苦豆子总碱及其两种单体生物碱对PC12细胞活性的抑制作用  

Inhibitory Effect of Sophora alopecuroides Total Alkaloids and Two Monomer Alkaloids on PC12 Cell Activity

作　　者：高莉 罗福祥 阿娜古丽·马合木提 罗静莺 闫明 GAO Li;LUO Fuxiang;ANAER Mahemuti;LUO Jingying;YAN Ming(Xinjiang Key Laboratory of Uighur Medical Prescription,Xinjiang Institute of Traditional Uighur Medicine,Urumqi 830011,China)

机构地区：[1]新疆维吾尔自治区维吾尔医药研究所新疆维吾尔自治区维吾尔医方剂学重点实验室,乌鲁木齐830011

出　　处：《中国实验方剂学杂志》2023年第17期126-133,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：天山雪松计划项目(2019XS18);新疆维吾尔自治区自然科学基金项目(2019D01A77)。

摘　　要：目的:研究比较从苦豆子中提取分离的苦豆子总碱、苦参碱、槐定碱分别对肾上腺嗜铬细胞瘤细胞(PC12细胞活性的影响。方法:采用噻唑蓝(MTT)比色法检测2、1、0.5、0.25、0.125、0.062 5 g·L^(-1)苦豆子总碱、苦参碱、槐定碱分别对PC12细胞增殖的影响;酶标法检测细胞乳酸脱氢酶(LDH)渗漏率;流式细胞术检测细胞凋亡率、细胞周期、细胞内Ca^(2+)水平;实时荧光定量聚合酶链式反应(Real-time PCR)检测B细胞淋巴瘤-2(Bcl-2)和Bcl-2相关X蛋白(Bax)mRNA转录水平;蛋白免疫印迹法(Western blot)检测细胞凋亡相关蛋白胱天蛋白酶-3(Caspase-3)、胱天蛋白酶-8(Caspase-8)及Bcl-2、Bax蛋白的表达。结果:与空白组比较,苦豆子总碱、苦参碱、槐定碱均能抑制PC12细胞增殖,使细胞LDH渗漏率显著增加,细胞内Ca2+荧光强度显著增强,诱导细胞的凋亡,并呈现一定的浓度依赖性(P<0.05,P<0.01),其中苦豆子总碱对PC12细胞的增殖抑制作用和诱导细胞凋亡作用最强(P<0.01);经苦豆子总碱、苦参碱、槐定碱处理后,苦豆子总碱组细胞周期进程G1/G0被阻滞,苦参碱、槐定碱组细胞周期进程G1/S被阻滞;PC12细胞Bax mRNA表达量均明显升高(P<0.05,P<0.01),Bcl-2 mRNA表达量均明显降低(P<0.05,P<0.01);均能明显下调细胞抗凋亡蛋白Bcl-2的表达(P<0.05,P<0.01),明显上调促凋亡蛋白Bax、Caspase-3、Caspase-8的表达(P<0.05,P<0.01),其中苦豆子总碱组蛋白表达变化最为显著。结论:苦豆子总碱、苦参碱、槐定碱中,苦豆子总碱对PC12细胞活性的抑制作用最强,其作用机制可能与其抑制抗凋亡蛋白表达,上调促凋亡蛋白表达,激活细胞线粒体Caspase-8凋亡通路有关。Objective:To compare the effects of total alkaloids,matrine,and sophoridine extracted from Sophora alopecuroides on the activity of pheochromocytoma cells(PC12 cells).Method:The effect of S.alopecuroides total alkaloids,matrine,and sophoridine at concentrations of 2,1,0.5,0.25,0.125,and 0.0625 g·L^(-1) on the proliferation of PC12 cells was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)assay.The lactate dehydrogenase(LDH)leakage rate was measured by enzyme-linked immunosorbent assay(ELISA).Flow cytometry was used to assess cell apoptosis rate,cell cycle distribution,and intracellular Ca^(2+) levels.Real-time quantitative polymerase chain reaction(Real-time PCR)was performed to determine the mRNA transcription levels of B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X protein(Bax).Protein expression levels of apoptosis-related proteins Caspase-3,Caspase-8,Bcl-2,and Bax were detected by Western blot.Result:Compared to the control group,S.alopecuroides total alkaloids,matrine,and sophoridine inhibited the proliferation of PC12 cells,increased LDH leakage rate,enhanced intracellular Ca2+fluorescence intensity,and induced cell apoptosis in concentration-dependent manner(P<0.05,P<0.01).Among them,S.alopecuroides total alkaloids had the strongest inhibitory effect on cell proliferation and induction of apoptosis in PC12 cells(P<0.01).After treatment with S.alopecuroides total alkaloids,matrine,and sophoridine,the cell cycle progression of PC12 cells was arrested at G1/G0 in the S.alopecuroides total alkaloids group,and at G1/S in the matrine and sophoridine groups.The expression levels of Bax mRNA were significantly increased(P<0.05,P<0.01),while the expression levels of Bcl-2 mRNA were significantly decreased(P<0.05,P<0.01).All treatments significantly downregulated the expression of the anti-apoptotic protein Bcl-2(P<0.05,P<0.01)and upregulated the expression of the pro-apoptotic proteins Bax,Caspase-3,and Caspase-8(P<0.05,P<0.01),with the most significant protein expression chan

关 键 词：苦豆子生物碱 PC12细胞 抑制率 乳酸脱氢酶(LDH)漏出率 细胞凋亡 细胞周期 

分 类 号：R2-0[医药卫生—中医学] R22R285.5R289R33