Xu Lisi, Zhang Ruonan, Zhang Xiaolin, Liu Bing, Shang Xiuli, Huang Daifa
Department of the Second Cadre Ward, General Hospital of Northern Theater Command, Shenyang, China.
Department of Neurology, The First Hospital of China Medical University, Shenyang, China.
Mol Med. 2025 Sep 2;31(1):284. doi: 10.1186/s10020-025-01326-y.
Neuroinflammation mediated by microglia activation is the key pathological mechanisms for cerebral ischemia-reperfusion injury (CIRI). This study investigated the role and underlying molecular mechanism of Rhomboid 5 homolog 2 (RHBDF2) in neuroinflammation during CIRI.
The in vivo middle cerebral artery occlusion and reperfusion (MCAO/R) mouse model and in vitro HMC3 microglia subjected to oxygen glucose deprivation and reperfusion (OGD/R) were established to mimic CIRI. Real-time PCR, western blot, immunohistochemistry, immunofluorescence, flow cytometry, and co-immunoprecipitation assays were used to confirm RHBDF2 expression and explore the molecular mechanism of microglia-specific RHBDF2 knockdown in CIRI. Methylated RNA immunoprecipitation was used to detect the m6A methylation level of RHBDF2 mRNA both in vivo and in vitro. RNA sequencing analysis was performed in OGD/R-treated HMC3 cells with or without RHBDF2 knockdown.
Our finding showed that RHBDF2 expression increased in both in vivo and in vitro CIRI models. Microglial-specific RHBDF2 knockdown reduced brain injury in MCAO/R mice, as evidenced by the reduction in the cerebral infarct volume and amelioration of the neurological deficits. Furthermore, we demonstrated that RHBDF2 knockdown alleviated neuroinflammation by inhibiting microglial M1 polarization and promoting microglial M2 polarization in MCAO/R mouse ischemic penumbra. Mechanistically, RHBDF2 interacted with STING and promoted the activation of the STING-TBK1-IRF3/p65 signaling pathway. Rescue experiments confirmed that RHBDF2 knockdown suppressed inflammation via the inhibition of STING-TBK1 signaling pathway. In addition, the m6A methylation level of RHBDF2 mRNA was significantly increased in the MCAO/R mouse brain tissues and OGD/R-treated HMC3 cells. YTHDF1 recognized the m6A sites of RHBDF2 and promote its expression in an m6A manner. Through RNA-seq, the possible downstream effectors of RHBDF2 in CIRI was predicted.
Microglial-specific RHBDF2 knockdown inhibits neuroinflammation in CIRI via STING-TBK1 signaling pathway, and is positively regulated by the m6A reader YTHDF1. This suggests RHBDF2 as a potential therapeutic target in ischemic stroke.
小胶质细胞激活介导的神经炎症是脑缺血再灌注损伤（CIRI）的关键病理机制。本研究探讨了菱形蛋白5同源物2（RHBDF2）在CIRI神经炎症中的作用及潜在分子机制。
建立体内大脑中动脉闭塞再灌注（MCAO/R）小鼠模型和体外氧糖剥夺再灌注（OGD/R）处理的HMC3小胶质细胞模型以模拟CIRI。采用实时定量PCR、蛋白质印迹、免疫组织化学、免疫荧光、流式细胞术和免疫共沉淀实验来确认RHBDF2的表达，并探索小胶质细胞特异性敲低RHBDF2在CIRI中的分子机制。采用甲基化RNA免疫沉淀法检测体内外RHBDF2 mRNA的m6A甲基化水平。对敲低或未敲低RHBDF2的OGD/R处理的HMC3细胞进行RNA测序分析。
我们的研究结果表明RHBDF2在体内和体外CIRI模型中表达均增加。小胶质细胞特异性敲低RHBDF2可减轻MCAO/R小鼠的脑损伤，脑梗死体积减小和神经功能缺损改善证明了这一点。此外，我们证明敲低RHBDF2可通过抑制MCAO/R小鼠缺血半暗带中小胶质细胞的M1极化和促进M2极化来减轻神经炎症。机制上，RHBDF2与STING相互作用并促进STING-TBK1-IRF3/p65信号通路的激活。挽救实验证实敲低RHBDF2通过抑制STING-TBK1信号通路抑制炎症。此外，MCAO/R小鼠脑组织和OGD/R处理的HMC3细胞中RHBDF2 mRNA的m6A甲基化水平显著增加。YTHDF1识别RHBDF2的m6A位点并以m6A方式促进其表达。通过RNA测序，预测了RHBDF2在CIRI中可能的下游效应分子。
小胶质细胞特异性敲低RHBDF2通过STING-TBK1信号通路抑制CIRI中的神经炎症，并受到m6A阅读蛋白YTHDF1的正向调控。这表明RHBDF2是缺血性中风的潜在治疗靶点。
