摘要
目的:探讨石蒜碱诱导H_(22)荷瘤小鼠体内肿瘤细胞凋亡的活性及其机制。方法:以昆明种小鼠为对象,通过前肢腋部皮下接种H_(22)肝癌小鼠腹水构建小鼠实体瘤模型,将造模后的小鼠随机分为阴性对照组、阳性对照组(羟基喜树碱6 mg/kg)和石蒜碱低、中、高剂量组(10、20、40 mg/kg),每组10只。阴性对照组小鼠灌胃等体积生理盐水,各给药组小鼠灌胃相应药物,每天1次,连续给药7天。末次给药后,检测小鼠的瘤体质量并计算抑瘤率。另通过腹腔注射H_(22)肝癌小鼠腹水构建小鼠腹水瘤模型,同法分组、给药。末次给药后,记录小鼠的生存时间并计算生存延长率,采用流式细胞术检测小鼠肿瘤细胞的早期凋亡率;在设立正常对照组(未荷瘤正常小鼠)的基础上,采用荧光探针钙黄绿素乙酰甲酯染色法考察各组小鼠肿瘤细胞的线粒体膜通透性,采用Rhodamine 123染色法考察其线粒体电位变化,采用比色法和Western blot法分别检测其胱天蛋白酶3(Caspase-3)活性和凋亡相关蛋白[Bcl-2、Bax、细胞色素C(Cyt-C)、Caspase-9]的表达情况。结果:与阴性对照组比较,阳性对照组和石蒜碱各剂量组小鼠瘤体质量均显著降低,生存时间均显著延长,肿瘤细胞的早期凋亡率均显著升高(P<0.05或P<0.01);其抑瘤率分别为39.41%、23.36%、36.50%、56.93%,生命延长率分别为49.23%、29.09%、50.19%、69.08%。与正常对照组比较,阴性对照组小鼠肿瘤细胞线粒体膜通透性、Caspase-3蛋白活性以及Cyt-C、Caspase-9蛋白的表达水平均显著升高,线粒体膜电位、Bcl-2/Bax比值显著降低(P<0.05或P<0.01);与阴性对照组比较,各药物组小鼠肿瘤细胞线粒体膜通透性和Bcl-2/Bax比值均显著降低,线粒体通透性、Caspase-3蛋白活性和Cyt-C、Caspase-9蛋白的表达水平均显著升高(P<0.05或P<0.01)。结论:石蒜碱可诱导H_(22)荷瘤小鼠体内肿瘤细胞凋亡,这种作用可能与其开放线粒体膜通透性转换孔以增加线粒体通透性、降低线粒体膜电位、诱导凋亡相关蛋白表达上调有关。
OBJECTIVE:To investigate the activity of lycorine to the in vivo apoptosis of tumor cells in H_(22)-bearing mice and its mechanism.METHODS:Kunming mice were inoculated subcutaneously with ascites of H_(22) hepatoma mice in the armpit of forelimb to establish solid tumor model.After modeling,mice were randomly divided into negative control group,positive control group(hydroxycamptothecin 6 mg/kg),lycorine low-dose,medium-dose and high-dose groups(10,20,40 mg/kg),with 10 mice in each group.Negative control group was given constant volume of normal saline intragastrically,and administration groups were given relevant medicine intragastrically,once a day,for consecutive 7 days.After last medication,the weight of tumor was detected and anti-tumor rate was calculated.Ascites tumor model of mice was established by intraperitoneal injection of H_(22) hepatoma mice ascites,and then were grouped with same method and given relevant medicine as above.After last medication,survival time of mice was recorded and the life prolongation rate was calculated.The early apoptotic rate of tumor cells in mice was detected by flow cytometry.On the basis of normal control group(normal mice without tumor),the mitochondrial membrane permeability of tumor cells in each group was investigated by Calcein AM staining.The changes of mitochondrial potential were investigated by Rhodamine 123 staining.Colorimetry and Western blot assay were adopted to detect the Caspase-3 activity and expression of apoptosis-related protein(Bcl-2,Bax,Cyt-C and Caspase-9).RESULTS:Compared with negative control group,the tumor weight of positive control group and lycorine groups were decreased significantly,while the survival time was significantly prolonged,and the early apoptotic rate of tumor cells was significantly increased(P<0.05 or P<0.01);the anti-tumor rates were 39.41%,23.36%,36.50%,56.93%,and life prolongation rates were 49.23%,29.09%,50.19%,69.08%.Compared with normal control group,the mitochondrial membrane permeability,Caspase-3 protein activity and protein expression of Cyt-C and Caspase-9 were significantly increased,while the mitochondrial membrane potential and Bcl-2/Bax ratio were decreased significantly(P<0.05 or P<0.01).Compared with negative control group,mitochondrial membrane permeability and Bcl-2/Bax ratio were decreased significantly in administration groups,while mitochondrial permeability,Caspase-3 protein activity and protein expression of Cyt-C and Caspase-9 were significantly increased(P<0.05 or P<0.01).CONCLUSIONS:Lycorine can induce the apoptosis of tumor cells in H_(22)-bearing mice,the effects of which may be associated with opening mitochondrial membrane permeability transition pore to increase mitochondrial permeability,decreasing mitochondrial membrane potential and up-regulating the expression of apoptosis-related proteins.
作者
辛国松
张晶
李吉业
季宇彬
刘斌
李钧
陈鹰翔
于淼
XIN Guosong;ZHANG Jing;LI Jiye;JI Yubin;LIU Bin;LI Jun;CHEN Yingxiang;YU Miao(Engineering Research Centre for Medicine,Harbin University of Commerce,Harbin 150076,China;Engineering Research Centre of Natural Anticancer Drugs,Ministry of Education,Harbin 150076,China)
出处
《中国药房》
CAS
北大核心
2021年第5期571-577,共7页
China Pharmacy
基金
黑龙江省自然科学基金资助项目(No.LH2019H066)
黑龙江普通本科高等学校青年创新人才培养计划(No.UNPYSCT-2017208)
中国博士后科学基金资助项目(No.2019M651296)
哈尔滨商业大学“青年创新人才”支持计划(No.2019CX37)
哈尔滨商业大学校级科研项目(No.18XN024)。
关键词
石蒜碱
荷瘤小鼠
线粒体
抗肿瘤
细胞凋亡
机制
Lycorine
Tumor-bearing mice
Mitochondrial
Anti-tumor
Apoptosis
Mechanism
