Shen Hangqi, Gong Qiaoyun, Zhang Jingting, Wang Haiyan, Qiu Qinghua, Zhang Jingfa, Luo Dawei
Department of Ophthalmology, Shanghai General Hospital (Shanghai First People's Hospital), Shanghai Jiao Tong University School of Medicine, 100 Haining Road, Hongkou District, Shanghai, 200080, China.
Eye Vis (Lond). 2022 Sep 5;9(1):35. doi: 10.1186/s40662-022-00305-2.
Diabetic retinopathy (DR) as a severe diabetic complication contributes to blindness. The increased permeability of retinal capillary endothelial cells (RCECs) as well as the production of inflammatory markers are closely related to DR occurrence. We recently revealed that TRIM46 promotes high glucose (HG)-caused ferroptosis in human RCECs (HRCECs). The current study aims to explore the molecular mechanism of how TRIM46 plays its role in DR progression.
Western blot was utilized to determine protein expression. The cell counting kit-8 assay was used to observe cell viability. The permeability of the cell layer was determined by measuring the transepithelial electrical resistance and fluorescein isothiocyanate (FITC)-dextran leak. Enzyme-linked immunosorbent assay was used to quantify the protein level of pro-inflammatory cytokines and co-immunoprecipitation was employed to verify the relationship between TRIM46 and IκBα.
HG dramatically upregulated TRIM46 protein expression in a dose-dependent way. Silencing TRIM46 effectively reversed HG-induced cell growth inhibition, cell cycle arrest, hyper permeability and pro-inflammatory cytokines secretion in HRCECs, while overexpression of TRIM46 exhibited an opposite effect. Furthermore, TRIM46 was able to interact with IκBα and promote the ubiquitination and degradation of IκBα. IκBα overexpression recovered the effects of TRIM46 overexpression in HRCECs. Furthermore, inhibiting the activation of NF-κB partially recovered HG-induced HRCEC injury, whereas TRIM46 overexpression reversed these effects.
This study demonstrates that TRIM46 interacts with IκBα to activate the NF-κB signaling pathway, thereby enhancing cell proliferation inhibition, hyper permeability and the inflammatory response of HRCECs in a HG state.
糖尿病视网膜病变（DR）作为一种严重的糖尿病并发症可导致失明。视网膜毛细血管内皮细胞（RCECs）通透性增加以及炎症标志物的产生与DR的发生密切相关。我们最近发现TRIM46促进高糖（HG）诱导的人RCECs（HRCECs）铁死亡。本研究旨在探讨TRIM46在DR进展中发挥作用的分子机制。
采用蛋白质免疫印迹法测定蛋白质表达。使用细胞计数试剂盒-8法观察细胞活力。通过测量跨上皮电阻和异硫氰酸荧光素（FITC）-葡聚糖渗漏来测定细胞层的通透性。采用酶联免疫吸附测定法量化促炎细胞因子的蛋白质水平，并采用免疫共沉淀法验证TRIM46与IκBα之间的关系。
HG以剂量依赖的方式显著上调TRIM46蛋白表达。沉默TRIM46可有效逆转HG诱导的HRCECs细胞生长抑制、细胞周期阻滞、高通透性和促炎细胞因子分泌，而TRIM46过表达则表现出相反的效果。此外，TRIM46能够与IκBα相互作用并促进IκBα的泛素化和降解。IκBα过表达恢复了TRIM46过表达对HRCECs的影响。此外，抑制NF-κB的激活部分恢复了HG诱导的HRCEC损伤，而TRIM46过表达则逆转了这些影响。
本研究表明，TRIM46与IκBα相互作用以激活NF-κB信号通路，从而增强HG状态下HRCECs的细胞增殖抑制、高通透性和炎症反应。
