Yang Juan, Yao Yingying, Fan Shuo, Li Xiaoyan
Department of Respiratory and Critical Care Medicine, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, Hubei, China.
Department of Emergency and Intensive Care, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, Hubei, China.
Shock. 2025 Feb 1;63(2):210-216. doi: 10.1097/SHK.0000000000002442.
Background: Ubiquitination and deubiquitination are involved in the progression of human diseases, including acute pneumonia. In this study, we aimed to explore the functions of ubiquitin-specific peptidase 9X-linked (USP9X) in lipopolysaccharide (LPS)-treated WI-38 cells. Methods: WI-38 cells were treated with LPS to induce the cellular damage and inflammation. 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay, and 5-ethynyl-2'-deoxyuridine (EdU) assay were performed to examine the proliferation of LPS-treated WI-38 cells. Flow cytometry analysis was conducted to detect LPS-treated WI-38 cell apoptosis. ELISA kits were utilized to determine the concentrations of inflammatory factors (IL-1β and TNF-α). Superoxide dismutase activity and reactive oxygen species level were examined with related kits. Ubibrowser (http://ubibrowser.bio-it.cn/ubibrowser/), ubiquitination assay, and co-immunoprecipitation assay demonstrated the interaction between USP9X and transducin β-like 1X related protein 1 (TBL1XR1). qRT-PCR assay and western blot assay were manipulated to determine the expression of USP9X and TBL1XR1. TBL1XR1 and USP9X knockdown experiments were conducted to explore their functions on LPS-induced WI-38 cell injury and inflammation. Results: TBL1XR1 expression was upregulated in LPS-treated WI-38 cells. TBL1XR1 knockdown promoted cell proliferation and repressed apoptosis, inflammation, and oxidative stress in LPS-treated WI-38 cells. Moreover, USP9X deubiquitinated TBL1XR1 to regulate TBL1XR1 expression. USP9X knockdown restored the effects of LPS on WI-38 cell proliferation, apoptosis, inflammation, and oxidative stress, but these effects of USP9X knockdown were further abolished by TBL1XR1 overexpression. In addition, USP9X promoted the NF-κB signaling pathway by the deubiquitination of TBL1XR1. Conclusion: USP9X promoted the apoptosis, inflammation, and oxidative stress of LPS-stimulated WI-38 cells through the deubiquitination of TBL1XR1.
泛素化和去泛素化参与包括急性肺炎在内的人类疾病进展。在本研究中，我们旨在探讨泛素特异性蛋白酶9X连锁（USP9X）在脂多糖（LPS）处理的WI-38细胞中的功能。方法：用LPS处理WI-38细胞以诱导细胞损伤和炎症。进行3-（4,5-二甲基-2-噻唑基）-2,5-二苯基-2-H-四氮唑溴盐（MTT）试验和5-乙炔基-2'-脱氧尿苷（EdU）试验以检测LPS处理的WI-38细胞的增殖。进行流式细胞术分析以检测LPS处理的WI-38细胞凋亡。使用ELISA试剂盒测定炎症因子（IL-1β和TNF-α）的浓度。用相关试剂盒检测超氧化物歧化酶活性和活性氧水平。Ubibrowser（http://ubibrowser.bio-it.cn/ubibrowser/）、泛素化试验和免疫共沉淀试验证明了USP9X与转导素β样1X相关蛋白1（TBL1XR1）之间的相互作用。进行qRT-PCR试验和蛋白质印迹试验以测定USP9X和TBL1XR1的表达。进行TBL1XR1和USP9X敲低实验以探讨它们对LPS诱导的WI-38细胞损伤和炎症的作用。结果：在LPS处理的WI-38细胞中TBL1XR1表达上调。TBL1XR1敲低促进LPS处理的WI-38细胞的增殖并抑制其凋亡、炎症和氧化应激。此外，USP9X使TBL1XR1去泛素化以调节TBL1XR1表达。USP9X敲低恢复了LPS对WI-38细胞增殖、凋亡、炎症和氧化应激的影响，但USP9X敲低的这些作用被TBL1XR1过表达进一步消除。此外，USP9X通过使TBL1XR1去泛素化促进NF-κB信号通路。结论：USP9X通过使TBL1XR1去泛素化促进LPS刺激的WI-38细胞的凋亡、炎症和氧化应激。
