| BackgroundType Ⅱ diabetes mellitus(T2DM)is a systemic metabolic disorder characterized by insulin resistance and impaired pancreatic β-cell function.Chronic hyperglycemia in individuals with T2 DM can result in compromised functionality of various tissues,including the heart,blood vessels,nerves,and skin.Among these complications,chronic non-healing diabetic ulcers represent one of the most prevalent and severe manifestations that can lead to bacteremia,amputation,and even mortality.Wound healing in diabetic patients is influenced by multiple factors;however,high glucose levels within tissue microenvironment along with persistent uncontrolled inflammation are considered as primary contributors.These factors impede normal reparative functions of fibrocytes and angiogenic cells within wound tissue thereby hindering Epithelial-mesenchymal transition(EMT)process as well as neovascularization which ultimately inhibits efficient wound healing.Currently,the treatment approaches for diabetic wound healing primarily revolve around traditional Chinese medicine extracts and exosomes derived from mesenchymal stem cells(MSC-exo).Chinese medicine extracts,sourced from natural plants or medicinal materials,exhibit potent anti-inflammatory and antioxidant effects without inducing toxic side effects on the body,thereby playing a constructive role in promoting the healing of diabetic wounds.However,only a limited number of TCM extracts have demonstrated applicability in this domain;hence further exploration is required to identify additional TCM extracts with potential for enhancing diabetic wound healing.Paeonol(Pae),an active compound extracted from the dried root bark of peony belonging to the goldenseal family,exhibits significant therapeutic efficacy in treating hypertension,coronary atherosclerosis,ulcerative colitis,and diabetes complications.Due to its cytoprotective and vascular protective properties,Pae holds promise as a potential drug for promoting diabetic wound healing;however,there remains a dearth of studies elucidating its mechanism in diabetic skin regeneration.MSC-exo,a micro vesicle structure released by mesenchymal stem cells,contains active ingredients such as lipids,proteins,and nucleic acids that facilitate epidermal regeneration and angiogenesis while accelerating wound healing.Moreover,MSC-exo exhibits excellent biocompatibility and low immunogenicity,rendering it a safer and more reliable option for clinical applications.By utilizing Paeloaded MSC-exo(Pae/exo),we anticipate achieving a synergistic effect between the two to expedite the healing process of diabetic wounds.The temperature-sensitive hydrogel Pluronic F127(PF-127)plays a pivotal role in diabetic wound healing due to its high-water content and exceptional hydrophilic polymer characteristics.This material rapidly transitions into a gel state upon exposure to human body temperature,thereby enhancing adhesion to skin wounds and facilitating controlled release of drugs or carrier particles.PurposeThis study focused on the promoting effect of PF-127 loaded with Pae/exo on T2 DM wound healing and its related mechanisms.First,the optimal concentration of Pae was selected and successfully loaded into MSC-exo to form Pae/exo.The morphology and biological function of Pae/exo were fully characterized.To explore the regulatory effects of Pae/exo on cell behavior,EMT,and angiogenesis in cell experiments,and to explore the mechanisms of upstream related mirnas.By embedding Pae/exo into PF-127,the therapeutic effect of Pae/exo was evaluated in T2 DM mouse models of full-thickness skin defects,and the mechanism of promoting skin healing was investigated.MethodsThe present study employed 3D skin cell mass to screen Pae,a Chinese herbal extract with potential wound healing properties.Subsequently,MSC-exo was extracted from mesenchymal stem cell culture medium and subjected to repeated freeze-thaw cycles to form Pae/exo.Comprehensive characterization of the morphology and physicochemical properties of Pae/exo was conducted,followed by investigation of its effects on fibroblast and vascular endothelial cell behavior through in vitro cell experiments.(1)Mesenchymal stem cells were utilized for the construction of 3D skin cell clusters using the cell chamber method,and their intracellular components were identified.Subsequently,the ATP luminescence method was employed to assess the impact of different Chinese herbal extracts on the viability of 3D skin cell clusters,leading to the screening of Pae at an effective concentration that promotes cellular vitality.(2)MSC-exo was extracted through ultrafiltration,followed by loading Pae into MSC-exo via repeated freeze-thaw cycles.The ratio between them and the number of freeze-thaw cycles were adjusted iteratively to optimize Pae release rate.Characterization of Pae/exo was performed using electron microscopy,Western blotting,and high-performance liquid chromatography(HPLC).(3)Application of Pae/exo on fetal prepuce fibroblasts(HSF)and human umbilical vein endothelial cells(HUVEC)enabled assessment of changes in cellular behaviors such as proliferation and migration.Subsequently,we conducted a comprehensive investigation into the impact of Pae/exo on EMT and angiogenesis,leading to the identification of miR-424-5p as a relevant micro RNA.The functional role of miR-424-5p in EMT and angiogenesis was validated through cellular transfection experiments.Furthermore,we generated miR-424 knockout mice to establish the correlation between miR-424-5p and diabetic wound healing using animal models.(1)The effects of Pae/exo on EMT and angiogenesis were validated through Western blot analysis,quantitative real-time polymerase chain reaction(q RT-PCR),and immunofluorescence experiments.Subsequently,HSF and HUVEC cells were transfected with miR-424-5p mimics and inhibitors to confirm the alterations in EMT and angiogenesis.(2)CRISPR/Cas9 technology was employed to generate miR-424 gene knockout mice for establishing a full-layer skin defect model in T2 DM.The impact of miR-424 on diabetic wound healing was investigated by assessing changes in wound area as well as conducting immunofluorescence and immunohistochemical experiments.Subsequently,Pae/exo was encapsulated in PF-127 and subjected to comprehensive physicochemical characterization,followed by investigation of the release kinetics of Pae/exo.The therapeutic efficacy of Pae/exo@PF-127 on wound healing,EMT,and angiogenesis in T2 DM mouse models with full-layer skin defects were subsequently evaluated.Results and conclusions(1)In this study,3D skin cell clusters were initially constructed and confirmed to contain both epidermal and fibroblast components.Subsequently,drug screening verified that a concentration of 20 μmol Pae could enhance cell viability.Following the successful synthesis of Pae/exo,effective loading of Pae was confirmed through electron microscopy,HPLC,and Western blot analysis.The release rate of Pae reached an optimal value of 97.075 % after adjusting the proportion and frequency of freezing and thawing.Cell experiments further validated that Pae/exo exhibited promotion effects on proliferation,migration,and scratch healing in HSF and HUVEC.(2)The promoting effect of Pae/exo on EMT and angiogenesis was confirmed through Western blot,q RT-PCR,and immunofluorescence experiments.Subsequently,it was demonstrated that Pae/exo induced a high expression of miR-424-5p in HSF and HUVEC.By transfecting miR-424-5p mimics and inhibitors,followed by the detection of factors associated with EMT and angiogenesis,it was further confirmed that Pae/exo promotes EMT and angiogenesis by upregulating miR-424-5p expression.Next,miR-424 gene knockout mice were generated to verify the inhibitory effect of miR-424 knockout on diabetic wound healing using a diabetes model.(3)Pae/exo was encapsulated within PF-127,and the optimal concentration of Pae/exo@PF-127 was determined through rheological experiments.It was verified that Pae/exo@PF-127 effectively promotes EMT and angiogenesis in full-thickness skin defect models of T2 DM,thereby facilitating T2 DM wound healing. |
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