Study Of Effects And Mechanism On Skin Wound Healing In Mice Regulated By Hst 1

Objective:Acute wound is one of the common diseases in clinical departments,especially in surgical departments.The delayed healing of acute wound will eventually turn into chronic wound,which will seriously affect the health and quality of life of patients.The process of wound healing is affected by many factors.At present,Although the research on the treatment of acute wound has achieved some results,the existing treatment methods still have their own defects.Hst 1,which is a small molecular polypeptide extracted from saliva,has been proved to accelerate the healing of acute wounds in animals.However,the role of HST 1 in wound healing and its specific mechanism are still lack of systematic research.Previous views believed that the promoting effect of Hst 1 on wound healing is mainly realized through its promoting migration of epithelial cells and promoting the formation of neovascularization.Epidermal cells and vascular endothelial cells are the main research objects,but there is still a lack of relevant research on whether Hst 1 regulates other important cells involved in the process of wound healing.The purpose of this study is to further explore the regulatory mechanism and role of Hst 1 on fibroblasts and macrophages in acute wound healing through experiments in vivo and in vitro.And construct the release vector of Hst 1 to make Hst 1 more effectively promote wound healing and improve the skin quality after wound healing and provide new ideas and directions for future research,and provide a theoretical basis for the transformation of Hst 1 in clinical application.Methods:1)The acute wound model was established in C57 BL / 6 mice,and then the wound was treated with Hst 1 solution.The skin was taken on the 3rd,5th and 7th day after wound operation and 7 days after wound healing.The wound healing rate after different treatment methods was evaluated by photos and hematoxylin and eosin staining;the secretion of various proinflammatory and anti-inflammatory factors during wound healing in different groups of mice was studied by immunohistochemical staining;the distribution of cells at different periods of wound healing was studied by immunofluorescence staining;2)Masson staining,Sirius red staining and Victoria blue staining were used to study the fiber deposition in the wounds of different groups of mice;the wound skin was taken 7 days after healing and the skin quality of different groups of mice after wound healing was compared by tensile fracture test and stress relaxation test.3)Using different cells as target cells in vitro,Hst 1 was added to the culture medium to study the regulatory effect of Hst 1 on different cells involved in the process of wound healing in vitro.Using fibroblasts as target cells,the effect of Hst 1 on fibroblast activity was detected by CCK8 experiment;the effect of Hst 1 on fibroblast proliferation and apoptosis was detected by flow cytometry;the migration ability of different groups of cells was detected by scratch test;the markers of myofibroblasts were detected by immunofluorescence staining,Western blotting and real-time fluorescence quantitative PCR,and the transformation function of fibroblasts into myofibroblasts was compared between different groups;the collagen secretion function of fibroblasts with different composition was detected by immunoblotting and real-time fluorescence quantitative PCR;the contractile function of fibroblasts was detected by collagen contraction test;the activation function of Hst 1 on m TOR signal pathway was detected by Western blot.4)The acellular dermal matrix was used as the carrier to load Hst 1,and the acute fullthickness wound skin model was constructed in mice.The wound healing rate,the number of local angiogenesis and collagen fiber deposition were measured.5)The hydrogel sustained release system was constructed by using methacrylamide acrylamide fibroin protein and methacrylamide as hyaluronic acid.The modification of hydrogel was verified by nuclear magnetic resonance spectrum and infrared spectrum,and the pore size,mechanical strength and rheological properties of the gel were detected.The results showed that it could meet the basic needs of wound dressing and sustained-release carrier.6)Macrophages were used as target cells,and the release of Hst 1 in hydrogels was used to study the regulation of macrophage function by the highest and lowest concentration of Hst1 in vitro.The migration ability of macrophages in different groups was compared by Transwell experiment;the transformation function of macrophages from M0 to M2 was detected by flow cytometry;The secretion of M2 macrophage related anti-inflammatory factors was detected by real-time fluorescence quantitative PCR.Results:1)In vivo experiments showed that after Hst 1 treated acute wounds in mice,the wound healing speed was significantly accelerated,and myofibroblasts and collagen deposition increased during the healing process.Hst 1 can reduce the number of macrophages and inhibit the secretion of related inflammatory factors during wound healing in vivo;2)7 days after healing,the mechanical properties of the wound skin of mice treated with Hst 1 were significantly better than those of the negative control group,and were closer to those of the back skin of normal mice;3)In vitro experiments showed that Hst 1 could promote the migration ability of fibroblasts,but had no effect on their proliferation,apoptosis and cell activity.In addition,Hst 1 can promote the transformation of fibroblasts into myofibroblasts and enhance their collagen secretion and contraction function;Hst 1 could activate key signaling molecules in m TOR signaling pathway;4)The combined application of acellular dermal matrix and Hst 1 could not improve the wound treatment effect.The healing speed,neovascularization and collagen deposition of mice treated with Hst 1 alone were higher than those of mice treated with acellular dermal matrix alone;5)The hydrogel prepared by methacrylamide acrylamide and hyaluronic acid can be crosslinked and the mechanical properties of the hydrogel can be used to treat acute wounds of the skin;6)The drug release experiment showed that the hydrogel had good sustained release effect on Hst 1.The lowest and highest concentrations of Hst 1 according to the Hst 1 release curve both could promote the migration of macrophages and the transformation of macrophages from M0 to M2,and the high concentration of Hst 1 could promote the secretion of vascular endothelial growth factor by macrophages.Conclusion: Hst 1 can not only accelerate the healing speed of acute wound,but also improve the mechanical properties of skin after wound healing.This phenomenon is mainly realized through a series of regulation of fibroblasts and macrophages by Hst 1 activating m TOR signaling pathway.We can continue in-depth research in this direction,clarify the regulation of Hst 1 on various procedures of wound healing,and promote the transformation and application of Hst 1 in clinic.