| Difficult to heal wound is one of the common complications of diabetes.Its treatment mainly depends on etiology treatment,debridement,antibiotics and dressings,but the treatment effect is not satisfactory.It is urgent to develop new therapeutic drugs and methods.In recent years,studies have found that polypeptides have the prospect of treating refractory wounds of diabetes.In the preliminary research,our research team discovered a new family of peptide Dybowskin-2CDYa in the skin of Northeast Forest Frog and obtained AWRK6 through modification.AWRK6 has the functions of antibacterial,regulating metabolism and promoting acute wound healing,which enlightens us to study its effect and mechanism on diabetes wound healing.This study first evaluated the effect of AWRK6 on wound healing in diabetes mice with full-thickness skin excision wound model.High fat diet and streptozotocin were used to induce type 2 diabetes in Kunming mice.A ring was placed on the back of the mice and the full-thickness skin with a diameter of 6 mm was excised to build a full-thickness skin resection wound model in diabetes mice.The experimental group,blank control group,and positive control group model mice were administered 10 nmol AWRK6,physiological saline,and 1 nmol Exendin-4 to the wound edge every 3 days,respectively.Non high-fat diet and streptozotocin induced mice were administered physiological saline as normal wound control.Take photos of the wound every 3 days and calculate the healing rate.On the 12 th day,execute the mice and take the skin tissue from the wound for hematoxylin eosin staining.The results showed that the mice fed a high-fat diet and streptozotocin induced fasting blood glucose levels were higher than11.1 mmol/L,and their wound healing rate was significantly lower than that of the normal wound control group mice.On the 9th and 12 th day of wound healing,compared with the blank control group mice,the AWRK6 treated group mice showed a significant increase in wound healing rate(p<0.05).Histological observation revealed that on the 12 th day of wound healing,the thickness of the epidermal layer in the AWRK6 group mice was closer to that of the normal group.The above research shows that AWRK6 can improve the speed and quality of skin wound healing in diabetes mice.Keratinocytes are the main components of the epidermis,and their migration,proliferation,and differentiation are important processes in wound healing,playing a crucial role in the wound healing mechanism.This study further established a high glucose induced model of human keratinocyte Ha Ca T,using CCK-8,cell scratch,and flow cytometry techniques to detect cell proliferation,migration,cell cycle distribution,and apoptosis.Real time fluorescence quantitative PCR was used to detect m RNA levels of keratinocyte differentiation related genes,and the effect of AWRK6 on keratinocyte proliferation,migration,and differentiation was analyzed.The results showed that the proliferation(p<0.001)and migration(p<0.05)of Ha Ca T cells were significantly inhibited under high glucose environment,and AWRK6 treatment could significantly improve the proliferation and migration inhibition of high glucose induced keratinocytes Ha Ca T(p<0.001).Flow cytometry analysis showed that there was no difference in the percentage of cells in G0/G1 phase,G2/M phase and S phase between the high glucose induction group and the high glucose induction+AWRK6 treatment group compared with the normal control group.AWRK6 did not affect the cell cycle of human keratinocytes.The above results indicate that AWRK6 improves the proliferation and migration of high glucose induced keratinocytes Ha Ca T independently of its regulation of cell cycle.Real time quantitative PCR results showed that compared with the control cells,high glucose induction significantly increased the m RNA level of the final differentiation protein of Ha Ca T cells(IVL)(p<0.01).AWRK6 treatment could reverse the expression of high glucose induced IVL(p<0.05),and significantly up regulate the expression of keratin 14(K14)(p<0.01).In order to further explore the cellular and molecular mechanisms of AWRK6 promoting diabetes wound healing,this study used the second generation sequencing technology to analyze the transcriptome of Ha Ca T cells in each group,and screened and verified the differentially expressed genes by real-time fluorescent quantitative PCR.At the same time,si RNA technology was used to silence the differentially expressed genes,and analyze the effects of the differentially expressed genes on cell migration,proliferation,differentiation and other functions.The results showed that AWRK6 treatment significantly reduced the upregulation of MYCL,OLFM4,and CAV1 gene m RNA levels induced by high glucose.The si RNA silencing of MYCL,OLFM4,and CAV1 genes did not affect the proliferation,cell cycle,and apoptosis of Ha Ca T cells(p>0.05),but significantly upregulated the expression of proteins related to Ha Ca T cell differentiation and keratinized cell membrane formation.Compared with the control group,MYCL gene silencing significantly increased the m RNA levels of K14,keratin 10(K10),filaggrin(FLG),and IVL genes.OLFM4 gene silencing significantly upregulated the m RNA levels of K10 and IVL genes.After CAV1 gene silencing,the m RNA levels of K14,K10,FLG,IVL,and loricin(LOR)genes significantly increased.The results indicate that MYCL OLFM4 and CAV1 genes may have a synergistic regulatory effect on the differentiation and keratinization process of Ha Ca T in keratinocytes.MYCL,OLFM4,and CAV1 genes may affect Ha Ca T cell differentiation by inhibiting the expression of K14 and K10,as well as the final differentiation related genes FLG,IVL,and LOR.The above results indicate that AWRK6 can improve the inhibition of high glucose induced proliferation and migration of Ha Ca T cells.By downregulating the expression of MYCL,OLFM4 and CAV1 genes,AWRK6 can promote the differentiation of Ha Ca T cells and the formation of keratinized capsule under high glucose environment,thereby promoting the healing of skin wounds in diabetes mice. |
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