摘要
目的探究蒲公英甾醇(TAR)对Erastin诱导的C28/I2软骨细胞铁死亡的作用。方法用Erastin诱导C28/I2软骨细胞系构建体外软骨细胞铁死亡模型,实验分为Control组、Erastin组、TAR组、TAR+Erastin组。CCK-8法检测细胞活力;乳酸脱氢酶(LDH)试剂盒和Calcein/PI细胞活性与细胞毒性检测试剂盒检测细胞毒性;流式细胞术检测脂质活性氧(ROS)含量;谷胱甘肽(GSH)试剂盒检测细胞内GSH含量;JC-1染色和RH123染色检测线粒体膜电位;Western blot法检测铁死亡关键指标酰基辅酶A合成酶长链家族成员4(ACSL4)和谷胱甘肽过氧化物酶4(GPX4)蛋白表达变化。结果TAR可以恢复Erastin处理导致的C28/I2软骨细胞细胞活力降低以及减少Erastin诱导的细胞毒性(P<0.01);与Control组比较,Erastin处理后细胞内脂质ROS水平升高(P<0.01),GSH含量降低(P<0.01),而TAR可以减少脂质ROS产生(P<0.01),增加GSH含量(P<0.01);TAR可以恢复铁死亡的C28/I2软骨细胞线粒体膜电位,减少ACSL4蛋白的表达(P<0.01),增加GPX4蛋白的表达(P<0.01);此外,TAR可以恢复IL-1β刺激导致的软骨细胞活力降低。结论TAR可以抑制Erastin诱导的C28/I2软骨细胞铁死亡,这一过程可能与调控ACSL4、GPX4蛋白表达有关。
Objective To investigate the role of Taraxasterol(TAR)on ferroptosis in chondrocytes induced by Erastin.Methods The C28/I2 chondrocyte line was treated with Erastin to construct the ferroptosis model of chondrocytes in vitro and the experiments were divided into Control,Erastin,TAR,and TAR+Erastin groups.Cell viability was detected by the CCK-8 assay.Cytotoxicity was detected by the lactate dehydrogenase(LDH)kit and the Calcein/PI cytokinesis kit.Flow cytometry was used to detect lipid reactive oxygen species(ROS).The intracellular glutathione(GSH)content was detected by GSH kit.Mitochondrial membrane potential was detected by JC-1 staining and RH123 staining.ACSL4 and GPX4 protein expression and the key indicators of ferroptosis were detected by Western blot.Results TAR restored the decreased cell viability of C28/I2 chondrocytes induced by Erastin treatment as well as reduced Erastin-induced cytotoxicity(P<0.01).Compared with the control group,the level of intracellular lipid ROS increased(P<0.01)and the content of GSH decreased(P<0.01)after treatment with Erastin,while TAR could reduce the production of lipid ROS(P<0.01)and increase the content of GSH(P<0.01).TAR restored mitochondrial membrane potential in C28/I2 chondrocytes ferroptosis,decreased ACSL4 protein expression(P<0.01)and increased GPX4 protein expression(P<0.01).In addition,TAR restored the reduced cell viability caused by IL-1βtreatment.Conclusion TAR can inhibit Erastin induced ferroptosis in C28/I2 chondrocytes,which may be related to the regulation of ACSL4 and GPX4 protein expression.
作者
周富丽
王浩
朱仁弟
赵英杰
杨雅茹
周仁鹏
胡伟
鲁超
Zhou Fuli;Wang Hao;Zhu Rendi;Zhao Yingjie;Yang Yaru;Zhou Renpeng;Hu Wei;Lu Chao(School of Pharmacy,Anhui Medical University,Hefei 230032;Dept of Clinical Pharmacology,The Second Affiliated Hospital of Anhui Medical University,Hefei 230601;Dept of Clinical Pharmacology,The First Affiliated Hospital,Anhui University of Science&Technology,Huainan 232007)
出处
《安徽医科大学学报》
CAS
北大核心
2024年第6期1053-1059,共7页
Acta Universitatis Medicinalis Anhui
基金
安徽省自然科学基金(编号:2208085MH215)
安徽省中医药传承创新科研项目(编号:2020cczd05)
安徽省重点研究与开发计划项目(编号:202204295107020035)。
