Mechanism of oil of pills for moistening muscle in promoting diabetes ulcer healing via regulating macrophage polarization
Objective It is to investigate whether oil of pills for moistening muscle can promote diabetic ulcer healing through macrophage polarization,and to provide more theoretical evidences for the treatment of diabetic foot ulcers with lu-bricant oil.Methods Thirty-five SD rats were taken,in which 10 rats were randomly selected as the blank group,and the remaining 25 rats were induced by high-sugar,high-fat diet combined with streptozotocin(STZ)to establish diabetes mod-els.Then a 3.0 cm ×3.0 cm defective wound was surgically made on the backs of rats in the blank group and modeled rats,and the wounds of the modeled rats was applied with 50％glacial acetic acid to establish diabetic ulcer models,20 successfully modeled rats with diabetic ulcers were randomly divided into diabetic ulcer group and oil of pills for moistening muscle group,with 10 rats in each group.The blank group and diabetic ulcer group were treated with saline gauze to cover the wound,while the oil of pills for moistening muscle group was treated with oil of pills for moistening muscle to cover the wounds,all treated for 14 days.During the treatment,the rats of the blank group were fed with regular standard chow,and the rats of the rest groups were fed with high-sugar and high-fat chow.After successful modeling,the blank control group rats were given conventional standard feed,while the other groups of rats were given high sugar and high fat feed.The ulcer areas before treatment,after 7 days and 14 days of treatment were observed,and the ulcer healing rates after 7 days and 14 days of treatment were calculated.At the end of the experiment,the ulcer tissues were taken to observe their pathological changes by HE staining,and the protein expressions of vascular endothelial growth factor B(VEGFB),fibroblast growth factor 1(FGF1),and platelet-derived growth factor subunit B(PDGFB)in the ulcer tissues were detected by Western blot method,the levels of pro-inflammatory cytokines[interleukin-6(IL-6),interleukin-12(IL-12),and tumor necrosis fac-tor-alpha(TNF-α)]secreted by Ml-type macrophages,anti-inflammatory cytokines[interleukin-4(IL-4)]secreted by M2-type macrophages,and growth factors[transforming growth factor-beta(TGF-β),vascular endothelial growth factor A(VEGFA)]were detected by ELISA,the distribution of M1 and M2 macrophages in the ulcer tissues was detercted by im-munofluorescence assay,the classification of M1-type and M2-type macrophages in the ulcer tissues of the diabetic ulcer group and oil of pills for moistening muscle group were detected by flow cytometry,and the ratios of F4/80/CCR7 and F4/80/CD206-positively stained cells(M1/M2-type macrophage ratios)in the ulcer tissues were calculated.Results The ul-cer areas of the diabetic ulcer group and oil of pills for moistening muscle group were significantly larger,while the ulcer healing rate was significantly lower than that of the blank group after 7 days and 14 days of dressing change(all P＜0.05);the ulcer areas of the oil of pills for moistening muscle group were significantly smaller,while the ulcer healing rate was sig-nificantly higher than that of the diabetic ulcer group after 7 days and 14 days of dressing change(all P＜0.05).After 14 days of dressing change,a large number of inflammatory cells infiltrated the ulcer tissue of the diabetic ulcer group,with generation of a small amount of fibroblasts and capillaries and severe inflammatory reaction;the inflammatory cell infiltra-tion in the ulcer tissue of the oil of pills for moistening muscle group was in between the blank group and diabetic ulcer group,with a large number of neonatal capillary and fibroblasts and a small amount of granulation tissue.The relative pro-tein expressions of VEGFB,FGF1 and PDGFB in the ulcer tissues of the oil of pills for moistening muscle group were sig-nificantly higher than those of the blank group and diabetic ulcer group(all P＜0.05).Compared with the blank group,the levels of IL-6,IL-12,TNF-α,IL-4,TGF-β and VEGFA in the ulcer tissues of the diabetic ulcer group were signifi-cantly increased(all P＜0.05);compared with the diabetic ulcer group,the levels of IL-6,IL-12 and TNF-α in the ulcer tissues of the oil of pills for moistening muscle group were significantly decreased(all P＜0.05),while the levels of IL-4,TGF-β and VEGFA were significantly increased(all P＜0.05).The absolute cell counts of M1-type and M2-type macro-phages of the diabetic ulcer group were significantly higher than those of the blank group(both P＜0.05);the absolute cell count of M1-type macrophages of the oil of pills for moistening muscle group was significantly lower,while the count of M2-type macrophages was significantly higher than those of the diabetic ulcer group(both P＜0.05).There were significantly more M2-type macrophages in the single-cell suspension of ulcer tissue cells of the oil of pills for moistening muscle group than that of the diabetic ulcer group(P＜0.05);the ratio of M1/M2-type macrophages of the diabetic ulcer group was sig-nificantly higher than that of the blank group(P＜0.05),and the ratio of M1/M2-type macrophages in the oil of pills for moistening muscle group was significantly lower than that of the diabetic ulcer group(P＜0.05).Conclusion There is an imbalance of M1/M2 type macrophage ratio in dDiabetic ulcers;Oil of pills for moistening muscle can promote macrophage polarization to M2 type macrophages,reduce M1 type macrophages,and improve the imbalance of M1/M2 type macropha-ges,thus to reduce the pro-inflammatory cytokines secreted by M1 type macrophages and increase the anti-inflammatory cy-tokines and growth factors secreted by M2 type macrophages,which can inhibit inflammatory reactions and promote diabetic ulcer healing.
diabetic foot ulcerulcer healingOil of pills for moistening musclemacrophage polarizationinflamma-tory response
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