| BackgroundCutaneous wound healing is a dynamic and complex process following skin injury and is regulated by a variety of factors including cutaneous innervation.Skin nerve fibres can regulate the function of skin cells,including dermal fibroblasts,through direct contact with skin cells or indirect secretion of nerve growth factors or neuropeptides,thereby maintaining skin homeostasis and regulating wound healing.During skin wound healing,different populations of dermal fibroblasts perform different functions including proliferation,differentiation,contraction,secretion,synthesis and remodelling of the extracellular matrix,which together contribute to wound healing.Studying the regulatory mechanisms of fibroblast function is necessary to understand and regulate effective wound healing(timely closure and high quality healing).Receptor activity modifying protein1(RAMP1),together with calcitonin receptor-like receptor(CRLR)and receptor component protein(RCP),form the receptor for the classical neuropeptide calcitonin gene related peptide(CGRP).Modulation of RAMP 1 function can effectively avoid the side effects caused by direct CGRP administration,making it a therapeutic target.RAMP1 is involved in wound healing by regulating angiogenesis and lymphangiogenesis,but the direct relationship between RAMP1 and fibroblasts has not been adequately described.Bibliometric analysis is a novel method of visualising scientific information,providing quantitative and qualitative analysis of the literature,thus helping researchers to obtain the overall metrological characteristics of the published literature in the field of study and to identify emerging areas.Many basic and clinical studies have been published in the field of cutaneous nerve effects on wound healing;however,there is a lack of bibliometric analysis of the current field.Proteomics has become an important tool for the elucidation of biological processes at the protein level,which,combined with bioinformatic analysis tools,can contribute to the in-depth study of the mechanisms of cellular function regulation.ObjectiveThe first aim is to analyse the trends,current status and hotspots of research in the field by means of bibliometric analysis,to obtain a macroscopic description of the research area and to clarify the significance of current research.The focus is then on RAMP1,a component of the classical neuropeptide CGRP receptor,and fibroblasts,one of the most important cells involved in wound healing,to investigate the effects of RAMP1 on the function of mouse skin fibroblast cells(MSFs).Finally,we focus on the regulation of fibroblast differentiation,which is important for high quality and timely healing of wounds,and investigate the mechanisms by which RAMP1 regulates the differentiation of MSFs.MethodsIn the first part,all relevant reviews and articles published in English were extracted from the Web of Science Core Collection(WoSCC)database using a "subject term" search.The subject term are related to related to "skin" and "nerve" and "wound healing".Publication date,journal,country or region,institution,author and author keywords were visualised and analysed using Microsoft Office Excel,VOS viewer and CiteSpace.In the second part we first examined the expression levels and trends of RAMP1 during the healing process of mouse skin wounds,Then,MSFs stably transfected with Tet-on-FlagRAMP1 overexpression(OE)and Tet-on-Flag control(Ctrl)lentiviruses were constructed to examine the effects of RAMP1 on MSFs function through various experimental techniques.Finally,the potential mechanisms by which RAMP1 regulates MSFs function were explored using Tandem mass tag(TMT)-labelled quantitative proteomics combined with bioinformatic analysis.In the third part,immunohistochemical staining was used to measure the dynamic changes in RAMP1 and α-Smooth muscle actin(α-SMA)expression in mouse skin wound tissue.The MSF overexpression model described above was used for in vitro experiments.The high mobility group AT-hook 1(HMGA1)plasmid and the α-SMA plasmid were used to overexpress HMGA1 and α-SMA in RAMP1 OE MSFs,respectively.Si-α-SMA was used to silence αSMA in RAMP1 OE+HMGA1 MSFs.qPCR,Western blot and immunofluorescence staining analyses were used to determine the levels of mRNA and protein in the different cell groups.Scratch wound healing assays were used to assess the migratory capacity of the different groups of cells.CUT&RUN(cleavage under targets and release using a nuclease)assays and dual luciferase reporter gene assays were used to predict and validate the relationship between HMGA1 and the α-SMA promoter.ResultsA total of 368 papers published between 1959 and 2022 were included in the analysis.Although there was a pulsation during this period,there was an overall upwards trend in studies related to the effect of skin innervation on wound healing.The USA,particularly the University of Washington,and Gibran,Nicole S.from the University of Washington,was the most active in this field.Wound Repair and Regeneration published the most relevant literature,and The literature "Calcitonin gene-related peptide:physiology and pathophysiology" had the highest total number of citations."Diabetic foot ulcer","epidermal stem cells","mesenchymal stem cells" and "mast cells" are current and potential future research hotspots.Research into the regulation of stem cells by neural factors and interventions in diabetic foot ulcers through neuroimmune regulation will flourish.RAMP1 levels dynamically changed during mouse skin wound healing,and RAMP1 overexpression acting alone promoted proliferation,migration,and inhibited differentiation and apoptosis of MSFs.Proteomics identified 337 differentially expressed proteins(DEPs)in MSFs caused by RAMP1 overexpression,including proteins involved in the regulation of glucose and lipid metabolism in cells(e.g.lipocalin receptor 1,CD36,etc.),protein kinases(e.g.serine/threonine protein kinase PAK3,etc.).And these DEPs were enriched for a variety of biological processes,molecular functions,cellular components and signaling pathways,including transcriptional regulator activity(ARID5B,transcription factor AP-1,HMGA1,etc.),oxidoreductase activity,signaling receptor activity,cytokine binding function,plasma membrane composition,receptor complex composition and multiple signaling pathways(pathways in cancer,pathways of neurodegeneration-multiple disease,PI3K-Akt signaling pathway,metabolism of xenobiotics by cytochrome p450,chemical carcinogenesis pathways and microRNAs in cancer).RAMP1 and α-SMA protein expression was dynamically changed and negatively correlated during mouse dorsal skin wound healing.RAMP1 overexpression in vitro decreasedα-SMA expression and inhibited the migration of MSFs by inhibiting MSFs differentiation through downregulating HMGA1,which was shown for the first time to bind to the α-SMA promoter and increase the transcription of α-SMA.ConclusionThe number of studies related to the influence of cutaneous nerves on wound healing is generally on the rise,and many scholars from many research institutions in different countries are continuing to delve into this field.The regulation of "stem cells",including "mesenchymal stem cells",by neurological factors and the neuroimmune regulation of diabetic foot ulcers will become a hot topic of research.Future research on the mechanisms of neurological factors regulation of cells that play a role in wound healing needs to be intensified.RAMP1 is involved in wound healing in mouse skin,and RAMP 1 alone affects a variety of functions in MSFs,and proteomic studies combined with biomimetic analysis can reveal the relevant regulatory mechanisms and provide directions for further mechanistic experiments.RAMP1 overexpression reduced the differentiation of MSFs and promoted the migration of MSFs by down-regulating α-SMA expression through suppressing HMGA1,revealing a new mechanism for the regulation of α-SMA transcription,providing a new theoretical basis for RAMP1-mediated regulation of fibroblast function and providing insight into the precise neuromodulatory targets for skin wound repair. |
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