Cell Biology and Toxicology ( IF 5.3 ) Pub Date : 2022-08-19 , DOI: 10.1007/s10565-022-09748-8 Kang Geng 1, 2, 3 , Xiumei Ma 1, 2 , Zongzhe Jiang 2 , Junling Gu 4 , Wei Huang 2 , Weiming Wang 1, 5 , Yong Xu 1, 2 , Youhua Xu 1
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Diabetic foot ulcer (DFU) is a devastating component of diabetes progression, leading to decreased quality of life and increased mortality in diabetic patients. The underlying mechanism of DFU is not completely understood. Hence, this study aims to elucidate the mechanism involved in wound healing in mouse models of DFU. Gain- and loss-of-function studies were performed to study the roles that WDR74 and TGF-β play in mouse models of DFU and primary bone marrow–derived mouse macrophages. M1 and M2 macrophage phenotypic markers, extracellular matrix (ECM) components, and angiogenic makers were determined by RT-qPCR and/or Western blot analysis. Localization of these proteins was determined by immunofluorescence staining and/or immunohistochemistry. Interaction between WDR74 with Smad2/3 in macrophages was detected by co-immunoprecipitation. We found that WDR74 and M2 macrophages were decreased in wound tissues from DFU mice. TGF-β/Smad pathway activation increased the expression of M2 macrophage markers (arginase-1 and YM1), IL-4, while decreased expression of M1 macrophage marker (iNOS). TGF-β/Smad pathway activation also increased the production of ECM and promoted the wound closure in diabetic mice. We also noticed that WDR74 overexpression increased Smad2/3 phosphorylation, elevated the population of M2 macrophage and ECM production, and alleviated DFU. LY2109761 treatment normalized effects of TGF-β or WDR74 overexpression. In conclusion, WDR74 promoted M2 macrophage polarization, leading to improved DFU in mice, through activation of the TGF-β/Smad pathway.
Graphical abstract
中文翻译：
WDR74 促进 TGF-β/Smad 通路激活，促进小鼠 M2 巨噬细胞极化和糖尿病足溃疡伤口愈合
糖尿病足溃疡（DFU）是糖尿病进展的一个破坏性组成部分，导致糖尿病患者的生活质量下降和死亡率增加。DFU 的底层机制尚不完全清楚。因此，本研究旨在阐明 DFU 小鼠模型伤口愈合的机制。进行功能获得和功能丧失研究，以研究 WDR74 和 TGF-β 在 DFU 小鼠模型和原代骨髓来源的小鼠巨噬细胞中发挥的作用。通过 RT-qPCR 和/或蛋白质印迹分析确定 M1 和 M2 巨噬细胞表型标记物、细胞外基质 (ECM) 成分和血管生成标记物。通过免疫荧光染色和/或免疫组织化学确定这些蛋白质的定位。通过免疫共沉淀检测巨噬细胞中 WDR74 与 Smad2/3 之间的相互作用。我们发现 DFU 小鼠伤口组织中 WDR74 和 M2 巨噬细胞减少。TGF-β/Smad 通路激活增加了 M2 巨噬细胞标志物（精氨酸酶-1 和 YM1）、IL-4 的表达，同时降低了 M1 巨噬细胞标志物（iNOS）的表达。TGF-β/Smad 通路激活还增加了 ECM 的产生并促进糖尿病小鼠的伤口闭合。我们还注意到 WDR74 过表达增加了 Smad2/3 磷酸化，增加了 M2 巨噬细胞数量和 ECM 产生，并减轻了 DFU。LY2109761 治疗使 TGF-β 或 WDR74 过表达的影响正常化。总之，WDR74 通过激活 TGF-β/Smad 通路促进 M2 巨噬细胞极化，从而改善小鼠的 DFU。
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