METHODS: To evaluate the protective effects of EMPA, the drug was injected into the vitreous body of mice post-retinal IR. Single-cell RNA sequencing (scRNA-seq) analysis was conducted to uncover the underlying mechanisms, and the results were further validated through in vivo and in vitro experiments.
RESULTS: EMPA effectively protected retinal ganglion cells (RGCs) from IR injury by attenuating local retinal inflammation. The scRNA-seq analysis revealed that EMPA downregulated the nucleotide-binding domain and leucine-rich repeat containing protein 3 (NLRP3) signaling pathway and restored mitochondrial dynamics by upregulating the expression of mitochondrial fusion-related genes, Mitofusin 1 (Mfn1) and optic atrophy 1 (Opa1). These findings were further corroborated by Western blotting. In vitro experiments provided additional insights, demonstrating that EMPA suppressed lipopolysaccharide (LPS)-induced cell inflammation and NLRP3 inflammasome activation. Moreover, EMPA enhanced mitochondrial fusion, neutralized mitochondrial reactive oxygen species (mtROS), and restored mitochondrial membrane potential (MMP) in BV2 microglia. Notably, genetic ablation of Mfn1 or Opa1 abolished the anti-inflammatory effects of EMPA.
CONCLUSIONS: Our findings highlight the positive contribution of Mfn1 and Opa1 to the anti-inflammatory therapeutic effect of EMPA. By restoring mitochondrial dynamics, EMPA effectively mitigates microglia-mediated neuroinflammation and prevents RGC loss in retinal IR injury.
方法:为了评估EMPA的保护作用,将药物注射到视网膜后IR小鼠的玻璃体中。进行单细胞RNA测序(scRNA-seq)分析以揭示潜在的机制,并通过体内外实验进一步验证结果。
结果:EMPA通过减轻局部视网膜炎症有效保护视网膜神经节细胞(RGCs)免受IR损伤。scRNA-seq分析显示,EMPA下调核苷酸结合域和富含亮氨酸重复序列的蛋白3(NLRP3)信号通路,并通过上调线粒体融合相关基因的表达来恢复线粒体动力学,有丝分裂素1(Mfn1)和视神经萎缩1(Opa1)。通过Western印迹进一步证实了这些发现。体外实验提供了额外的见解,证明EMPA抑制脂多糖(LPS)诱导的细胞炎症和NLRP3炎性体激活。此外,EMPA增强线粒体融合,中和的线粒体活性氧(mtROS),并恢复BV2小胶质细胞的线粒体膜电位(MMP)。值得注意的是,Mfn1或Opa1的基因消融消除了EMPA的抗炎作用.
结论:我们的发现强调了Mfn1和Opa1对EMPA抗炎治疗作用的积极贡献。通过恢复线粒体动力学,EMPA有效缓解小胶质细胞介导的神经炎症并防止视网膜IR损伤中的RGC损失。