| Objective:The therapeutic effect and mechanism of deferoxamine/chitosan(DFO/CS)hydrogel on deep tissue pressure injury in mice were studied with the wet healing theory of clinical wound care.Methods:DFO/CS hydrogel was prepared according to the literature,and the morphology and drug release in vitro were investigated;The model of deep tissue stress injury was established by using SPF C57BL/6 male mice as the research object,which was randomly divided into four groups by random number method:model group,chitosan group,deferoxamine group and DFO/CS group,with 10 mice in each group,and the treatment period was 14 days.The digital camera was used to record the changes of mouse wounds and calculate the wound healing rate with the same conditions.Prussian blue staining and inductively coupled plasma mass spectrometry(ICP-MS)were used to determine the content of iron ions in wounds.HE staining was used to evaluate the infiltration degree of skin muscle cells,the regeneration of capillaries and skin accessory organs.The expression of HIF-1α,VEGF,SDF-1 and TNF-αwere detected by Western blot.The effect of DFO on reactive oxygen species(ROS)level in Ha Ca T cells and its polarization effect on RAW264.7 macrophages were detected by flow cytometry in vitro.Resuls:1.The chitosan hydrogel of deferoxamine was successfully prepared.The results of drug release in vitro showed that CS could effectively control the sustained release of DFO.2.Animal experiment:The model of deep tissue pressure injury in mice was established,and the results were compared with those in the model group.(1)The results of ICP-MS and Prussian blue staining showed that the concentration of free iron ions in muscle tissue of model group mice increased,and the phenomenon of"iron accumulation"appeared obviously.On the contrary,iron accumulation in DFO group decreased significantly.(2)The results of HE staining showed that the infiltration degree of inflammatory cells in DFO/CS group and DFO group decreased significantly(P<0.01),the regeneration number of blood vessels and skin accessory organs increased significantly(P<0.01),and collagen deposition was uniform and dense,with good connectivity,which had significant statistical difference compared with model group.Epithelial hyperproliferation and epidermal thickening appeared in the model group.The epithelization degree of DFO/CS group,DFO group and CS group was moderate and close to that of normal group.(3)The results of immunohistochemical staining showed that the expression of CD31 in DFO/CS group was significantly higher than that in model group and CS group(P<0.01).(4)The results of Western blot showed that the expressions of HIF-1αand VEGF were significantly increased in DFO/CS group and DFO group,while the expression of TNF-αwas significantly decreased,and SDF-1 was significantly increased only in DFO/CS group(P<0.01).3.Cell experiment:(1)The results of ROS detection showed that after pretreatment with DFO(10μMol/L,50μMol/L,100μMol/L)for 1h and 2h respectively,DFO could significantly inhibit the ROS level in Ha Ca T cells,which was significantly different from the positive control group(P<0.01).(2)Flow cytometry showed that the percentages of F4/80~+CD206~+(M2-phenotype)macrophages induced by DFO(50μMol/L,100μMol/L,200μMol/L)were 15.47±0.89%,20.93±0.97%and 29.72±1.84%.The percentages of F480~+/CD86~+(M1-phenotype)macrophages were 18.7±0.75%,15.6±0.71%and 12.0±1.11%respectively,showing a decreasing trend.Conclusion:1.The pressure injury model of C57BL/6 mice was successfully established.The concentration of free iron ions in skin and muscle tissue increased,and the phenomenon of"iron accumulation"appeared.There was no obvious iron accumulation in DFO group.2.2.DFO/CS hydrogel can stabilize HIF-1αexpression,promote VEGF and SDF-1expression,down-regulate TNF-αexpression,inhibit ROS production in Ha Ca T cells,regulate macrophage polarization to M2 type,reduce inflammatory cell infiltration,promote granulation tissue and blood vessel regeneration,and accelerate wound healing. |
