Inhibitory effects of different concentrations of auranofin on M1 macrophage function and its therapeutic potential in diabetic wound healing

doi:10.12307/2026.555

Abstract

Abstract: BACKGROUND: During diabetic wound healing, the sustained activation of M1 macrophages exacerbates the inflammatory response and hinders wound repair. Auranofin, an anti-inflammatory drug, has not been clearly studied for its effects on M1 macrophages and its potential role in diabetic wound healing.
OBJECTIVE: To investigate the effects of different concentrations of auranofin on the biological function of M1 macrophages and evaluate its potential application in diabetic wound healing.
METHODS: RAW264.7 and THP-1 cells were used as research models. M1 polarization was induced using different concentrations of interferon-γ and lipopolysaccharide. M1 macrophages were treated with 1 and 2 μmol/L auranofin. Cell counting kit-8 assay was used to evaluate the effect of auranofin on cell viability. Quantitative real-time PCR was performed to detect mRNA expression of interleukin-1β, interleukin-6, and tumor necrosis factor-α. ELISA was employed to measure the levels of interleukin-1β, interleukin-6, and tumor necrosis factor-α in the supernatant. Western blot analysis was used to assess the expression of nuclear factor-κB (p65), phosphorylated mitogen-activated protein kinases (MAPK), and total MAPK proteins. Additionally, 6-8-week-old male C57BL/6J and db/db diabetic mice were used for wound healing experiments, with the mice divided into C57 control, db/db control and auranofin treatment groups, each containing six animals. Dorsal skin defect modeling and treatment with intraperitoneal injection of auranofin were performed to observe wound healing in mice.
RESULTS AND CONCLUSION: (1) Cell experiments showed that co-treatment with interferon-γ (10 ng/mL) and lipopolysaccharide (100 ng/mL) significantly induced M1 polarization in RAW264.7 and THP-1 cells, resulting in increased mRNA expression of interleukin-1β, interleukin-6, and tumor necrosis factor-α. Treatment with auranofin (1 and 2 μmol/L) reduced the mRNA expression of these inflammatory factors in the cells and inhibited the secretion of inflammatory factors in the cell supernatant. (2) Auranofin treatment significantly suppressed the activation of nuclear factor-κB (p65) and phosphorylated MAPK signaling pathways. (3) Animal experiments showed that auranofin promoted wound healing in db/db diabetic mice, suggesting that auranofin has strong anti-inflammatory effects and may facilitate the healing of wounds in diabetic mice.

Key words: auranofin, M1 Macrophages, diabetes mellitus, skin defects, inflammatory factors, wound healing, engineered tissue construction

CLC Number:

Pan Hongfei, Zhuang Zhenbing, Xu Baiyun, Yang Zhangyang, Lin Kairui, Zhan Bingqing, Lan Jinghan, Gao Heng, Zhang Nanbo, Lin Jiayu. Inhibitory effects of different concentrations of auranofin on M1 macrophage function and its therapeutic potential in diabetic wound healing [J]. Chinese Journal of Tissue Engineering Research, 2026, 30(6): 1390-1397.

Figures/Tables 5

2.1 细胞存活率对于M1型极化的RAW 264.7细胞(图1A)，金诺芬在浓度为0.000 1-1 μmol/L时，细胞活力保持接近100%，表明金诺芬在这一浓度范围内对细胞活力影响较小。当金诺芬浓度超过1 μmol/L时，细胞活力开始显著下降，尤其在10 μmol/L及以上浓度，抑制效果明显，呈剂量依赖性。对于M1型极化的THP-1细胞(图1B)，低浓度金诺芬处理后细胞活力维持较高水平，随着浓度增加，细胞活力逐渐下降，10 μmol/L及以上浓度时，细胞活力显著降低，趋势与M1型极化的RAW 264.7细胞类似。金诺芬对M1型极化的RAW 264.7和THP-1细胞系均表现出剂量依赖性的抑制作用，且在高浓度时对细胞活力有显著的抑制效果，后续细胞实验选择1 μmol/L和2 μmol/L金诺芬进行干预。 2.2 金诺芬抑制M1型巨噬细胞炎症因子表达干扰素γ(10 ng/mL)与脂多糖(100 ng/mL)联合使用显著促进了两种巨噬细胞向M1型极化，表现为炎症因子mRNA表达显著增强(图2)。在此基础上，对M1型极化的RAW264.7细胞、THP-1细胞进行不同浓度金诺芬(1，2 μmol/L)处理24 h，结果显示金诺芬能显著抑制M1型巨噬细胞中炎症因子mRNA表达(图3)。通过ELISA检测细胞培养上清液中白细胞介素1β、白细胞介素6、肿瘤坏死因子α水平，结果显示，在M1状态下，这些炎症因子的水平显著高于M0状态，表明M1型巨噬细胞具有更高的炎症活性。金诺芬在1 μmol/L和2 μmol/L浓度下，能够显著降低M1型极化的RAW264.7细胞、THP-1细胞中炎症因子水平，见图4。 2.3 金诺芬对M1型极化的RAW264.7和THP-1细胞中NF-κB(p65)、p-MAPK和总MAPK蛋白表达的影响诱导的M1型巨噬细胞中，NF-κB和p-MAPK蛋白表达水平升高。金诺芬处理后，NF-κB(p65)和p-MAPK蛋白表达下降，见图5，提示金诺芬可能是通过抑制NF-κB以及MAPK信号通路，调节M1型巨噬细胞中白细胞介素1β、白细胞介素6、肿瘤坏死因子α免疫因子分泌。 2.4 各组小鼠背部创面愈合情况图6A为小鼠背部皮肤创面大体图像，在创伤后第3-7天，金诺芬治疗组的创面面积较db/db对照组显著缩小。图6B显示3组小鼠在饲养期间的体质量差异无显著性意义，表明在此浓度下金诺芬对小鼠没有明显毒性。"

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