膝痹宁Ⅱ方对膝骨关节炎软骨细胞铁死亡及Nrf2/GPX4信号通路的影响
Effects of Xibining Ⅱ Prescription on knee osteoarthritis chondrocytes ferroptosis and Nrf2/GPX4 signaling pathway
廖太阳 1马振源 1邢润麟 2李晓辰 2梅伟 2吴鹏 2徐波 3茆军 2丁亮 2王培民4
作者信息
- 1. 南京中医药大学附属医院/江苏省中医院,南京 210029;南京中医药学大学代谢病中医研究重点实验室,南京 210023
- 2. 南京中医药大学附属医院/江苏省中医院,南京 210029
- 3. 南京中医药大学附属苏州市中医医院,苏州 215007
- 4. 南京中医药大学附属医院/江苏省中医院,南京 210029;江苏省中医外用药开发与应用工程研究中心,南京 210023
- 折叠
摘要
目的:探究膝痹宁Ⅱ方对膝骨关节炎(KOA)原代小鼠关节软骨细胞(PMCs)铁死亡的影响及其保护机制.方法:使用叔丁基过氧化氢(TBHP)诱导PMCs铁死亡模型,随机分为对照组,模型组和膝痹宁Ⅱ低、中、高剂量组.采用CCK-8法和EDU染色检测细胞活力;流式细胞术检测活性氧(ROS)水平;FerroOrange染色检测Fe2+水平;试剂盒检测铁含量、丙二醛(MDA)、超氧化物歧化酶(SOD)和谷胱甘肽(GSH)水平,BODIPY 581/591 C11染色检测脂质过氧化水平;JC-1染色检测线粒体膜电位水平;Western Blot和qRT-PCR检测核因子E2相关因子2(Nrf2)、谷胱甘肽过氧化物酶4(GPX4)、溶质载体家族7成员11(SLC7A11)、长链脂酰辅酶A合成酶4(ACSL4)蛋白和mRNA表达水平.结果:与模型组比较,膝痹宁Ⅱ方各剂量组可显著增加PMCs细胞存活率,抑制ROS水平升高,降低Fe2+、MDA水平和铁含量,升高SOD、GSH水平,减少脂质过氧化损伤,恢复线粒体膜电位,上调Nrf2、GPX4和SLC7A11表达,下调ACSL4表达,差异均有统计学意义(P＜0.01,P＜0.05).结论:膝痹宁Ⅱ方在体外可能通过激活Nrf2/GPX4信号通路发挥抗氧化作用,实现抑制KOA小鼠PMCs铁死亡的作用.
Abstract
Objective:To investigate the effects and protective mechanism of Xibining Ⅱ Prescription on ferroptosis of primary mouse articular chondrocytes(PMCs)in knee osteoarthritis(KOA).Methods:A cellular ferroptosis model of PMCs was induced using tert-butyl hydroperoxide(TBHP)and randomly divided into control group,model group and low,medium and high dose groups of Xibining Ⅱ Prescription.The cell viability was detected by CCK-8 and EDU staining.Reactive oxygen species(ROS)levels were detected by flow cytometry.The Fe2+level was detected using FerroOrange staining.The levels of iron content,malondialdehyde(MDA),superoxide dismutase(SOD),and glutathione(GSH)were detected using corresponding reagent kits.The lipid peroxidation level was detected using BODIPY 581/591 C11 staining.The mitochondrial membrane potential level was detected using JC-1 staining.The protein and mRNA expression levels of Nrf2,GPX4,SLC7A11,and ACSL4 were detected by Western Blot and qRT-PCR,respectively.Results:Compared with the model group,Xibining Ⅱ Prescription all dose groups were able to significantly increase the survival rate of PMCs cells,inhibit the increase of ROS levels,reduce the levels of Fe2+,MDA,and iron content,increase the levels of SOD and GSH,reduce lipid peroxidation damage,restore mitochondrial membrane potential,upregulate the expression of Nrf2,GPX4,and SLC7A11,and downregulate ACSL4 expression(P＜0.01,P＜0.05).Conclusion:Xibining Ⅱ Prescription may potentially activate the Nrf2/GPX4 signaling pathway in vitro and exert antioxidant effects to inhibit ferroptosis of KOA mouse articular chondrocytes.
关键词
膝痹宁Ⅱ方/膝骨关节炎/软骨细胞/铁死亡/核因子E2相关因子2/谷胱甘肽过氧化物酶4信号通路/抗氧化Key words
Xibining Ⅱ Prescription/Knee osteoarthritis(KOA)/Chondrocytes/Ferroptosis/Nrf2/GPX4 signaling pathway/Antioxidant引用本文复制引用
基金项目
国家自然科学基金面上项目(82274545)
江苏省医学重点学科/实验室建设单位(JSDW202252)
江苏省中医院第三批高峰学术人才(y2021rc02)
江苏省中医院中医膝骨关节炎临床医学创新中心(Y2023zx05)
江苏省第四批中医临床优秀人才()
江苏省自然科学基金青年项目(BK20220260)
出版年
2024
NETL
NSTL
万方数据