分心木乙醇提取物对肝癌细胞增殖、侵袭、迁移和凋亡的影响

Effects of the Diaphragma Juglandis Fructus ethanol extract on the proliferation, invasion, migration and apoptosis of hepatocellular carcinoma cells

目的探讨分心木乙醇提取物(Diaphragma juglandis fructus ethanol extract,DJFEE)通过Wnt/β-catenin信号通路对肝癌细胞增殖、侵袭、迁移和凋亡的机制。方法以Huh7细胞为研究对象,采用不同浓度(50、100、200μg/mL)的分心木乙醇提取物处理Huh7细胞,分别为:空白对照组(Control)、分心木乙醇提取物低剂量组(L-DJFEE)、分心木乙醇提取物中剂量组(M-DJFEE)和分心木乙醇提取物高剂量组(HDJFEE)。然后采用200μg/mL的分心木乙醇提取物和10 mmol/L的Wnt/β-catenin激动剂氯化锂(LiCl)处理细胞,记为DJFEE组和DJFEE+LiCl组。噻唑蓝(MTT)、克隆形成实验检测Huh7细胞的增殖情况;Transwell小室检测Huh7细胞的迁移和侵袭能力;流式细胞术检测Huh7细胞的凋亡率;蛋白免疫印迹(Western blot)检测Huh7细胞的增殖细胞核抗原(PCNA)、基质金属蛋白酶-9(MMP-9)、基质金属蛋白酶-14(MMP-14)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved caspase3)、Bcl-2相关X蛋白(Bax)、B型白细胞/2型淋巴细胞样蛋白(Bcl-2)、Wnt/β-catenin通路相关蛋白的表达。结果与空白对照组相比,分心木乙醇提取物低、中、高剂量组处理Huh7细胞后,降低细胞增殖、迁移和侵袭能力,增加细胞凋亡率,下调PCNA、MMP-9、MMP-14、Bcl-2、Wnt1和β-catenin蛋白表达,上调Cleaved caspase3、Bax蛋白。DJFEE+LiCl组的迁移细胞数目、侵袭细胞数目、PCNA、MMP-9、MMP-14、Bcl-2蛋白表达明显高于DJFEE组,细胞抑制率、细胞凋亡率、Cleaved caspase3、Bax蛋白表达明显低于DJFEE组。结论分心木乙醇提取物可以抑制肝癌细胞的增殖、迁移和侵袭,促进其凋亡,其作用机制可能与Wnt/β-catenin信号通路有关。

Objective To explore the mechanism of Diaphragma Juglandis Fructus ethanol extract(DJFEE) on the proliferation, invasion, migration and apoptosis of liver cancer cells through the Wnt/β-catenin signaling pathway.Methods Huh7 cells were divided into blank control group(Control) and Diaphragma juglandis Fructus Ethanol extract low-dose group(L-DJFEE,50 μg/ml),middle-dose group(M-DJFEE,100 μg/ml) high dose group(H-DJFEE,200 μg/ml),and DJFEE(200 μg/ml) plus 10 mmol/L Wnt/β-catenin agonist lithium chloride(LiCl)(DJFEE+LiCl) group.The methylthiazolyldiphenyl-tetrazolium bromide(MTT) method and clone formation were used to detect the proliferation of Huh7 cells;Transwell chamber was used to detect the migration and invasion of Huh7 cells;flow cytometry to detect the apoptosis rate of Huh7 cells;Western blot was used to detect proliferating cell nuclear antigen(PCNA),matrix metalloproteinase-9(MMP-9),matrix metalloproteinase-14(MMP-14),activated cysteine-containing aspartate proteolytic enzyme 3(Cleaved caspase3),Bcl-2 related X protein(Bax),B-cell leukemia/lymphoma 2(Bcl-2),and Wnt/β-catenin pathway related protein expression.Results Compared with the blank control group, treatment of Huh7 cells with low, medium and high doses of DJFEE reduced the cell proliferation, migration and invasion ability;increased the rate of apoptosis, and downregulated PCNA,MMP-9,MMP-14,Bcl-2,Wnt1 and β-catenin protein expression.DJFEE also increased Cleaved caspase3 and Bax protein.The number of migrating cells, the number of invading cells, PCNA,MMP-9,MMP-14,and Bcl-2 protein expression in DJFEE+LiCl group were significantly higher than those in DJFEE group.DJFEE+LiCl reduced cell inhibition rate, apoptosis rate, Cleaved caspase3,and Bax protein expression compared to DJFEE group.Conclusion Diaphragma juglandis Fructus Ethanol extract(DJFEE) can inhibit the proliferation, migration and invasion of liver cancer cells, and promote cell apoptosis, its mechanism may be related to the Wnt/β-catenin signaling pathway.