Application And Mechanism Of Exosomes Combined With Botulinum Toxin A In Skin Wound Repair

Skin wound repair is a complex process of tissue repair and remodeling,which may lead to different complications,the most common long-term complication is scar.Scarring can be associated with depression,social avoidance and disfigurement,and can have devastating consequences for sufferers,including restricted movement,poor aesthetics,permanent disability and other effects.Reducing the complications caused by trauma repair can relieve a lot of physical and psychological pain for patients.Therefore,accelerating trauma repair and reducing long-term complications is a big dilemma faced by clinicians.In recent years,exosomes(EXO)have been the focus of translational medicine research and become A new therapeutic direction in wound repair.Botulinum toxin A(BTX-A)can reduce muscle traction caused by local muscle tension of skin and is considered as A new and effective treatment for wound repair.In this study,the multi-dimensional treatment of wound repair was studied to explore the combined effect of adipose tissue-derived stromal cells(ADSC)-EXO and BTX-A,so as to provide new therapeutic support for clinical treatment of skin wound repair.Objective:To study the effect of adipose-derived exosomes(ADSC-EXO)combined with Botulinum toxin Type A(BTX-A)on skin wound repair and its mechanism.Methods:(1)Human skin fibroblasts(HSF)were co-cultured with PKH67 labeled EXO for2h and photographed.(2)HSF cells were cultured on 6-well plates.The cells were vertically cut with 1000μl gun tip and randomly divided into group PBS,group EXO,group BTX-A and group EXO with BTX-A.They were observed at 0h,12h and 24h and photographed.(3)The whole skin layer was removed on the back of SD rats by aseptic operation to form A circular wound model with A diameter of 10mm.The wound model was randomly divided into group PBS,EXO,BTX-A and EXO with BTX-A with 12 rats in each group.(4)Group EXO was injected with EXO 100μl(2.5×10~7partical EXO)around the wound,group BTX-A was injected with BTX-A100μl(3U/ml),group EXO with BTX-A was injected with EXO and BTX-A mixture,the injection dose was the same as before.The control group was injected with PBS and normal saline at 0,3,5 and 7 days after EXO.(5)Wound healing was observed and photographs were taken.On the 16th day,skin tissues of the wound area of rats were collected,and the morphology of regenerative epithelium,infiltrating cells,collagen fiber bundles and angiogenesis were observed under HE staining microscope.(6)On the 16th day,the expression levels of VEGF,collagen I,collagen III,TGFβ1 and TGFβ3 in scar tissue were detected by Western Blot and immunohistochemistry.Results:(1)PKH67 fluorescence particles were found in HSF cells and The migration rate of HSF was significantly accelerated 12 and 24 hours after EXO and EXO with BTX-A treatment(compared with the group PBS,P<0.05).(2)From the 5th day after operation,the wound healing of group EXO treatment and group EXO combined with BTX-A was better than that of group botulinum toxin alone and PBS treatment.(3)Naked eye observation:the skin wound healing of EXO group and EXO+BTX-A group was better than PBS group and BTX-A group on the 5th day after surgery.The wound healing of d EXO group 16 was better,and the wound healing of EXO+BTX-A group was the best.(4)On day 9th,compared with group PBS,scar area in group EXO combined with BTX-A was the smallest(P<0.05);(5)Extensive infiltration of inflammatory cells was observed in group PBS,while fewer infiltrating cells were observed in group EXO and EXO with BTX-A,with uniform distribution of dermal collagen and less cross-linking.The group EXO with BTX-A was the best,and the number of newborn blood vessels was more than that in group PBS and BTX-A.(6)WB and immunohistochemistry showed that compared with group PBS,the expression of VEGF and TGF-β3 in group EXO and EXO with BTX-A was up-regulated;Compared with group EXO and BTX-A,VEGF expression was higher in group EXO with BTX-A(P<0.05).Compared with other groups,TGF-β1 expression was down-regulated and TGF-β3/TGF-β1 ratio was up-regulated in group BTX-A(P<0.05).Sixteen days after surgery,compared with group PBS,collagen I and III expressions in EXO group and group EXO with BTX-A were significantly down-regulated,and the collagen I/III ratio was up-regulated(P<0.05).Conclusions:EXO and BTX-A increase HSF migration,EXO promotes neovascularization,which is conducive to accelerating wound healing,and inhibits COLIII and COLI formation in the late stage.After combined application of BTX-A,the effect is better,reducing muscle and skin pull,thus reducing wound microtrauma and inflammation.The mechanism may also be related to the up-regulation of VEGF expression,TGF-β3/TGF-β1 and COL III/COL I ratio.