| Objective:The delayed healing of cutaneous wounds after trauma seriously affects the quality of life of patients.To establish the intervention criteria for different healing ratess of cutaneous wounds,the molecular mechanism of delayed healing of cutaneous wounds was investigated.We investigated the role of macrophage differentiation in the nonunion matrix remodeling of chronic cutaneous wounds.At the same time,through Next-generation sequencing and bioinformatics analysis,we further found that M2-type macrophages showed less infiltration in the cutaneous wound tissues with delayed healing,while AKT3(AKT Serine/Threonine Kinase 3)was also significantly down-regulated in the wound tissues with delayed healing.Therefore,we further investigated the role of AKT3 in macrophage differentiation and the remodeling of nonhealing matrix in chronic cutaneous wounds.Subsequently,we constructed a mouse model of AKT3 gene knockout and explored the changes in cutaneous wound healing and the effects on M2-type macrophage infiltration and differentiation.Methods:1.Cutaneous tissues of the lower leg of patients with delayed healing of wound cutaneous and normal healing rates were taken from the cutaneous tissues.After paraffin embedding and fixation,HE,Masson and EVG staining were carried out to observe the differences in morphology,collagen fiber and elastic fiber content of the tissues with delayed healing of wound cutaneous and normal healing rates.We used immunohistochemical method to observe the expression differences of the epithelial molecular indicator CK5,and the epithelial proliferation rates indicator PCNA,in the delayed healing and normal healing tissues of cutaneous wounds,and Tunel method was used to detect the changes in the apoptosis rates of the delayed healing and normal healing tissues of cutaneous wounds.2.RNA was extracted from cutaneous wound delayed healing and normal healing tissues by trizol method,followed by transcriptome sequencing and clustering analysis.GO(Gene Ontology)and KEGG functional analysis were conducted to find the functional clustering and signaling pathways that play a key role in cutaneous wound delayed healing and normal healing tissues.The key functions and signal pathways obtained by GO and KEGG clustering analysis were further analyzed by Venn method,and the heat maps were drawn and enriched to the key molecules.Further GSEA(Gene Set Enrichment Analysis)was conducted to look for Enrichment molecules related to key functions and signaling pathways obtained by GO and KEGG Analysis.3.Expression changes of AKT3,COL1A1 and COL11A1 in delayed and normal healing tissues of cutaneous wounds were detected by immunohistochemistry and western blot.We used western blotting to analyse the different expression of phosphorylated AKT3 in delayed and normal healing tissues of cutaneous wounds.4.GSEA analysis was performed on macrophage function and PI3K-AKT signaling pathway,and heat map was drawn to screen 10 molecules enriched significantly;We used tissue immunofluorescence method to lable CD68/CD206 positive macrophages and AKT3 to analyze the relationship between AKT3 expression location and M2-type macrophages.Flow cytometry was used to separate M2-type macrophages with double positive CD68/CD206 cells,and real-time quantitative PCR(q RT-PCR)was used to detect the expression changes of AKT3 in M2-type macrophages with double positive CD68/CD206 cells.The expression of AKT3 and phosphorylated AKT3 in M2-type macrophages with double positive CD68/CD206 were detected by western blot.The expression location and quantitative relationship between COL1A1,COL11A1 and CD68-positive macrophages were detected by tissue immunofluorescence labeling.5.Thp-1 was induced into M2-type macrophages,and the induction efficiency was detected by q RT-PCR.After thp-1 induction,AKT3 was knocked down in M2-type macrophages and the knockout efficiency was detected by western blot.A co-culture system of human dermal fibroblasts(HSF)and M2-type macrophages(THP-1induction and tissue sorting)was established in vitro,which was divided into four blank control groups(HSF),the THP-1+HSF co-culture group,the M2-type macrophages+HSF co-culture group,and the M2-type macrophages subtracting AKT3+HSF co-culture group.CCK-8 and Ed U detected HSF cells proliferation in all four groups.Migration rates experiment was used to detect the migration rates of HSF cells in four groups.Meanwhile,we used western blot to analyse the expression levels of COL1A1 and COL11A1 proteins in four groups of HSF cells.6.Mice with systemic AKT3 knockout were constructed and the knockout efficiency was detected by western blot.Cutaneous perforation was used to simulate the cutaneous wound in the back of mice with AKT3 systemic knockout,and the difference in the healing rates of cutaneous wound in the 0,7,14 day control group and the AKT3 systemic knockout group was observed.Histological staining HE,Masson and EVG were used to observe the differences of cutaneous wound tissues in morphology,collagen and elastin expression in the control group and AKT3 group.Immunofluorescence staining was used to observe the difference in the number of M2-type macrophages infiltration in the cutaneous wound f4/80 and CD206 mice in the control group and the AKT3 whole body knockout group.Western blotting was used to detect the expression changes of COL1A1 and COL11A1 proteins in cutaneous wounds of mice in the control group and AKT3 systemic knockout group,while immunohistochemistry was used to detect the expression differences of COL1A1 and COL11A1 in cutaneous wounds of mice in the control group and AKT3 systemic knockout group.We also used QRT-PCR to detect the differences of expression of TGF-expression and il-10 at 7 and 14 days in the cutaneous wounds of mice in the control group and the AKT3 systemic knockout group.7.Flow cytometry was used to separates M2-type macrophages from the cutaneous wound tissues of the control group and the AKT3 systemic knockout group,and the expression level of AKT3 in the M2-type macrophages was detected.The co-culture system of M2-type macrophages and JB6 cells in mice was established,and the proliferation capacity of JB6 cells was detected by CCK8 and Ed U methods.Changes in migration capacity of JB6 cells after co-culture were detected.Western blotting was used to detect COL1A1 and COL11A1 protein expression in JB6 cells after co-culture.Results:1.HE staining results showed that the cutaneous tissue structure with delayed cutaneous healing was looser and contained more inflammatory cells than that with normal healing rates;The results of Masson staining showed that the collagen fibers and elastic fibers of cutaneous tissues with delayed healing rate were decreased compared with those with normal healing rate.EVG staining also showed that cutaneous tissues with delayed healing rates were decreased when cutaneous tissues with normal healing rates were compared with elastic fibers of cutaneous tissues with normal healing rates.Immunohistochemical results showed that the expression of CK5 and PCNA in cutaneous tissues with delayed healing rates was significantly down-regulated compared with that in cutaneous tissues with normal healing rates,and Tunel results suggested that the apoptosis rates of cutaneous tissues with delayed healing rates was significantly increased compared with that with normal healing rates.2.delayed cutaneous wound healing and the normal healing tissue transcriptome study sequencing results suggest cut genes and 1570 raised a total of 1792,GO,delayed KEGG function analysis of cutaneous wound healing and the normal healing tissue contains 20 major function clustering and signaling pathways,including the ECM remodeling,ECM contact,cell adhesion,etc.Venn analysis of PI3K-AKT signaling pathway,ECM receptor contact and cell adhesion to three KEGG was performed to obtain 35.Venn analysis was performed on six GO enrichment functions including cell adhesion,cell migration,extracellular matrix remodeling,collagen fiber remodeling,and multicellular functions,and the results showed that AKT3,COL1A1,and COL11A1 were down-regulated in delayed healing tissues of cutaneous wounds.Further GSEA analysis of cell adhesion molecules,collagen metabolism,adhesion and extracellular structural remodeling found that cutaneous wound surface significantly down-regulated in delayed healing tissues.The results of immunohistochemistry and western blot indicated that the expressions of AKT3,COL1A1 and COL11A1 were down-regulated in the tissues with delayed healing of cutaneous wounds.The expression of phosphorylated AKT3 was down-regulated in delayed healing tissues of cutaneous wounds by western blotting.3.GSEA analysis was performed on macrophage function and PI3K-AKT signaling pathway,and heat map was drawn to screen 10 molecules enriched significantly;Tissue immunofluorescence assay indicated that CD68/CD206 positive macrophages and AKT3 co-located in the cutaneous wound tissue,and the number of CD68/CD206/AKT3 positive cells significantly decreased.The M2-type macrophages with double positive CD68/CD206 were sorted by flow cytometry,and real-time quantitative PCR(q RT-PCR)showed that the expression of AKT3 in M2-type macrophages with double positive CD68/CD206 was down-regulated significantly.The down-regulated expression of AKT3 and phosphorylated AKT3 in M2-type macrophages with double positive CD68/CD206 were detected by western blot.The expression levels of COL1A1,COL11A1 and CD68 were down-regulated in the tissues with tissue immunofluorescence labeling.5.The expression levels of M2-type macrophage markers CD206 and CD163 were up-regulated by q RT-PCR significantly.AKT3 was knocked down in M2-type macrophages after thp-1 induction,and the expression of AKT3 was significantly down-regulated by western blot detection.After AKT3 was knocked down,the M2 polarization was affected then.A co-culture system of human dermal fibroblasts(HSF)and M2-type macrophages(thp-1 induction and tissue sorting)was established in vitro,which was divided into four blank control groups(HSF),the thp-1 +HSF co-culture group,the M2-type macrophages +HSF co-culture group,and the M2-type macrophages subtracting AKT3+HSF co-culture group.Cck-8 and Ed U detected the proliferation capacity of HSF cells in the four groups and found that the M2-type macrophages +HSF co-culture group had the strongest proliferation capacity.After AKT3 was knocked down,the proliferation capacity of HSF was weakened.The migration rates test showed that HSF cells in the M2-type macrophages +HSF co-culture group had the fastest migration rates,and the migration ability was weakened after AKT3 was knocked down.Western blot was used to detect the expression levels of COL1A1 and COL11A1 proteins in all four groups of HSF cells,and the results showed that the levels of COL1A1 and COL11A1 proteins in the M2-type macrophages +HSF co-culture group were up-regulated significantly.After AKT3 was deleted,the protein expressions were decreased.6.The expression of AKT3 in cutaneous tissues of AKT3 knockout mice was significantly down-regulated by western blot.The cutaneous wound healing rates of mice with systemic AKT3 knockout slowed down compared with the control group at7 and 14 days.HE staining results showed that the cutaneous wound tissue structure of mice with AKT3 systemic knockout was looser and contained more inflammatory cells than that of control mice.The Masson staining result indicated that compared with the control group,the collagen fibers and elastic fibers of the cutaneous wound tissues of AKT3 were reduced in the mice with AKT3 body knockout.Meanwhile,EVG staining indicated that the elastic fibers of the cutaneous wound tissues of AKT3 body knockout in the control group were reduced.The results of immunofluorescence staining demonstrated that the number of M2-type macrophages infiltration in the cutaneous wound f4/80 and CD206 mice in the AKT3 group was decreased.Western blotting demonstrated that the COL1A1 and COL11A1 expression levels were down-regulated in cutaneous wounds of mice in the AKT3 systemic knockout group.Meanwhile immunohistochemistry showed that the COL1A1 and COL11A1 expression levels were down-regulated in cutaneous wounds of AKT3 systemic knockout mice.The differences in expression levels of TGF-mine and il-10 in cutaneous wounds of mice in the control group and the AKT3 systemic knockout group by QRT-PCR at 7 and 14 days,and the TGF-mine and il-10 expression levels in cutaneous wounds of mice in the AKT3 knockout group were detected.7.Flow cytometry was used to separates M2-type macrophages from the cutaneous wound tissue of the control group and the AKT3 systemic knockout group.The AKT3 expression was down-regulated in M2-type macrophages from the cutaneous wound separation of AKT3 knockout mice by Western blotting significantly.We established the co-culture system of M2-type macrophages and JB6 cells in mice.CCK8 and Ed U methods indicated that the proliferation capacity of JB6 cells was decreased after we selected the co-culture of M2-type macrophages from the cutaneous wound of AKT3 knockout mice compared with M2-type macrophages in the control group significantly.The results of the co-culture of JB6 cells and M2-type macrophages selected from the cutaneous wound of mice was removed by AKT3 showed that the migration ability of JB6 cells was decreased.Western blotting was used to detect the down-regulation of COL1A1 and COL11A1 proteins in JB6 cells after co-culture of M2-type macrophages selected from the cutaneous wound of AKT3 knockout mice Conclusions:1.Delayed healing of skin wounds is mainly caused by tissue remodeling obstacles;The delayed healing of skin wounds was caused by loose tissue structure,collagen fiber and elastic fiber formation disorder,and the increased apoptosis rate of residual cells in tissues.2.The main matrix leading to the abnormal skin wound matrix remodeling stage is the decreased infiltration of M2-type macrophages and the down-regulation of COL1A1 and COL11A1 expressions.3.AKT3 deletion in M2-type macrophages is the main mechanism leading to decreased infiltration and abnormal polarization of M2-type macrophages,while AKT3 deletion in M2-type macrophages also leads to decreased COL1A1 and COL11A1 expression and exocrine in wound fibroblasts. |
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