类风湿关节炎患者外周血单个核细胞System Xc-/GSH/GPX4铁死亡通路的表达及其对炎症因子分泌的影响

The expression of System Xc-/GSH/GPX4 ferroptosis pathway in peripheral blood mononuclear cells of rheumatoid arthritis patients and its effect on the secretion of inflammatory factors

目的探讨类风湿关节炎(RA)患者外周血单个核细胞(PBMC)胱氨酸/谷氨酸逆转运体(System Xc-)/谷胱甘肽(GSH)/谷胱甘肽过氧化物酶4(GPX4)铁死亡通路中相关基因与蛋白的表达及其对炎症因子分泌的影响。方法纳入RA患者及同期健康体检者各30例,采用Ficoll-hypaque密度梯度离心法分离PBMC,将细胞分为健康对照、RA、铁死亡抑制剂、铁死亡诱导剂组。采用细胞计数试剂盒8(CCK-8)检测细胞活力,流式细胞术FerroOrange荧光探针检测细胞内Fe 2+相对荧光强度、DHE荧光探针检测细胞内活性氧(ROS)阳性细胞比例,蛋白免疫印迹及实时荧光定量PCR(qPCR)检测核转录因子红系2相关因子(Nrf2)、溶质载体家族7成员11(SLC7A11)、GPX4蛋白与mRNA的表达,流式细胞术检测细胞上清液中肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-1、IL-6的含量。结果与健康对照组比较,RA组PBMC内Fe 2+浓度和ROS水平升高,Nrf2、SLC7A11、GPX4蛋白和mRNA的表达降低,PBMC上清液中TNF-α、IL-1、IL-6的含量升高,差异均有统计学意义。与RA组比较,铁死亡抑制剂组PBMC内Fe 2+浓度和ROS水平降低,SLC7A11、GPX4蛋白和mRNA的表达升高,Nrf2蛋白的表达升高,PBMC上清液中IL-6的含量降低、TNF-α的含量升高,差异均有统计学意义。与RA组比较,铁死亡诱导剂组PBMC内ROS水平升高,SLC7A11蛋白和mRNA的表达降低,Nrf2蛋白的表达降低、GPX4蛋白的表达升高,PBMC上清液中TNF-α、IL-1的含量升高,差异有统计学意义。与铁死亡诱导剂组比较,RA患者PBMC细胞活力降低,差异有统计学意义(P<0.0001)。结论RA患者PBMC中存在铁死亡,抑制或诱导RA患者PBMC铁死亡,将抑制或促进炎性因子分泌。抑制RA患者PBMC铁死亡可能有助于治疗RA。

Objective To investigate the expression of genes and proteins in the peripheral blood mononuclear cell(PBMC)cystine/glutamate antiporter system(System Xc-)/glutathione(GSH)/glutathione peroxidase 4(GPX4)ferroptosis pathway and its influence on inflammatory factors in patients with rheumatoid arthritis(RA).Methods 30 patients with RA and 30 healthy participants were enrolled.PBMCs were isolated using Ficoll-hypaque density gradient centrifugation.The cells were categorized into the healthy control,RA,ferroptosis inhibitor,ferroptosis inducer group.The cell viability was checked using the cell counting kit-8(CCK-8)method.Intracellular Fe 2+relative fluorescence intensity and reactive oxygen species(ROS)levels were detected using the FerroOrange and Dihydroethidium(DHE)fluorescent probes,respectively.Western blot and real-time quantitative PCR(qPCR)detected the expression of nuclear factor erythroid 2-related factor 2(Nrf2),solute carrier family 7 member 11(SLC7A11),GPX4 proteins and mRNA.And the flow cytometry quantified the levels of tumor necrosis factorα(TNF-α),Interleukin(IL)-1,and IL-6 in the supernatant of each cell group.Results Compared to the healthy control group,the RA group showed a significantly increased Fe 2+concentration and elevated ROS levels,reduced expression of Nrf2,SLC7A11 and GPX4 proteins and mRNA,and increased contents of TNF-α,IL-1 and IL-6 in PBMC supernatant,and the differences were statistically significant.The concentration of Fe 2+and ROS levels in the inhibitor group were lower than those in the RA group,the proteins expressions of Nrf2,SLC7A11 and GPX4 increased,the mRNA expressions of SLC7A11 and GPX4 increased,the content of IL-6 in the PBMC supernatant decreased but the content of TNF-αincreased,and the differences were statistically significant.In contrast,the inducer group,when compared to the RA group,displayed increased ROS levels,reduced expression of SLC7A11 protein and mRNA and decreased expression of Nrf2 protein,and the contents of TNF-αand IL-1 in the PBMC supernatant increased,but the expression of GPX4 protein increased,and the differences were statistically significant.The inducer group,compared to the RA group,showed increased cell viability,and the difference was statistically significant(P<0.0001).Conclusion The presence of ferroptosis in PBMC in RA patients,inhibiting or inducing PBMC ferroptosis in RA patients,will inhibit or promote the secretion of inflammatory factors.Inhibition of PBMC ferroptosis in RA patients may be helpful in the treatment of RA.