蓝萼甲素通过Bcl-2/Beclin1靶点调节人肝癌HCCLM3细胞自噬与凋亡的机制研究  

Study on regulation mechanism of glaucocalyxin A on the autophagy and apoptosis of HCCLM3 hepatocellular carcinoma cells through Bcl-2/Beclin1 target

作　　者：朱琳琳 张明明 郭格格 徐祉轩 ZHU Linlin;ZHANG Mingming;GUO Gege;XU Zhixuan(School of Laboratory Medicine,Xinxiang Medical University/Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine/Henan Key Laboratory of Immunology and Targeted Therapy,Henan Xinxiang 453003,China)

机构地区：[1]新乡医学院医学检验学院/河南省分子检验与医学检验技术协同创新中心/河南省免疫与靶向药物重点实验室,河南新乡453003

出　　处：《中国药房》2022年第9期1082-1087,共6页China Pharmacy

基　　金：河南省科技攻关项目(No.212102310753)。

摘　　要：目的研究蓝萼甲素(GLA)对人肝癌HCCLM3细胞自噬与凋亡的调节作用机制。方法HCCLM3细胞按不同实验目的主要设对照组、GLA 2.5μg/mL组、GLA 5μg/mL组、GLA 10μg/mL组。对照组仅加入完全培养基,各给药组分别加入含GLA相应终质量浓度的完全培养基。采用流式细胞术检测细胞周期分布与凋亡情况;采用透射电镜观察细胞线粒体形态和自噬情况(仅设对照组、GLA 5μg/mL组);采用JC-1染色和荧光倒置显微镜观察并检测细胞线粒体膜电位;采用Western blot法检测细胞中Bcl-2、Bax、Beclin1、活性半胱氨酸天冬氨酸蛋白酶3(cleaved caspase-3)蛋白的表达;采用免疫共沉淀法检测细胞中Bcl-2与Beclin1的结合与解离(仅设GLA 5μg/mL组、GLA 10μg/mL组)。结果与对照组相比,GLA 5、10μg/mL能够诱发细胞周期显著阻滞于G2~M期、诱导线粒体膜电位显著下降、增加促凋亡作用,还能显著促进Bax、cleaved caspase-3、Beclin1蛋白表达,显著抑制Bcl-2蛋白表达(P<0.01);GLA 5μg/mL还可引发线粒体形态显著改变,自噬小体增多。免疫共沉淀法结果显示,与GLA 5μg/mL比较,GLA 10μg/mL能够增强Bcl-2与Beclin1的结合。结论GLA可能通过Bcl-2/Beclin1靶点调节HCCLM3细胞自噬与凋亡,且作用效果与剂量密切相关。OBJECTIVE To study the regulatory mechanism of glaucocalyxin A(GLA)on autophagy and apoptosis of HCCLM3 hepatocellular carcinoma cells.METHODS HCCLM3 cells were taken,and control group,GLA 2.5μg/mL group,GLA 5μg/mL group and GLA 10μg/mL group were mainly set according to different experimental purposes.In control group,only complete medium was added;in each administration group,complete medium containing the corresponding final concentration of GLA was added.Cell cycle distribution and apoptosis were detected by flow cytometry;mitochondrial morphology and autophagy were observed by transmission electron microscope(only control group,GLA 5μg/mL group);JC-1 staining and fluorescence inverted microscope were used to observe and detect the mitochondrial membrane potential of the cells;Western blot assay was used to detect the protein expression of Bcl-2,Bax,Beclin1 and cleaved caspase-3 proteins in the cells;the co-immunoprecipitation method was used to detect the binding and dissociation of Bcl-2 and Beclin1(only GLA 5μg/mL group,GLA 10μg/mL group).RESULTS Compared with control group,GLA 5μg/mL and GLA 10μg/mL could induce a significant arrest of the cell cycle in the G2-M phase for HCCLM3,a significant decrease in mitochondrial membrane potential,an increase in apoptosis as well as significant promotion of the protein expression of Bax,cleaved caspase-3 and Beclin1,and significant inhibition of the protein expression of Bcl-2(P<0.01).GLA 5μg/mL also significantly changed mitochondrial morphology and increased autophagosomes.The results of co-immunoprecipitation showed that compared with GLA 5μg/mL,GLA 10μg/mL could enhance the binding of Bcl-2 and Beclin1.CONCLUSIONS GLA can regulate the autophagy and apoptosis of HCCLM3 cells by Bcl-2/Beclin1 target.The effect is closely related to the dose of GLA.

关 键 词：蓝萼甲素 靶点 机制 自噬 凋亡 BCL-2 BECLIN1 

分 类 号：R965[医药卫生—药理学] R735.7[医药卫生—药学]