| Research BackgroundWound healing is a very hot spot in biomedical research fields. A lot of studies have been done on wound healing from all aspects, such as, inflammatory cell, histocyte,cytokine, growth factor and extracellular matrix, which enables us to have a further understanding on the wound healing process in clinic.People are more and more concerned about the role of macrophages on wound healing.Recent studies show that macrophages can play an efficient role on each wound healing stage, such as inflammatory, proliferation and tissue remodeling. No macrophages no wound healing. Macrophages deficiency or function defect may have obvious effects on the process of wound healing.As an anti-inflammatory drug and immunosuppressor, glucocorticoid is frequently practiced in clinic. However, irregular or high dose medication for a long time may lead to a series of complication and adverse reaction, even life-threatening. The animal experiment and clinical study have confirmed that delayed wound healing is one of the adverse reactions about GC. Although many researches have been done, the detailed mechanisms are still unknow so far.Based on the above researches, we use dexamethasone, a typical GC, to explore the regulating effects of macrophage- GC signals on wound healing, so as to recognize the underlying molecular mechanism.Part Ⅰ Corticosteroid delayed mouse skin wound healing by reducing the number of macrophages in the wound siteObjective: 1.To confirm that GC can delay wound healing;2. To confirm that GC can reduce the number of macrophages in the wound site;3. Research the effect of GC on wound local tissue and local macrophages’ GR level.Methods: After the completion of model of mice skin wound healing, the experimental group and the control group were treated with Dex vs. PBS, respectively.Measure wound diameter daily on macroscopic level and take photos every other day.Seven days after injury, take the mice skin wound tissue of each group to HE staining,then measure the wound diameter with the microscope. Seven days after injury, detect the number of local macrophages through immunohistochemistry method and flow cytometry. And detect the GR’s m RNA expression of the mouse skin wound local tissue and macrophages through Real-time PCR.Results: 1. Six and nine days after injury, the mice wound diameter on macroscopic level of experimental group and control group were 3.2±0.2mm, 1.9±0.1mm(***,p<0.001)and 2.5±0.1mm, 1.0±0.2mm(***,p<0.001), respectively; seven days after injury, the mice wound diameter on histological level of experimental group and control group were 3.2±0.2mm, 1.2±0.4mm(**,p<0.01), respectively. It can be seen that the mice skin wound healing of experimental group delayed significantly.2. Seven days after injury, detect the number of local macrophages through immunohistochemistry method and results show that at higher magnification F4/80+(surface marker of macrophages) cell population of experimental group and control group were, 18.0±1.7, 37.3±3.5(**,p<0.01), respectively. Flow cytometry analysis get the consistent results that dexamethasone can obviously decrease the number of macrophages in the wound site.3. Seven days after injury, detect the GR’s m RNA expression of wound local tissue and the local macrophages, the results show that dexamethasone can enhance the GR’s m RNA expression significantly.Conclusion: GC can reduce the number of macrophages in wound site, consequently delayed skin wound healing in mouse and may militate through the GR signals.Part Ⅱ Glucocorticoid reduce the number of macrophages in wound site by inhibiting macrophage migrationObjective: To investigate the underlying mechanism of GC reduced the number of macrophages in wound site.Methods: First, we use ELISA to detect the expression of chemokine CXCL1, CXCL2 in mice’s serum of experimental group and control group respectively. Next, we detect the expression of CXCR2, chemokine receptor, expressed on the surface of macrophage by flow cytometry. Then the migration ability of macrophages was evaluated through in vivo cell migration experiment and Trans-well essay. We also detect the expression of macrophages’ CXCR2. Finally, we observed the two groups macrophages in vitro cultured from the morphology.Results: Compared with the control group, the chemokine CXCL1, CXCL2 expression in mice serum of the experimental group was decreased obviously. The MFI of CXCR2 in vitro cultured macrophage of experimental group was also decreased.Evaluating the ability of macrophage migration through the in vivo cell migration experiment and Trans-well essay, the results show that dexamethasone can inhibit macrophage migration significantly. Furthermore the migrated macrophages’ CXCR2 expression was also decreased in experimental group. In vitro cultured macrophage morphological observation showed that dexamethasone can restrain the growth of the pseudopodia of macrophages.Conclusion: GC is likely to suppress macrophage migration by down regulate CXCL1/2- CXCR2, this could finally result in reduced numbers of local macrophages in the wound site.Part Ⅲ Glucocorticoid reduce the number of macrophages in wound site and delayed skin wound healing in mouse by glucocorticoid receptor signalsObjective: To explore the signaling pathways on GC delayed mouse skin wound healing by decreasing the number of wound macrophages.Methods: We used mifepristone(RU486), the blocker of GR, to block the GC-GR signaling pathway, and operated the mouse skin wound healing experiment again. The mice were divided into three groups: PBS group with intraperitoneal injection of PBS daily; Dex group with intraperitoneal injection of Dex solution(5mg/kg) daily;Dex+RU486 group with Dex solution(5mg / kg) and RU486 solution(2mg / kg) daily.The same as the first part, assessed skin wound healing in mice from the level of macroscopic and histological level respectively. Detect the number of local macrophages through immunohistochemistry method and flow cytometry. At the same time we also detect the expression of GR of the macrophages in the wound site.Results: According to the results from macroscopic and histological level, the wound healing rate of Dex+RU486 group was accelerated significantly than that of Dex group,almost close to the PBS group. Immunohistochemistry and flow cytometry was used to detect the wound local macrophages number and the results show that the number of CD11b+ F4/80 + macrophages in Dex+RU486 group was similar to PBS group and all increased significantly than Dex group. Additionally the local wound macrophages’ GR expression detected by flow cytometry showed that the wound macrophages’ GR MFI of Dex+RU486 group was similar to PBS group. However, they were significantly less than Dex group.Conclusion: GC can actually inhibit macrophage migration and lead to delay skin wound healing through the GC-GR signals. |
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