Department of Anatomy and Neurobiology, School of Basic Medical Science, Central South University, Changsha 410008, China; The Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital), Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences, Hangzhou 310022, China.
Jiangxi Research Institute of Ophthalmology and Visual Sciences, Affiliated Eye Hospital of Nanchang University, Nanchang 330006, China.
Biochim Biophys Acta Mol Cell Res. 2023 Mar;1870(3):119433. doi: 10.1016/j.bbamcr.2023.119433. Epub 2023 Jan 24.
Ischemia/reperfusion (I/R) injury is one of the most common etiologies in many diseases. Retinal I/R leads to cytokine storm, resulting in tissue damage and cell death. Pyroptosis, a novel type of regulated cell death, occurs after cellular I/R injury. In this study, we established an oxygen glucose deprivation (OGD/R) cellular model (R28) to simulate retinal I/R injury. We conducted an LDH assay, and EthD-III and PI staining procedures to confirm pyroptosis. Mass spectrometry and bioinformatics analysis were used to identify the possible proteins interacting with NLRP3. Co-IP and various molecular biology techniques were used to investigate the possible modes regulating NLRP3 by DTX3L. EthD-III, PI staining and LDH assays demonstrated pyroptosis induced by OGD/R injury, mediated via NLRP3 pathway. Mass spectrometry and bioinformatics analysis screened out three candidate proteins interacting with NLRP3, and further Co-IP experiment indicated that DTX-3L may interact with NLRP3 to regulate its protein levels after injury. Co-IP experiments and various molecular biology methods demonstrated that DTX3L ubiquitinates NLRP3 resulting in pyroptosis after R28 OGD/R injury. Further, NLRP3 LRR and DTX3L RING domains interact with each other. Our study demonstrated that DTX3L may ubiquitinate NLRP3 to regulate OGD/R-induced pyroptosis globally in R28 cells.
缺血再灌注（I/R）损伤是许多疾病中最常见的病因之一。视网膜 I/R 导致细胞因子风暴，导致组织损伤和细胞死亡。细胞 I/R 损伤后会发生细胞焦亡，这是一种新型的调控性细胞死亡。在本研究中，我们建立了一个氧葡萄糖剥夺（OGD/R）细胞模型（R28）来模拟视网膜 I/R 损伤。我们进行了 LDH 测定、EthD-III 和 PI 染色程序以确认细胞焦亡。质谱和生物信息学分析用于鉴定可能与 NLRP3 相互作用的蛋白。免疫共沉淀（Co-IP）和各种分子生物学技术用于研究 DTX3L 可能调节 NLRP3 的方式。EthD-III、PI 染色和 LDH 测定表明，OGD/R 损伤诱导的细胞焦亡是通过 NLRP3 途径介导的。质谱和生物信息学分析筛选出与 NLRP3 相互作用的三个候选蛋白，进一步的 Co-IP 实验表明，DTX-3L 可能与 NLRP3 相互作用以调节其损伤后的蛋白水平。Co-IP 实验和各种分子生物学方法表明，DTX3L 使 NLRP3 泛素化，导致 R28 OGD/R 损伤后发生细胞焦亡。此外，NLRP3 的 LRR 和 DTX3L 的 RING 结构域相互作用。我们的研究表明，DTX3L 可能通过泛素化 NLRP3 来调节 R28 细胞中 OGD/R 诱导的细胞焦亡。
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