Li Yue, Yang Boyu, Chen Qi, Zhou Yuchen, Hu Wen, Huang Xiaojia, Huang Guangyi, Tang Ningning, Tang Fen, Huang Hui, Lan Qianqian, He Wenjing, Xu Fan, Ye Yiming, Jiang Li
Department of Ophthalmology, The People's Hospital of Guangxi Zhuang Autonomous Region, Guangxi Key Laboratory of Eye Health, Guangxi Health Commission Key Laboratory of Ophthalmology and Related Systemic Diseases Artificial Intelligence Screening Technology, Institute of Ophthalmic Diseases, Guangxi Academy of Medical Sciences, Guangxi, China.
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou, China.
Invest Ophthalmol Vis Sci. 2025 Jun 2;66(6):54. doi: 10.1167/iovs.66.6.54.
To reveal the role of receptor-interacting protein kinase 3 (RIPK3) in regulating macrophage inflammation and corneal neovascularization (CoNV) induced by alkali burn.
A corneal alkali burn (AB) model was established in C57BL/6J (wild-type) and RIPK3fl/flCx3cr1+/cre (RIPK3-/-, RIPK3 knockout [KO]) mice using sodium hydroxide. Anterior segment optical coherence tomography and hematoxylin and eosin staining were used to evaluate the impact of RIPK3 on corneal edema and morphology. CoNV was detected by slit-lamp microscopy and whole-mount immunofluorescence staining of cornea. Corneal macrophage and necroptotic cell death was analyzed through immunofluorescence staining and propidium iodide (PI) staining. Activation of the necroptosis pathway was examined after corneal AB by western blot.
Necroptosis-related proteins RIPK1, RIPK3, and mixed lineage kinase domain-like (MLKL) were upregulated and activated following corneal AB. Among these, RIPK3 demonstrated the most pronounced increase. Notably, the elevated level of RIPK3 was prominently colocalized with the infiltrating F4/80+ macrophages. RIPK3 KO significantly alleviated corneal edema and morphology defects. Additionally, as the corneal morphological defects progressed, macrophages became activated, and CoNV and lymphangiogenesis (LyG) were enhanced. RIPK3 KO markedly reduced AB-induced macrophage accumulation, as well as CoNV and LyG. RIPK3 KO mice also showed a meaningful decrease in PI+ necroptotic cells. Mechanistically, AB-induced necroptosis stimulated the expression of MLKL and fibroblast growth factor 2 (FGF2), whereas RIPK3 deficiency decreased their expression.
This study revealed that RIPK3-mediated necroptosis drives macrophage inflammation and CoNV. Targeting RIPK3 could effectively suppress these responses by inhibiting the MLKL/FGF2 pathway, making it a promising therapeutic strategy for corneal AB.
揭示受体相互作用蛋白激酶3（RIPK3）在调节碱烧伤诱导的巨噬细胞炎症和角膜新生血管形成（CoNV）中的作用。
使用氢氧化钠在C57BL/6J（野生型）和RIPK3fl/flCx3cr1+/cre（RIPK3-/-，RIPK3基因敲除[KO]）小鼠中建立角膜碱烧伤（AB）模型。采用眼前节光学相干断层扫描和苏木精-伊红染色评估RIPK3对角膜水肿和形态的影响。通过裂隙灯显微镜检查和角膜整装免疫荧光染色检测CoNV。通过免疫荧光染色和碘化丙啶（PI）染色分析角膜巨噬细胞和坏死性细胞死亡。通过蛋白质印迹法检测角膜AB后坏死性凋亡途径的激活情况。
角膜AB后，坏死性凋亡相关蛋白RIPK1、RIPK3和混合谱系激酶结构域样蛋白（MLKL）上调并激活。其中，RIPK3的增加最为显著。值得注意的是，RIPK3水平的升高与浸润的F4/80+巨噬细胞显著共定位。RIPK3基因敲除显著减轻了角膜水肿和形态缺陷。此外，随着角膜形态缺陷的进展，巨噬细胞被激活，CoNV和淋巴管生成（LyG）增强。RIPK3基因敲除显著减少了AB诱导的巨噬细胞积聚以及CoNV和LyG。RIPK3基因敲除小鼠的PI+坏死性凋亡细胞也有显著减少。机制上，AB诱导的坏死性凋亡刺激了MLKL和成纤维细胞生长因子2（FGF2）的表达，而RIPK3缺乏则降低了它们的表达。
本研究表明，RIPK3介导的坏死性凋亡驱动巨噬细胞炎症和CoNV。靶向RIPK3可通过抑制MLKL/FGF2途径有效抑制这些反应，使其成为角膜AB的一种有前景的治疗策略。
