Study On The Effect And Mechanism Of The Novel Peptide AWRK6 In Promoting Skin Wound Healing

The skin is the outermost part of the human body and is the organ that covers the largest area of the human body.Trauma,a surgical disease that causes changes in the skin structure,is closely related to the skin structure.Wound repair mainly includes hemostasis,inflammation,proliferation,and remodeling.Continued excessive inflammation will affect the transition from wound repair to the proliferative phase.The proliferation stage is mainly through the proliferation and migration of fibroblasts,endothelial cells and keratinocytes to form granulation tissue and new blood vessels,completing tissue formation and remodeling.Trauma seriously affects the lives of patients and brings a huge burden to the social medical system.Polypeptide drugs have the characteristics of small molecular weight,low cost,and long half-life in the body,which have antibacterial and anti-inflammatory effects,and play an important role in the process of wound repair.AWRK6(SWVGKHGKKFGLKKHKKH)is a new type of cationic peptide composed of 18 amino acids with biological activity,which is a new type of antimicrobial peptide modified by base substitution and optimization from skin antimicrobial peptide of Rana chensinensis Dybowskin-2CDYa(SAVGRHGRRFGLRKHRKH)Previous studies in the laboratory have proved that AWRK6 has antibacterial effects,inhibits inflammatory cell infiltration caused by lipopolysaccharide,and promotes collagen production.In this study,male Kunming mice weighing 18-22 g were selected and divided into blank group,AWRK6 experimental group and Exendin-4 positive control group.Mice in each group underwent full-thickness skin excision on the back to construct a skin injury model with a diameter of 0.8 cm.The mice in the experimental group were instilled with 1 nmol and 10 nmol AWRK6,respectively,and the mice in the positive control group were instilled with 1 nmol Exendin-4,and the wounds were instilled(30μL)every 3 days.The wounds were taken and recorded,and the healing rate was calculated.Tissues from wounds were taken on the 3rd,6th,and 12 th days,and the tissue morphology changes during wound healing were observed by HE staining,and IL-6 and IL10 and content changes of TNF-α were evaluated by ELISA kit and immunohistochemical staining during wound healing in mice.AWRK6(0.1 μM,0.5 μM,1 μM,5 μM,and 10 μM)was used to intervene in vitro culture of human keratinocytes HaCaT and human fibroblasts HSF.Exendin-4was used as a positive control to analyze the effects of AWRK6 on the proliferation and migration capacity of HaCaT and HSF cells.The immunoblotting technique was used to explore the effect of AWRK6 on the key proteins Erk1/2,JNK and p38 MAPK and their phosphorylation expression in the MAPK signal pathway related to human keratinocyte proliferation.At the same time,RT-q PCR was used to explore the effect of AWRK6 on the RNA transcription levels of human keratinocyte cyclins CCNA,CCNB,CCND and CCNE.Analyze the mechanism of AWRK6 in promoting wound healing.The results show that AWRK6 can significantly promote the skin wound healing of mice.The AWRK6 10 nmol group has the best healing effect that the wound healing rate on the third day after trauma can reach 80.1%,which is extremely different from the NC group(52.5%).(p(27)0.001).On the 6th day after trauma,the wound healing rate of mice in the AWRK6 10 nmol group(86.5%)was significantly higher than that of mice in the NC group(69.2%)(p(27)0.001).HE staining results showed that mice in the AWRK6 10 nmol group showed tissue re-epithelialization and hair follicle regeneration earlier.The results of immunohistochemistry showed that 10 nmol AWRK6 can inhibit the secretion of IL-6 in wounded skin tissues.Cell level analysis showed that 5 μM AWRK6 can significantly promote the proliferation and migration of HaCaT cells.After culturing HaCaT cells with 5 μM AWRK6 for 24 h,the levels of JNK/p-JNK protein were significantly increased,and the RNA transcription of cyclins CCNB,CCND and CCNE were significantly up-regulated.Conclusion: AWRK6 can up-regulate the expression of p-JNK/JNK protein by activating the MAPK pathway,promote the proliferation of HaCaT cells,accelerate the re-epithelialization of wound tissue,and promote skin wound healing.AWRK6 may regulate the cell cycle by up-regulating the RNA transcription of cyclins CCNB,CCND and CCNE,promote the proliferation of HaCaT cells,and promote skin wound healing.