| BackgroundCurrently,the clinical outcome of chronic wounds remains unsatisfactory and the cost of chronic wounds care has increased dramatically,adding to the economic pressure associated with wounds treatment.Common diseases that cause or combine with chronic wounds include diabetes mellitus,pressure ulcers,post-radiation lesions,peripheral vascular lesions,and venous stasis-like lesions,which have different underlying disease types,age and gender distributions,and different pathophysiological environments for their chronic wounds.This poses a great challenge for clinical treatment.Diabetic patients and obese people are a high-risk group for chronic wounds.Chronic inflammation is an important feature of diabetic chronic wounds due to the long-term effects of high blood sugar and free fatty acids,which lead to a chronic inflammatory state in various tissues.In the actual clinical treatment work,in addition to the treatment targeting the etiology,as we further study the microenvironment of the wounds,we found that these chronic wounds have vascular damage and the wounds have a hypoxic state with an impaired oxygen supply and an increased oxygen demand.As with chronic inflammation,ischemia and hypoxia are important features of chronic wounds and directions for intervention.Diabetic mouse models have been widely used to study diabetic wounds,but the specific gene profile of immune cells in diabetic wounds has not been well characterized,and most of the existing studies on diabetic wounds models are limited to single observation time results.The existing studies on diabetic wounds models are mostly limited to single observation time results,which do not provide a comprehensive picture of the temporal changes of immune cells during the wounds healing process.Single-cell sequencing is a technology that has emerged and rapidly developed in recent years.It provides a method to classify and functionally study cell populations in a specific environment,is not subject to biases that may arise from previous experience,is better able to characterize cellular changes,and has good potential for the discovery of new rare cells and cell-specific states.This technique has become an important technical aid in other fields of cell lineage characterization and cell developmental differentiation studies.In addition to the treatment of the primary disease and general symptomatic support for the ischemic and hypoxic factors and chronic inflammation present in chronic wounds,stem cell therapy is currently a highly anticipated development.Stem cells have become an important breakthrough point in the field of medicine due to their unique self-renewal and multi-directional differentiation ability.Basic research on induced pluripotent stem cells,mesenchymal stem cells,epidermal stem cells,and adipose stem cells in wound healing has made some progress.However,there is still some controversy about the safety and ethical issues of stem cell therapy.It has not been possible to carry out related work on a large scale in China.Exosomes are biological entities enclosed by phospholipid bilayers and are found in a wide range of physiological fluids.Secretion of exosomes can be seen in various cell types and can carry important biomolecules from their parent cells.With recent advances in exosome research,stem cell-derived exosomes can be used as an alternative or complementary means of stem cell therapy,avoiding the safety risks associated with stem cell therapy,greatly enhancing the safety of stem cell use and broadening the avenues for stem cell applications.ObjectiveFirstly this study aimed to map the general state and characteristic spectrum of immune cells in a diabetic mouse model,and to characterize the distribution differences,genetic differences and possible differentiation differences between immune cell populations in a linear and continuous manner by means of time series.Secondly,by constructing a mouse ischemic wound model to simulate the immune environment of ischemic wound,the effects of epidermal stem cell-derived exosomes on wound healing and macrophage function and polarization were investigated.Finally,by analyzing the differential gene expression of specific cell populations in the diabetic wound microenvironment and the differential influence of exosomes on macrophage polarization in ischemic wound,we provide possible therapeutic ideas and intervention targets for both pathogenic factors of high glucose and ischemia in chronic wound.MethodsIn the first part,streptozotocin(STZ)-induced diabetic mice were constructed and whole skin defect wounds were created.woundtic single cell suspensions were prepared from woundtic tissue sampled on days 1,3,5,and 7 and CD45-positive immune-related cells were sorted out by flow cytometry.The cells were analyzed by single cell sequencing and data visualization.In the second part,we constructed a mouse ischemic wound model with a double-tipped dorsal flap and extracted exosomes of primary representative dermal stem cell origin to examine their effects on macrophage function and polarization in vitro,and in vivo tests to examine the effects of exosomes on ischemic wound healing,blood flow and macrophage polarization at the wound margin.The mechanism of exosome content and its effect on macrophage polarization was determined by proteomics.ResultsWe performed single-cell sequencing of CD45+cells collected from woundtic tissues of wild-type and STZ-induced diabetic C57BL/6J mice by excluding low-quality cells and normalizing the single-cell data of the obtained samples to eliminate batch effects,and obtained data from a total of 9240 cells.A total of 17 cell populations were identified based on their gene expression profile and after filtering for low-quality cells,including 4 neutrophil populations,2 monocyte populations,3 macrophage populations,2 DC cell populations,1 NK cell population,1 T cell population,1 mast cell population,1 fibroblast population,and 1 undescribed population of macrophages expressing osteoclast marker genes that were predominant in the control acute wound group.There was a temporal variation in the differential distribution of genes in the number of immune cells between the two groups,with the peak of the difference occurring at day 5,and significant differences in the inflammatory response,defense mechanisms,cellular metabolic patterns,and angiogenesis between the two groups.In contrast,the specific macrophage cells expressing osteoclast markers were not primarily involved in the inflammatory process,but may be involved in the wound healing process by balancing tissue homeostasis,tissue remodeling,and collagen metabolism in the extracellular matrix.Ischemic wounds showed delayed healing compared to their own control acute wounds,along with a thinning of the epidermis and dermis,and a reduced blood flow signal was found in the ischemic wounds compared to the control wounds as measured by the laser scatter blood flow instrument.The macrophage dermis within the wound margin was biased towards Ml-type polarization state.Epidermal stem cell-derived exosomes were able to promote macrophage proliferation and migration in vitro.In macrophage polarization assays,the M1 macrophage polarization marker iNos was reduced after exosome intervention application,while the M2 macrophage marker Argl was elevated.The rate of wound healing was increased after exosome intervention on ischemic wounds in mice,while the blood flow signal was significantly higher in ischemic wounds with exosome intervention than in ischemic wounds with intervention.The macrophage M2 macrophage marker protein Argl was elevated and the M1 macrophage marker iNOS was decreased in the post-intervention wound.In proteomic assays,M1 macrophages with exosome intervention expressed inflammation-related proteins including interleukin-1(IL-1)tumor necrosis factor alpha(TNF-α)was decreased,while multiple differential proteins suggested that NF-KB,an important pathway for M1 macrophage polarization,was inhibited.differential protein changes in M2 macrophages after intervention were enriched in metabolism and necroptosis,cytokine binding,and many differential proteins related to STAT6-related signaling pathways emerged.ConclusionThere are significant temporal differences in the population changes of immune cells in the diabetic mouse wound model.We characterized the population of wound-associated immune cells in terms of quantity,gene expression,and functional changes at different times,and mapped the dynamic changes in the spectrum of immune cells in mouse diabetic wound for the first time.We also identified a population of macrophages with giant osteoclast-associated genes that had not been previously characterized in the field of cutaneous wound,and suggested a possible mechanism for their promotion of wound healing.A clearer understanding of the specific process of wound healing is provided.It provides an important reference for the study of immune cell-related marker genes and the mechanisms affecting diabetic wound healing.In this study,we demonstrated for the first time that exosomes can promote the healing of ischemic wounds and improve the blood flow around the wounds in mice.Also,epidermal stem cell-derived exosomes were able to promote macrophage proliferation and inhibit M1-type macrophage polarization and promote M2-type macrophage cell polarization in vitro.It is also proposed that the effect of epidermal stem cell-derived exosomes on macrophage polarization may be achieved by inhibiting the activation of NF-KB signaling pathway to inhibit Ml-type macrophage polarization,while promoting STAT6-related signaling pathway to promote M2-type macrophage polarization.It provides new evidence and theoretical basis for the application of epidermal stem cell exosomes in wound and sheds light on the search for targets of immune cell involvement in chronic wound targeting ischemic 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