藏药雪灵芝提取物对肝癌细胞HepG2增殖及凋亡的影响及机制研究  

Effect and mechanism of Arenaria kansuensis Maxim. Extracts on the Proliferation and Apoptosis of Human Hepatocellular Carcinoma HepG2 Cells

作　　者：雷宁 许本善 吕亚丽[2] LEI Ning;XU Ben-shan;LV Ya-li(Department of Pharmacy,PLA Rocket Force Characteristic Medical Center,Beijing 100088,China;Department of Pharmacy,Beijing Chao-Yang Hospital,Capital Medical University,Beijing 100020,China)

机构地区：[1]火箭军特色医学中心药剂科,北京100088 [2]首都医科大学附属北京朝阳医院药事部,北京100020

出　　处：《中国新药杂志》2022年第7期693-698,共6页Chinese Journal of New Drugs

基　　金：国家自然科学基金资助项目(81403184)。

摘　　要：目的:研究雪灵芝提取物对肝癌细胞HepG2增殖及凋亡的影响,并探讨其潜在作用机制。方法:采用硅胶柱层析法对雪灵芝乙醇提取物的乙酸乙酯萃取部位进行分离,以二氯甲烷-甲醇-水为流动相进行梯度洗脱,分别得到子部位Z1,Z2,Z3和Z4。运用CCK-8法检测不同质量浓度的4个子部位对HepG2增殖活性的作用,Hoechst 33258染色观察给药组HepG2细胞形态的变化,Annexin V-FITC/PI双染流式细胞技术检测细胞凋亡情况。采用Western blot法检测各组细胞中B淋巴细胞瘤-2蛋白(Bcl-2)、Bcl-2相关x蛋白(Bax)、细胞周期蛋白D1(Cyclin D1)的表达水平。结果:4个子部位对HepG2细胞增殖具有不同程度的抑制作用,且子部位Z3,Z4的抑制作用相对更为显著;Hoechst 33258染色观察到经子部位Z3,Z4作用的HepG2细胞出现典型的细胞凋亡形态;Annexin V-FITC/PI双染流式细胞术证明了其对于HepG2细胞具有诱导凋亡作用;Western blot结果显示,在12.5~25μg·mL^(-1)浓度范围内,子部位Z3能够下调Bc1-2表达并增加Bax的表达,使Bax/Bcl-2比值升高,从而促进凋亡发生;在6.25~25μg·mL^(-1)浓度范围内,子部位Z4可通过下调Bcl-2和Cyclin D1的表达,实现促凋亡的作用。结论:雪灵芝乙醇提取物的乙酸乙酯萃取部位的子部位能够抑制HepG2细胞增殖并诱导其凋亡,其机制可能与抑制Bcl-2,Cyclin D1蛋白表达有关。Objective: To study the effects of Arenaria kansuensis Maxim. extracts on the proliferation and apoptosis of hepatocellular carcinoma HepG2 cells, and to explore its mechanism. Methods: The ethyl acetate extracted fraction of Arenaria kansuensis Maxim. ethanol extracts was separated by silica gel columnchromatography using CH;Cl;-methanol-H;O as mible phase. The fractions of Z1, Z2, Z3, and Z4 were obtained. CCK-8 assay was used to detect the effect of different concentrations of Z1~Z4 on the proliferation activity of HepG2 cells. After treated for 24 h, the morphological changes of the cells were observed by Hoechst 33258 fluorescence staining. The apoptosis was analyzed by flow cytometry using annexin V-FITC/PI double staining. Western blot was used to detect the expression of genes including Bcl-2, Bax and Cyclin D1. Results: The results showed that different concentrations of fractions Z1 to Z4 inhibited the proliferation of HepG2 cells, especially fractions Z3 and Z4. Typical apoptosis morphology of HepG2 cells with Hoechst 33258 staining was observed under fluorescence microscope after treated with fractions Z3 and Z4. The result of Annexin V-FITC/PI staining certificated furtherly the effect on promoting apoptosis of both fractions on HepG2 in vitro. Western blot results showed that the expression of Bcl-2/Bax was up-regulated by fraction Z3 over the concentration range of 12.5~25 μg·mL^(-1) in HepG2 in vitro. Fraction Z4 promoted apoptosis over the concentration range of 6.25~25 μg·mL^(-1) by down-regulating the expression of Bcl-2 and Cyclin D1 in HepG2 in vitro. Conclusion: Arenaria kansuensis Maxim. extracts can inhibit the proliferation of hepatocellular carcinoma HepG2 cells while inducing its apoptosis. The mechanism may be related to the inhibition of the expression of Bcl-2 and Cyclin D1.

关 键 词：雪灵芝 肝癌细胞HEPG2 增殖 凋亡 机制 

分 类 号：R965[医药卫生—药理学]