对叶百部总生物碱对人肝癌SMMC-7721细胞凋亡及Bcl-2,Bax和cleaved Caspase-3蛋白表达的影响  

Effect of Stemona tuberosa Alkaloids on Apoptosis of SMMC-7721 Cells and Expression of Bcl-2,Bax,and Cleaved Caspase-3

作　　者：林思 朱华[1,2] 秦慧真 王孝勋[1] 许立拔 邓玲玉 LIN Si;ZHU Hua;QIN Hui-zhen;WANG Xiao-xun;XU Li-ba;DENG Ling-yu(Guangxi University of Chinese Medicine,Nanning 530200,China;Guangxi Key Laboratory of Zhuang and Yao Ethnic Medicine,Guangxi University of Chinese Medicine,Nanning 530200,China)

机构地区：[1]广西中医药大学,南宁530200 [2]广西中医药大学壮瑶药重点实验室,南宁530200

出　　处：《中国实验方剂学杂志》2021年第19期73-79,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：广西壮瑶药重点实验室项目(桂科基字[2014]32号);广西壮瑶药协同创新中心项目(桂教科研[2013]20号);广西重点学科(壮药学)(桂教科研[2013]16号);广西第八批特聘专家项目(壮瑶药质量标准研究,桂人才通字[2019]13号);广西一流学科中药学(民族药学)(桂教科研[2018]12号)。

摘　　要：目的:研究对叶百部总生物碱对人肝癌SMMC-7721细胞凋亡及B细胞淋巴瘤-2(Bcl-2),Bcl-2相关X蛋白(Bax和切割型半胱氨酸天冬氨酸蛋白水解酶-3(cleaved Caspase-3)蛋白表达的影响。方法:选择人肝癌SMMC-7721细胞,常规培养及传代,对叶百部总生物碱设置添加干预的质量浓度为50,75,112,167,250 mg·L^(-1),另设置仅添加10%胎牛血清作为空白组。采用噻唑蓝(MTT)比色法和细胞克隆实验观察细胞增殖的作用,Hoechst 33258染色法观察细胞凋亡,蛋白免疫印迹法(Western blot)检测3种凋亡相关蛋白Bcl-2,Bax和cleaved Caspase-3的表达。结果:对叶百部总生物碱可抑制SMMC-7721细胞增殖,与空白组比较,细胞增殖抑制率显著升高(P<0.01),作用24,48,72 h的50%细胞抑制浓度(IC_(50))分别为(173.36±8.75),(112.14±16.50),(96.41±2.60)mg·L^(-1),细胞集落形成抑制率显著升高(P<0.01),存在浓度依赖性。与空白组比较,对叶百部总生物碱可促进细胞凋亡,SMMC-7721细胞凋亡数目和凋亡率显著增加(P<0.01),在荧光正置显微镜下可观察到典型细胞凋亡形态,如亮蓝色细胞核染色,细胞质浓缩和细胞核固缩等。与空白组比较,抑凋亡蛋白Bcl-2表达显著下调(P<0.01);与空白组比较,对叶百部总生物碱75,112,167,250 mg·L^(-1)促凋亡蛋白Bax,cleaved Caspase-3蛋白表达显著上调(P<0.01)。结论:对叶百部总生物碱有很好的抑制SMMC-7721细胞增殖和促进凋亡的作用,其机制可能与抑制Bcl-2蛋白表达和促进Bax,cleaved Caspase-3蛋白表达有关。Objective:To investigate the effect of Stemona tuberosa alkaloids on the apoptosis of human hepatoma SMMC-7721 cells and the expression of apoptosis-related proteins including B lymphocytoma-2(Bcl-2),Bcl-2-associated X protein(Bax),and cleaved cysteinyl aspartate-specific protease-3(cleaved Caspase-3).Method: SMMC-7721 cells were routinely cultured,passaged,and treated with various concentrations(50,75,112,167,and 250 mg·L^(-1))of S. tuberosa alkaloids,while those in the blank control group were only treated with 10% fetal bovine serum. The cell proliferation was determined by tetrazolium bromide(MTT)colorimetry and colony assay and the cell apoptosis by Hoechst 33258 staining. The protein expression levels of Bcl-2,Bax,and cleaved Caspase-3 were detected by Western blot. Result: S. tuberosa alkaloids inhibited the proliferation of SMMC-7721 cells,and the inhibition rate was significantly increased in comparison with that in the blank control group(P<0.01),with the half maximal inhibitory concentrations(IC_(50))at 24 h,48 h,and72 h being(173.36±8.75),(112.14±16.50),and(96.41±2.60)mg·L^(-1),respectively. The cell colony-inhibitory activity was significantly increased in a dose-dependent manner(P<0.01). Compared with the blank control group,S. tuberosa alkaloids promoted the apoptosis of SMMC-7721 cells,manifested as increased number of apoptotic cells and elevated apoptotic rate(P<0.01). The typical morphological changes such as brightly bluefluorescent condensed nuclei, cytoplasmic shrinking, and karyopyknosis were found under the upright fluorescence microscope. As revealed by comparison with the blank control group,the expression of Bcl-2 was significantly down-regulated(P<0.01),while the protein expression levels of pro-apoptotic protein Bax and cleaved Caspase-3 in the 75,112,167,and 250 mg·L^(-1) S. tuberosa alkaloids groups were significantly upregulated(P<0.01). Conclusion: S. tuberosa alkaloids inhibit the proliferation of SMMC-7721 cells and promote their apoptosis possibly by inhibiting Bcl-2 prot

关 键 词：对叶百部总生物碱 人肝癌 SMMC-7721细胞 凋亡 B细胞淋巴瘤-2(Bcl-2) Bcl-2相关X蛋白(Bax) 切割型半胱氨酸天冬氨酸蛋白水解酶-3(cleaved Caspase-3) 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学]