大黄素联合雷替曲塞抑制胰腺癌细胞增殖及对血管紧张素转换酶2、血管紧张素Ⅱ的影响

Inhibition of Emodin Combined with Raltitrexed on the Proliferation of Pancreatic Cancer Cells and Its Effect on Angiotensin Converting Enzyme 2 and Angiotensin Ⅱ

目的:探讨大黄素联合雷替曲塞抑制胰腺癌PANC-1细胞系增殖的作用及对血管紧张素转换酶2(ACE2)、血管紧张素Ⅱ(AngⅡ)的影响。方法:将PANC-1细胞分为对照组(0.1%DMSO)、雷替曲塞组(雷替曲塞)、大黄素组(大黄素)和联合用药组(大黄素+雷替曲塞)。使用四甲基偶氮唑盐比色法(MTT)检测大黄素、雷替曲塞和联合用药对PANC-1细胞增殖能力的影响;使用酶联免疫吸附试验测定细胞上清液白细胞介素(IL)6、IL-1β和肿瘤坏死因子α(TNF-α)水平;使用Annexin-V FITC/PI双染法检测大黄素组、雷替曲塞组以及联合用药组的细胞凋亡情况;使用蛋白质印迹法、免疫荧光法检测各组细胞ACE2、AngⅡ蛋白表达情况。结果:MTT检测结果显示,大黄素组(100μmol/L)、雷替曲塞组(40μmol/L)和联合用药组PANC-1细胞抑制率分别为46.73%、42.39%和68.71%,与对照组相比,差异均有统计学意义(P<0.05)。大黄素组、雷替曲塞组和联合用药组PANC-1细胞上清液中IL-6、IL-1β和TNF-α水平显著低于对照组,而雷替曲塞组显著低于大黄素组,联合用药组显著低于大黄素组、雷替曲塞组,差异均有统计学意义(P<0.05)。大黄素组、雷替曲塞组及联合用药组PANC-1细胞凋亡率均显著高于对照组,差异均有统计学意义(P<0.01)。蛋白质印迹法、免疫荧光法结果证实,大黄素组、雷替曲塞组和联合用药组PANC-1细胞ACE2的表达高于对照组,AngⅡ的表达低于对照组,差异均有统计学意义(P<0.001);且联合用药组ACE2的表达最高、AngⅡ的表达最低。结论:大黄素、雷替曲塞单药能够有效抑制胰腺癌PANC-1细胞增殖,提高ACE2水平、降低AngⅡ水平,具有促进肿瘤细胞凋亡的作用,而2种药物联合应用具有协同效应。

OBJECTIVE: To probe into the inhibitory effect of emodin combined with raltitrexed on the proliferation of pancreatic cancer PANC-1 cell line and its effect on angiotensin converting enzyme 2(ACE2) and angiotensin Ⅱ(AngⅡ). METHODS: The PANC-1 cells were divided into control group(treated with 0.1% DMSO), raltitrexed group(treated with raltitrexed), emodin group(treated with emodin) and combination group(treated with emodin combined with raltitrexed). The methyl thiazolyl tetrazolium method(MTT) was used to detect the effect of emodin, raltitrexed and drug combination on the proliferative capacity of PANC-1 cells;the enzyme linked immunosorbent assay was used to measure the interleukin(IL) 6, IL-1β and tumor necrosis factor α(TNF-α) levels in cell supernate;the Annexin-V FITC/PI double-staining method method was used to detect the cell apoptosis in the emodin group, raltitrexed group and combination group;the Western blotting and immunofluorescence methods was used to detect the expression of ACE2 and AngⅡ protein in each group. RESULTS: The MTT result showed that the inhibition rates of emodin group(100 μmol/L), raltitrexed group(40 μmol/L) and combination group on PANC-1 cells were respectively 46.73%, 42.39% and 68.71%, the differences were statistically significant compared with the control group(P<0.05). The IL-6, IL-1β and TNF-α levels in the supernate of PANC-1 cells were significantly lower in the emodin group, raltitrexed group and combined group than those in the control group, while those of raltitrexed group were significantly lower than the emodin group, and those of the combination group were significantly lower than the emodin group and raltitrexed group, with statistically significant differences(P<0.05). The PANC-1 cell apoptosis rates of the emodin group, raltitrexed group and combination group were significantly higher than that of the control group, with statistically significant difference(P<0.01). The Western blotting and immunofluorescence method confirmed that the expression of ACE2 in PANC-1 cells was higher and the expression of AngⅡ in PANC-1 cells was lower in the emodin group, raltitrexed group and combination group than those in the control group, with statistically significant differences(P<0.001), the expression of ACE2 was the highest and the expression of AngⅡ was the lowest in the combination group. CONCLUSIONS: Single use of emodin or raltitrexed can effectively inhibit the proliferation of pancreatic cancer PANC-1 cells, increase ACE2 level and decrease AngⅡ level, which has the effect of promoting apoptosis of tumor cells, while the combination of two drugs has a synergistic effect.