Abstract:

Objective · To investigate the effects of insulin on high glucose-cultured human mononuclear cell line THP-1 and macrophage phenotype transformation in diabetic wounds. Methods · THP-1 cells were cultured with normal (5.6 mmol/L) and high (25 mmol/L) glucose, respectively, stimulated with PMA for differentiation, and induced to M1 macrophages with LPS. After treated with insulin for 6 h, expression changes of M1 type macrophage markers inducible nitric oxide synthase (iNOS), tumor necrosis factor α (TNF-α), and interleukin-1β (IL-1β), as well as M2 type macrophage markers arginase1 (Arg1) and IL-10 were detected using real-time PCR and Western blotting. High fat diet feeding plus multiple intraperitoneal injections of low dose streptozotocin (STZ) were used to induce type II diabetes rat model. After blood glucose level has been stable for five weeks, two full-thickness skin wounds with the diameter of 1cm were made on the back of DM rats. Wounds were randomly assigned to being treated with insulin (0.2 U insulin /20 μL saline) or saline (20 μL saline) using the random number table. Characteristics of macrophage phenotypes were observed 3, 7, and 25 days after wounds were made. Normal rats (n=3) served as controls. Results · After being cultured with high glucose, the mRNA levels of M1 markers iNOS and TNF-α were up-regulated in LPS-induced THP-1 cells, while the mRNA levels of M2 markers Arg1 and IL-10 were down-regulated. After being treated with insulin for 6 h, mRNA levels of iNOS and TNF-α were down-regulated, protein levels of iNOS, IL-1β were down-regulated too, while mRNA and protein levels of Arg1 and IL-10 were up-regulated. In addition, the expression level of phosphorylated NF-κB-p65 was significantly increased after high glucose culture and was significantly decreased after insulin intervention. Compared to normal rat skin wounds, the expression of iNOS in macrophages was significantly increased in wounds of diabetic rats. The expression of iNOS in macrophages was high in saline treated wounds 3 and 7 days after the wounds were made and the expression of Arg1 was low 25 days after the wounds were made. In insulin treated wounds, the expression of iNOS started to decrease on day 7 after the wounds were made and the expression of Arg1 was significantly higher than that in saline treated wounds on day 25 after the wounds were made. Conclusion · Insulin can induce macrophage phenotype transformation from M1 to M2 under high glucose condition and the mechanism may be associated with the phosphorylation of NF-κB-p65.

Key words: macrophage phenotype, insulin, diabetic wound