Small Extracellular Vesicles Derived From Human Umbilical Cord Mscs Regulate Calcium Homeostasis To Promote Diabetic Wound Healing

Objective:Diabetic foot ulcer（DFU）is one of the most significant complications of diabetes mellitus and the number one cause of nontraumatic amputation for DFU refractory wounds.Current treatments are effective,but they do not yet address the problem of prolonged healing.The imbalance of Ca²⁺homeostasis is closely related to the progression of type 2 diabetes,and Ca²⁺,as an important intracellular second messenger,plays a critical role in the maintenance of cellular function.Our previous study demonstrated that human umbilical cord mesenchymal stem cells derived from small extracellular vesicles（hucMSC-sEVs）can effectively promote tissue damage repair.This study aimed to explore whether hucMSC-sEVs could be an effective new solution for DFU repair,and to analyze new mechanisms of diabetic wound repair from a new perspective of calcium homeostasis regulation,providing new targets and new ideas for DFU treatment.Methods:1.Bovine serum albumin modified with advanced glycosylation end products（AGE-BSA）was used to stimulate rat dermal fibroblasts（DFs）to mimic the hyperglycemic environment of diabetes.Protein profiling was performed on the traumatized surfaces and marginal tissues of DFU patients to cluster and analyze the changes in Ca²⁺homeostasis and the changes in Ca²⁺levels in DFs after in vitro AGE-BSA stimulation.q RT-PCR and Western blot were performed several times to verify the changes in important Ca²⁺channels mediating calcium transport under the condition of DFs injury,and the preliminary screening was performed to identify the stable low expression of Ca²⁺channel TRPC6.The changes of TRPC6 in DFU were further verified by targeted mass spectrometry analysis of 13 DFU samples and 8 control samples.Gene correlation analysis was performed to analyze the relationship of TRPC6with genes regulating the cell cycle,glucose transporter protein,and collagen production.Subsequently,additional DFU clinical samples were taken for immunofluorescence assay to verify traumatic TRPC6 expression.2.Small interference RNA（si RNA）,TRPC6 channel-specific inhibitor,and overexpression plasmid were used to knock down,inhibit,and overexpress TRPC6,respectively.calcium fluorescent probes Fura-2 AM and Fluo-4 AM were used to detect changes in Ca²⁺levels in DFs;q RT-PCR,western blot,and cellular immunofluorescence to detect the expression of inflammatory factors and the ability to secrete extracellular matrix such as collagen in DFs;flow cytometry to detect the level of apoptosis in DFs;CCK8 assay and scratch assay to detect the proliferation and migration ability of DFs.3.Laser confocal microscopy was used to observe the uptake of hucMSC-sEVs by DFs.q RT-PCR and cellular immunofluorescence were used to detect the ability of hucMSC-sEVs on an extracellular matrix such as collagen secretion by DFs,and flow cytometry was used to detect the apoptosis level of DFs.We constructed an SD rat DFU model,added hucMSC-sEVs intervention,HE staining to observe skin structure,immunohistochemistry to detect neovascularization index CD31,and Sirius red staining to detect collagen distribution,and comprehensively evaluated the therapeutic effects of hucMSC-sEVs on DFU.4.q RT-PCR,western blot,and cellular immunofluorescence were used to detect the expression of TRPC6 after the hucMSC-sEVs intervention;immunohistochemistry and tissue immunofluorescence was used to detect the expression of TRPC6 after hucMSC-sEVs intervention in DFU model skin wounds;Fura-2 AM,Fluo-4 AM intracellular Ca²⁺levels in DFs after the hucMSC-sEVs intervention.5.RNA-seq and bioinformatics were used to analyze the transcription factor protein families involved in DFU,and the transcription factor SP2 involved in the regulation of TRPC6 expression was screened by database comparison;dual luciferase reporter gene assay and chromatin immunoprecipitation（Ch IP）assay were used to verify the binding of SP2 to The binding of SP2 to the TRPC6 promoter and the specific site of binding was verified using dual luciferase reporter gene assays and Ch IP assay.The DFs were treated with hucMSC-sEVs knocked down by si RNA,and the proliferation and migration ability of DFs were detected by CCK8 assay and scratch assay.q RT-PCR,western blot,and cellular immunofluorescence were used to detect the expression of TRPC6 and collagen.6.Short or prolonged overexpression of TRPC6（24h,48h,72h,96h）and addition of exogenous Ca²⁺（Ca Cl₂）simulated the continuous calcium inward flow of cells with hucMSC-sEVs to detect the calcium content,apoptosis level,and migration ability.JC-1 detected the change of mitochondrial membrane potential of DFs after hucMSC-sEVs intervention;mitochondrial calcium fluorescent probe Rhod-2 AM was used to detect mitochondrial calcium levels in DFs after the hucMSC-sEVs intervention;western blot was used to detect mitochondrial calcium uniporter（MCU）and sodium-calcium exchanger（NCLX）in DFs after the hucMSC-sEVs intervention.After using MCU inhibitor ruthenium red 360（Ru360）and NCLX inhibitor（CGP37157）,hucMSC-SEVs intervention was performed.The mitochondrial Ca²⁺level,ROS level,and apoptosis level of DFs were measured by Rhod-2 AM,reactive oxygen species（ROS）detection reagent,and flow cytometry,respectively.Results:1.Protein profiling and clustering analysis revealed an imbalance of calcium homeostasis and impaired mitochondrial function in the traumatic tissues of DFU patients;AGE-BSA significantly induced apoptosis and inflammatory factor expression in DFs,while intracellular calcium inward flow was reduced.Further screening of calcium channels mediating calcium inward flow in skin cells revealed a significant and stable decrease in TRPC6 channel protein,which was verified by targeting protein profiles,and gene correlation analysis showed a significant positive correlation between TRPC6 andα-SMA and collagen expression,important indicators of DFs activation.Tissue immunofluorescence showed significantly lower TRPC6expression in traumatic tissues in DFU clinical samples than in the corresponding traumatic rim tissues.2.Knockdown and inhibition of TRPC6 channels reduced intracellular calcium levels in DFs,decreased the ability of DFs to secrete extracellular matrix such as collagen,impaired the proliferation and migration of DFs,and promoted the expression of apoptosis and inflammatory factors;overexpression of TRPC6 channels increased intracellular calcium levels,enhanced the biological functions of DFs such as proliferation,migration,and collagen secretion,and decreased the expression of inflammatory factors.3.Cell uptake assays showed that hucMSC-sEVs could be efficiently endocytosed by DFs;hucMSC-sEVs significantly reduced the apoptosis of DFs caused by AGE-BSA and improved the ability of DFs to secrete collagen;in vivo experiments showed that hucMSC-sEVs effectively shortened the healing time of diabetic rat skin wounds,and accelerated neovascularization and dermal collagen rearrangement.4.In vitro and in vivo experiments showed that hucMSC-sEVs significantly increased TRPC6 expression,and cellular immunofluorescence showed that hucMSC-sEVs increased TRPC6 expression on cell membranes;meanwhile,hucMSC-sEVs increased intracellular calcium ion levels in DFs.5.Combined with RNA-seq of clinical DFU patient samples and predictions from NCBI,UCSC,JASPAR,and other databases,SP2 proteins in the zinc finger structural（zf-C2H2）transcription factor protein family were screened for targeting.SP2 was found to be highly expressed in hucMSC-sEVs,and its treatment significantly increased SP2 expression in the nuclei of target cells,with a corresponding increase in TRPC6expression.Dual luciferase reporter gene and Ch IP assays detected that SP2 could bind to the TRPC6 promoter and positively regulate TRPC6 gene expression in the region from-1519bp to-1725bp of the TRPC6 gene promoter.hucMSC-sEVs with knockdown of SP2 could not effectively promote TRPC6 expression and biological functions of DFs.6.HucMSC-sEVs can restore cellular damage caused by sustained calcium inward flow,as well as restore mitochondrial function and mitochondrial Ca²⁺levels,promote mitochondrial MCU and NCLX expression,and restore mitochondria-mediated calcium metabolic function.Conclusion:In the high glucose toxicity environment,there is an imbalance in calcium homeostasis in diabetic ulcer wounds,and both TRPC6 protein,which mediates calcium inward flow,and mitochondrial function,an important organelle that mediates calcium metabolism,are impaired.HucMSC-sEVs upregulate TRPC6 gene expression by binding to a specific region of the TRPC6 gene promoter（-1519bp to-1725bp）via the transporter transcription factor SP2,and restore mitochondrial function,which is an important organelle mediating calcium metabolism.It also restores mitochondrial function and promotes DFU wound repair by restoring calcium homeostasis.