Mechanism Of Egg White Derived Peptide KPHAEVVLR Accelerating Wound Healing Of Oral Soft Tissue

BackgroundSoft tissue is the barrier inside the oral cavity,The oral soft tissue often suffers from injury and defect due to diseases,surgery,inflammation,trauma and other factors.There are plentiful bacteria colonized in the oral environment and the wound is easy to be infected after trauma.Due to chewing the secondary trauma is more likely to occur during the process of wound healing.The healing of oral soft tissue is also affected by some other systemic factors,such as systemic diseases（diabetes）,tumor radiotherapy and so on.These reasons may lead to greater challenges in the repair of oral soft tissue injury.If the wound does not heal for a long time,there may be systemic symptoms such as local infection and even bacteremia.Delayed healing after some surgeries,such as rotatory surgery and bone graft surgery,may affect the postoperative outcome and lead to surgical failure.At present,the methods of promoting and repairing oral soft tissue trauma include the application of biological agents such as growth factors,physical therapy and soft tissue grafts.Although these methods can achieve decent efficacy,there are still some limitations.For example,the continuous use of growth factors may lead to malignant proliferation.The application of the drug in high frequency and lasts for long time in oral cavity which may cause side effect due to swallowing.Autologous mucosal transplantation requires a donor operation area,which is more traumatic and painful.Allogeneic tissue transplantation may cause immune rejection.Synthetic graft material is easy to have poor vascularization during the healing process,which affects its normal healing.In the previous research,we have found a peptide KPHAEVVLR（KR-9）.It is an active peptide obtained by hydrolysis and purification of egg white which can accelerate skin wound healing in mice.The main function is to promote the secretion of heat shock protein 90αof cell membrane,migration and mild proliferation of human skin fibroblasts.Egg white polypeptide（KR-9）,as an active peptide that can promote soft tissue repair,has the advantages of convenient preparation,low cost and high biosafety.Its main function is to promote cell migration,which makes it more advantageous in accelerating soft tissue repair.The use of KR-9 on soft tissue repair can avoid scar and malignant proliferation.KR-9 derived from food,which makes it safe and more advantageous as an oral drug.Therefor we will explore whether KR-9 can promote the wound healing of oral tissue and its mechanism.The application of KR-9 will provide a new treatment strategy and method for solving the delayed healing of oral soft tissue and the preparation of soft tissue repair materials.Method（1）Human gingival fibroblasts（HGF）were extracted.HGF was treated with gradient concentration of KR-9,and the effect of KR-9 on cell viability was detected by CCK-8.The subsequent experimental concentration of polypeptide was confirmed according to the results.The effects of peptides on cell migration were detected by scratch assay and Transwell assay.（2）Confocal microscope was used to detect the localization of KR-9 in cells.RNA sequencing was used to detect the different gene expression between the HGF group treated with KR-9 and the control group.The possible mechanism of KR-9 was analyzed based on the sequencing results.The effect of KR-9 on the expression of PI3K/AKT/mTOR pathway protein of HGF cells was detected by Western blot.After PI3K inhibitor was applied to cells,AKT/mTOR protein expression was detected in control group,inhibitor group,and inhibitor+KR-9.The effect of peptide with inhibitor on cell migration and proliferation was examined.（3）The effect of KR-9 on viability of human umbilical vein vascular endothelial cell（HUVEC）was detected by CCK-8,and the appropriate concentration was screened.The effect of KR-9 on the expression of PI3K/AKT/mTOR pathway protein in HUVEC cells was detected by Western blot.The effect of KR-9 on HUVEC migration was detected by scratch assay and Transwell assay.The effect of KR-9 on tubule formation of HUVEC cells was detected by tubule formation in matrix glue.After cell interaction with PI3K inhibitor,HUVEC proliferation,migration and tubule formation in control group,inhibitor group and inhibitor+KR-9 group were detected.（4）The ability of KR-9 peptide to clear away ABTS⁺and DPPH free radicals was detected,and its antioxidant capacity was determined in vitro.Different concentrations of H₂O₂solution were applied to HUVEC,and CCK-8 experiment was performed to detect cell viability.CCK-8 was used to detect the cell viability of the control group,the injured group and the peptide group with different concentrations after H₂O₂treatment.DCFH-DA fluorescent probe was used to detect the effect of KR-9 on intracellular ROS content after oxidative damage of HUVEC.（5）The rat model of maxillary hard palatal mucosa defect was constructed by the punch.The experimental group was treated with 200μM KR-9 peptide solution,while the control group was treated with the same volume of saline.The rats were sacrified at 3,5,7,11 days after surgery,respectively.The distance between the wound was detected by H&E staining,and CD31 was detected by immunohistochemistry to observe the angiogenesis in the repaired tissue.Result（1）Human gingival fibroblasts were successfully extracted.KR-9 at concentrations of 50～200μM could promote the proliferation of human gingival fibroblasts.The cell viability was inhibited at concentrations of 300μM and 400μM.The highest concentration of peptides was selected as 200μM in subsequent experiments.KR-9 concentration of 50～200μM can promote the migration of human gingival fibroblasts,and the promotion effect is enhanced with the increase of concentration.（2）The peptide was able to enter HGF,and the RNA sequencing results showed that the experimental group and the control group had the most different genes in PI3K/AKT pathway.Westernblot assay results showed that KR-9 could promote the phosphorylation of PI3K,AKT and mTOR proteins,and activate the PI3K/AKT/mTOR signaling pathway.With the increase of peptide concentration,the promoting effect was strengthened.PI3K inhibitor LY294002 can reduce the phosphorylation of AKT and mTOR,and KR-9 can partially increase the reduced phosphorylation level.The effect of KR-9 on cell proliferation and migration can be inhibited by LY294002.（3）The concentration of KR-9 at 50～200μM can promote the proliferation of HUVEC,and the activity of HUVEC was inhibited at 300μM.The concentration of KR-9 at 50～200μM can promote HUVEC migration and tube formation in vitro,and the effect is gradually enhanced as the concentration increasing.KR-9 can promote the phosphorylation of PI3K/AKT/mTOR signaling pathway marker protein and activate PI3K/AKT/mTOR signaling pathway in HUVEC cells.The effect of KR-9 on cell proliferation,migration and tubularization could be inhibited by LY294002.（4）KR-9 with concentrations of 25～200μM have antioxidant activity which can promote the scavenging of ABTS⁺and DPPH free radicals in vitro.KR-9 peptide at concentrations of 25-200μM can protect HUVEC cells from H₂O₂-induced injury,and the protective effect strenghtened as the concentration increasing.Egg white peptide KR-9 can reduce ROS content produced by damaged HUVEC and alleviate oxidative stress.With the increase of concentration,the effect is gradually enhanced.（5）KR-9 can accelerate wound healing of maxillary mucosal defects of hard palatal in rats,and promote the angiogenesis of repaired tissues during the process of healing.Conclusion（1）KR-9 promotes HGF proliferation and migration by activating PI3K/AKT/mTOR signaling pathway.（2）KR-9 peptide promotes HUVEC proliferation,migration and tubule formation by activating PI3K/AKT/mTOR signaling pathway.（3）KR-9 has antioxidant effect,which can improve the cell survival rate of HUVEC injured by H₂O₂ and reduce the ROS content in injured HUVEC.（4）KR-9 accelerates wound healing of hard palatal mucosa defects in rats and promote the formation of blood vessels during the process of healing.