Amoupour Moein, Brouki Milan Peiman, Barati Mahmood, Hivechi Ahmad, Rajabi Fomeshi Motahareh, Kiani Ghalesardi Omid, Ahmadvand Davoud, Karkuki Osguei Nushin, Samadikuchaksaraei Ali
Department of Medical Biotechnology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran.
Cellular and Molecular Research Centre, Iran University of Medical Sciences, Tehran, Iran; Department of Tissue Engineering and Regenerative Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran.
Int J Biol Macromol. 2024 Mar;262(Pt 1):129876. doi: 10.1016/j.ijbiomac.2024.129876. Epub 2024 Feb 2.
Impaired polarization of M1 to M2 macrophages has been reported in diabetic wounds. We aimed to improve this polarization by down-regulation of expression of the "Suppressor of Cytokine Signaling 3" (SOCS3) gene in macrophages. Two oligodeoxynucleotide (ASO) sequences were designed against SOC3 mRNA and were loaded to mannosylated-polyethyleneimine (Man-PEI). The optimum N/P ratio for Man-PEI-ASO was determined to be 8 based on loading efficiency, particle size, zeta potential, cellular uptake and cytotoxicity assay. pH stability of ASO in Man-PEI-ASO and its protection from DNase I was confirmed. After in vitro treatment of macrophages with Man-PEI-ASO, SOCS3 was downregulated, SOCS1 upregulated, and SOCS1/SOCS3 ratio increased. Also, expressions of macrophage markers of M2 (IL-10, Arg1, CD206) increased and those of M1 (IL-1β, NOS2, CD68) decreased, and secretion of pro-inflammatory cytokines (TNF-α and IL-1β) decreased while that of anti-inflammatory cytokine IL-4 increased. All suggested a polarization into M2 phenotype. Finally, the Man-PEI-ASO was loaded in hydrogel and applied to a diabetic wound model in mice. It improved the healing to the level observed in non-diabetic wounds. We show that using antisense sequences against SOC3 mRNA, macrophage polarization could be directed into the M2 phenotype and healing of diabetic wound could be highly improved.
据报道，糖尿病伤口中M1巨噬细胞向M2巨噬细胞的极化受损。我们旨在通过下调巨噬细胞中“细胞因子信号转导抑制因子3”（SOCS3）基因的表达来改善这种极化。设计了两条针对SOC3 mRNA的寡脱氧核苷酸（ASO）序列，并将其加载到甘露糖基化聚乙烯亚胺（Man-PEI）上。基于负载效率、粒径、zeta电位、细胞摄取和细胞毒性测定，确定Man-PEI-ASO的最佳N/P比为8。证实了ASO在Man-PEI-ASO中的pH稳定性及其对DNase I的抗性。用Man-PEI-ASO对巨噬细胞进行体外处理后，SOCS3表达下调，SOCS1表达上调，SOCS1/SOCS3比值增加。此外，M2巨噬细胞标志物（IL-10、Arg1、CD206）的表达增加，而M1巨噬细胞标志物（IL-1β、NOS2、CD68）的表达减少，促炎细胞因子（TNF-α和IL-1β）的分泌减少，而抗炎细胞因子IL-4的分泌增加。所有这些都表明巨噬细胞向M2表型极化。最后，将Man-PEI-ASO负载到水凝胶中，并应用于小鼠糖尿病伤口模型。它将伤口愈合改善到了非糖尿病伤口所观察到的水平。我们表明，使用针对SOC3 mRNA的反义序列，可以将巨噬细胞极化导向M2表型，从而显著改善糖尿病伤口的愈合。
