| ObjectiveBased on the theory of combination of disease and syndrome,the representative prescription of traditional Chinese medicine surgical ulcer with Semi-yin and Semi-yang syndrome-Chonghe Ointment was used as experimental research prescription to explore its mechanism of angiogenesis in diabetic ulcer with Semi-yin and Semi-yang syndrome.MethodsThis paper consists of two experiments:1.Experiment 1:Based on the theory of combination of disease and syndrome,the animal model of diabetic ulcer was established by the method of animal model of diabetic ulcer combined with the prescription of bitter cold purging Fanxieye granules.The currently recognized animal model groups of diabetic ulcer with Yang syndrome and diabetic ulcer with Yin syndrome were set up for comparison.The macroscopic characterization information of rats was collected and scored according to the scale.Combined with the microscopic indicators of modern medicine,the curative effect of Chonghe Ointment,the representative prescription of Semi-yin and Semi-yang syndrome in the experiment,was verified,and the evaluation model was established.2.Experiment 2:180 Wistar rats were divided into six groups:normal group(group Z),model group(group M),Chonghe ointment group(group C),Jinhuang ointment group(group J),Huiyang Yulong ointment group(group H),and erythromycin ointment group(group X),with 30 rats in each group.In addition to the normal group,the rest of the groups were established diabetic ulcer with Semi-yin and Semi-yang syndrome model.Each treatment group was given the corresponding external drug with Sterilized Sanfu plaster,daily morning and afternoon dressing once.After debridement on the 1st,3rd,7th,14 th and 21 st day,the fixed focal length and pixels were taken by digital camera,and the wound pictures with rulers were taken perpendicularly to the wound.The collected pictures were measured by NIH Image J1.48 software to calculate the wound healing rate.HE staining was performed on the wound tissue of rats for histopathological observation and Masson staining was used to evaluate collagen fiber generation during wound healing.Immunohistochemical staining was used to detect the expressions of CD31,MPO,CD163 and CD68.The changes of vascular endothelial cells,neutrophils and macrophages during treatment were evaluated.The expressions of VEGF and VEGFR1 were detected by Western blot to observe the changes of Angiogenic cytokines and relative receptor.The expressions of miR-126 and PIK3R2 were detected by RT-PCR,and the expressions of angiogenesis-related genes were evaluated from the perspective of molecular biology.ResultsExperiment 1:1.The ’Intraperitoneal injection of STZ-skin defect-staphylococcus aureus infections-gavage of prescription for purgation of bitter cold’ composite factor modeling method can establish animal model of diabetic ulcer with Semi-yin and Semi-yang syndrome.Macroscopic syndrome differentiation basically conforms to the characteristics of disease and syndrome.2.Wound healing rate:There was no statistical significance between the diabetic ulcer with Semi-yin and Semi-yang syndrome group and the diabetic ulcer with Yang group(P>0.05),and the difference was statistically significant with the diabetic ulcer with Yin group(P<0.05).3.Statistical results of CD31 microvessel density by immunohistochemistry:Microvessel density in diabetic ulcer with semi-yin and semi-yang syndrome group was lower than that in diabetic ulcer with Yang syndrome group,and the difference was statistically significant(P<0.05).There was no statistical significance to compare with diabetic ulcer with Yin syndrome group(P>0.05).Experiment 2:1.General condition of rats:Rats in normal group have white and shiny hair,normal water intake,food intake and activity.The water intake of rats in the other groups was higher than that in the normal group,and the urine volume was larger.The pads needed to be replaced every 2–3 days.The hair color of rats in the model group was slightly yellow and dull with cold tail,crouching,low activity,slow wound healing and poor overall state.The overall state of Chonghe ointment and Jinhuang ointment group was better than that of the model group,and the wound healing of Chonghe ointment group was the fastest,followed by erythromycin ointment group,but the wound of erythromycin ointment group had septic subsidence,and the overall state was not as good as the Chonghe ointment and Jinhuang ointment group.2.The wound healing rate:The wound healing time of the Chonghe ointment group was the shortest,and the wound healing rate was the highest on the 7th and 21 st day of medication,which was statistically significant compared with the model group,Jinhuang ointment,Huiyang Yulong ointment,and erythromycin ointment group(P<0.05).3.HE,Masson staining results:The skin structure of the normal group was normal,with collagen fibers arranged orderly.There is no obvious pathological changes.The skin structure of the model group was seriously damaged.Compared with the Chonghe Ointment,Jinhuang Ointment and erythromycin ointment groups,the infiltration time of inflammatory cell was longer,the neovascularization was less,and the reepithelialization time was longer.On the third day,the damage area of epidermis and dermis in Chonghe ointment group began to scab,and there were a large number of infiltrations of inflammatory cell in each group.Collagen fiber damage was serious,and the arrangement was scattered.The difference was not obvious.On the 7th day,a large number of new blood vessels were seen under the microscope in the Chonghe ointment group,the collagen fibers were arranged regularly,and the content was increased compared with the 3rd day.The arrangement of collagen fibers in the other groups was disordered.The epidermis and dermis in the Jinhuang ointment group were stripped.There were continuous edema and inflammation,accompanied by bleeding red blood cells.Cell edema was serious in erythromycin ointment group.On the 14 th day,Chonghe ointment group had the best epidermal repair,basically completed reepithelialization,reduced number of new blood vessels,reduced inflammatory infiltration,and formed relatively coarse collagen fiber bundles and arranged regularly.In the Jinhuang ointment group,pathological scar tissue was formed,and blood coagulation was observed in the epidermis after bleeding.The granulation tissue in the other groups was still mature,and collagen fibers began to be arranged orderly.On the 21 st day,the skin structure of the Chonghe ointment group was normal,and the collagen fibers were repaired well.The dense scar tissue was formed in the Jinhuang ointment group,and the collagen fibers of the other three groups were not fully mature.Overall,the Chonghe ointment group had the best skin tissue repair and the shortest reepithelialization time.4.Statistical results of CD31 microvessel density,MPO cumulative optical density value,CD163 positive cell are counted by Immunohistochemical:In normal group,immunohistochemical staining of CD31,CD163,MPO was negative.On the 7th day,the CD31 microvessel density in the Chonghe ointment group was higher than that in the model group,Jinhuang ointment,Huiyang Yulong ointment and erythromycin ointment group,and the difference was statistically significant(P<0.05).On the 7th day,the expression of MPO in the Chonghe ointment group was lower than that in the model group,Jinhuang ointment group and erythromycin ointment group,the difference being statistically significant(P<0.05).There was no statistical significance between the Chonghe ointment group and Huiyang Yulong ointment group(P>0.05).On day 3 and 7,the number of CD163 positive cells in the Chonghe ointment group was higher than that in model group,Jinhuang ointment,Huiyang Yulong ointment and erythromycin ointment group,and the difference was statistically significant(P<0.05).5.Expression of VEGF and VEGFR1 protein by Western blot:On the third day,Expression of VEGF in Chonghe ointment group was higher than the other groups.The difference was statistically significant(P<0.05).Expression of VEGFR1 was higher than the normal group,model group,Jinhuang ointment group,Huiyang Yulong ointment group,but lower than the erythromycin ointment group.The difference was statistically significant(P<0.05).On the 7th day,the expressions of VEGF and VEGFR1 in the Chonghe ointment group were higher than those in other groups,and the difference was statistically significant(P<0.05).6.Detection of miR-126 and PIK3R2 gene expression by RT-PCR:Expression of miR-126 in the Chonghe ointment group was higher than that in other groups,expression of PIK3R2 gene was lower than that in other groups,and the difference was statistically significant(P<0.05).ConclusionExperiment 1:’intraperitoneal injection of STZ-skin defect-staphylococcus aureus infection-gavaging by prescription of bitter cold diarrhea’ complex factor modeling method can simulate clinical Yang syndrome of ulcer mistreatment into Semi-yin and Semi-yang syndrome.Local syndrome differentiation of sore surface fit the characteristics of diabetic ulcer with Semi-yin and Semi-yang syndrome.Experiment 2:Chonghe ointment may be upregulation of miR-126 and negative regulation of PIK3R2,activating VEGF/VEGFR signaling pathway to enhance expression of VEGF to promote angiogenesis. |
