Research Into The Repair Effect And Mechanism Of Total Flavone Of Abelmoschus Manihot On Chronic And Refractory Wound Healing

Background:Chronic and refractory wound is a common clinical disease,which is difficult to solve with long time treatment and poor curative effect,and thus seriously affects the patient's quality of life and recovery.Angiogenesis is an important part of wound healing.A large number of new blood vessels can provide sufficient nutritional basis and secrete effective substances,which are beneficial to the proliferation of granulation and accelerate the repair of the wound.The traditional Chinese medicine has unique curative effect for treating wound and ulcers with a long history.The theory of "blood producing" and "muscle generation" in traditional Chinese medicine is in line with the concept and connotation of angiogenesis in modern medicine.The Chinese herb Abelmoschus Manihot is usually called "Chuang Jia Yao Yao" for treating wound and ulcers effectively.Phytochemical researches have indicated the total flavonoids extracted from flowers of Abelmoschus Manihot(TFA)are the major constituents of the flowers,possessing the similar pharmacological action.Our previous study found that the total flavones of Abelmoschus Manihot exhibits pro-angiogenic activity.Therefore,it is presumed the total flavones of Abelmoschus Manihot may also improve wound healing.However,the underlying molecular mechanism remains unclear.Objective:To investigate the effects of TFA on chronic and refractory wound healing,and further to explore the underlying mechanism and target of action in aspect of promoting angiogenesis,for the purpose of providing experimental basis for the clinical application of total flavone of Abelmoschus manihot.Methods:Experiment 1:To observe the curative effect of TFA on chronic and refractory wound healing,and explor the possible underlying mechanism in vivo.Firstly,we established hormone-induced chronic refractory skin ulcer rats model.Then rats were randomly divided into 5 groups:Negative control group(normal saline),Positive control group(rb-bFGF),Low dose TFA group(50mg/ml TFA),Middle dose TFA group(100mg/ml TFA)and High-dose TFA group(200mg/ml TFA).The wound surface area,the rate of wound healing and histopathology were observed in each group on day-3,7,14,and 21,respectively.At the same time,expression of angiogenesis related cytokines such as VEGF,VEGFR2,Ang ?,Ang ? and eNOS in granulated tissue of wound surface in rats were also measured by immunohistochemistry,immunofluorescence assay,enzyme-linked immunosorbent assay(ELISA)and Real-Time PCR methods,respectively.Experiment 2:To investigate the pro-angiogenic activity of TFA,and explor the underlying mechanism in vitro.Human umbilical vein endothelial cells(HUVECs)were cultured and then divided into 4 groups:normal control group(complete culture medium),inhibitor group(10 mol/L LY294002 pretreatment),TFA groups(5 g/mL,10 g/mL and 20 g/mL concentrations of TFA represent low,middle and high-dose,respectively),the inhibitor + TFA groups(10 mol/L LY294002 pretreatment prior to 5 g/mL,10 g/mL and 20 g/mL concentrations of TFA).The cell proliferation,migration and tube formation were performed using CCK 8 chemical assay,scratch assay,Transwell assay and tube formation methods.Levels of PI3K and Akt phosphorylation were measured by Western blot.Expression of VEGF-A,VEGFR2,PI3K and Akt was evaluated by RT-qPCR and Western blot assays.Experiment 3:To investigate the pro-angiogenic activity of TFA and the relationship with the PI3K/Akt signaling pathway in vivo.Firstly,the chick chorioallantoic membrane(CAM)model was established.And then randomly divided into 4 groups:normal control group(complete culture medium),inhibitor group(10 mol/L LY294002 pretreatment),TFA groups(5 g/mL,10 g/mL and 20 g/mL concentrations of TFA represent low,middle and high-dose,respectively),the inhibitor + TFA groups(10 mol/L LY294002 pretreatment prior to 5 g/mL,10 g/mL and 20 g/mL concentrations of TFA).The numbers of vessel branches as index of angiogenesis in CAM model were counted using microscope.Results:1.The hormone-induced chronic refractory skin ulcer rats model and the chick chorioallantoic membrane(CAM)model were successfully established in this work.2.TFA exhibits the curative effect on chronic and refractory wound healing by reducing the wound surface area and improving the rate of wound healing.3.TFA can promote chronic wound granulation tissue hyperplasia,fibroblast proliferation and collagen deposition,as well as capillary density and maturity.4.TFA upregulates the expression of angiogenesis related cytokines such as VEGF,VEGFR2,Ang ?,Ang ? and eNOS in granulated tissue of wound surface in rats,suggesting that TFA promote chronic wounds healing is likely through promoting angiogenesis and/or regulating the the related angiogenic signal pathway.5.Experiments in HUVECs culture in vitro indicated TFA treatment obviously promoted HUVECs proliferation,migration,invasion and tube formation.Further investigation on underlying mechanism showed that TFA markedly augmented PI3K and Akt phosphorylation,and up-regulated VEGF-A and VEGFR2 expression in HUVECs by RT-qPCR and Western blot assays.6.In contrast,pretreatment of HUVECs with a PI3K inhibitor LY294002 prior to TFA stimulation not only markedly attenuated TFA-induced cells proliferation,migration,invasion and tube formation,but also significantly abolished TFA-induced VEGF-A and VEGFR2 over-expression as well as PI3K and Akt phosphorylation in HUVECs.7.TFA evidently promoted the formation of branched blood vessels in CAM model.8.PI3K inhibitor LY294002 also dramatically suppressed TFA-stimulated formation of branched blood vessels in CAM model in vivo.Conlusion:Taken together,results demonstrated TFA exhibits the curative effect on chronic and refractory wound healing based on the pro-angiogenic activity,and at least in part,the underlying mechanism of action by activating the VEGF-A/VEGFR2-PI3K/Akt signaling axis.These findings provide the first pharmacological rationale for the use of TFA as an angiogenic promoter and suggest that TFA could be developed into a potential candidate for treatment of angiogenesis-related human diseases,such as wound healing and ischemic injuries.At the same time,it provides theoretical support for the modernization of TFA.