摘要
目的探究姜黄素(Cur)调控过氧化物酶6(Prdx6)蛋白表达在抑制C28/I2人正常软骨细胞系铁死亡中的作用及可能机制。方法番红固绿和HE染色观察骨关节炎(OA)大鼠膝关节病理学改变。免疫组化和Western blot法分析检测Prdx6、GPX4蛋白在OA软骨组织中的表达。用不同浓度Cur处理C28/I2软骨细胞系,噻唑蓝(MTT)法检测细胞活力,乳酸脱氢酶(LDH)检测细胞毒性。流式细胞术检测软骨细胞中脂质活性氧(ROS)的产生,总谷胱甘肽(GSH)检测试剂盒检测软骨细胞中GSH含量。Western blot法检测软骨细胞中Prdx6和铁死亡相关蛋白的表达。分子对接技术探究Cur分子与Prdx6蛋白之间的相互作用。结果OA发病进程中,OA大鼠和OA患者出现软骨损伤、软骨细胞数量减少等病理学改变。与正常人相比,OA患者软骨组织中Prdx6、GPX4蛋白表达降低。进一步研究显示20μmol/L Cur可以抑制铁死亡诱导剂(Erastin)诱导的C28/I2软骨细胞系铁死亡,提高细胞活力,降低细胞毒性,抑制脂质ROS的产生,提高细胞内GSH含量,并增加Prdx6、SLC7A11、FTH、GPX4蛋白表达,降低ACSL4蛋白表达。此外,Cur分子与Prdx6蛋白分子对接过程中存在范德华力和π键相互作用。结论姜黄素可能通过上调Prdx6蛋白表达抑制Erastin诱导的C28/I2软骨细胞系铁死亡。
Objective To investigate the role and possible mechanism of curcumin(Cur)regulating Peroxiredoxin-6(Prdx6)expression in inhibiting Erastin-induced ferroptosis in C28/I2 chondrocytes.Methods Safranin O/Fast Green and hematoxylin-eosin(HE)staining were performed to observe the pathological changes in the knee joint of rats with osteoarthritis(OA).The expression levels of Prdx6 and GPX4 proteins in cartilage tissues with OA were detected by immunohistochemistry and Western blot.C28/I2 chondrocytes were treated with different concentrations of Cur,cell viability was detected by thiazolyl blue tetrazolium bromide(MTT)assay and cytotoxicity was measured by lactic dehydrogenase(LDH)assay.The production of lipid reactive oxygen species(ROS)in chondrocytes was detected by flow cytometry,and the total glutathione(GSH)assay kit was used to detect the GSH level in chondrocytes.Western blot was performed to detect the expression level of Prdx6 and ferroptosis-related proteins in chondrocytes.The interaction between the Cur molecule and Prdx6 was analyzed through the molecular docking technique.Results During the OA progression,OA rats and OA patients showed pathological changes such as damage to the cartilage and a decrease in the number of chondrocytes.The expression levels of Prdx6 and GPX4 were reduced in the cartilage tissues of OA patients compared with healthy people.Further study revealed that the treatment of Erastin-induced ferroptosis in C28/I2 chondrocytes in a mouse model with 20μmol/L of Cur could improve cell viability,decrease cytotoxicity,inhibit lipid ROS production,and increase the level of intracellular GSH.Western blot results showed decreased expression of Prdx6,SLC7A11,FTH,and GPX4 and increased expression of ACSL4.In addition,Cur molecules interacted with Prdx6 protein by van der Waals forces andπbond.Conclusion Cur may inhibit Erastin-induced ferroptosis in C28/I2 chondrocytes by upregulating the Prdx6 expression level.
作者
陈凡
周富丽
陈勇
付万进
周仁鹏
胡伟
鲁超
Chen Fan;Zhou Fuli;Chen Yong;Fu Wanjin;Zhou Renpeng;Hu Wei;Lu Chao(School of Pharmacy,Anhui Medical University,Hefei 230032;Dept of Clinical Pharmacology,The Second Hospital of Anhui Medical University,Hefei 230601;Dept of Clinical Pharmacology,The First Affiliated Hospital,Anhui University of Science&Technology,Huainan 232001)
出处
《安徽医科大学学报》
CAS
北大核心
2023年第12期2106-2112,共7页
Acta Universitatis Medicinalis Anhui
基金
安徽省自然科学基金(编号:2208085MH215)
安徽省重点研究与开发计划(编号:202204295107020035)。
