The Role And Mechanism Of MicroRNA-155 In Macrophage Phenotype Transition Of Diabetic Wound Healing

Objective To analyse the role and mechanism of microRNA-155 in macrophage phenotype transition of diabetic wound healing.Methods 1)THP-1 cells were turned into macrophages after PMA stimulation in vitro.Flow cytometry was used to test macrophage marker(CD68),M1 marker(CD86)and M2marker(CD206).In addition,the phagocytosis was tested after marcrophages engulfed the fluorescent beads.2)RT-PCR and Western Blot were employed to observe the expression of down stream genes.ELISA was used to determine macrophage related cytokines,such as IL-6 and IL-10.3)The diabetic rat model was created using STZ.Afterwards,10 mm punch was employed to set up diabetic wound.Day 0,3,7,13 were considered to be the observing time points.The experimental group was injected with miR-155 inhibitor,while,the control group was injected with saline.Then,the wound healing rate was recorded.4)Immunohistochemistry was used to observe the markers(CD68,CD86 and CD206)of macrophages.RT-PCR and Western Blot were employed to test the genes(SOCS1 and STAT1)related to the plasticity of macrophage phenotypes.ELISA was used to compare the cytokines between the two groups(IL-6 and IL-10).Results 1)Compared to the control group,macrophages in high glucose showed a higher miR-155 and CD86 level.However,CD206 and phagocytosis remained almost the same between the two groups.2)In high glucose environment,after miR-155 inhibition,CD206 and phagocytosis wereelevated in the experimental group,while where was no significant change in terms of CD86.SOCS1 and STAT1 were increased in miR-155 inhibition group on mRNA level.On protein level,SOCS1 was higher and p-STAT1 was lower in miR-155 inhibition group.However,STAT1 was similar in both groups.3)In diabetic rats,there was no significant diffidence of wound healing rate between the experimental and control groups.4)In diabetic rats,CD206 was increased in the experimental group while there was no significant difference in both groups.On mRNA level,SOCS1 and STAT were elevated.On protein level,SOCS1 was increased and p-STAT1 was decreased in the experimental group.STAT was similar in both groups.In accordance with in vitro results,IL-6 was higher and IL-10 was lower in the miR-155 inhibition group.Conclusions In diabetic wound or high glucose condition,M1 was the main macrophage phenotype.After the inhibition of miR-155,the level of M2 macrophages increased.This phenotype change was mediated by SOCS1/STAT1 pathway.Therefore,miR-155 might play a role in the plasticity of macrophage phenotypes.