Mechanism Study On Promoting Diabetic Wound Healing Of Svf-gel Conditioned Medium By Rapid Adipose Tissue Regeneration

Objective:To explore the mechanism of stromal vascular fraction gel conditioned medium(SVF-gel-CM)to promote wound healing in diabetic mice,and to clarify the effect of adipogenic signals on diabetic wound healing,and to provide a theoretical basis for the clinical application of SVF-gel-CM.Methods:1.To explore the mechanism of SVF-gel-CM promoting wound healing in diabetic mice:SVF-gel-CM was obtained in vitro;the skin wound model of diabetic mice was constructed,and then SVF-gel-CM alone or in combination with the PPARγ inhibitor GW9662 was transplanted into the wound;Photographs were taken on d0,d3,d7,d10,and d14 to observe wound healing;HE,CD31 immunohistochemistry,perilipin and CD206 immunofluorescence staining were used to observe wound healing tissue structure,angiogenesis,and adipocyte regeneration,respectively,and infiltration of M2 macrophages;the m RNA expression of adipose differentiation genes(PPARγ,Fabp4,C/EBPα),inflammatory genes(TNF-α,IL-1β,IL-10)and macrophage markers(i NOS,Arg1)were detected by q PCR.2.Exploring the effect of adipogenic signaling on wound healing: The skin wound model of diabetic mice was constructed,and the PPARγ agonist Rosiglitazone(RGZ)and the inhibitor GW9662 were used to intervene,and photographs were taken at different time points to observe the wound healing;The pathological mechanism was explored by HE and immunofluorescence;the m RNA expressions of adipogenic differentiation genes,inflammatory genes and macrophage markers were detected by q PCR.Statistical analysis of the data was performed using one-way ANOVA.Results:1.The results of wound healing in diabetic mice showed that SVF-gel-CM significantly accelerated wound healing from d3,and the wound healed completely on d14(P<0.05);however,SVF-gel-CM+GW9662 delayed wound healing(P<0.05),incomplete wound healing on d14.HE staining results showed that: SVF-gel-CM accelerated healing,decreased inflammatory cell infiltration was observed on d7,and wounds healed completely on d14,re-epithelialization had been completed,and the tissue structure was dense and complete;on the contrary,the SVF-gel-CM combined with GW9662 group delayed the wound healing,the wound was severely infiltrated by inflammatory cells on the 7th day,the wound was not completely healed and re-epithelialization was incomplete by the 14 th day,the tissue structure was incomplete and the connection was not tight.Immunofluorescence showed that SVF-gel-CM significantly promoted the formation of perilipin-positive cells(2.962 %±0.610 %)and the early recruitment of CD206-positive cells(12.320 %±1.369 %)on d7;on the contrary,SVF-gel-CM+GW9662 inhibited the formation of perilipin-positive cells and early recruitment of CD206-positive cells;perilipin cells and CD206-positive cells were reduced in each group on d14.CD31 immunohistochemistry showed that SVF-gel-CM promoted wound angiogenesis in diabetic mice,while the proportion of angiogenesis decreased after GW9662 blockade(P<0.05).q PCR results showed that compared with the SVF-gel-CM combined with GW9662 group,SVF-gel-CM significantly promoted the expression of the adipogenic gene PPARγ as well as its upstream and downstream genes,increased the expression of Arg1 m RNA,and decreased the expression of i NOS m RNA;at the same time,it can also reduce the expression of pro-inflammatory gene TNF-α and IL-1β m RNA,and promote the expression of anti-inflammatory gene IL-10 m RNA,the difference was statistically significant(P<0.01).2.The results of wound healing showed that RGZ significantly accelerated wound healing from d3,the most significant effect of promoting healing was on d7(P<0.05),and the wound healed completely by d14;on the contrary,GW9662 delayed wound healing(P<0.05),and the wound did not heal completely by d14.HE results showed that RGZ accelerated wound healing,the subcutaneous tissue structure was more dense on d7,and the wound was completely healed on d14,achieving complete re-epithelialization,complete and dense tissue structure;on the contrary,GW9662 delayed wound healing,and the subcutaneous tissue structure was loose on d7,the wound healing was incomplete on d14,the tissue re-epithelialization was incomplete,and the tissue structure was loose and the connection was incomplete.Perilipin immunofluorescence showed that the number of perilipin positive cells(0.879 %±0.096 %)in RGZ group increased on d7(P<0.01),while the number of GW9662 perilipin positive cells(0.092 %±0.020 %)decreased on d14(P<0.01).CD206 immunofluorescence showed that RGZ significantly promoted the recruitment of CD206-positive cells(12.433 %±1.956 %)in the early stage of the wound,while GW9662 significantly inhibited the recruitment of CD206-positive cells(1.363 %±0.159 %).q PCR results showed that RGZ promotes the expression of adipogenic genes,especially PPARγ,while the inhibitor does the opposite;RGZ significantly decreased the expression of i NOS m RNA and increased the expression of Arg1 m RNA(P<0.01);At the same time,RGZ also significantly reduced the pro-inflammatory gene TNF-α and IL-1β m RNA expression(P<0.01),while up regulating the expression of anti-inflammatory gene IL-10 m RNA(P<0.01),GW9662 group showed the opposite.Conclusion:1.SVF-gel-CM promotes wound healing in diabetic mice by improving wound inflammation,rapidly inducing adipose regeneration and angiogenesis.2.Activating adipogenic signals may promote wound healing in diabetic mice by mediating the early recruitment of M2 macrophages,inducing adipocyte regeneration.