Liu Xiaorong, Jin Xiaomei, Wang Xianhui, Yan Xiaodan, Wang Chi, Wang Kaiyue, He Xiaoyan, Zhai Wanqing
Department of Neurology, The First People's Hospital of Taicang, Taicang Affiliated Hospital of Soochow University, Suzhou, Jiangsu, P.R. China.
Department of Neurosurgery, The First People's Hospital of Taicang, Taicang Affiliated Hospital of Soochow University, Suzhou, Jiangsu, P.R. China.
Immunopharmacol Immunotoxicol. 2023 Oct;45(5):539-548. doi: 10.1080/08923973.2023.2189061. Epub 2023 Jun 6.
The A20 protein is considered to have a potent anti-inflammatory effect, but its mechanism of action in the regulation of ferroptosis and inflammation after stroke is still unknown. In this study, the A20-knockdown BV2 cell line (sh-A20 BV2) was constructed at first, and the oxygen-glucose deprivation/re-oxygenation (OGD/R) cell model was constructed. Both the BV2 and sh-A20 BV2 cells were treated with the ferroptosis inducer erastin for 48 h, the ferroptosis-related indicators were detected by western blot. The mechanism of ferroptosis was explored by western blot and immunofluorescence. Under OGD/R pressure, the oxidative stress level of sh-A20 BV2 cells was inhibited, but the secretion of the inflammatory factors TNF-α, IL-1β, and IL-6 was significantly upregulated. And sh-A20 BV2 cells had higher expression levels of GPX4 and NLRP3 proteins under OGD/R induction. Western blot further confirmed that sh-A20 BV2 cells inhibited OGD/R-induced ferroptosis. Under the effect of erastin of the ferroptosis inducer (0-1000 nM), sh-A20 BV2 cells had higher cell viability than wild-type BV2 cells and significantly inhibited the accumulation of ROS and the level of oxidative stress damage. It was confirmed that A20 could promote the activation of the IκBα/NFκB/iNOS pathway. It was confirmed by an iNOS inhibitor that iNOS inhibition could reverse the resistance effect of BV2 cells to OGD/R-induced ferroptosis after A20 knockdown. In conclusion, this study demonstrated that inhibition of A20 mediates a stronger inflammatory response while enhancing microglial resistance by knocking down A20 in BV2 cells.
A20蛋白被认为具有强大的抗炎作用，但其在中风后铁死亡和炎症调节中的作用机制仍不清楚。在本研究中，首先构建了A20基因敲低的BV2细胞系（sh-A20 BV2），并构建了氧糖剥夺/复氧（OGD/R）细胞模型。将BV2和sh-A20 BV2细胞用铁死亡诱导剂erastin处理48小时，通过蛋白质免疫印迹法检测铁死亡相关指标。通过蛋白质免疫印迹法和免疫荧光法探讨铁死亡的机制。在OGD/R压力下，sh-A20 BV2细胞的氧化应激水平受到抑制，但炎症因子TNF-α、IL-1β和IL-6的分泌显著上调。并且sh-A20 BV2细胞在OGD/R诱导下具有更高的GPX4和NLRP3蛋白表达水平。蛋白质免疫印迹法进一步证实sh-A20 BV2细胞抑制了OGD/R诱导的铁死亡。在铁死亡诱导剂erastin（0-至1000 nM）作用下，sh-A20 BV2细胞比野生型BV2细胞具有更高的细胞活力，并显著抑制了活性氧的积累和氧化应激损伤水平。证实A20可促进IκBα/NFκB/iNOS途径的激活。通过iNOS抑制剂证实，抑制iNOS可逆转A20基因敲低后BV2细胞对OGD/R诱导的铁死亡的抵抗作用。总之，本研究表明，抑制A20可介导更强的炎症反应，同时通过敲低BV2细胞中的A20增强小胶质细胞的抗性。
