| Objective: Based on the previous research,this project plans to explore the part mechanisms of MEBT/MEBO to accelerate the chronic wounds repair through PI3K/AKT/HIF-1? signaling pathway,it aims to improve the pathological mechanism of chronic wounds,and provide some ideals to the treat of the disease.At the same time,it also improves the mechanism of action of moist exposed burn therapy/hydro exposed burn ointment(MEBT/MEBO)on chronic wounds,and enhances the scientific theoretical basis for the promotion and application of MEBT/MEBO technology in clinical practice.Methods: 90 SPF rats purchased from Guangxi Medical University,they are all male rats.After purchase,they were raised at the SPF Level Animal Experiment Center of Youjiang Medical University for Nationalities.They were randomly divided into blank group,acute group and chronic wound group.The blank group without any treatment with injury,the acute group with full-thickness skin defect treatment,the chronic wound group is based on the treatment of full-thickness skin defect,with reference to Fu Xiaobing's full-thickness skin defect method and Shen's modified plastic ring granuloma quantitative method,It has been improved and made into chronic wounds in rats.In short,hydrocortisone is injected after full-thickness skin resection.The chronic wounds were divided into the chronic wound group(the modle group),the Beifuxin group and the Meibao group according to the treatment method.The chronic wound group was treated without drug intervention after modeling.The Beifuxin group was used Beifuxin after modeling.The Meibao group was treated with MEBT/MEBO.Every group was treated immediately after modeling.Before each dressing change,the wounds were cleaned with 1/5000 nitrofurazone solution before each dressing change,and the dressing was changed twice a day.The wound surface was taken with SONY camera every day to analyze the wound healing rate after the preparation of wound.In each group,6 rats were randomly selected to anesthetize at 3days,7days and 14 days.The entire wound granulation tissues were defect.The part of wound granulation tissue were fixed in formaldehyde,and then used to HE staining and CD31 immunohistochemistry.The remaining samples were quickly frozen in a-80°C refrigerator,and all the wound granulation tissue were collected and then subjected to Western blot detection.From the macroscopic wound healing rate,inflammatory cell infiltration and angiogenesis of pathological sections,to the microscopic protein molecular signal transduction pathway,which aims to explore the pathological mechanism of chronic wounds and the potential mechanism of MEBT/MEBO techniques in chronic wounds.Results:(1)wound healing rate,the wound healing rate was observed at 14 days.The healing rate of the wound in the acute group was 0.965±0.019,the healing rate of the wound in the chronic wound group was 0.468±0.013,the healing rate of the wound in the Beifuxin group was 0.975±0.007,and the healing rate in the wound of the MEBO group was 0.974±.0.002.P<0.001 in the chronic wound group and the acute group,P<0.001 in the chronic wound group and the Beifuxin group,and P<0.001 in the chronic wound group and the MEBO group,there were statistical difference.P=0.630 in the acute group and the Beifuxin group,P=0.709 in the acute group and the MEBO group,and P=0.935 in the Beifuxin group and the MEBO group,and there was no statistical difference.(2)HE staining.In the acute group,acute inflammatory cells infiltrated-neutrophils.In the acute group,wound edema appeared on the 3rd day after operation.The subsequent edema of the wound disappeared,and angiogenesis occurred.The hair follicle appeared in 14 days,indicating that the wound healing was completed.Chronic wounds showed chronic inflammatory cell infiltration-lymphocytes,and in the comparison between the Befuxin group,the MEBO group with the chronic wounds group,it can be seen that the chronic inflammatory cell infiltration was significantly reduced after intervention.The chronic group continued to have lymphocytic infiltration and tissue edema characteristics.At 7 days after surgery,Beifuxin showed an increase in collagen,and epithelial cells appeared on the 14 th day,suggesting that the epithelial migration of the wound has covered the wound.On the 7th day after the operation,the angiogenesis occurred in the Meibao group,and the hair follicle appeared in 14 days,indicating that the wound healing was completed.There were no significant changes in the blank group.(3)CD31 immunohistochemical staining was used to observe the neovascularization.At the third day,the blood pressure count of the blank group,the acute group,the chronic wound group,the Beifuxin group,the MEBO group was 2.610±0.979,3.720±0.826,2.610±1.043,4.060±1.056,4.220 ± 1.309,respectively.there was no significant difference between the blank group and the chronic wounds group,P=0.209.However,there was no significant difference between the acute group and the Beifuxin group and the MEBO group,P=0.345,P=0.158,respectively;there was no significant difference in blood vessel count between Beifuxin group and MEBO group.P=0.636.At the 7th day,the blood pressure count of the blank group,the acute group,the chronic wound group,the Beifuxin group,the MEBO group was 2.560±0.784,10.610±1.290,7.280±1.873,8.330±1.749,9.670 ± 1.847,respectively.There was no significant difference in the blood vessel count between the chronic wound group and the Beifuxin group,P=0.420.There was no significant difference in the blood vessel count between the acute group and the MEBO group,P=0.404;Beifuxin group and MEBO group,There were no statistical differences in blood vessel counts between the groups,P=0.196.At the 14 th day,the blood pressure count of the blank group,the acute group,the chronic wound group,the Beifuxin group,the MEBO group was 2.670±0.840,6.500±1.295,5.220±0.732,6.390±1.243,6.500 ± 0.985,respectively.There was no significant difference in the blood vessel counts between the acute group and the Beifuxin group and the MEBO group,P=0.999,P=1.0,respectively;there was no significant difference in the blood vessel count between the Beifuxin group and the MEBO group,P =0.998.(4)Western-Blot detection of PI3K/AKT/HIF-1? signal pathway expression,PI3 K,P-AKT results trend we have published in the early stage,according to experimental design,the detection of PI3 K / AKT downstream molecules,HIF-1?/VEGFR2.(1)HIF-1? protein expression results,at the third day,HIF-1? protein expression of the blank group,the acute group,the chronic wound group,the Beifuxin group,the MEBO group was 0.472±0.023,1.066±0.092,0.976±0.042,1.540±0.075,1.484±0.056,respectively.Only the Beifuxin group and the MEBO group were no statistical difference,p = 0.133;at the 7th day,HIF-1? protein expression of the blank group,the acute group,the chronic wound group,the Beifuxin group,the MEBO group was 0.455±0.016,0.963±0.033,0.839±0.021,1.394±0.051,1.227±0.055,respectively.There were statistical differences between the groups;at the 14 th day,HIF-1? protein expression of the blank group,the acute group,the chronic wound group,the Beifuxin group,the MEBO group was 0.458±0.026,0.862±0.032,0.830±0.064,1.047±0.092,1.000±0.086,respectively.There was no significant difference in the protein expression of HIF-1? between the acute group and the chronic wound group,p=0.407;there was no difference between the Beifuxin group and the MEBO group,p=0.234.The protein expression trend of HIF-1? was as follows: there was no significant change in the expression level of the blank group without any special treatment,p>0.05;HIF-1? in the acute group,chronic wound group,Beifuxin group and MEBO group The protein expression trend was continuously decreased,and the protein expression level of HIF-1? was 3 days > 7 days > 14 days.(2)The protein expression of VEGFR2,at the 3rd day,VEGFR2 protein expression of the blank group,the acute group,the chronic wound group,the Beifuxin group,the MEBO group was 0.746±0.018,1.156±0.098,0.971±0.053,1.119±0.061,1.096±0.069,respectively.The acute group and the Befuxin group,MEBO group no difference,respectively p = 0.335,p = 0.125.There was no difference between the Beifuxin group and MEBO groups,p=0.551.At the 7th day,VEGFR2 protein expression of the blank group,the acute group,the chronic wound group,the Beifuxin group,the MEBO group was 0.736±0.044,2.085±0.077,1.616±0.037,1.970±0.070,1.902±0.029,respectively.There was no difference between the acute group and the Beifuxin group,p=0.124.There was no difference between the Befuxin group and the MEBO group,p=0.286;At the 14 rd day,VEGFR2 protein expression of the blank group,the acute group,the chronic wound group,the Beifuxin group,the MEBO group was 0.742±0.043,0.956±0.059,0.948±0.056,0.937±0.051,0.943±0.038,respectively.The protein expression levels of VEGFR2 in the acute group,the chronic wound group,the Beifuxin group and the MEBO group were not statistically different,p=0.786,p=0.515,p=0.645,respectively.There was no significant difference in the protein expression of VEGFR2 between the chronic wound group and Beifuxin group,MEBO group,p=0.703,p=0.849,respectively.there was no difference between the Befuxin group and the MEBO group,p=0.848.The protein expression trend of VEGFR2 was as follows: there was no significant change in the expression level of the blank group without any special treatment,p>0.05;the protein expression of VEGFR2 in the acute group,the chronic wound group,the Beifuxin group and the MEBO group were dynamic change,the protein expression of VEGFR2 was 7 days > 3 days > 14 days.Conclusions:(1)MEBT/MEBO can promote the healing of chronic wounds and has a similar healing rate in the rat model of chronic wounds by Beifuxin;(2)MEBT/ MEBO can promote chronic wounds Angiogenesis,increased blood circulation,reduced lymphocyte infiltration,and improved chronic inflammatory response in chronic wounds;(3)MEBT/MEBO can Activate PI3K/AKT/HIF-1? signal pathway to promote the expression of VEGFR2,a key factor in angiogenesis,to promote angiogenesis of wounds,improve hypoxic state of wounds,and achieve healing of chronic wounds;(4)The technical application of MEBT/MEBO in chronic wounds can be further promoted in the clinic. |
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