College of Veterinary Medicine, Shandong Agricultural University, Tai`an, China.
Qingdao Animal Disease Prevention and Control Center, Qingdao Municipal Bureau of Agriculture and Rural Affairs, Qingdao, China.
Front Immunol. 2022 Jun 30;13:934084. doi: 10.3389/fimmu.2022.934084. eCollection 2022.
M1-polarized macrophages can improve the body's immune function. This study aimed to explore the mechanism of polysaccharide (PGPS) degrading SOCS1/2 protein through autophagy and promoting M1 polarization in 3D4/21 cells. Immunoprecipitation, confocal laser scanning microscopy, flow cytometry, and intracellular co-localization were used to detect the expression of related phenotypic proteins and cytokines in M1-polarized cells. The results showed that PGPS significantly promoted the mRNA expression of IL-6, IL-12, and TNF-α and enhanced the protein expression of IL-6, IL-12, TNF-α, IL-1β, iNOS, CD80, and CD86, indicating that PGPS promoted M1 polarization in 3D4/21 cells. Next, the effect of the PGPS autophagy degradation of SOCS1/2 on the M1 polarization of 3D4/21 cells was detected. The results showed that PGPS significantly downregulated the expression level of SOCS1/2 protein, but had no obvious effect on the mRNA expression level of SOCS1/2, indicating that PGPS degraded SOCS1/2 protein by activating the lysosome system. Further research found that under the action of 3-MA and BafA1, PGPS upregulated LC3B II and downregulated SOCS1/2 protein expression, which increased the possibility of LC3B, the key component of autophagy, bridging this connection and degrading SOCS1/2. The interaction between SOCS1/2 and LC3 was identified by indirect immunofluorescence and Co-IP. The results showed that the co-localization percentage of the two proteins increased significantly after PGPS treatment, and LC3 interacted with SOCS1 and SOCS2. This provides a theoretical basis for the application of PGPS in the treatment or improvement of diseases related to macrophage polarization by regulating the autophagy level.
M1 极化的巨噬细胞可以改善机体的免疫功能。本研究旨在探讨多糖（PGPS）通过自噬降解 SOCS1/2 蛋白并促进 3D4/21 细胞 M1 极化的机制。采用免疫沉淀、共聚焦激光扫描显微镜、流式细胞术和细胞内共定位检测 M1 极化细胞中相关表型蛋白和细胞因子的表达。结果表明，PGPS 显著促进了 IL-6、IL-12 和 TNF-α 的 mRNA 表达，并增强了 IL-6、IL-12、TNF-α、IL-1β、iNOS、CD80 和 CD86 的蛋白表达，表明 PGPS 促进了 3D4/21 细胞的 M1 极化。接下来，检测了 PGPS 自噬降解 SOCS1/2 对 3D4/21 细胞 M1 极化的影响。结果表明，PGPS 显著下调了 SOCS1/2 蛋白的表达水平，但对 SOCS1/2 的 mRNA 表达水平没有明显影响，表明 PGPS 通过激活溶酶体系统降解 SOCS1/2 蛋白。进一步研究发现，在 3-MA 和 BafA1 的作用下，PGPS 上调了 LC3B II 并下调了 SOCS1/2 蛋白表达，增加了自噬关键成分 LC3 桥接这一连接并降解 SOCS1/2 的可能性。通过间接免疫荧光和 Co-IP 鉴定了 SOCS1/2 与 LC3 的相互作用。结果表明，PGPS 处理后两种蛋白的共定位百分比显著增加，LC3 与 SOCS1 和 SOCS2 相互作用。这为通过调节自噬水平应用 PGPS 治疗或改善与巨噬细胞极化相关的疾病提供了理论依据。
