Effect Of Autophagy Activity On Macrophage Polarization And Wound Healing In Diabetic Mice

Object iveDiabetes is a chronic metabolic disease caused by multiple causes.Its accompanying multi-system damage is extremely irritating to people.The main reason for the complicated pathogenesis of refractory diabetic wounds is the persistent chronic inflammation of the wound.At present,this inflammatory state is related to the balance of macrophages with different polarization states in the wound.At the same time,autophagy participates in various aspects of wound healing and plays an important role.However,the relationship between the two in the healing of diabetic wounds has not been studied.This study aimed to investigate whether autophagy activity is related to the polarity status of macrophages in diabetic wounds and the effect on wound healing efficiency by using autophagy regulators to intervene in diabetic mouse wound models.MethodsNinety-six SPF male BALB/C mice were established by intraperitoneal injection of 150 mg/kg streptozotocinto establish a diabetic mouse model,then they were randomly divided into 4 groups:High-dose rapamycin group(HR group),low-dose rapamycin group(LR group),3-Methyladenine inhibition group(3-MA group),and blank control group(Control group).The corresponding drug solutions were injected intraperitoneally every day and we observe the rates of wound closure.The skin tissue samplesfrom wounds of diabetes mice were taken at 1,3,5,7,10 and 14 days after the injury.We retained the preservation of fresh skin tissue liquid nitrogen in wounds of diabetic mice and the preservation of 4%par formaldehyde fixed paraffin in intact wound tissue.In the later stage,the protein was extracted by grinding homogenate to detect the expression of LC3,CD16 and CD163 protein in wound skin tissue.The other part of the skin tissue in liquid nitrogen storage was homogenatedfor RNA extraction,and weused qPCR to detect the TNF-? and VEGF mRNA expression levels in wound tissue.Paraffin sections were used to detect M1 and M2 macrophages in wound tissue labeled with CD16 and CD163 by immunofluorescence staining.Results1.Compared with the diabetic mice in the control group,the low-dose rapamycin group showed higher wound healing efficiency in the whole process of wound healing.High-dose rapamycin healing rate is higher than the control group in the early stage of wound healing,then gradually reduced,the overall healing efficiency was lower than the control group(P<0.05);The healing rate of the 3-MA group was lower than that of the control group in the early,then gradually increases in the middle and late stages of healing.The overall healing efficiency was lower than that of the low-dose rapamycin group(P<0.05).2.At each time point in the wound healing process of diabetic mice,the autophagyactivity induced by rapamycin improved,the expression of M1 macrophage-associated protein CD16 was increased and the expression of M2 macrophage-associated protein CD163 was decreased.The expression of M1 macrophage-associated protein CD163 was increased and the expression of M1 macrophage-associated protein CD16 was decreased by 3-MA inhibition of autophagy activity(P<0.05).3.Immunofluorescence staining test results show that M1 type macrophage marker CD16 positive expression was higher in HR group and the positive expression of CD163 in M2macrophage marker was lower than that in the control group at various points in the healing process of diabetic mice(P<0.05).The positive expression of CD16 in the 3-MA group was significantly lower than that in the control group and the rapamycin-induced group,and the positive e.xpression of CD163 was significantly higher than other groups(P<0.05).4.Peak expression of TNF-? mRNA on the 7th day after injury in the control group of diabetic mice during wound healing;Compared with the normal blank control group,the TNF-? mRNA expression peaked in the wound surface of the mice in the rapamycin group,and the inflammation level was enhanced;The expression level of VEGF mRNA in the wound of rapamycin group was significantly lower than that of the control group,indicating that the increased autophagy induction could promote the level of wound inflammation(P<0.05).ConclusionRapamycin can induce the increase of autophagy activity in streptozotocin-induced diabetic mice,promote the transformation of macrophages into M1 type polar state in woundsand improve the inflammatory level of wounds.Inhibition of autophagy can promote polarity conversion of M2 macrophages;Low-dose rapamycin has a promoting effect on wound healing in diabetic mice.