首页|实脾消积饮含药血清对肝癌HepG2细胞线粒体动力学平衡和铁死亡的影响

实脾消积饮含药血清对肝癌HepG2细胞线粒体动力学平衡和铁死亡的影响

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目的 从细胞线粒体动力学平衡和铁死亡角度探讨实脾消积饮治疗原发性肝癌的可能作用机制.方法 制备实脾消积饮含药血清,体外培养肝癌HepG2细胞.设置空白组、对照组、顺铂组(10μg/ml)及5%、10%、15%含药血清组,每组设4个复孔.各组加入相应的药物,37 ℃、5%CO2培养24 h后采用CCK-8实验计算细胞存活率,Transwell小室侵袭实验培养48 h后计算穿过基质胶到达上层小室膜的细胞数量,划痕实验培养24 h、48 h计算划痕迁移率,筛选含药血清浓度进行后续实验.实验设置对照组、10%含药血清组(筛选出的浓度)、10%含药血清+Mdivi-1组,培养24 h后检测线粒体、活性氧(ROS)平均荧光强度及三磷酸腺苷(ATP)含量,检测细胞胞浆和线粒体动力相关蛋白1(Drp1)、线粒体磷酸化动力相关蛋白1(p-Drp1)蛋白表达.实验设置对照组、10%含药血清组、铁死亡激活剂组(Erastin,10 μmol/L)、10%含药血清+铁死亡抑制剂组(ferrostatin-1,10 μmol/L),各组加入相应药物后培养24 h,检测细胞内谷胱甘肽(GSH)、丙二醛(MDA)含量以及亚铁离子水平,检测细胞中溶质载体家族7成员11(SLC7A11)、谷胱甘肽过氧化物酶4(GPX4)蛋白表达.结果 与对照组比较,各浓度的含药血清组和顺铂组细胞存活率、侵袭个数及24 h、48 h划痕迁移率均降低(P<0.01);随着含药血清浓度的增加,含药血清组细胞存活率、侵袭个数及48h划痕迁移率逐渐降低(P<0.01),最终筛选出10%实脾消积饮含药血清作为后续实验干预浓度.与对照组相比,10%含药血清组线粒体平均荧光强度及ATP含量降低,ROS平均荧光强度升高(P<0.05);与10%含药血清组比较,10%含药血清+Mdivi-1组线粒体平均荧光强度、ATP含量升高,ROS平均荧光强度降低(P<0.05或P<0.01).与对照组比较,其余各组细胞中GSH含量及SLC7A11、GPX4蛋白表达降低,MDA含量及亚铁离子水平升高(P<0.05或P<0.01);与铁死亡激活剂组比较,10%含药血清组和10%含药血清+铁死亡抑制剂组MDA含量及亚铁离子水平降低,SLC7A11、GPX4蛋白表达升高(P<0.05或P<0.01);与10%含药血清组比较,10%含药血清+铁死亡抑制剂组GSH含量亦升高(P<0.01).结论 实脾消积饮含药血清可能通过引起线粒体动力学失衡及诱导铁死亡,抑制肝癌HepG2细胞的增殖、侵袭与迁移,从而发挥抗肝癌作用.
Effect of Shipi Xiaoji Beverage(实脾消积饮)-containing Serum on Mitochondrial Dynamics Imbalance and Ferroptosis of Liver Cancer HepG2 Cells
Objective To explore the possible mechanism of Shipi Xiaoji Beverage(实脾消积饮)in treating pri-mary liver cancer from the perspective of mitochondrial dynamics imbalance and ferroptosis.Methods The Shipi Xiaoji Beverage-containing serum was prepared,and liver cancer HepG2 cells were cultured in vitro.The blank group,control group,cisplatin group(10 μg/ml),and 5%,10%,and 15%medicated serum groups were set,with 4 duplicate holes in each group.After adding the corresponding medicinals to each group,the cell survival rate was calculated using the CCK-8 test after 24-hour culture at 37 ℃ with 5%CO2.Using transwell invasion assay,the number of cells that reached the upper chamber membrane through penetrating the Matrigel was calculated after culturing for 48 hours.The scratch migration rate was calculated after incubation for 24 h and 48 h in the scratch experiment,and the concentration of the medicated serum was screened for subsequent experiments.The control group,10%medicated serum group(the screened concentration)and 10%medicated serum+Mdivi-1 group were set up.After culturing for 24 hours,the average fluorescence intensity of mitochondria and reactive oxygen species(ROS),adenosine triphos-phate(ATP)content and level were measured,and the protein expression of cytoplasmic and mitochondrial dynein-related protein 1(Drp1)and mitochondrial phosphorylated dynamin-related protein 1(p-Drp1)was detected.The control group,10%medicated serum group,ferroptosis activator group(Erastin,10 μmol/L)and 10%medicated serum+ferroptosis inhibitor group(fer-1,10 μmol/L)were set up,and after adding the corresponding medicinals to each group and culture for 24 hours,the intracellular glutathione(GSH),malondialdehyde(MDA)content and ferrous ion level were detected,as well as the protein expression of solute carrier family 7 member 11(SLC7A11)and glutathione peroxidase 4(GPX4)in cells.Results Compared to those in the control group,the cell survival rate,invasion number,24 h and 48 h scratch migration rate in all medicated serum groups and cisplatin group significantly decreased(P<0.01);as the concentration of medicated serum increased,the cell survival rate,invasion number and 48h scratch migration rate of each medicated serum group gradually decreased(P<0.01),and finally,10%of Shipi Xiaoji Beverage-containing serum was selected for subsequent experimental concentration.Compared to the control group,the 10%medicated serum group had decreased average fluorescence intensity of mitochondria and ATP content and increased average fluorescence intensity of ROS(P<0.05);compared to the 10%medicated serum group,the 10%medicated serum+Mdivi-1 group had higher average fluorescence intensity of mitochondria and ATP content and lower average fluorescence intensity of ROS(P<0.05 or P<0.01).Compared to those in the control group,GSH content and protein expression of SLC7A11 and GPX4 in other groups significantly decreased,while MDA content and ferrous ion level increased(P<0.05 or P<0.01);compared to the ferroptosis activator group,the 10%medicated serum group and the 10%medicated serum+ferroptosis inhibitor group had decreased MDA content and ferrous ion level,and increased protein expression of SLC7A11 and GPX4(P<0.05 or P<0.01);the 10%medicated serum+ferroptosis inhibitor group had higher GSH level than the 10%medicated serum group(P<0.01).Conclusion Shipi Xiaoji Beverage may inhibit the proliferation,infestation,migration of HepG2 cells by causing mitochondrial dynamics imbalance and inducing ferroptosis,thereby playing against liver cancer.

primary liver cancerShipi Xiaoji Beverage(实脾消积饮)HepG2cellmitochondriadynamicsferroptosis

翦慧颖、李克雄、曾普华

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湖南中医药大学,湖南省长沙市岳麓区学士路300号,410208

湖南省中西医结合医院/湖南省中医药研究院附属医院

原发性肝癌 实脾消积饮 HepG2细胞 线粒体 动力学 铁死亡

国家自然科学基金国家中医药局青年岐黄学者支持项目湖南省科技创新项目湖南省自然科学基金湖南省中医药科研项目湖南省中医药研究院科研项目

820744252023JJ40400D2022010202115

2024

中医杂志
中华中医药学会 中国中医科学院

中医杂志

CSTPCD北大核心
影响因子:1.464
ISSN:1001-1668
年,卷(期):2024.65(6)
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