Mechanism Of MEBO Activated Macrophages Activating EGF Signal Promoting Diabetic Wound Healing

Part Ⅰ: Study on the polarization types of macrophages and key signaling pathways affecting wound healing during chronic wound healing in miceObjective:Single cell transcriptome sequencing(sc RNA seq)was used to explore the relationship between the polarization type of macrophages(Mac)and fibroblasts(Fib),epithelial cells(EC),as well as the main signal pathways affecting wound healing.Methods:Exploring the different manifestations of cell subpopulation communication intensity,quantity,and interaction between wound healing(Wound)and unhealed wound(Un＿Wound)tissues in mice using sc RNA seq method;Clarify the specific signaling pathways and their ligand receptors in two types of wound tissues,and conduct comparative analysis;And explore the relationship between mouse Wound and Un＿Monocyte subgroup types and several key subgroup types in the WOUND organization.Results:(1)The single cell quality control Counts values of mouse type 2 wounds(Wound,Un＿Wound)showed no abnormalities,and there was a good overall correlation between cells.The cell subpopulations in Wound tissue are mainly composed of skin tissue stem cells(Tissues SC),EC,chondrocytes(Chondrocytes),B cells(B cells),monocytes(Monocytes),FIB,endothelial cells(Endo),keratinocytes(KC),and T cells(T cells);Un＿The relevant cell subpopulations in WOUND tissue are mainly skin tissue SC,T cells,corrugated tube cells(CMP),Monocyte,Neurons,Endo,EC,KC,Fibroclasts,Chondrocytes,and B cells;In the Wound tissue,there is a close association between Monocyte and the subpopulations of EC,FIB,and Tissue sc cells;At Un＿In terms of the communication strength and number of Monocyte with EC and FIB cell subpopulations within the Wound tissue,compared to the Wound tissue,they are relatively low;(2)For the two cell subpopulations of chronic wound tissue in mice,FIB and Epihelial,both are affected by the activation effect of the CXCL1-Akr1 signaling axis of the EGF signaling pathway contained in Monocyte.The interaction between different ligands receptors in the CXCL signaling pathway is the highest for the entire signaling pathway,with CXCL16-Akr6 and CXCL1-Akr1 ligand receptors having the highest value.The analysis of the regulatory function of a single ligand receptor in different cell communication shows that the signal correlation between Cxcl3-Ark1 ligand receptor and vascular endothelial cells(VEC)in Mac is more significant and crucial;(3)The Monocyte subgroup is composed of five categories: 0,1,2,3,4,and 5.The first two categories occupy the main position in the Wound tissue,and the CD68 and CD162 specific expression subgroups have a significant advantage.At the same time,the number of Monocytes labeled with CD163 is higher than that labeled with CD68.The Monocyte subgroup in the Wound tissue is M1 type Mac,M2 type Mac,and the latter occupies the main position.Conclusion:(1)There is a close relationship between Wound and the functions of EC,Monocyte,and FIB cell subpopulations contained in the tissue;The intensity and quantity of interactions between Monocyte cell subpopulations and EC and FIB cell subpopulations in the Wound tissue are compared to Un＿The WOUND organization is significantly higher;(2)The activation function of CXCL1-Akr1 signal axis in the EGF signal pathway of M2 type Mac and the molecular mechanism of enhancing angiogenesis play a role in the regulation of EC and Fib by Mac subgroups;(3)The subtypes of Monocyte cells are mainly M1 type Mac and M2 type Mac,which play a major role in the Wound tissue.Part Ⅱ: The Mechanism of CXCL1-Ackr1 Signal Axis in MEBO MEBO Activating M2 Macrophage Activating EGF Signal Pathway to Promote diabetes HealingObjective: Analysis of MEBO Moist Burn Ointment on Un in Mice＿The activation efficiency of the CXCL1-Akr1 signaling axis in the M2 type Mac signaling pathway EGF within the sound,as well as the molecular promotion mechanism for wound epithelialization and fibrosis.Methods: The experimental subjects were 50 C57BL/6Jnifdc mice,which were randomly assigned to the Acute group(acute wound group),Model group(model group),MEBO group(Meibao group),GF group(inhibition group),and bixin group(Befuxin group);In addition to the Acute group,the other four groups of experimental animals were given intraperitoneal injection of STZ for 5 days at a dose of 50 mg/kg.After 8 weeks of continuous feeding,their tail venous blood was collected to confirm the completion of model construction.A defect wound model was constructed in the full thickness skin area of the experimental animal’s back.The Model group,MEBO group,and bixin group all added0.02 ml of physiological saline to the wound surface,while the GF group of experimental animals added 0.02 ml of CXCL1-Ackr1 signaling pathway inhibitor to the wound surface,while ensuring full drug absorption.Animal wound healing tissues were taken at 3 days,1week,and 2 weeks after the preparation of the DFU model.Clarify the wound healing rate of each group of experimental animals;Observe the wound healing status of 5 groups at 3 days,1 week,and 2 weeks through a low-power microscope;The sorting of Macs was performed using flow cytometry,and the expression levels of CD163/CXCL1 Ackr1 in Macs were analyzed using immunofluorescence(IF)staining;The expression levels of CXCL1 Ackr1 and CD163 in the wounds of 5 groups of mice at 3 days,1 week,and 2 weeks were analyzed using Western blotting(WB).Results:(1)As time increased,the wound tissues of all 5 experimental animals showed a slow healing performance,and the healing status of the Acte group showed an advantage compared to the DFU model group;The application of CXCL1 Ackr1 signaling pathway inhibitors can reduce the wound healing speed of DFU mice,while the application of MEBO and Befuxin can improve the wound healing speed of DFU mice,with the MBEO group having the best therapeutic effect.In terms of M1(CD68+)fluorescence intensity in wound tissue,DFU mice showed a higher fluorescence intensity compared to the Acte group,while in terms of M2(CD163+)fluorescence intensity,they showed a lower fluorescence intensity compared to the Acte group;The application of CXCL1 Ackr1 signaling pathway inhibitors can once again increase the fluorescence intensity of M1(CD68+)in the wound tissue of DFU mice,while reducing the fluorescence intensity of M2(CD163+);The application of MEBO and Beifuxin can increase the fluorescence intensity of M1(CD68+)and reduce the fluorescence intensity of M2(CD163+);In M1(CD68+)fluorescence intensity,the MEBO group had the lowest,while in M2(CD163+)fluorescence intensity,the group had the highest,showing significant differences(P<0.05).(2)In terms of fluorescence intensity of CD163 and CD163/CXCL1 Ackr1 in wound tissue,DFU mice showed lower fluorescence intensity compared to the Acute group;By intervening,the fluorescence intensity of both is reduced;The application of MEBO and Beifuxin can both enhance the intensity of CD163/CXCL1-Ackr1 and CD163 in the wound tissue of DFU mice.In terms of fluorescence intensity,the highest was observed in the MEBO group,showing a significant difference(P<0.05);Inflammatory mediator IFN in wound tissue-γ、 IL-1、TNF-α、 The levels of IL-6 and apoptosis index(AI)in the model group were higher than those in the Acute group;The application of inhibitors can induce IFN in DFU mice-γ、 IL-1、TNF-α、 Improved levels of IL-6 and AI;Both MEBO and Beifuxin can enable IFN-γ、 DFUIL-1、TNF-α、Downregulation of IL-6 and AI levels,especially in the MEBO group of experimental animals,in IFN-γ、 IL-1、TNF-α、 The decrease in IL-6 and AI levels was the largest,showing a significant difference(P<0.05);In the expression of PCNA,COL1A1,CK5,COL11A1 in the wound tissue,the model group was lower than the Acute group;The application of CXCL1 Ackr1 signaling pathway inhibitors can lower the expression levels of these four factors;The application of MEBO and Beifuxin can both increase the expression level of these four factors,especially in the MEBO group,showing significant differences(P<0.05).Conclusion: For mouse wounds,a high glucose environment can lead to delayed healing;(2)The M1/M2 disorder in the wound tissue of DFU mice can be reversed by using MEBO,increasing the proportion of M2 type Mac,and inhibiting inflammatory activity and cell apoptosis in the wound tissue;(3)MEBO activates the activation signal pathway of EGF CXCL-Akr1 signal axis through activating M2 type Mac pathway,which has a positive impact on angiogenesis,epithelization and fibrosis of wound tissue,thus facilitating wound healing as soon as possible.