基于GSH/GPX4/ROS通路探讨独活寄生汤抑制软骨细胞铁死亡的机制研究  

Mechanism of Duhuo Jisheng Decoction Inhibiting Chondrocyte Ferroptosis Based on GSH/GPX4/ROS Signaling Pathway

作　　者：许丽梅 汪银枝 董丹宇 林叶青 王水良 李西海 林木南 XU Limei;WANG Yinzhi;DONG Danyu;LIN Yeqing;WANG Shuiliang;LI Xihai;LIN Munan(The 900th Hospital of Joint Logistic Support Force of the Chinese People's Liberation Army,Fuzhou,Fujian 350025,China;Fuzong Teaching Hospital(900th Hospital),Fujian University of Traditional Chinese Medicine,Fuzhou,Fujian 350025,China;Academy of Integrative Medicine,College of Integrative Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou,Fujian 350122,China)

机构地区：[1]中国人民解放军联勤保障部队第九〇〇医院,福建福州350025 [2]福建中医药大学福总教学医院(第九〇〇医院),福建福州350025 [3]福建中医药大学中西医结合学院中西医结合研究院,福建福州350122

出　　处：《康复学报》2025年第2期152-159,共8页Rehabilitation Medicine

基　　金：国家自然科学基金项目(82074461);福建省自然科学基金项目(2021J011258);福建中医药大学校管课题(XB2022141)。

摘　　要：目的基于GSH/GPX4/ROS通路探讨独活寄生汤抑制erastin诱导软骨细胞铁死亡的机制。方法选择4周龄SPF级雄性SD大鼠30只,采用机械-Ⅱ型胶原酶消化法提取大鼠原代软骨细胞进行体外培养。软骨细胞随机分为对照组、模型组、独活寄生汤组、Fer-1组,对照组用正常培养基培养;模型组用含1μmol/L erastin的培养基培养;独活寄生汤组用含1μmol/L erastin和300μg/mL独活寄生汤培养基培养;Fer-1组用含1μmol/L erastin和1μmol/L Ferrostatin-1培养基培养,每组干预24 h。干预后,采用透射电镜观察各组软骨细胞超微结构;采用比色法检测细胞内丙二醛(MDA)含量;采用微量法检测铁离子(Fe2+)含量;采用微板法检测谷胱甘肽(GSH)含量;采用Western blot检测谷胱甘肽过氧化物酶4(GPX4)、溶质载体家族7成员11(SLC7A11)和酰基辅酶A合成酶长链家族成员4(ACSL4)蛋白表达水平;采用荧光显微镜观察细胞内GPX4蛋白表达水平;采用荧光探针检测法观察细胞内ROS和脂质过氧化水平。结果①软骨细胞超微结构:与对照组比较,模型组线粒体嵴减少甚至消失,线粒体外膜破裂。与模型组比较,独活寄生汤组线粒体膜相对完整,线粒体变窄,嵴数量增加。②MDA、Fe2+、GSH含量:与对照组比较,模型组MDA、Fe2+含量均明显升高(P<0.05),GSH含量明显降低(P<0.05);与模型组比较,独活寄生汤组和Fer-1组MDA、Fe2+含量均明显降低(P<0.05),GSH含量均明显升高(P<0.05)。③软骨细胞铁死亡相关蛋白表达水平:与对照组比较,模型组GPX4、SLC7A11蛋白表达水平明显降低,ACSL4蛋白表达水平明显上升,GPX4蛋白荧光表达量明显降低,差异均具有统计学意义(P<0.05);与模型组比较,独活寄生汤组GPX4、SLC7A11蛋白表达水平均明显上升(P<0.05),Fer-1组GPX4、SLC7A11蛋白表达水平差异无统计学意义(P>0.05),独活寄生汤组、Fer-1组ACSL4蛋白表达水平明显降低,GPX4蛋白荧光表达量明显增加,差异均�Objective To explore the mechanism of Duhuo Jisheng decoction inhibiting erastin-induced chondrocyte ferroptosis through the glutathione/glutathione peroxidase 4/reactive oxygen species(GSH/GPX4/ROS)signaling pathway.Methods A total of 304-week-old SPF-grade male SD rats were selected.Primary chondrocytes were isolated by mechanical typeⅡcollagenase digestion method and cultured in vitro.Chondrocytes were randomly divided into control group,model group,Duhuo Jisheng Decoction group,and Fer-1 group.The control group was cultured with normal medium;the model group was cultured with medium containing 1μmol/L erastin;the Duhuo Jisheng Decoction group was cultured with medium containing 1μmol/L erastin and 300μg/mL Duhuo Jisheng Decoction;the Fer-1 group was cultured in medium containing 1μmol/L erastin and 1μmol/L Ferrostatin-1.All groups were treated for 24 hours.After intervention,transmission electron microscope was used to observe the ultrastructure of the chondrocytes;colorimetric method was used to detect the content of malondialdehyde(MDA);micromethod was used to detect the content of iron ion(Fe2+);microplate assay was used to detect the content of glutathione(GSH);Western blot was used to detect the protein expression levels of glutathione peroxidase 4(GPX4),solute carrier family 7 member 11(SLC7A11)and long-chain acylcoenzyme A(CoA)synthase 4(ACSL4);fluorescence microscopy was used to detect the expression level of GPX4 protein;fluorescence probe was used to detect the levels of intracellular ROS and lipid peroxidation.Results(1)Ultrastructure of chondrocytes:compared with the control group,the mitochondrial cristae in the model group decreased or even disappeared,and the outer membranes of mitochondria were broken.Compared with the model group,the mitochondrial membranes of the Duhuo Jisheng Decoction group were relatively complete,the mitochondria became narrow,and the number of cristae increased.(2)Contents of MDA,Fe2+and GSH:compared with the control group,the contents of MDA and Fe2+in the model

关 键 词：骨关节炎 软骨细胞 铁死亡 GSH/GPX4/ROS通路 独活寄生汤 

分 类 号：R285.5[医药卫生—中药学]