Exosomes Derived From Adipose-derived Mesenchymal Stem Cells Facilitate Wound Healing Of Frostbite Injury By Regulating SOCS3/TGF-?1 Signaling

BackgroundFrostbite is a kind of skin and subcutaneous tissue damage caused by low temperature,which is common in northeast China and has the characteristics of seasonal onset.Without timely and effective treatment,deep frostbite can lead to amputation and even death.At present,the research on frostbite mainly focuses on the prevention and treatment of hypothermia injury during in vitro preservation of transplanted organs,while the research on frostbite wound healing focuses on clinical review and lacks basic exploration.The research on the healing mechanism of frostbite wound can provide theoretical support for clinical treatment and help reduce the amputation rate after frostbite.In the past,frostbite brought us a painful lesson in the Korean War,and in the sino-Indian border conflicts in recent years,our soldiers also faced a huge risk of frostbite.The research on frostbite wound healing can effectively guarantee the health of the soldiers,and is of great strategic significance to our country.Adipose-derived mesenchymal stem cells(ADSCs)have been proved to have significant therapeutic effects on a variety of refractory and chronic wounds,which can affect the proliferation and migration of fibroblasts(HSFs)and promote the synthesis and secretion of collagen fibers.The proteins,m RNA,mi RNA and other substances contained in exosomes can regulate cell life activities and signal transduction between cells,and avoid ethical disputes brought by stem cell transplantation,thus having broad application value.At present,basic research on frostbite wound healing is still weak.Moreover,by exploring the related mechanism of adipose-derived stem cells exosomes(ADSCs-Exos)regulating frostbite wound healing,relevant blanks will be filled and treatment methods for frostbite can be enriched.ObjectiveTo explore the related mechanism of ADSCs-Exos regulating SOCS3/TGF-?1signaling pathway and affecting frostbite wound healing.Methods1.The mouse skin frostbite wound model was constructed,and the m RNAs differentially expressed between frostbite tissue and normal skin tissue were screened by second-generation RNA-seq.By analyzing relevant biological information and referring to previous research results,the key gene of interest was identified.2.SOCS3 was taken as the target gene to verify the gene function of SOCS3 in regulating frostbite wound healing.In vitro,SOCS3 was silenced by lentivirus infection or SOCS3 and TGF-?1 were silenced simultaneously.The effects of SOCS3 on the proliferation and migration of HSFs were observed by CCK-8 assay,scratched test and Transwell test.Western blot was used to detect the expression levels of related proteins,including SOCS3,TGF-?1,Collagen I and Collagen ?,verifying the negative regulation effect of SOCS3 on TGF-?1.In animal experiments,SOCS3 expression in frostbite wound tissues was interfered,and the expression of SOCS3 in the wound tissues was detected by RT-q PCR.The wound healing rate was calculated and the effect of SOCS3 inhibition on the wound healing rate was evaluated.HE and Masson staining were used to observe the tissue morphological changes and collagen fiber deposition on the wound tissue.Western blot was used to detect the protein expression level of TGF-?1 in the wound tissue after SOCS3 silencing.3.The adipose tissue was obtained from clinical harvest,ADSCs were isolated by collagenase digestion method,the multidirectional differentiation potential of stem cells was verified by adipogenic,osteogenic and chondrogenic induced differentiation experiments,and related markers of stem cells were detected by flow cytometry.In vitro continuous subculture,cell culture supernatant was collected,and ADSCs-Exos were obtained by supercentrifugation method.NTA,TEM and Western blot were used for further identification of exosomes.4.In vitro experiments,CCK-8 assay,scratch test and Transwell test were used to detect the proliferation and migration of HSFs,and to observe the promoting effect of ADSCs-Exos on proliferation and migration of HSFs.In animal model,the effect of ADSCs-Exos on frostbite wound was observed by calculating wound healing rate and pathological staining.During the experiment,the expression of SOCS3,TGF-?1and other related molecules in HSFs and peripheral wound tissue after ADSCs-Exos intervention were detected.5.The upstream regulatory factor miR-143 on SOCS3 was predicted and screened by bioinformatics website,and the targeting relationship between mi R-143 and SOCS3 was verified by dual luciferase assay.In vitro and in vivo experiments,the regulation of mi R-143 carried by ADSCs-Exos on SOCS3 and TGF-?1 was observed through overexpression of mi R-143 in ADSCs-Exos,so as to explore the regulation mechanism of ADSCs-Exos on frostbite wound healing.Results1.RNA-seq obtained differentially expressed m RNAs after frostbite,and it was found that SOCS3 expression was significantly increased after frostbite injury.Bioinformatics analysis results and previous literature reports indicate that SOCS3 plays an important negative regulatory role in the process of wound healing.2.In vitro experiments,it showed that SOCS3 silencing up-regulated the expressions of TGF-?1,Collagen I and Collagen ? in HSFs,and promoted the cell proliferation and migration,while both SOCS3 and TGF-?1 silencing could counteract the promoting effect of SOCS3 silencing.In animal models,silencing SOCS3 expression after frostbite injury was beneficial for collagen fibers deposition in the wound edge and accelerated frostbite wound healing.3.ADSCs were successfully isolated from human adipose tissue by collagenase digestion method.The obtained ADSCs had multidirectional differentiation potential and expressed stem cell-related surface markers.ADSCs-Exos were isolated from cell culture supernatant by supercentrifugation.The exosomes obtained had a typical "tea tray" structure,the particle size distribution was consistent with the distribution of exosomes,and the expression of CD63,CD81 and Alix were also detected.4.It was found that ADSCs-Exos could promote proliferation and migration of HSFs and inhibit SOCS3 expression in HSFs in vitro.In animal models,subcutaneous injection of ADSCs-Exos could promote frostbite wound healing in mice,inhibit the expression of SOCS3 in frostbite tissues,and up-regulate the expression of TGF-?1,Collagen I and Collagen ?.5.The prediction results of bioinformatics website and dual luciferase assay showed that there was a regulatory relationship between mi R-143 and SOCS3.It was found that Exos-mi R-143 mimic significantly inhibited SOCS3 expression in HSFs and promoted the cell proliferation and migration.In animal models,Exos-mi R-143 mimic could significantly reduce SOCS3 expression after frostbite and promote frostbite wound healing.ConclusionIn the process of frostbite wound healing,SOCS3 can regulate the expression of TGF-?1 and affect the proliferation and migration of fibroblasts,thus playing a negative role in wound healing.mi R-143 carried by ADSCs-Exos up-regulated the expression level of TGF-?1 by regulating SOCS3,promoted the proliferation and migration of HSFs,and accelerated frostbite wound healing.