| Background:Wound healing is a highly coordinated pathophysiological process that usually goes through four stages: coagulation,inflammation,proliferation and remodeling.Normal wound healing requires the participation of fibroblasts,endothelial cells,immune cells and other cells,and secretes various cytokines to regulate the proliferation,migration and apoptosis of repair cells in an orderly manner.Meanwhile,the dynamic balance between the deposition and degradation of extracellular matrix was maintained,and wound healing was finally completed.However,scar formation occurs due to prolonged non-healing or excessive healing for some reasons,which is difficult in clinical treatment.The regulation of inflammatory response to wound healing is a double-edged sword.In the process of wound inflammation,monocytes,mast cells and other inflammatory cells remove harmful substances such as pathogenic bacterias,and release cytokines to promote wound healing.However,the disorder of wound inflammation regulation is the main cause of the occurrence of chronic refractory wounds.It has been proved that if inflammatory response persists on the wounds,the transition from inflammatory stage to proliferative stage cannot be achieved,and the wound is refractory.In addition,persistent chronic inflammation can induce excessive healing and produce pathological scarring.Therefore,it is of great scientific significance and clinical value to study the mechanism of inflammation regulation in wound healing.The resolution of wound inflammation is a complex process requiring the synergistic effects of neutrophils,macrophages and fibroblasts.Fibroblasts,as the main functional cells during wound repair,are involved in the regulation of inflammation as well as the secretion of extracellular matrix(ECM)in the formation of granulation tissue.At the early stage of wound,fibroblasts secrete cytokines to collect white blood cells and promote their infiltration and activation.In addition,fibroblasts can directly contact neutrophils to promote their apoptosis and clearance to restrict wound inflammation.These results suggest that fibroblasts are involved in the formation of inflammation in the early stage of wound as well as the resolution in the later stage through secretion factors and direct contact with inflammatory cells.However,the specific mechanism,especially how does the resolution of inflammation occur,is not completely clear.Immune response or inflammatory response of the body is the result of dynamic balance of immune regulation.Recently,immune checkpoint is discovered as a negative immune regulation mechanism of the body to fight against excessive immune response or inflammatory response,which plays an important role in tumor avoidance immune surveillance and the occurrence of autoimmune diseases.The PD-1 / PD-L1 pathway is one of the key pathways of the immune checkpoint pathway,which is involved in the maintenance of peripheral immune tolerance.However,it is still unclear whether immune checkpoints are involved in the regulation of inflammatory regression in wound healing.In previous studies,we confirmed that the expression of PD-L1 increased during wound healing,thus we raise the hypothesis that fibroblasts may participate in the regulation of inflammation in wound healing by PD-L1 signal pathway.However,the mechanism of up-regulation expression of PD-L1 in fibroblasts during wound healing and PD-L1 signaling pathway involved in inflammation regulation remain yet to be proven.A variety of cytokines(such as TGF-beta,PDGF,EGF,FGF-2,IL-1 and TNF,etc.)exist to precisely coordinate repair cells and promote wound healing.However,whether the expression of PD-L1 in fibroblasts during wound healing is regulated by some factors still needs to be further explored.Meanwhile,it has been proved that the expression of PD-L1 is mainly regulated at the translation level,so whether the expression of PD-L1 in fibroblasts during wound healing is dependent on protein translation regulation needs to be further studied.There is evidence that macrophages are involved in the inflammatory response throughout the wound healing process,especially in the late stage of the inflammatory response.Macrophages not only phagocytic apoptotic cells,but also transform the phenotype and function,polarization from M1 type(pro-inflammatory)to M2 type(pro-inflammatory),and PD-1 / PD-L1 pathways play an important role in the macrophage polarization.Thus,we further speculated that fibroblasts may play an important role in the polarization of macrophages by expressing PD-L1,and participate in wound inflammation resolution,eventually promote wound healing.Dimethyl sulfoxide(DMSO)is a widely recognized solvent due to the presence of highly polar subunits,which can dissolve a variety of low-solubility compounds,such as drugs,inhibitors,etc.As a potential drug,DMSO has been used in the treatment of various tumors and acute and chronic inflammation.However,since the safety of DMSO has not been fully confirmed,there are still few in vivo data on DMSO and it cannot be widely used as a treatment in clinical practice.Studies have confirmed that DMSO can reduce the level of local inflammation by reducing the level of pro-inflammatory factors,and DMSO can promote wound healing,but the mechanism is not completely clear.At the early stage,we used DMSO as a solvent to dissolve eIF4 E inhibitor 4EGI-1,and we were surprised to find that DMSO alone could effectively promote the proliferation of fibroblasts.Therefore,we hypothesized that DMSO may promote the proliferation of fibroblasts,which are stimulated by cytokines to express high levels of PD-L1 to regulate the regression of wound inflammation and ultimately promote wound healing.Objectives:The purpose of this study is to clarify the mechanism by which fibroblasts participate in wound healing by expressing immune checkpoint receptor PD-L1,which mediates inflammation resolution.The details are as follows :(1)confirm the expression of PD-L1 in wound healing and its role in wound healing;(2)clarify the regulatory factors for the expression of PD-L1 in fibroblasts;(3)explore the mechanism of the involvement of PD-L1 positive fibroblasts in wound inflammation resolution;(4)study the specific mechanism of DMSO promoting the healing of refractory wounds,and finally provides a new target and theoretical basis for the treatment of refractory wound or pathological scar caused by the disorder of inflammation regulation.Methods:1.The wound model of WT mice was prepared.Immunohistochemistry,WB and qRT-PCR were used to detect the expression levels of PD-L1 protein and mRNA in wounds of different days,and the expression of pd-l1 during wound healing were analyzed.Wound chronic healing model was prepared by subcutaneous injection of LPS in WT mice and PD-L1-/-mice,and the function of PD-L1 in wound healing process was analyzed.2.PD-L1 positive cells in the wound tissue were detected by FACS and immunofluorescence;Spatial localization of PD-L1 positive fibroblasts and macrophages by immunofluorescence analysis;qRT-PCR was used to analyze inflammatory factors TNF-αand IL-6,FACS was used to detect the number of macrophages,and WB was used to detect the dynamic changes of macrophage surface markers CD16 and CD206 with the wound healing process.3.The expression of PD-L1 in MEF cells was detected by FACS stimulated with wound exudate and then combination with TGF-beta and FGF-2 to antibody treatment;PD-L1 protein and mRNA expression in MEF cells treated with TGF-beta and FGF-2 were detected by FACS,WB and qRT-PCR.MEF cells treated with TGF-beta and FGF-2 were co-cultured with LPS activated macrophages.Expressions of CD16 and CD206 of macrophages were detected by FACS and immunofluorescence,and the expressions of TNF-α,IL-6 and IL-10 were detected by ELISA.RECELL technique was used to analyze the wound healing process in pd-l1-/-mouse chronic wound healing model by supplementing TGF-β1 and FGF-2 pretreated MEF cells.4.Polyribosome was used to detect the changes of PD-L1 mRNA content on the poly ribosomes of TGF-β1 and FGF-2 co-treated MEF cells,and the activation and expression of key proteins in the translation pathway were detected by WB.To explored the mechanism of TGF-β1 and FGF-2 regulating PD-L1 expression at the translation level by specific pathway inhibitors.5.The proliferation and migration of fibroblasts treated with DMSO at different concentrations were investigated by CCK-8 detection and scratch test,and the relevant mechanisms were discussed.6.The pull-down experiment of 7m-GTP was used to explore the effect of DMSO on pathway proteins and translation initiation complexes,and the specific pathway inhibitors were used to reveal the mechanism of DMSO on fibroblasts.7.The refractory wound surface model on the back of mice was prepared,and the influence of different concentrations of DMSO on wound healing process was analyzed.Results1.PD-L1 presents dynamic expression in wound healing process,and PD-L1 knockout significantly inhibits wound healing.1)PD-L1 protein expression in wound tissue showed a dynamic change of first increasing and then decreasing with the healing process,reaching a peak 5 days after injury,while PD-L1 mRNA level showed no significant change.2)LPS stimulation significantly slowed down the healing speed of acute inflammatory wound in mice,among which the wound healing speed of PD-L1-/-mice was significantly slowed down,indicating that PD-L1 knockout aggravated the chronic refractory degree caused by inflammation.2.PD-L1 plays an important role in the regulation of inflammation in wound healing.1)compared with DC and macrophages,the number of PD-L1 positive fibroblasts in mouse tissues increased significantly 5 days after injury,indicating that PD-L1 was mainly located in fibroblasts during the transformation from inflammatory stage to proliferative stage of wound healing,and there was spatial correlation between macrophages and fibroblasts in this process.2)in wound healing process,compared with WT mice,the levels of inflammatory factors TNF-α and IL-6 mRNA and the number of recruited macrophages in wound tissues of PD-L1-/-mice were significantly increased,suggesting that PD-L1 is involved in regulating inflammatory responses in wound healing process.In addition,the expression of CD16 in wound tissues of PD-L1-/-mice was increased and the resolution time was delayed,while the expression of CD206 was decreased,suggesting that PD-L1 plays an important role in macrophage polarization.3.TGF-β1,together with FGF-2,up-regulated the expression of PD-L1 in MEF cells,affected the polarization of macrophages,and participated in the regulation of wound inflammation.1)wound seepage on day 5 up-regulated the expression of PD-L1 in MEF cells,and combined application of TGF-β1 and FGF-2 neutralizing antibody significantly reversed the expression of PD-L1.TGF-β1 and FGF-2 up-regulated the expression of PD-L1 in MEF cells,but the mRNA expression level did not change significantly.2)TGF-β1 and FGF-2 pretreated MEF can reduce the M1 ratio of macrophages and increase the M2 ratio after co-culture,reduce the expression of pro-inflammatory factor TNF-α and IL-6,and increase the expression of anti-inflammatory factor IL-10,indicating that TGF-β1 and FGF-2 treated MEF can promote the transformation of macrophages from M1 to M2.On the contrary,TGF-β1 and FGF-2 treatment of PD-L1-/-MEF cells significantly reduced the transformation ability of macrophages from M1 to M2,indicating that TGF-β1 and FGF-2 stimulated fibroblasts to regulate macrophage polarization dependent PD-L1 signal.When the pretreated WT and PD-L1-/-MEF cells were fixed,its ability to regulate macrophage polarization is reduced,indicating that fibroblasts may regulate macrophage polarization through other channels,such as releasing soluble factors.However,there is still a significant difference in its level of regulation,indicating that PD-L1 pathway plays an important role in regulating macrophage polarization.3)TGF-β1 and FGF-2 preconditioning MEF cells promoted wound healing of PD-L1-/-mice under inflammatory conditions,confirming that TGF-β1 and FGF-2 up-regulated PD-L1 expression of MEF cells,regulated inflammatory response,and promoted wound healing.4.FGF-2 in collaboration with TGF-β1 up-regulated the expression of PD-L1 in fibroblast through the eIF4 E protein translation pathway.1)It was found that the mRNA of PD-L1 in the polyribosome was significantly increased in fibroblasts when treated by TGF-β1 and FGF-2,suggesting that the regulation of PD-L1 was mainly dependent on translation level.2)FGF-2 combined with TGF-β1 significantly up-regulated the activation and expression of PI3K-Akt-mTOR and p38-Erk-MNK signaling pathways in fibroblast,and phosphorylated the expression of eIF4 e,while the addition of eIF4 E blocker significantly inhibited the above up-regulation,suggesting that TGF-β1 and FGF-2 activated the translation signal to regulate the expression of PD-L1 at the translation level.5.DMSO activates protein translation by Akt/mTOR to promote fibroblast proliferation1)With the stimulation of different concentrations of DMSO by fibroblasts,the proliferation ability of fibroblasts was significantly enhanced at different time points due to the low concentration of DMSO,and the proliferation ability was the strongest at 5mM.However,DMSO did not affect the migration ability of fibroblasts.It suggested that DMSO could promote fibroblast proliferation2)When fibroblasts were stimulated by DMSO for 24 h,the phosphorylation level of AKT/mTOR was significantly increased.It is suggested that DMSO can promote fibroblast proliferation through the AKT/mTOR signaling pathway.Phosphorylation of p70S6 K was inhibited by the addition of Rapamycin,an inhibitor of the mTOR/AKT signaling pathway.It is suggested that DMSO can promote fibroblast proliferation through the AKT/m TOR signaling pathway.3)The 7m-GTP pull-down assay showed that DMSO induced the phosphorylation of 4EBP1 and promoted the formation of the translation initiation complex eIF4 F,while the addition of Rapamycin inhibited the phosphorylation of 4EBP1 by DMSO and the formation of the active translation initiation complex eIF4 F.It is suggested that DMSO can activate protein translation through the AKT/mTOR signaling pathway to promote fibroblast proliferation.6.DMSO promoted the healing of refractory wounds in mice,while Rapamycin,an inhibitor added to the m TOR signaling pathway,inhibited its healing effect.Conclusion1.During wound healing,especially in the transformation from inflammatory stage to proliferative stage,PD-L1 positive fibroblasts affect wound healing by contacting macrophages to regulate inflammatory response;2.TGF-β1 and FGF-2 are key factors to induce the expression of PD-L1 in fibroblasts.TGF-β1,together with FGF-2,up-regulates the expression of PD-L1 in fibroblasts at the translation level,promoting the polarization of macrophages from M1 to M2,leading to the transformation from inflammatory stage to proliferative stage,and promoting wound healing.3.This study proved that PD-L1 promotes wound healing by regulating inflammatory response during wound healing,providing a new target and theoretical basis for the treatment of refractory wounds or pathological scars caused by inflammatory disorders.4.DMSO activates protein translation through the Akt/m TOR signaling pathway,significantly promoting the proliferation of fibroblasts,and a large number of proliferating fibroblasts can regulate wound inflammation through the highly expressed PD-L1 ligand on their surfaces,thus promoting wound healing.It provides a new mechanism for the regulation of wound healing. |
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