黄芪-白花蛇舌草抑制肺癌A549细胞增殖机制

Mechanism of Scutellariae Radix-Hedyotidis Herba Against Proliferation of Lung Adenocarcinoma A549 Cells

目的:从细胞水平观察黄芪-白花蛇舌草对人肺腺癌A549细胞增殖及白细胞介素-6(IL-6),磷脂酰肌醇3-激酶(PI3K),蛋白激酶B(Akt),磷酸化蛋白激酶B(p-Akt),雷帕霉素靶蛋白(m TOR),缺氧诱导因子-1α(HIF-1α),细胞周期蛋白D_(1)(Cyclin D_(1))表达量的影响,并探讨其作用的分子机制。方法:设立空白组(完全培养基),黄芪-白花蛇舌草低、中、高质量浓度组(20,40,60 mg·L^(-1)),顺铂低、中、高质量浓度组(5,10,20 mg·L^(-1)),采用噻唑蓝(MTT)比色法检测黄芪-白花蛇舌草(0,20,40,60 mg·L^(-1))和顺铂(0,5,10,20 mg·L^(-1))干预A549细胞24,48,72 h后的细胞活性,计算各组细胞增殖能力,筛选出各药物组最佳浓度;分别用完全培养基,黄芪-白花蛇舌草(40 mg·L^(-1)),顺铂(10 mg·L^(-1)),联合药物(40 mg·L^(-1)+10 mg·L^(-1))干预肺腺癌A549细胞,设置为空白组、黄芪-白花蛇舌草组、顺铂组、联合组,计算各组在24,48,72 h后对A549细胞增殖抑制影响;采用酶联免疫吸附测定(ELISA)检测各组72 h后细胞培养上清中IL-6的水平;实时荧光定量聚合酶链式反应(Real-time PCR)检测HIF-1α,Cyclin D_(1)m RNA水平;蛋白免疫印迹法(Western blot)检测各组蛋白PI3K,Akt,p-Akt,m TOR,HIF-1α,Cyclin D_(1)表达的变化情况。结果:在作用细胞24 h后,各黄芪-白花蛇舌草组对细胞增殖未表现出明显抑制,作用48 h,与空白组比较,黄芪-白花蛇舌草组对肿瘤细胞有明显增殖抑制作用(P<0.05),表现出时间依赖性,在作用细胞72 h,黄芪-白花蛇舌草各浓度组未表现出明显差异,故选用40 mg·L^(-1)为中药最佳浓度组,用于后续实验。与空白组比较,经过不同浓度顺铂干预后,细胞增殖均受到不同程度抑制(P<0.05),表现出明显的时间依赖性,基于细胞存活率考虑,选用顺铂10 mg·L^(-1)为最佳浓度。与空白组比较,黄芪-白花蛇舌草与顺铂联合应用明显抑制肺腺癌A549细胞增殖,增殖抑制率表现出明显的时间依赖性(P<0.05),且药物作用72 h,联合组与其他两组比较差异更加显著(P<0.01);与空白组比较,各用药组细胞培养液IL-6分泌显著降低(P<0.01),以联合组下降更加突出。与空白组比较,黄芪-白花蛇舌草、顺铂均能降低HIF-1α,Cyclin D_(1)m RNA水平(P<0.05,P<0.01),且联合用药降低幅度更加明显;各用药组PI3K,p-Akt,m TOR,HIF-1α,Cyclin D_(1)蛋白均明显降低(P<0.05,P<0.01),且联合组各蛋白表达量明显低于顺铂组(P<0.01)。结论:黄芪-白花蛇舌草对肺癌A549细胞增殖具有抑制作用,潜在机制可能与其抑制IL-6/PI3K/Akt/m TOR-HIF-1α轴的表达与分泌有关。

Objective:To observe the effects of Scutellariae Radix-Hedyotidis Herba on the proliferation of human lung adenocarcinoma A549 cells and the expression of interleukin-6(IL-6),phosphatidylinositol3-kinase(PI3K),protein kinase B(Akt),p-protein kinase B(p-Akt),mechanistic target of rapamycin(m TOR),hypoxia-inducible factor-1α(HIF-1α),and Cyclin D_(1)at the cellular level,and to explore their molecular mechanism.Method:Following the set-up of the blank group(complete medium),low-,moderate-,and high-dose(20,40,and 60 mg·L^(-1))Scutellariae Radix-Hedyotidis Herba groups,and low-,moderate-,and high-dose(5,10,and 20 mg·L^(-1))cisplatin groups,the cell were treated with the corresponding drugs for 24,48,and 72 h for detecting their viability by tetrazolium bromide(MTT)colorimetry.A549 cells were then divided into the blank group,Scutellariae Radix-Hedyotidis Herba group,cisplatin group,and combined medication group and intervened with the complete medium,40 mg·L^(-1)Scutellariae Radix-Hedyotidis Herba,10 mg·L^(-1)cisplatin,and 40 mg·L^(-1)Scutellariae Radix-Hedyotidis Herba+10 mg·L^(-1)cisplatin,respectively,for24,48 and 72 h,followed by the measurement of inhibitory effects against the proliferation of A549 cells in each experimental group.The level of IL-6 in cell culture supernatant was determined by enzyme-linked immunosorbent assay(ELISA)after 72 h.The m RNA expression levels of HIF-1αand Cyclin D_(1)in each group were assayed by real-time polymerase chain reaction(Real-time PCR),and the protein expression levels of PI3K,Akt,p-Akt,m TOR,HIF-1α,and Cyclin D_(1)by Western blot.Result:After 24 h intervention,Scutellariae Radix-Hedyotidis Herba did not significantly inhibit the proliferation of A549 cells.However,48 h later,the inhibitory effect in Scutellariae Radix-Hedyotidis Herba groups were significantly enhanced in comparison with that in the blank group(P<0.05),exhibiting a time-dependent response.After 72 h of action,no significant change was present in the inhibitory effect of each Scutellariae Radix-Hedyotidis Herba group,so the optimal concentration of Scutellariae Radix-Hedyotidis Herba was set at 40 mg·L^(-1)for follow-up experiments.As demonstrated by the comparison with the blank group,cisplatin at each concentration inhibited the cell proliferation in a time-dependent manner (P<0.05).Considering the cell survival rate,the best concentration of cisplatin was set at 10 mg·L^(-1).Compared with the blank group,Scutellariae Radix-Hedyotidis Herba combined with cisplatin remarkably inhibited the proliferation of A549 cells in a time-dependent manner(P<0.05),and the differences between the combined medication group and the other two groups became more significant after 72 h of medication(P<0.01).The IL-6 level in each experimental group,especially in the combined medication group,significantly declined in contrast to that in the blank group(P<0.01).The m RNAexpression levels of HIF-1αand Cyclin D_(1)in all experimental groups were obviously lower than those in the blank group,with the most significant changes observed in the combined medication group(P<0.05,P<0.01).The protein expression levels of PI3K,p-Akt,m TOR,HIF-1α,and Cyclin D_(1)in each experimental group was significantly down-regulated(P<0.05,P<0.01),and the levels in the combined medication group were even lower than those in the cisplatin group(P<0.01).Conclusion:Scutellariae Radix-Hedyotidis Herba has an inhibitory effect on the proliferation of A549 cells,which may be related to its inhibition against the expression and secretion of IL-6/PI3K/Akt/m TOR-HIF-1αaxis.