Yu Ming-Chen, Li Xiao-Lin, Ning Ming-Liang, Yan Zhen-Zhong, Yu Wan-Tao
Department of Orthopedics, Changzhou Geriatric hospital affiliated to Soochow University, Changzhou 213000, China; Department of Orthopedics, Changzhou NO. 7 People's Hospital, Changzhou 213000, China.
Department Of Orthopdics, The First People's Hospital of Changzhou, Changzhou 213000, China.
Brain Res Bull. 2025 Jan;220:111157. doi: 10.1016/j.brainresbull.2024.111157. Epub 2024 Dec 2.
This study aimed to investigate the effect of Ubiquitin-Specific Peptidase 22 (USP22) on the inflammatory response mediated by BV-2 mouse microglia and explore the role of the PU box binding protein 1 (PU.1)/NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome in the USP22-induced polarization of BV-2 cells.
The BV-2 mouse microglia line was cultured in vitro, and plasmid and siRNA transfection was performed to overexpress or knockdown USP22. Subsequently, BV-2 cells were treated with lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) and interleukin (IL)-4 to induce M1 and M2 polarization, respectively. Western blot was used to detect the expression levels of USP22, PU.1, M1 polarization markers [inducible nitric oxide synthase (iNOS), and cluster of differentiation (CD) 86], M2 polarization markers [arginase 1 (Arg1), and CD206], in BV-2 cells from different treatment groups. Additionally, measurement was performed on the inflammasome NLRP3, and its activation-related proteins [NIMA-related kinase7 (NEK7), cleaved-caspase 1, apoptosis-associated speck-like protein containing a CARD (ASC)]. Enzyme-linked immunosorbent (ELISA) assay was employed to determine the levels of inflammatory cytokines tumor necrosis factor-alpha (TNF-α), IL-1 β, and IL-10 in the cells. Furthermore, immunofluorescence was utilized to analyze the levels of iNOS and Arg1-positive BV-2 cells in different treatment groups. Moreover, the ubiquitination level of PU.1 was detected using immunoprecipitation.
The protein expression level of USP22 was significantly down-regulated in BV-2 cells treated with M1 polarization. Overexpression of USP22 remarkably reduced the protein levels of iNOS and CD86, but markedly increased the protein levels of Arg1 and CD206 in cells. Besides, there was a notable reduction in the levels of TNF-α and IL-1 β in the cell culture medium, while a remarkable increase was observed in the level of IL-10. Additionally, the level of iNOS-positive cells was significantly decreased, while the level of Arg1-positive cells was considerably increased. However, up-regulation of PU.1 expression could reverse the above results and promoted the expression of NLRP3 and its activation-related proteins. Notably, overexpression of USP22 significantly down-regulated the protein expression and ubiquitination level of PU.1.
USP22 inhibits the M1 polarization of BV-2 mouse microglia. The PU.1/NLRP3 inflammasome pathway may be a critical regulatory pathway of USP22 in BV-2 cell polarization.
本研究旨在探讨泛素特异性蛋白酶22（USP22）对BV-2小鼠小胶质细胞介导的炎症反应的影响，并探讨PU盒结合蛋白1（PU.1）/含核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）炎性小体在USP22诱导的BV-2细胞极化中的作用。
体外培养BV-2小鼠小胶质细胞系，进行质粒和小干扰RNA（siRNA）转染以过表达或敲低USP22。随后，分别用脂多糖（LPS）、干扰素-γ（IFN-γ）和白细胞介素（IL）-4处理BV-2细胞以诱导M1和M2极化。采用蛋白质免疫印迹法检测不同处理组BV-2细胞中USP22、PU.1、M1极化标志物[诱导型一氧化氮合酶（iNOS）和分化簇（CD）86]、M2极化标志物[精氨酸酶1（Arg1）和CD206]的表达水平。此外，检测炎性小体NLRP3及其激活相关蛋白[NIMA相关激酶7（NEK7）、裂解的半胱天冬酶1、含CARD结构域的凋亡相关斑点样蛋白（ASC）]。采用酶联免疫吸附测定（ELISA）法测定细胞中炎性细胞因子肿瘤坏死因子-α（TNF-α）、IL-1β和IL-10的水平。此外，利用免疫荧光分析不同处理组中iNOS和Arg1阳性BV-2细胞的水平。此外，采用免疫沉淀法检测PU.1的泛素化水平。
在经M1极化处理的BV-2细胞中，USP22的蛋白表达水平显著下调。USP22的过表达显著降低了细胞中iNOS和CD86的蛋白水平，但显著提高了Arg1和CD206的蛋白水平。此外，细胞培养基中TNF-α和IL-1β的水平显著降低，而IL-10的水平显著升高。此外，iNOS阳性细胞的水平显著降低，而Arg1阳性细胞的水平显著升高。然而，PU.1表达上调可逆转上述结果，并促进NLRP3及其激活相关蛋白的表达。值得注意的是，USP22的过表达显著下调了PU.1的蛋白表达和泛素化水平。
USP22抑制BV-2小鼠小胶质细胞的M1极化。PU.1/NLRP3炎性小体途径可能是USP22在BV-2细胞极化中的关键调节途径。
