| Background:Diabetic wounds are often difficult to heal.One of the reasons is that the wounds show a persistent chronic inflammatory state,in which abnormal macrophage phenotype and function play an important role.Aloe polysaccharideo（AP）not only has anti-inflammatory and wound healing effects,but also affects the function of macrophages and the secretion of cytokines.Its positive effect on wound healing may be different from the polarization state of wound macrophages of transformation.Objective:To investigate the effect of AP on the healing of diabetic wounds and the effect of the polarization of macrophages in the wound tissue.Methods:1.Establishment of diabetic rat model and grouping:30 SPF male SD rats were adaptively fed with standard diet for 3 days,and 7 rats were selected as blank control group（BLANK group）by random number table method and fed for 4weeks.The remaining 23 rats were used to prepare the diabetes model:they were fed with high-fat and high-sugar diet for 4 weeks,and a one-time intraperitoneal injection of 1%streptozotocin solution 30 mg/kg,with fasting blood glucose≥16.7 mmol/L accompanied by obvious symptoms.Symptoms such as polydipsia,polyphagia,and polyuria were used as modeling criteria.Diabetic rats were randomly divided into aloe polysaccharide group（AP group）,control group（b FGF group）and model group（MODEL group）,7 rats in each group（excluding 2 rats:1 rat with fasting blood glucose<16.7mmol/L,the other 1 rat die）.2.Skin wound preparation and intervention:use electric clippers to shorten the back hair of rats and depilate them with depilatory cream.Each rat was anesthetized by intraperitoneal injection of 10%chloral hydrate at a dose of 3.0 ml/kg,and four wounds with a diameter of 10.0 mm and a depth of 20.0 mm were prepared on the back of the rat with a 10.0 mm skin punch.AP group and b FGF group applied 10%aloe vera polysaccharide solution（prepared by aloe vera polysaccharide powder and normal saline in a mass ratio of 1:9）0.1ml and basic fibroblast growth factor 0.1ml to each wound respectively,MODEL group and BLANK Each wound in the group was smeared with 0.1 ml of normal saline.Once a day for 14 consecutive days.3.On the 1st,4th,7th,11th,and 14th days of the intervention,one rat in each group was selected by random number table method as the object of observation and sampling（after the last sampling,there were two remaining experimental rats in each group which can prevent data loss due to accidental death of experimental rats during the experiment）.The general condition and wound healing of the rats were observed,the wounds were photographed,and the wound area was delineated and marked with Image J image analysis software,and the wound healing rate was calculated,and four10.0 mm deep muscles were cut from the original surface with a 10.0 mm skin punch.Layers of wound tissue samples.Enzyme-linked immunosorbent assay（ELISA）was used to detect the expressions of TNF-αand TGF-β1 in the wound tissue.HE staining was used to observe the pathological changes of the wound.Immunohistochemical staining was used to detect the surface markers CD86 and CD206 of M1 and M2macrophages in the wound.Expression,Image J image analysis software counted the number of immunohistochemically stained positive cells.4.Statistical analysis:The blood glucose value,body weight,cytokine content,and the ratio of positive cells in immunohistochemical staining were expressed as±s.Analysis of variance was used for comparison between multiple groups,and SNK-q test was used for pairwise comparison at the same time point.IBM SPSS Statistics25.0 software and Graph Pad Prism 9.0 software were used for data processing analysis and graph drawing.P<0.05 indicated a statistically significant difference.Results:1.General situation:The vital signs of the rats in each group were stable during the experiment.The rats in the AP group,b FGF group and MODEL group showed symptoms of diabetes,with higher fasting blood glucose and obvious weight loss during the experiment;the living habits of the BLANK group No significant changes were seen,fasting blood glucose was normal,and body weight increased slowly during the experiment.2.Wound healing:There was no obvious infection in the wounds of the rats in each group during the experiment.On the 4th day,the AP group showed obvious granulation tissue proliferation.By the 14th day,most of the wounds in the AP group were closed,and the new epidermis on the edge of the wound gradually increased.The wound was thick and the color was similar to the surrounding normal skin,while the wound in the MODEL group was dark red.Although the epithelialization trend was visible on the edge of the wound,the unhealed area was still large.The wound healing rate of AP group was significantly better than that of b FGF group and MODEL group.On the 14th day,the wound healing rate of AP group was（86.05±1.35）%,which was better than that of b FGF group（75.37±2.37）%and MODEL group（58.03±3.74）.%（P<0.001）.3.The expression of TNF-αin the wound:On the first day,the expression of TNF-αin the wound of AP group,b FGF group and MODEL group was the same（P>0.05）.From 1 to 7 days,the expression of TNF-αin the wounds of AP group,b FGF group and MODEL group gradually increased,but the MODEL group increased the most;higher level.On the 14th day,the expression of TNF-αin AP group was less than that in b FGF group（P<0.05）and MODEL group（P<0.001）.4.The expression of TGF-β1 in the wound:On the first day,the expression of TGF-β1 in the wounds of the MODEL group,the AP group and the b FGF group was the same（P>0.05）.Afterwards,the expression of TGF-β1 in the wounds of AP group gradually increased and remained at a high level,while the expression of TGF-β1 in the wounds of MODEL group increased but was always lower than that of AP group（P<0.001）.On the 14th day,the expression of TGF-β1 in the wounds of AP group was the same as that of b FGF group（P>0.05）,but significantly higher than that of MODEL group and BLANK group（P<0.001）.5.Histological changes:On day 1,wound tissue necrosis,swelling and inflammation were more obvious in AP group,b FGF group and MODEL group,while BLANK group showed acute exudative inflammation;on day 4-7,AP group and b FGF group The inflammation of the wound was slightly relieved,and granulation tissue and new microvessels began to appear,while a large amount of necrotic tissue was still seen in the wound of the MODEL group;from 7 to 11 days,more collagen fibers and fibroblasts appeared in the wound of the AP group,accompanied by more red blood cells.On the 14th day,the wounds in the BLANK group had basically completed epithelization,and the wounds in the AP group had obvious epithelization,with a large number of Collagen fiber formation,inflammation was significantly reduced in b FGF group,partial epithelialization and scattered new microvessels and collagen fiber formation were seen.The wound in MODEL group showed that there were still a lot of red blood cells,inflammatory cells in the tissue increased slightly,blood vessel formation was difficult,tissue necrosis and vacuoles phenomenon is more serious.6.Changes of CD86+cells:The CD86+cells in the wounds of the MODEL group were few in the early stage,gradually increased and maintained a high level from the 7th to the 14th day of the experiment.On the 14th day,the ratio of CD86+cells in the AP group was（14.23±3.12）%,which was lower than（20.12±2.71）%（P<0.05）in the b FGF group and（31.53±3.19）%（P<0.001）in the MODEL group.But it was higher than（6.35±0.91）%of BLANK group（P<0.05）.7.Changes of CD206+cells:On the first day,scattered CD206+cells were found in the wounds of BLANK group,AP group,b FGF group and MODEL group,and their cell ratios were basically the same（P>0.05）.After that,the wound surface in the BLANK group first increased and then decreased,while that in the AP group increased gradually.The b FGF group and the MODEL group increased slowly,and the changes were basically the same（P>0.05）.On the 14th day,the ratio of CD206+cells in the wound of AP group was（32.62±1.89）%,which was significantly higher than that of the other three groups（P<0.001）.Conclusions:1.AP can inhibit the secretion of TNF-αin the wound of diabetic rats,promote the secretion of TGF-β1,reduce the inflammatory response of the wound and promote the proliferation of granulation tissue,which has obvious effect of promoting healing;2.AP may promote wound healing in diabetic rats by promoting the transformation of wound macrophage phenotype M1 to M2. |
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