Effect And Mechanism Of CHUANG LING YE Lotion On Healing And Angiogenesis Of Chronic Diabetic Wound

Background and objective:A chronic wound is a wound that does not heal in an orderly set of stages and in a predictable amount of time the way most wounds do.Chronic wounds may never heal or may take years to do so.These wounds cause patients severe emotional and physical stress and create a significant financial burden on patients and the whole healthcare system.In the management of chronic wounds,the most common chronic wounds are leg ulcers,which are usually vascular or diabetic,accounting for 98%of all lower limb wounds.Angiogenesis is the prerequisite and foundation of tissue repair.It is an important connection in the process of wound repair.The granulation tissue that repairs the wound contains high contents of neovascularization.With a long history of treatment in angiogenic defects and ischemic diseases,traditional Chinese medicine has a unique effect,which emphasis on holistic treatment and syndrome differentiation.Chuangling Ye is a mercury-free external Chinese medicine liquid preparation,which is made from Abelmoschus manihot,rhubarb,myrobalan,safflower.Our previous study found that total flavones of abelmoschus manihot has the effect on promoting the proliferation of vascular endothelial cells.Therefore,we propose a scientific hypothesis that CLY can improve wound healing by regulating the activity of the neovascular endothelial cells,promoting angiogenesis and improving the balance of cytokines in the wound microenvironment.Methods:The research can be divided into three parts.In the first part,the experiment was mainly based on the therapeutic effect of Chungling Ye(CLY)on full-thickness skin wound model in diabetic rats.Normal control group(NC group),diabetes group(DM group),low-dose of CLY group(L-CLY),high-dose of CLY group(H-CLY)and positive control group(bFGF,PC group)were set up.The diabetic criteria model can be defined as random blood glucose levels≥ 16.7 mmol/L(300 mg/dL),with polyuria,weight loss,listlessness,greasy fur performance appeared and water and food consumption increased after a one-time high-dose intraperitoneal injection of streptozotocin in a rat.Full-thickness skin wound model was carried out on Day 14 after streptozotocin injection.The general status,wound healing area,wound healing time,wound healing rate,post-injury survival curve was observed and analyzed during the experiment.Based on the curative effect observation and the preliminary results of our studies,we put forward the hypothesis that Chuanglingye can improve angiogenesis in chronic wound healing of rats.The structure of wound tissue and the number and distribution of inflammatory cells,fibroblasts,collagen and capillaries in wound healing process were observed by H&E staining and Masson staining at different time points.Transmission electron microscopy was used to observe the ultrastructure of the wound.Fluorescein-labeled CD31,CD34 and aSMA were used to perform immunofluorescent staining of frozen sections of the wounds of rats in each group in order to observe the number and distribution of vascular endothelial cells and the density and activity of blood vessel during the healing process in each group.In addition,the expression of vascular endothelial growth factor(VEGF)and its receptor(VEGFR2),Nitrogen synthase(eNOS),nitric oxide(NO)and endothelin(ET)were detected and analyzed by immunohistochemistry,enzyme-linked immunosorbent assay and Real-time PCR.In the second part,chicken embryo chorioallantoic membrane,an ideal model of angiogenesis was used as a carrier.By observing the effects of CLY on the growth,the number and the density of blood vessels in the embryo chorioallantoic membrane,the fact that CLY can promote the angiogenesis of chronic wounds will be confirmed.In the third part,based on the results from the first and second part,focus was turned to the effects of CLY on vascular endothelial cells and angiogenesis.Human umbilical vein endothelial cells(HUVECs)impaired model was induced by high glucose stimulation.The impact of CLY on HUVECs impaired model induced by high glucose was explored from the aspects of cell viability,morphology and apoptosis by CCK-8,microscopy and westernblot methods.In studying the relationship between CLY and angiogenesis during wound healing,cell scratch assay,transwell chamber migration assay,and tube formation assay were used to explore the migration of endothelial cells and tube formation.The expression of VEGF/VEGFR 2 and aSMA in HUVEC impaired model induced by high glucose were detected by immunofluorescence and real-time PCR.In mechanism research,the PI3K inhibitor LY 294002 was used to intervene the endothelial cells to clarify the role of PI3K/Akt pathway in the angiogenesis of endothelial cells protected by CLY.Results:1.Diabetic rat model was successfully established by one-time high-dose intraperitoneal injection of streptozotocin,and then a full-thickness skin wound model was performed by surgically resecting the dorsal skin and subcutaneous connective tissue.The random blood glucose level of STZ-induced diabetic rats(diabetic model group,experimental low-dose group,experimental high-dose group and positive control group)was higher than 16.7 mmol/L at various time points.Polyuria,weight loss,listlessness,greasy fur performance and increased water and food consumption were also the criteria of STZ-induced diabetic rats model.The wound gross appearance shows that diabetic wound healing was significantly worse than the normal rat wound group.2.CLY can promote wound healing in the diabetic wound model,reduce the wound area,improve the wound healing rate and shorten the wound healing time.3.During the period of drug treatment,there was no significant difference in the blood glucose between the diabetic groups,suggesting that CLY can promote wound healing independent of the hypoglycemic effect,which may relate to improving angiogenesis,cell migration and a series of the physiological process.4.CLY can promote the proliferation and maturation of granulation tissue,the proliferation of fibroblasts,the deposition of collagen fibers and the closely and orderly organization of cells in diabetic chronic wound model,suggesting that CLY can accelerate the wound healing process.5.CLY can promote the proliferation of vascular endothelial cells and myofibroblasts,microvascular maturation and increase the microvascular density in the wound,suggesting that CLY can improve angiogenesis in diabetic chronic wound healing process.6.CLY increases the expression of VEGF,VEGFR and their downstream pathways in diabetic chronic wound model,suggesting that the wound healing treated by CLY is likely to be mediated by VEGF/VEGFR,PI3K/Akt signaling pathway.7.Chicken embryo chorioallantoic membrane,an ideal model of angiogenesis was used as a carrier.It was observed that CLY can increase the number of angiogenesis in chick chorioallantoic membrane model.8.HUVECs impaired model was induced by 33mM glucose for 72 hours incubation.Cell viability decreased and typical apoptotic morphology was observed.9.CLY has a protective effect on high glucose-induced HUVECs impaired model.10.CLY can increase cell migration,tube formation and expression of aSMA and VEGF/VEGFR 2 in impaired HUVECs model,which demonstrated that CLY could promote angiogenesis in wound healing process.11.CLY can promote the phosphorylation of PI3K/Akt pathway in high glucose-induced HUVECs impaired model.Conlusion:Based on the diabetic full-thickness skin wound model,high glucose-induced HUVECs impaired model and chick chorioallantoic membrane model,we found that CLY can regulate the activity of neovascular endothelial cells,promote angiogenesis,improve the balance of cyfokines in the wound microenvironment,which may improve the ischemic state,shorten the healing time and promote wound healing process.The mechanism may relate to VEGF/VEGFR 2 and PI3K/AKT signaling pathway.