摘要
目的从细胞自噬和线粒体动力学途径探究千金子制霜前后提取物对人胚胎肾细胞(HEK293)和大鼠小肠上皮细胞(IEC-6)的影响。方法将HEK293和IEC-6细胞分为对照组及千金子生品提取物(100、200、400μg/mL)组和千金子霜品提取物(100、200、400μg/mL)组,分别采用MTT法和流式细胞术测定细胞活力、细胞凋亡、活性氧(reactive oxygen species,ROS)水平和线粒体膜电位;使用丹酰尸胺(dansylcadaverine,MDC)染色法观察细胞自噬囊泡数量;利用Western blotting检测细胞中微管相关蛋白1轻链3(microtubule-associated protein1 light chain 3,LC3)、p62、线粒体融合蛋白2(mitofusin 2,Mfn2)、视神经萎缩蛋白1(optic atrophic protein 1,OPA1)、动力相关蛋白1(dynamin-related protein 1,Drp1)的表达。结果与对照组比较,千金子制霜前后提取物作用于HEK293细胞、IEC-6细胞后存活率显著降低(P<0.01),细胞凋亡率显著升高(P<0.05、0.01),自噬囊泡显著增多(P<0.01),细胞内ROS水平显著升高(P<0.01),线粒体膜电位显著降低(P<0.05、0.01),p62、Mfn2、OPA1蛋白表达水平显著降低(P<0.01),LC3-II/I、Drp1蛋白表达水平显著升高(P<0.01)。与相同剂量的生品比较,千金子制霜后细胞存活率升高(P<0.01),诱导HEK293和IEC-6细胞凋亡的能力减弱(P<0.01),细胞自噬囊泡的数量减少(P<0.01),线粒体膜电位升高(P<0.05、0.01),ROS水平降低(P<0.05、0.01),并能减弱对p62、Mfn2、OPA1蛋白表达的下调作用及LC3-II/I、Drp1蛋白表达的上调作用(P<0.05、0.01)。结论千金子制霜减毒的作用机制可能与通过调控细胞自噬途径和线粒体动力学平衡、影响细胞增殖及凋亡有关。
Objective To investigate the effects of extracts from Qianjinzi(Euphorbiae Semen)before and after frosting on human embryonic kidney cells(HEK293)and rat intestinal epithelial cells(IEC-6)through the pathways of cell autophagy and mitochondrial dynamics.Methods The HEK293 and IEC-6 cells were divided into control group,Euphorbiae Semen extracts(100,200,400μg/mL)groups and Euphorbiae Semen Pulveratum extracts(100,200,400μg/mL)groups.The MTT method and flow cytometry were employed to determine cell viability,cell apoptosis,reactive oxygen species(ROS)level and mitochondrial membrane potential;The number of autophagic vesicles in cells was observed using dansylcadaverine(MDC)staining method;Western blotting was used to detect the expressions of microtubule-associated protein 1 light chain 3(LC3),p62,mitofusin 2(Mfn2),optic atrophic protein 1(OPA1)and dynamin-related protein 1(Drp1)in cells.Results Compared with control group,the extracts from Euphorbiae Semen before and after frosting significantly reduced the cell viability rate of HEK293 cells and IEC-6 cells(P<0.01),significantly increased the apoptosis rate(P<0.05,0.01)and the number of autophagic vesicles(P<0.01),elevated the intracellular ROS level(P<0.01),decreased the mitochondrial membrane potential(P<0.05,0.01),and down-regulated the expression levels of p62,Mfn2 and OPA1(P<0.01),while up-regulated the protein expression levels of LC3-II/I and Drp1(P<0.01).Compared with the same dose of Euphorbiae Semen,after frosting,the cell viability rate in HEK293 and IEC-6 cells was increased(P<0.01),the ability to induce apoptosis of HEK293 and IEC-6 cells was weakened(P<0.01),the number of autophagic vesicles was decreased(P<0.01),the mitochondrial membrane potential was increased(P<0.05,0.01),ROS level was reduced(P<0.05,0.01),and the effect of downregulation of p62,Mfn2 and OPA1 proteins as well as the up-regulation of LC3-II/I and Drp1 proteins were weaken(P<0.05,0.01).Conclusion The attenuation mechanism of processing for Euphorbiae Semen may be closely related to its regulation of cell autophagy pathways and mitochondrial dynamic balance,ultimately affecting cell proliferation and apoptosis.
作者
姜明瑞
马思媛
朱海婷
张俊丽
王晓宇
陈文静
毛易凡
徐鹏
张新宁
王英姿
JIANG Mingrui;MA Siyuan;ZHU Haiting;ZHANG Junli;WANG Xiaoyu;CHEN Wenjing;MAO Yifan;XU Peng;ZHANG Xinning;WANG Yingzi(School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 102488,China)
出处
《中草药》
北大核心
2025年第21期7829-7839,共11页
Chinese Traditional and Herbal Drugs
基金
北京市自然科学基金项目(7232286)
国家中医药管理局高水平建设学科(zyyzdxk-2023272)。
