Effects Of Royal Jelly On The In Vitro Wound-healing Model

Royal jelly possesses a wide range of biological activities and medical values,and has been used for the treatment of diseases in many areas since ancient times.Studies have reported that royal jelly has the function of wound repairing;however,the mechanisms of action underlaying such activity is still unclear.In this study,royal jelly derived from different nectariferous plants were collected to study the effects on the in vitro wound healing model at both cytological and molecular levels to explore the mechanisms of promoting wound healing,and to compare the functional and molecular differences among different types of royal jelly.It will facilitate the isolation of molecules with potential wound-healing activities from royal jelly,and provide clues for the development of royal jelly in the respect of wound repair.Here,different categories of royal jelly were screened with MTT cell viability assay,in which royal jelly collected during Chinese chestnut flowering could significantly promote the proliferation of keratinocytes compared with that collected from rapeseed flowering.The water-soluble extracts and lipid-solubility extracts(RJLE)demonstrated the similar activities to the fresh royal jelly that extracts of royal jelly collected from rapeseed flowering did not display any proliferative activities.Based on the results of MTT cell viability assay,we investigated the mechanisms of RJ-ZJ-BL1(collected in Zhejiang Province during Chinese chestnut flowering)and RJ-ZJ-YC1(collected in Zhejiang Province during rapeseed flowering)in the wound healing process.1.The migration ability of keratinocytes was evaluated using in vitro scratched wound healing model and Transwell assay,in which RJ-ZJ-BL1 significantly promoted cell migration at the concentration of 31.25 μg/m L and 62.25 μg/m L,while RJ-ZJ-YC1 did not demonstrated any activity at testing concentrations.Furthermore,the inhibitors of ERK signaling pathway(PD98059,10μM)and p38-MAPK signaling pathway(SB203580,15 μM)dramatically inhibited the cell migration induced by RJ-ZJ-BL1.2.When exploring the effects of RJ-ZJ-BL1 and RJ-ZJ-YC1 on AQPs,it was found that these two types of royal jelly exhibited significantly distinct impacts on the APQ1 m RNA expression.They showed dramatically different effects on the AQP5 m RNA expression at 31.25μg/m L;RJ-ZJ-BL1 remarkably facilitated the expression of AQP3 m RNA at 7.8125～62.5 μg/m L,which inhibited the expression of AQP3 protein at the concentration of 62.5 μg/m L and 125 μg/m L.3.The LPS-induced inflammatory RAW264.7 macrophages were employed to explore the function of royal jelly and its extracts at the inflammatory stage of the wound healing process.Both kinds of royal jelly and their extracts could inhibit the generation of NO with a dose-dependent manner.Water-soluble extracts of both royal jelly(WRJE1 and WRJE2)significantly promoted the secretion of TGF-β1 at 125～2000 μg/m L,while lipid-solubility extracts inhibited the production of TGF-β1 remarkably.Besides,water-soluble extracts of royal jelly derived from rapeseed flowering(WRJE2)inhibited the generation of TNF-α.It is speculated that royal jelly and its extracts may exert the wound healing activity by antiinflammation via inhibiting the production of NO or other inflammatory factors at inflammatory stage.4.Label-free protein quantification and untargeted metabolomics were employed to analyze the proteins and small molecules present in RJZJ-BL1 and RJ-ZJ-CY1.A total of 63 proteins and 957 small molecules were detected in RJ-ZJ-BL1,and 71 proteins and 917 small molecules were found in RJ-ZJ-YC1,among which eight proteins were special.To sum up,this study suggested royal jelly collected during Chinese chestnut flowering might promote wound healing by facilitating cell proliferation and migration via mediating AQP3.On the other hand,royal jelly collected during rapeseed flowering mainly processed antimicrobial activity.It will provide theoretical foundation for the development of specific royal jelly.