to observe the effect of GS-Rd on the cell viability of NSCLC cell line A549 in vitro.High content analysis software was used to observe the fluorescence intensity of A549 cell line at different concentrations and times to evaluate the apoptosis of A549 cells.Transwell invasion assay was used to observe the invasion capability.Flow cytometry method was used to observe the cell cycle induced by different concentrations of GS-Rd in A549 cells.Western blotting was used to detect the expression of A549 cycle-related proteins after GS-Rd treatment.Results MTT assay showed that the 24 h proliferation inhibition rates of A549 cells treated with 12.5,25,50 and 100μmol/L GS-Rd were 3.54%,18.25%,26.58%and 32.38%respectively,and the 48 h proliferation inhibition rates were 21.91%,27.15%,40.37%and 46.44%respectively.The inhibition effect also increased with increasing concentration in a dose-to time-dependent manner(P<0.05).The high content analysis showed that GS-Rd also had significant pro-apoptotic effect on cells,presenting time-effect and dose-effect relationship.The number of membrane cells of 12.5,25,50μmol/L GS-Rd was 78.2±16.32,57.0±10.37 and 42.8±15.98 respectively,which lower than 89.8±12.87 of the blank group(P<0.05).Compared with the blank group,the invasion rate of GS-Rd decreased significantly in a concentration dependent manner(P<0.05).Flow cytometry showed that compared with the blank group,the proportion of cells in G_(0)/G_(1) phase gradually increased after GS-Rd treated A549 cells for 24 hours,while the proportion of cells in S phase gradually decreased,and the cell cycle was arrested at G_(0)/G_(1) phase.Western blotting showed that the expression of phosphorylated AKT decreased and p21,p27 expression increased.Conclusion GS-Rd can directly inhibit the proliferation of the NSCLC cell line A549,and it can also inhibit the cell proliferation by blocking the cell cycle in the G_(1) phase.In addition,the invasive capacity of the A549 cell line could also be reduced.