基于Notch1通路探讨苍术酮对肝癌细胞化疗耐药的作用机制研究

To Explore the Mechanism of Atractylon on Chemotherapy Resistance of Hepatocellular Carcinoma Cells Based on Notch1 Pathway

目的:基于Notch1通路探讨苍术酮对肝癌细胞化疗耐药的影响。方法:不同浓度苍术酮作用人肝癌氟尿嘧啶耐药细胞株Bel/Fu 48 h,采用CCK8法检测细胞存活情况,并选择最适抑制浓度。人肝癌耐药细胞株Bel/Fu随机分为空白组、5-氟尿嘧啶组、苍术酮组、联合组,各组分别以对应培养液培养48 h。光学显微镜下观察各组细胞状态;CCK-8法、流式细胞术检测细胞存活情况及凋亡水平,Transwell实验检测细胞侵袭能力;RT-qPCR技术检测各组细胞中P糖蛋白(P-gp)和多药耐药相关蛋白1(MRP1)的mRNA相对表达量;Western blotting法检测各组细胞中P-gp、MRP1、Notch1、Hes1、Jagged1、B细胞淋巴瘤/白血病-2基因(Bcl-2)及Bcl-2相关X蛋白(Bax)蛋白相对表达量。结果:Bel/Fu细胞存活率随苍术酮作用浓度增大而降低(P<0.05),苍术酮作用浓度为32μmol/L时,细胞存活率接近50%。与空白组比较,5-氟尿嘧啶组细胞存活率,凋亡率,侵袭数,P-gp、MRP1的mRNA和蛋白相对表达量,以及Notch1、Hes1、Jagged1、Bcl2、Bax蛋白相对表达量均无明显变化(P>0.05),镜下细胞形态、结构及密度无较大差异。与5-氟尿嘧啶组比较,苍术酮组及联合组细胞存活率,侵袭数,P-gp、MRP1的mRNA和蛋白相对表达量,以及Notch1、Hes1、Jagged1、Bcl2蛋白相对表达量均显著降低,细胞凋亡率及Bax蛋白相对表达量显著升高(P<0.05),镜下细胞变形,密度变小,大量呈凋亡状态;5-氟尿嘧啶对Bel/Fu细胞的作用效果不及苍术酮,苍术酮单独作用效果不及5-氟尿嘧啶联合苍术酮时的作用效果(P<0.05)。结论:苍术酮可促进Bel/Fu细胞凋亡,抑制其存活及侵袭,增强肝癌细胞的化疗敏感性,其作用机制可能与抑制Notch1通路激活有关。

Objective:To investigate the effect of atractylone on chemoresistance of hepatocellular carcinoma cells based on Notch1 pathway.Methods:Different concentrations of atractylon were used to treat human hepatocellular carcinoma fluorouracil-resistant cell line Bel/Fu for 48 h.The survival of the cell was detected by CCK8 method,and the optimal inhibitory concentration was selected.Human hepatocellular carcinoma drug-resistant cell line Bel/Fu was randomly divided into blank group,5-fluorouracil group,atractylon group and combination group.Each group was cultured with corresponding culture medium for 48 h.The cell status of each group was observed under optical microscope.CCK-8 method and flow cytometry were used to detect cell survival and apoptosis,and Transwell assay was used to detect cell invasion ability.The relative expression of P-glycoprotein(P-gp)mRNA and multidrug resistance associated protein 1(MRP1)mRNA was detected by RT-qPCR.The relative expression levels of P-gp,MRP1,Notch1,Hes1,Jagged1,B cell lymphoma/leukemia-2 gene(Bcl2)and Bcl-2-associated X protein(Bax)were detected by Western blotting.Results:The survival rate of Bel/Fu ells decreased with the increasing concentration of atractylone (P<0.05), and when the concentration of atractylone was 32 μmol/L, the cell survival rate was close to 50%. Compared with the blank group, there was no significant change in the cell survival rate, apoptosis rate, invasion number, mRNA and protein expression of P-gp and MRP1, and protein expression of Notch1, Hes1, Jagged1, Bcl2 and Bax in the 5-fluorouracil group (P>0.05). There was no significant difference in cell morphology, structure and density under the microscope. Compared with the 5-fluorouracil group, the cell survival rate, invasion number, mRNA and protein expression of P-gp and MRP1, and protein expression of Notch1, Hes1, Jagged1, Bcl2 were significantly decreased in the atractylon group and the combined group, and the apoptosis rate and and protein expression of of Bax were siclgnificantly increased (P<0.05). In addition, the cells were deformed, the density became smaller, and a large number of cells were in the state of apoptosis under the microscope. The effect of 5-fluorouracil on Bel/Fu cells was not as good as that of atractylon, and the effect of atractylon alone was not as good as that of 5-fluorouracil combined with atractylon (P<0.05). Conclusion: Atractylon can promote the apoptosis of Bel/Fu cells, inhibit their survival and invasion, and enhance the chemosensitivity of hepatocellular carcinoma cells. The mechanism may be related to the inhibition of Notch1 pathway activation.