Wound Healing Effect Of An Astragalus Membranaceus Polysaccharide And Its Mechanism

ObjectiveAstragalus polysaccharin is the most important active ingredients of astragalus membranaceus.It has been used as an important component of herbal prescriptions to reduce swelling,drain pus,invigorate health,resist disease,antioxidant,antiviral,anticancerand expel toxin for thousands years.Astragalus membranaceus was proved have certain effect on wound healing,but there is no definite analysis and report for its mechanism.This paperwill examine the effect of astragalus polysaccharides promote skin wound healing and the wound healing mechanism.MethodsThe first part is the extraction and purification of Astragalus polysaccharides and the preparation of astragalus polysaccharides ointment.Crude astragalus polysaccharides were extracted form astragalus membranaceus through a series of steps degrease,water extraction,ethanol precipitation,and deproteinization.The crude APS fractionated on DEAE-cellulose anion-exchange column giving the two fractions APS-1 and APS-2,respectively.APS-2 was further purified on Sephadex G-100 columnobtaining a homogeneous fraction APS2-1.The IR spectrum from 4000 cm^-1 to 400cm^-1 was recorded of APS2-1.The APS2-1 was scanned with the wavelength from 190 nm to 400nm on a UV-vis spectrophotometer.The second part is the astragalus polysaccharides APS2-1 promoting skin wound healing in mice.The mice were randomized into 3 groups.The 10-mm full-thickness excisional skin wound was made on the back of each mouse.Each wound was received 0.5g ointment（APS2-1:Vaseline:SDS:H₂O=2:60:2:65 as the APS2-1 groups,PBS:Vaseline:SDS:H₂O=2:60:2:65 as the control,Jing Wan Hong ointment as the positive control）for 3 continuous weeks and dressed with fresh gauze.The weight and state,wound healing and histopathology of three groups of mice were analyzed.The expression of cytokines TGF-beta1,FGF-2 and EGF and cell cycle regulatory proteins Cyclin D1 were detected by ELISA.The third part is the astragalus polysaccharides APS2-1 effect on fibroblast cells and its mechanism.The effect of APS2-1 on human skin fibroblastic cells was examined.The effect of APS2-1 on proliferation of CCC-HSF-1 was detected by MTT methods.The effect of APS2-1 on migration of CCC-HSF-1 was detected by Transwell methods.The effect of APS2-1 on cell cycle of CCC-HSF-1 was detected by flow cytometry.The effect of APS2-1 on cyclin D1 mRNA of CCC-HSF-1 was detected by qRT-PCR.The effect of APS2-1 on the expression of NF-κB p65,IκBαand Cyclin D1 in CCC-HSF-1 was detected by western blot.ResultsThe crude astragalus polysaccharide isolated from the root of astragalus membranaceus by a series of experimental procedures,including ethanol infusion,water extraction,deproteination,dialysis,ethanol precipitation,and lyophilization.After purified utilizing the DEAE-cellulose ion-exchange charomatography,the polysaccharide aqueous solution was separated into two fractions.The main fraction,APS2,was then collected and purified with Sephadex G-100 charomatography.As a result,two purified fractions were generated,considering the APS2-1easy accessed,the following experiments would be focused on APS2-1 fraction.No peak was appeared at 280 nm,suggesting that APS2-1 carried no protein or polypeptide.No peak was appeared at 254 nm,suggesting that APS2-1 carried nonucleic acid.The FT-IR spectrum of APS2-1,APS2-1 in 3390,2930,1640,1420,1160,1010,914,1010 cm^-1 has obvious absorption peak.The APS2-1 treated wound groups exhibitedaccelerating weight compared with the controls at 7,14 and 21days,and had no significant difference with the positive groups.The APS2-1 treated wound groups exhibited accelerating wound closure compared with the controls,and had no significant difference with the positive groups.Massive inflammatory cells infiltration was observed in control group.While in APS2-1 treated mice,the infiltration was reduced and some fibroblasts appeared.Moreover,new blood vessels and new skin were both appeared in APS2-1and positive groups,suggesting that APS2-1 was capable of healing wound by promoting re-epithelialization and revascularization.The levels of cyclin D1,TGF-β1,FGF-2 and EGF in APS2-1 treated mice were significantly higherthan control mice,and there was no significantly difference between APS2-1 and positive group.Adherent CCC-HSF-1 cells were spindle,radiate,triangle,polygon,and suspending CCC-HSF-1cells were circular.In 0-48 hours the CCC-HSF-1 cells growth slowly and belongs to incubation period.In 48-96 hours the CCC-HSF-1 cells growth fastly and belongs to logarithmic phase.In96-144 hours the CCC-HSF-1 cells growth weakly and belongs to platform phase.APS2-1,at the concentration of 1.0 mg/l,5.0 mg/l,and 25.0 mg/lcould stimulate the proliferation of the human fibroblast cells CCC-HSF-1in a dose dependent manner compared to 0 mg/l.The fibrolast cells CCC-HSF-1 under the treatment of 25.0 mg/lAPS2-1 for 48 h were the best concentration and time.The numbers of migrated cells after the treatment of 25.0 mg/l APS2-1 for 24 h,48 h and 72h were more than control.As the cultured time increased,the migration cell number was also increased.The results suggested that APS2-1 promote cell cycle progression by increasing percentage of S and G2/M phase cells and decreasing percentage of G0/G1 phase cells,leading to promotion of cell division cycle at the concentration of 25.0 mg/l.As compared with control,25.0mg/l APS2-1 can significantly regulate cyclin D1 mRNA expression in CCC-HSF-1 cells and accelerate cell cycle progression and promoting cell proliferation.APS2-1could significantly decreased the level of phosphorylation of IκBα,and inhibited the translocation of NF-κB p65subunit from cytoplasm to the nucleus.Conclusions1.The extraction and purification methods for astragalus polysaccharide were established in ourlaboratory.Ultraviolet and infrared spectroscopy identification methods for astragaluspolysaccharide were also established in our laboratory.2.Astragalus polysaccharidepromotes the healing of skin wounds in mice through cttractingfibroblasts,promoting reepithelialization,promoting revascularization.3.APS2-1could stimulate the proliferation,migration of the human fibroblast cells.APS2-1promote cell cycle progression by increasing percentage of S and G2/M phase cells anddecreasing percentage of G0/G1 phase cells,leading to promotion of cell division cycle.APS2-1 can significantly regulate cyclin D1 mRNA expression in CCC-HSF-1 cells andaccelerate cell cycle progression and promoting cell proliferation.APS2-1 promotingexpression of cytokine TGF-beta 1,FGF-2 and EGF and promoting expressionof cell cycleregulatory proteins Cyclin D1.APS2-1could significantly decreased the level of phosphorylation of IκBα,and inhibited the translocation of NF-κB p65 subunit from cytoplasm to the nucleus.