首页|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

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

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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.
Effects of Xibining Ⅱ Prescription on knee osteoarthritis chondrocytes ferroptosis and Nrf2/GPX4 signaling pathway
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.

Xibining Ⅱ PrescriptionKnee osteoarthritis(KOA)ChondrocytesFerroptosisNrf2/GPX4 signaling pathwayAntioxidant

廖太阳、马振源、邢润麟、李晓辰、梅伟、吴鹏、徐波、茆军、丁亮、王培民

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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

南京中医药大学附属苏州市中医医院,苏州 215007

江苏省中医外用药开发与应用工程研究中心,南京 210023

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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

国家自然科学基金面上项目江苏省医学重点学科/实验室建设单位江苏省中医院第三批高峰学术人才江苏省中医院中医膝骨关节炎临床医学创新中心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

82274545JSDW202252y2021rc02Y2023zx05BK20220260

2024

中华中医药杂志
中华中医药学会

中华中医药杂志

CSTPCD北大核心
影响因子：1.135
ISSN：1673-1727
年,卷(期)：2024.39(10)