Histatin-1 Regulates Ras-Raf-MEK-ERK Signaling Pathway To Reverse The Inhibitory Effect Of High Glucose On The Proliferation And Migration Of Keratinocytes And Fibroblasts

Objective: Diabetic wound healing is delayed,which seriously affects the quality of life of patients.The damage of skin tissue cells such as keratinocytes and fibroblasts caused by high-glucose environment is an important reason for the poor healing ability of diabetic wound.The healing ability of oral mucosal is better than that of skin tissue,the presence of salivary mucin may be an important factor causing the difference in healing ability between oral mucosa and skin tissue.As a salivary polypeptide,histatin-1 has been found to have a stimulating effect on important cells involved in the wound healing process in oral mucosa and non-oral mucosal tissues,and was initially found to be related to the regulation of ERK.Ras-Raf-MEK-ERK(MAPK/ERK pathway for short)signaling pathway is involved in the regulation of various biological behavior of cells,and is closely related to cell proliferation,migration and differentiation,cytoskeleton construction and maintenance of cell morphology.However,whether histatin-1 also plays a role and the possible molecular mechanism in wound healing of high-glucose injury has not been reported.The aim of this study is to investigate the effect of histatin-1 on skin keratinocytes and fibroblasts in the high-glucose microenvironment,and the regulatory role of Ras-Raf-MEK-ERK signaling pathway in this process.Methods:(1)Firstly,the model of keratinocytes with high-glucose injury was constructed,and mRNA-seq and bioinformatics methods were used to preliminarily obtain the overall effect of histatin-1 on the mRNA expression profile of keratinocytes with high-glucose injury,and real-time PCR was used to verify the sequencing results.At the same time,bioinformatics methods were used to predict the possible target receptors and interacting proteins of histatin-1,and determine the direction for further mechanism research.(2)The high glucose-injured model of human keratinocytes and human skin fibroblasts was established.And CCK-8 method was used to detect cell proliferation.Cell migration was detected by scratch assay.Flow cytometry was used to detect cell apoptosis level.The expression levels of 8-OHd G were detected by ELISA.Western blot was used to further detect the expression and activation levels of Ras-Raf-MEK-ERK signaling pathway proteins.Results:(1)The mRNA expression profiles of high glucose-injured keratinocytes treated with histatin-1 were different from those of high-glucose injured keratinocytes.The differences were mainly enriched in biological processes such as epidermal growth and cell cycle,and the cell components were mainly located in the cell components related to cell migration and adhesion.Signaling pathways were mainly enriched in MAPK signaling pathway,inflammatory response related pathways and so on.(2)Histatin-1 promoted the proliferation of normal keratinocytes and significantly antagonized the inhibitory effect of high glucose on the proliferation and migration of keratinocytes,and decreased the apoptosis level and the content of 8-OHd G of keratinocytes in high-glucose environment.The expressions of Ras,MEK,p-MEK,ERK,p-ERK in keratinocytes in high glucose microenvironment were up-regulated.In contrast,histatin-1 could down-regulate the levels of phosphorylated MEK,and Ras,which were elevated by high glucose.(3)Histatin-1 significantly attenuated the inhibitory effect of high glucose on the proliferation of fibroblasts and decreased the apoptosis level and the content of 8-OHd G of fibroblasts in high glucose microenvironment.The protein molecules of Ras-Raf-MEK-ERK signaling pathway could be abnormally up-regulated or activated in fibroblasts under high glucose microenvironment,including Ras,total MEK,phosphorylated MEK,total ERK and phosphorylated ERK.In contrast,histatin-1 down-regulated the expression levels of phosphorylated MEK,total ERK and Ras in high-glucose injured fibroblasts.Conclusion: Histatin-1 could promote the proliferation and migration of keratinocytes and fibroblasts and reduce the cell apoptosis and the level of cell oxidative damage by regulating the overexpression and activation of Ras-Raf-MEK-ERK signaling pathway molecules in high-glucose microenvironment,so as to alleviate the inhibitory effect of high glucose on keratinocytes and fibroblasts in wound healing.