The Effect And Mechanism Of Angelica Dahurica(AD) On Skin Ulcer In Type 2 Diabetic Mice

ObjectiveIn this study diabetic C57BLKS/Jdb/db（db/db）male mice and homologous male C57BLKS/Jdb/+（db/m）mice of the same age were punched on the back to establish a diabetic skin ulcer mouse model.After interventional treatment of Angelicae dahuricae,evaluate its effects on wound inflammation dissipation,angiogenesis and maturation,collagen deposition,etc.The mechanism of action of Angelica dahurica was further explored in vitro.Method1.144 male,8-week-old db/db mice and 36 male homologous male db/m mice.Anesthetize with phenobarbital after 1 week of adaptive feeding.A skin ulcer model was prepared by symmetrical perforation of the skin on the back with a diameter of6 mm.The dead mice were removed.The mice was randomly divide into db/m group,db/db group and Angelica administration group（db/db+ADL,db/db+ADM,db/db+ADH）.The low-dose,middle-dose and high-dose groups were given 1.8g/kg,3.6g/kg and 7.2g/kg of Angelica dahurica respectively.The db/m and db/db groups were given the same amount of normal saline.Once a day for 21 days.A group of mice were sacrificed at 4,7,11,21 days after trauma,and wound tissues were taken for analysis.HE,Masson,and immunohistochemical staining were used to observe changes in granulation tissue structure such as wound inflammation,angiogenesis,and collagen precipitation;immunofluorescence double staining and Western Bloting were used to detect the changes of macrophage polarization and neovascularization in db/db mouse wound tissue after AD intervention.2.After pretreatment of macrophages with AD in vitro,the macrophages are induced to polarize.Cell immunofluorescence and Western Bloting are used to observe the effect of AD on the polarization of macrophages to M1 and M2.At the same time,the transwell chamber was used to co-culture macrophages and endothelial cells,and the chemotaxis of polarized macrophages after AD pretreatment to endothelial cells was observed.3.Human umbilical vein endothelial cells and human brain perivascular cells were cultured in vitro.and give 1umol/LCo Cl₂ to make the endothelial cells chemically hypoxic.Endothelial cells are divided into normal glycemia hypoxia group（NG+C）,hyperglycemia and hypoxia group（HG+C）,hypertonic hypoxia group（MA+C）,hyperglycemia and hypoxia+ADgroup（HG+C+AD）.The transwell co-cultivation experiment and matrigel tube formation experiment were used to observe the effect of AD intervention on endothelial cells in hyperglycemia and hypoxia and the ability of pericytes to co-tube formation,and the expression of FOXO1 and Ang-2 was detected in endothelial cells under hyperglycemia and hypoxia after AD treatment.Result1.Effects of AD on inflammation,neovascularization and collagen precipitation on the wound tissue of db/db miceThe general situation and pathological changes of the wound surface:from 4days to 21 days after trauma,the healing rate of each dose group of AD was higher than that of the db/db group（P<0.05）.Early stage of wound healing,inflammatory exudation and the number of inflammatory cells on the wound tissue of db/db mice are significantly reduced after AD treatment.In the stage of wound proliferation,neovascularization,collagen fiber formation and wound granulation tissue formation increased on the wound tissue of db/db mice after AD intervention.Among them,the healing rate and healing situation of the db/db+ADL group was always significantly better than that of the other two groups（P<0.05）.2.Effect of AD on the polarization of macrophages in the early inflammatory phase of wound healing in db/db diabetic miceImmunofluorescence staining and cell fluorescence shown AD treatment decreased M1 phenotype marker（iNOS）expression,and increased M2 phenotype marker（Arg-1）and endothelial cell marker（CD31）expression.iNOS,IL1-β,TNF-αand SOCS3 was inhibited by AD in vivo and vitro.While the expressions of Arg-1,VEGF and SOCS1 was increased.We also found in vitro M1 macrophages after AD intervention accelerated endothelial cell migration compared with M1 by transwell co-culture experiment.3.Effects of AD on the maturation of neovascularization in the proliferation stage of db/db diabetic miceAfter AD treatment,the fluorescent staining ofα-SMA in the pericellular markers of neovascularization in db/db mice was significantly enhanced.After AD intervention,the expression levels of FOXO1 and Ang-2 protein in db/db mouse wound tissues and endothelial cells under high glucose hypoxia were significantly reduced（P<0.05）,and p-AKT and p-FOXO1 protein expression increased（P<0.05）.Co-culture of endothelial cells and pericytes under high glucose hypoxia after AD intervention in vitro found that AD can promote the chemotaxis of endothelial cells to pericytes under high glucose hypoxia（P<0.05）,and promote high glucose hypoxia Tubular structures of endothelial cells and pericytes were formed in vitro（P<0.05）.Conclusions1.AD can reduce the inflammation and infiltration of the wounds in the early stage of repair,and promote the angiogenesis and maturation of the wounds in the proliferation stage of the wound repair.Among them,the treatment effect of Angelica dahurica low dose is better than that of middle and high dose.2.AD relieves early inflammation and infiltration in db/db mice wound repair,which may be related to the inhibition of hyperpolarization of macrophages from diabetic mice wounds to M1 by regulating the expression of SOCS1/SOCS3.3.The positive effect of AD on the formation and maturation of neovascularization on wound tissure of db/db mice,as well as on the formation of pericytes and endothelial cells under high glucose and hypoxia in vitro,may be up-regulated with p-AKT to promote the phosphorylation of FOXO1 and inhibit the activation of FOXO1and reduce Ang-2 expression.