铁死亡在HIV-1 gp120 V3环致小胶质细胞炎症中的作用机制研究

Role and mechanism of ferroptosis in microglial inflammation induced by HIV-1 gp120 V3 loop

目的:探讨人类免疫缺陷病毒1型(human immunodeficiency virus type 1,HIV-1)gp120 V3环致CHME-5小胶质细胞炎症反应与铁死亡的关系,并观察p53和铁死亡对该炎症反应的影响及可能机制。方法:体外培养人源CHME-5小胶质细胞,设立空白组、随机肽段组、HIV-1 gp120 V3环组、HIV-1 gp120 V3环+ferrostatin-1(Fer-1;铁死亡抑制剂)组和HIV-1 gp120 V3环+pifithrin-α(p53抑制剂)组。分别采用HIV-1 gp120 V3环(终浓度2 mg/L)和随机肽段(终浓度2 mg/L)处理CHME-5细胞24 h;Fer-1(终浓度20μmol/L)和pifithrin-α(终浓度10μmol/L)预处理CHME-5细胞2 h,HIV-1 gp120 V3环(终浓度2 mg/L)再处理24 h。ELISA法检测各组细胞上清液中炎症因子水平;Western blot法检测铁死亡相关蛋白[转铁蛋白受体1(transferrin receptor-1,TFR-1)、溶质载体家族7成员11(solute carrier family 7 member 11,SLC7A11)和谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)]及p53的蛋白表达;酶标仪法检测细胞内亚铁离子(Fe2+)和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活性。结果:(1)ELISA结果显示,与对照组相比,gp120 V3环组炎症因子白细胞介素1β(interleukin-1β,IL-1β)、IL-6和肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)水平显著升高(P<0.01);与gp120 V3环组相比,gp120 V3环+Fer-1组和gp120 V3环+pifithrin-α组炎症因子IL-1β、IL-6和TNF-α水平显著下降(P<0.01);(2)Western blot结果显示,与对照组相比,gp120 V3环组蛋白p53显著上调(P<0.01),铁死亡相关蛋白TFR-1显著上调(P<0.01),SLC7A11和GPX4显著下调(P<0.01);与gp120 V3环组相比,gp120 V3环+pifithrin-α组铁死亡相关蛋白TFR-1显著下降(P<0.05),SLC7A11和GPX4蛋白显著升高(P<0.05);(3)与对照组相比,gp120 V3环组Fe2+含量显著增加(P<0.01),GSH-Px活性显著降低(P<0.01);与gp120 V3环组相比,gp120 V3环+Fer-1组和gp120 V3环+pifithrin-α组Fe2+含量显著下降(P<0.05),GSH-Px活性显著升高(P<0.01)。结论:HIV-1 gp120 V3环致CHME-5小胶质细胞炎症中存在铁死亡,且抑制铁死亡能减轻炎症。HIV-1 gp120 V3环致CHME-5小胶质细胞炎症与p53蛋白调控铁死亡有关,抑制p53可减轻铁死亡和炎症反应。

AIM:To investigate the relationship between the inflammatory response and ferroptosis induced by human immunodeficiency virus type 1(HIV-1)gp120 V3 loop in CHME-5 microglia,and to observe the effects of p53 and ferroptosis on inflammatory response and its possible mechanisms.METHODS:Human CHME-5 microglia were cul⁃tured in vitro,and were dividied into 5 group:blank group,random peptide group,HIV-1 gp120 V3 loop group,HIV-1 gp120 V3 loop+ferrostatin-1(Fer-1;an inhibitor of ferroptosis)group and HIV-1 gp120 V3 loop+pifithrin-α(an inhibitor of p53)group.The CHME-5 cells were treated with HIV-1 gp120 V3 loop(final concentration:2 mg/L)and random pep⁃tide(final concentration:2 mg/L)for 24 h,or pretreated with Fer-1(final concentration:20μmol/L)or pifithrin-a(final concentration:10μmol/L)for 2 h,and then HIV-1 gp120 V3 loop for another 24 h.ELISA was used to detect the expres⁃sion levels of inflammatory factors in the cell supernatant of each group.Western blot was used to measure the expression levels of ferroptosis-related proteins[glutathione peroxidase 4(GPX4),transferrin receptor-1(TFR-1)and solute carrier family 7 member 11(SLC7A11)]and p53.The level of intracellular ferrous ion(Fe2+)and the activity of glutathione per⁃oxidase(GSH-Px)were measured.RESULTS:Compared with blank group and random peptide group,the levels of inter⁃leukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and IL-6 in HIV-1 gp120 V3 loop group were significantly increased(P<0.01).Compared with HIV-1 gp120 V3 loop group,the levels of IL-1β,IL-6 and TNF-αwere significantly decreased in the HIV-1 gp120 V3 loop+Fer-1 group and HIV-1 gp120 V3 loop+pifithrin-αgroup(P<0.01).Western blot showed that compared with blank group and random peptide group,protein p53 was significantly upregulated(P<0.01),ferropto⁃sis-related proteins TFR-1 was up-regulated(P<0.01),SLC7A11 and GPX4 were significantly downregulated(P<0.01)in HIV-1 gp120 V3 loop group.Compared with HIV-1 gp120 V3 loop group,ferroptosis-related protein TFR-1 was signifi⁃cantly decreased(P<0.01),GPX4 and SLC7A11 were significantly up-regulated in HIV-1 gp120 V3 loop+pifithrin-αgroup(P<0.05).Compared with blank group and random peptide group,the level of Fe2+was significantly increased(P<0.01),and the activity of GSH-Px was significantly decreased(P<0.01)in HIV-1 gp120 V3 loop group.Compared with HIV-1 gp120 V3 loop group,the level of Fe2+was reduced(P<0.05)and the activity of GSH-Px was significantly im⁃proved in HIV-1 gp120 V3 loop+Fer-1 group and HIV-1 gp120 V3 loop+pifithrin-αgroup(P<0.01).CONCLUSION:Ferroptosis exists in CHME-5 microglial inflammation induced by HIV-1 gp120 V3 loop,and inhibition of ferroptosis can attenuate inflammation.The CHME-5 microglial inflammation induced by HIV-1 gp120 V3 loop is related to the regulation of ferroptosis by p53,and inhibition of p53 can attenuate ferroptosis and inflammatory.