The Mechanism Of Angelica Dahurica On Diabetic Wound Healing And Angiogenesis

Objective:The causes of diabetic refractory wound are very complex,vascular dysplasia can hinder the healing of the wound.Angiogenesis is a key stage in wound healing,the maturity of new blood vessels is an important guarantee for the effective healing of chronic wounds.However,there is no effective treatment methods.Angelica dahurica as the traditional Chinese medicine,it is used for carbuncle and sore ulcers in the clinical,but,the mechanism of Angelica dahurica promoting the healing of diabetic wounds is still unclear.This study aims to explore the effect of Angelica dahurica treating diabetic refractory wound,looking for the target and mechanism of Angelica dahurica at the level of experimental animals and cells,it expands that there is promising therapeutic potential for Angelica dahurica in diabetic wounds,and,promote the traditional Chinese medicine combine with the modern medicine.Methods:1.db /db Eight-week-old male db/db(C57BLKS/J-lepr /lepr)mice were used as an animal model,and heterozygous control db/m mice were used as a control.A sterile 6-mm punch biopsy tool was used to create two wounds on the back of the mice.The db/db mice(low,middle,high experimental groups,n = 9)received A.dahurica at a dose of 1.8 g/kg,3.6 g/kg,7.2g/kg once daily by oral gavage,while mice in the db/m normal control(n = 9)and db/db experimental control(n = 9)groups received an equal amount of physiological saline once daily.Some of the animals was euthanized at day4,7,11,21 post-injury.The wounds were taken for analysis by use immunofluorescence double staining,q PCR and Western Blotting etc.In order to explore the mechanism of Angelica dahurica in treating diabetic wound healing.2.HUVECs as the research object,A Cell Counting Kit 8(CCK-8)was used to investigate the viability of A.dahurica on HUVECs,Cell proliferation was assessed with an EdU test.The activation of Angelica dahurica on the PI3K/AKT signaling pathway by use the Western Blotting,as well as the expression of PDGF-β,to verify the mechanism of Angelica dahurica to promote angiogenesis by use LY294002;Western Blotting and immunofluorescence techniques were used to detect the expression of HIF-1α under high glucose and hypoxia conditions.Using the Western Blotting to detect the expression of PDGF-β after HUVECs were transfected with HIF-1α-si RNA kits and p Receiver-M11-HIF-1α kits.3.HBVPs as the research object,A Cell Counting Kit 8(CCK-8)was used to investigate the viability of A.dahurica on HBVPs,Cell proliferation was assessed with an EdU test and Transwell.The activation of Angelica dahurica on the PI3K/AKT signaling pathway by use the Western Blotting,as well as the expression of PDGFR-β,to verify the mechanism of Angelica dahurica to promote angiogenesis by use LY294002;Western Blotting and immunofluorescence techniques were used to detect the expression of HIF-1α under high glucose and hypoxia conditions.Using the Western Blotting to detect the expression of PDGFR-β after HBVPs were transfected with HIF-1α-si RNA kits and p Receiver-M11-HIF-1α kits.Results:1.Compared with db/m mice,the healing rate of db/db mice was significantly slower.A.dahurica treatment accelerated wound closure in db/db mice at a rate that was faster than that of db/m mice.There was no significant difference in blood glucose,liver and kidney function,and body weight of diabetic mice during drug intervention.2.RT-PCR and Western Blotting results revealed that A.dahurica significantly enhanced the expression of HIF-1α and PDGF-β/ PDGFR-β.The levels of HIF-1α and PDGF-β/ PDGFR-βwere reduced in the db/db group compared with those of the db/m group,but HIF-1α and PDGF-β/ PDGFR-βlevels were upregulated in the A.dahurica treatment group compared to the db/db group,especially the A.dahurica.L.3.HIF-1α and PDGF-β/ PDGFR-β in the A.dahurica group was higher than that in the db/db group.CD31+ CD146 double staining showed that the numbers of newly formed and mature blood vessels were higher in the A.dahurica group than in the db/db group.4.Cell experiments show that high glucose and hypoxia can induce the damage of HUVECs and HBVPs,and it can be seen that the viability of these two cells decreases;Angelica dahurica has a protective effect on the HUVECs and HBVPs which induced by high glucose and hypoxia.5.In HUVECs and HBVPs,A.dahurica promoted cell proliferation and tube formation,which was accompanied by increased nuclear translocation of HIF-1α under hypoxic conditions and led to elevated PDGF-β/PDGFR-β protein expression.A.dahurica activated the PI3K/AKT signaling pathway in human HUVECs and HBVPs,which were abrogated by the PI3 K inhibitor LY294002.Furthermore,the cellular expression of PDGF-β/PDGFR-β decreased significantly after treatment with a HIF-1α-si RNA,and PDGF-β/PDGFR-β expression were increased in HIF-1α-overexpressing cells.Conclusions:1.Angelica dahurica can significantly improve the healing of diabetic wounds.2.Angelica dahurica accelerated wound healing may related to the expression of HIF-1α,PDGF-β/PDGFR-β increased,which suggested that A.dahurica has a proangiogenic function.3.A.dahurica has potential as a therapy for promoting angiogenesis in diabetic wounds through mediated regulation of HIF-1α/PDGF-β through PI3K/AKT signaling pathways.