| Background and Objective:Diabetic refractory wound is one of the most serious and costly chronic complications of diabetes mellitus.At present,the treatment of diabetic refractory wound is mainly accomplished by wound nurses through debridement,inflammation control,wound moistening and granulation tissue proliferation.The healing promoting materials and medicines used include functional dressings,growth factors,traditional Chinese medicine healing agents,etc.Despite the development,there are still some problems,such as the shortening of wound healing cycle and the high cost of long-term treatment.Therefore,it is still a difficult and hot spot to adopt efficient and economical treatment and nursing methods in clinical research.Studies have shown that the main site of diabetic refractory wounds is inflammation-promoting M1 macrophages,which are difficult to polarize into M2 macrophages.In the relatively abnormal microenvironment of diabetic wounds,macrophages have phenotypic transformation dysfunction,and their anti-inflammatory and repair-promoting functions are difficult to play,leading to the difficulty of wound repair.Quercetin,as a common flavonoid with strong anti-oxidation and anti-inflammatory effects,widely exists in hawthorn,safflower and other more than 100 kinds of Chinese herbal medicines.In vitro studies have shown that quercetin can promote macrophage phenotype polarization to M2.Therefore,on this basis,this study intends to observe the effect of quercetin on wound healing in streptozotocin-induced type 1 diabetic rats,and collect the wound tissues at different time points for histological and molecular biological analysis to explore the mechanism of quercetin in promoting wound healing.Toprovide experimental basis for further research and development of wound healing agent containing quercetin,as well as animal and molecular biological basis for clinical diabetic wound care,to provide more scientific reference for clinical diabetic refractory wound care,and to change the current clinical wound care experience summary model.Methods:1.Establishment of diabetic skin model in SD rats under long-term high glucose stimulation: the skin of diabetic rats stimulated by high glucose for 8 weeks and the skin of normal rats in the same period were collected,and the histopathological changes were observed by HE staining;the expression levels of related vascular genes and inflammatory factors genes in the skin were detected by RT-qPCR.2.Study on the promoting effect of quercetin on the healing of refractory wounds in diabetic rats: Full-thickness skin excision was used to construct the model of refractory wounds in diabetic rats.Different concentrations of quercetin were administered locally.The wound healing rate was monitored dynamically and the changes of wound state were recorded by photographs to observe the effect of quercetin.The wound tissues were collected at different time points in the course of administration and stained with HE and Mason.The changes of wound histology and collagen deposition were analyzed.3.The mechanism of quercetin promoting wound healing in diabetic rats: the wound tissues were collected at different time points during the course of administration and labeled with immunohistochemistry(M2macrophage specific markers CD206 and M1 macrophage specific markers iNOS),and the proportion of CD206 and iNOS positive cells was counted to explore the regulation of quercetin on macrophage polarization;Quantitative RT-qPCR was used to detect the effects of quercetin on the expression of related vascular genes and to analyze the angiogenesisactivity of quercetin-stimulated animal models.RT-qPCR was used to detect the effects of quercetin on the expression of pro-inflammatory factors,anti-inflammatory factors,M2 macrophage specific marker genes and M1 macrophage specific marker genes at the level of gene,and to further explore the effects of quercetin on wound inflammation and macrophage.Phenotypic effects of macrophages.Results :1.Establishment of diabetic skin model in SD rats under long-term high glucose stimulation: Compared with normal rat skin,the infiltration of inflammatory cells in the skin of diabetic rats increased significantly.The levels of IL-6,TNF-?,iNOS and Arg-1 of M1 macrophage specific marker gene and M2 macrophage specific marker gene in diabetic group were significantly higher than those in normal group(P < 0.01).The expression levels of vascular-related CD31 and VEGF-? genes in the skin of normal rats were significantly higher than those in diabetic rats(P <0.01).2.Study on the effect of quercetin on the repair of refractory wounds in diabetic rats: The area of wounds in quercetin treatment group was significantly smaller than that in model group,and the area of wounds in high concentration quercetin group decreased the most.Up to the 14 th day of wound healing,the wound healing rate of high concentration quercetin group was the highest,while that of 10% dimethyl sulfoxide solvent control group was the lowest.Histopathology showed that the number of inflammatory cells and granulation tissue in quercetin treatment group decreased significantly on the 7th day after treatment.The staining intensity,angiogenesis rate,collagen maturity,collagen distribution score and fibroblast distribution of quercetin treatment group were higher than those of model group.There was no significant difference between model group and 10% DMSO control group.3.Study on the mechanism of quercetin promoting wound healing in diabetic rats: The wound tissues were collected on the 3rd and 7th day after administration for immunohistochemistry.The results showed that the number of CD206 positive cells in quercetin treatment group was significantly higher than that in model group.In addition,the expression of CD206 increased in a dose-dependent manner in quercetin-treated group.Immunohistochemistry showed that the number of CD206 positive cells in Q-LD(P < 0.01),Q-MD(P < 0.01)and Q-HD(P < 0.01)control groups was significantly higher than that in model group.The number of iNOS positive cells in Quercetin treatment group was significantly less than that in model group(P < 0.01).Immunohistochemistry showed that the distribution of iNOS positive cells in Q-LD(P < 0.01),Q-MD(P < 0.01)and Q-HD(P < 0.01)control groups was significantly lower than that in model group.The expression level of iNOS in quercetin treatment groups were significantly different from that in model group,and with the increase of concentration,the expression level of iNOS decreased(P < 0.01).The expression levels of Msr-1 and Arg-1 in quercetin treatment groups were significantly different from those in model group,and with the increase of concentration,the expression levels of Msr-1 and Arg-1 increased(P <0.01).The expression levels of IL-6 and TNF-? in quercetin treatment groups were significantly different from those in model group.With the increase of concentration,the expression levels of IL-6 and TNF-?decreased(P < 0.01).The expression level of anti-inflammatory factor IL-10 in quercetin treatment groups was significantly different from that in model group,and with the increase of concentration,the expression level of IL-10 increased(P < 0.01).The expression levels of vascular related genes CD31 and VEGF-? in quercetin treatment groups were significantly different from those in model group,and with the increase of concentration,the expression levels of CD31 and VEGF-? increased(P < 0.01).Conclusion:1.After 8 weeks of high glucose stimulation,the skin of diabetic rats had obvious histopathological changes,inflammatory cells increased,the skin was in an inflammatory state,and blood vessels decreased.It means that SD rat diabetic skin model is successfully constructed.2.Quercetin can promote angiogenesis,granulation tissue proliferation and collagen deposition in wound site,and can significantly promote the healing of refractory wounds in diabetic rats.3.Quercetin can promote the polarization of macrophages to M2,inhibit the polarization of macrophages to M1,alleviate the excessive inflammation in the wound site of diabetes mellitus. |
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