| The cause of diabetic foot ulcer(DFU)is complex,Because of long-term hyperglycemia with complications such as angiopathy and neuropathy or co-infection,It leads to the DFU is difficult to be cured,the treatment effect is not very well,and has a high rate of disability or even death.The polysaccharide of Pseudostellaria heterophylla(PHP)is a kind of macromolecular substance with various biological activities.It can reduce blood sugar,improve glucose tolerance,moisturize,repair skin tissue,and provide a wet closed environment to accelerate wound healing.Bone marrow mesenchymal stem cell(BM-MSCs)transplantation can promote the secretion of various cytokines,target damaged tissues and organs,repair microcirculation,reconstruct the function of tissues and cells,and provide a new technical means for the treatment of various tissue injury diseases.In this study,Extrapolation of PHP,PHP external application or oral administration,BM-MSCs transplantation,Extrapolation of PHP and Transplantation of BM-MSCs was used to improve the DFU,and to discuss the possible mechanism of PHP and its combination with BM-MSCs transplantation in treating diabetic foot ulcer in rats.It lays the foundation for the research and development of new drug products of Pseudostellaria heterophylla and promote the clinical application of stem cells.Chapter I Polysaccharide Preparation of Pseudostellaria heterophyllaIn this study,the PHP was prepared by water extraction and alcohol precipitation method.A total of 492.50 g of polysaccharides were obtained from Radix Pseudostellariae,the yield was 9.85%,and the obtained polysaccharides were yellowish-white powder.The average content of polysaccharide in Radix Pseudostellariae determined by phenol sulfuric acid method was 43.60%.The relative molecular weight of polysaccharides in Pseudostellaria heterophylla was determined by Gel Permeation Chromatography.The molecular weight was3.0×10~3~2.1×10~5 Da.Chapter II pharmacodynamic study on the treatment of DFU in rats with PHP and its combination with BM-MSCs transplantation2.1 Laboratory animals and BM-MSCsThe rat hyperglycemia model was prepared by intraperitoneal injection of streptozotocin(STZ).after 72h of injection,fasting blood glucose values?16.7mmol/L were monitored for3 consecutive days as successful rats in the hyperglycemia model.On this basis,the rat DFU model was prepared by whole-layer skin resection.In vitro culture of BM-MSCs to the third generation by whole bone marrow adherent method,DAPI fluorescent markers BM-MSCs,The growth activity of DAPI labeled BM-MSCs was detected by MTT assay.2.2 Study on the dose effect and time effect relationship of the external application of the PHP in the treatment of diabetic foot ulcer in rats48 SPF male Wistar rats were randomly divided into 6 groups,8 rats in each group.Blank group,model group,positive group((Recombinant Human Epidermal Growth Factor Derivative for External Use Liquid,RHEGFDEUL)and PHP-HD group?PHP-MD group?PHP-LD group(0.30,0.20,0.14g/mL),twice a day,local administration 1ml.The fasting blood glucose of rats was monitored 7 and 14 days after administration,the wounds of DFU were photographed,the image was analyzed with ImageJ software,the wound area was measured and the wound healing rate was calculated,and the best dosage and treatment course of external administration of PHP were determined.In the experimental period,Fasting blood glucose levels in blank control group were normal(4.25±0.48~4.53±0.48),fasting blood glucose levels in the hyperglycemia model group remained high(19.20±4.90~29.70±3.81),P<0.01,the difference was statistically significant,the results showed that the rat model of hyperglycemia was successful,External use of RHEGFDEUL and PHP did not reduce the blood sugar value of hyperglycemia rats,and the blood sugar of hyperglycemia rats did not recover naturally.Compared with the model group,the scab formation and healing speed of the blank group were significantly faster than that of the diabetic group(P<0.01).Compared with the model group(47.06%±0.23),the DFU of PHP-MD group(92.10%±0.10)healed faster(P<0.01),the difference was statistically significant;than PHP-LD group(82.39%±0.09),PHP-HD group(69.99%±0.12).The wound healing rate of PHP-MD group was similar to that of positive group(P>0.05).That 0.20g/mL PHP was the best dosage for external use,and the course of administration was about 14 days.In the experimental period,2.3 Pharmacodynamic experiment of PHP combined with BM-MSCs transplantation in the treatment of DFU in rats96 SPF male Wistar rats were randomly divided into 9 groups,Blank group,model group,positive group,Metformin+PHP-MD group,PHP-MD group,PHP-MD oral and external group,BM-MSCs group,BM-MSCs+PHP-MD group,PBS group.In the pharmacodynamic experiment,blood glucose levels in the positive control group,BM-MSCs+PHP-MD group,BM-MSCs group,PBS group remained high(19.64±3.46~31.98±2.65),compared with model group,there was no significant difference(p>0.05),That is to say,BM-MSCs transplantation could not ideally reduce the blood glucose of hyperglycemic rats as well as the external application of RHEGFDEUL and PHP,meanwhile,there was no natural recovery in hyperglycemia rats.The average blood glucose of the Metformin+PHP-MD group was 10.21±2.43 and 11.08±1.39 respectively,all of which were at lower levels,compared with the blood sugar values of the blank control group,the difference was not statistically significant(p>0.05),for PHP-MD group,there was no significant difference in blood glucose compared with the model group(p>0.05).Glucose levels have been decreased in PHP-MD oral and external group,and at 14 day,there were significant statistical differences compared with the mean blood glucose in the model group,it shows that PHP oral has a certain hypoglycemic effect,oral metformin has a significant hypoglycemic effect.In the 14th day after administration,PHP-MD group,PHP-MD oral and external group,BM-MSCs group,BM-MSCs+PHP-MD group(91.40%±0.17~94.77%±0.06),which wound scab and healing speed were faster than model group,compared with the model group(28.97%±0.12),the difference was statistically significant(p<0.01),compared with the positive group,there was no significant difference in wound healing rate(p?0.05).The results showed that PHP and its combination with BM-MSCs transplantation could produce synergistic effect to improve the therapeutic effect of DFU in rats.Chapter III the mechanism of the treatment of DFU in rats with PHP and its combination with BM-MSCs transplantation3.1 Histopathological studyHE staining analysis of wound tissue in each group.endothelial cells were labeled with CD31 immunohistochemistry to confirm tissue angiogenesis.HE staining results showed that after 14 days of administration,except model group and PBS group,The epidermal structure of each administration group and blank group was continuous and complete,the new granulation tissue is thicker,a large number of fibroblast proliferation and collagen deposition were observed,there are many new capillaries and new hair follicles.The number of new hair follicles in BM-MSCs+PHP-MD group was the most.It is suggested that BM-MSCs+PHP-MD may promote the formation of DFU by promoting the process of wound epithelization.CD31 immunohistochemistry showed,in the 14th day after administration,The microvasculature density of each administration group was no significant difference,The mean number of positive microvessels in the BM-MSCs+PHP-MD group(15.97±3.58)was significantly higher than that in the BM-MSCs group,PHP-MD oral+external group,PHP-MD group(15.37±3.56~15.94±4.17),compared with the model group and PBS group(11.08±3.23~11.75±3.64)(P<0.01).It is suggested that BM-MSCs+PHP-MD may promote the healing of DFU in rats,which may be related to promoting the neovascularization of ulcer surface.3.2 expression of VEGF mRNA in DFU of rats in each groupVEGF is an effective vascular endothelial growth factor,which can induce vascular proliferation in vivo and is the key factor to restore endothelial function and integrity.qPCR detection of VEGF mRNA expression in the ulcer area of rats showed that,in the 14th day after administration,The relative expression of VEGF mRNA in the wound of foot ulcer in each group(1.03±0.38~2.08±0.17)was higher than that in the model group(0.66±0.05),p<0.05,and the difference is statistically significant.This result indicates that hyperglycemia inhibits the expression of VEGF mRNA in the DFU wounds,which leads to the difficulty of diabetic foot ulcer healing.The effect of PHP and its combination with BM-MSCs transplantation on the healing of DFU may be to enhance the expression of VEGF mRNA,promote wound angiogenesis,increase blood supply,blood oxygen and tissue temperature of the wound,so as to promote the healing of DFU.In this study,Extrapolation of PHP,Extrapolation and Oral Administration of PHP,BM-MSCs transplantation,Extrapolation of PHP combined with BM-MSCs transplantation were used in the treatment of DFU.It give full play to the characteristics of"wet sealing tissue wound"with polysaccharide external application,pharmacological effect of oral polysaccharide on Hypoglycemia,combined with the characteristics of self-renewal,multidirectional differentiation and immunomodulation after transplantation of BM-MSCs,to improve the repair of DFU wound effectively in rats.And the mechanism may be to promote the healing of DFU wound by increasing the expression of VEGF,promoting the process of angiogenesis and epithelialization. |
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