Mechanism Of MEBO Regulated CCL2-ACKR1 Signaling Pathway To Promote Diabetic Wound Healing

Objective:(1)By single-cell sequencing(single-cell RNA-sequencing,The sc RNA-seq)technique,we analyzed the relationship of CC class chemokine ligand 2(c-c motif ligand 2,CCL2)and the atypical chemokine receptor 1(atypical chemokine receptors 1,ACKR 1)with tissue stem cells and endothelial cells in Healing DFU and DFU and explored the mechanisms affecting wound healing;(2)To investigate the activation of CCL 2-ACKR 1 signaling axis in tissue cells and the molecular mechanism of promoting wound healing by affecting the activity of endothelial cells.Methods:(1)The results of gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis of tissue stem cells and endothelial cells in Healing DFU and DFU wound tissues were resolved by single-cell sequencing;the number of cell subpopulation communication,intensity and variability of interactions;further determination of two wound tissue-specific signaling pathways and their ligand-receptor comparative analysis;(2)We divided 48 C57 BL / 6Jnifdc mice as subjects and randomly divided them into four groups: acute wound group(Acute group,n=12),model group(Model group,n=12),befuxin group(GF group,n=12),and wet burn cream group(MEBO group,n=12).Except for the acute wound group,the other three groups were injected with streptozotocin(50mg / kg)for 5 consecutive days,and tail venous blood was collected for 8 weeks to determine the success of the model.After the successful establishment of the diabetes model,the defect wound model was prepared at the full thickness skin of the back of mice,physiological saline(20μL)was added in the Acute group and Model group mice,beifuxin(20μL)in the GF group,and wet burn cream(20μL)in the MEBO group until the drug was fully absorbed.Mice wound healing tissues were collected 3d,7d and 14 d after the DFU model establishment,and the specimens included wound surface,new granulation and some normal skin tissue.Put a part of the tissue cut at each point into the frozen storage tube and placed in liquid nitrogen,and transferred to the-80℃ refrigerator in time,the other part of the frozen embedding agent treatment,and stored in the-20℃refrigerator.We determined the wound healing rate of each group of mice and observed the wound healing of each group of mice at 3d,7d and 14d;immunofluorescent chemical staining of CCL2 and ACKR1 after 14d;expression levels of CCL2 and ACKR1 in 3d,7d and 14 d by western blotting(Western blot,WB).Results:(1)Diabetic healing-related differentially expressed genes(DEGs)analysis showed that there were 1,948 differential genes between tissue stem cells in healing and non-healing wounds,of which 1,198 genes were up-regulated and 685 genes were down-regulated.The results of GO functional enrichment analysis in tissue stem cells showed that they were closely related to wound healing.The CCL2-ACKR1 signaling pathway activity in tissue stem cells influenced the biological activity of endothelial cell subpopulation,which ultimately promoted the healing of DFU wounds;(2)With the extension of time,the traumatic tissues of each group of mice showed a trend of gradual healing,and the healing of mice in the acute wound group was better than that in the DFU model group;the intervention of beifuxin and MEBO moist burn cream could accelerate the healing of traumatic tissues in DFU mice,and the MBEO group showed the best healing effect;(3)The fluorescence intensity of CCL2 and ACKR1 in the wound tissues of DFU mice was lower than that of the acute trauma group;the intervention of both Befuzi and MEBO moist burn cream could upregulate the intensity of CCL2 and ACKR1 in the wound tissues of DFU mice,and the fluorescence intensity of CCL2 and ACKR1 in the wound tissues of the MEBO group was the highest,and the difference was statistically significant(P<0.05);(4)The Acute group showed a continuous increase in CCL2 and ACKR1 protein expression in their wound tissues over time,and the expression was significantly higher than that of the Model group.The intervention of beifuxin and MEBO could both upregulate the intensity of CCL2 and ACKR1 in the wound tissues of DFU mice,and the difference was statistically significant.Conclusion:Conclusion:(1)CCL2 and ACKR1 expression was dysregulated in wound tissues of DFU mice,and MEBO intervention could reverse the dysregulation of CCL2 and ACKR1 in wound tissues of DFU group mice;(2)MEBO activates the CCL2-ACKR1 signaling axis through activation of tissue stem cells and promotes the generation of vascular endothelial cells in wound tissue,which ultimately promotes wound healing.