| Objective To observe the effect of the simplified formula of Tuo-Li-Xiao-Du powder which contained Astragalus membranaceus,Angelica sinensis,Radix angelicae,Gleditsia sinensis on the wound angiogenesis in diabetic rats.Analyzed the chemical components.Used network pharmacology methods to explore the target and potential mechanism of angiogenesis in the diabetic rat wounds and futher verified it.Methods 1.Diabetic rats were induced by intravenous injection of a single high dose of STZ.The rats subsequently underwent procedures of dorsal skin puncturing to establish a diabetic animal model of cutaneous wound.Rats were randomly assigned to a normal control group(NC),diabetic group(DM)and a simplified formula of TLXDS group(DM+TLXDS).Randomized blood glucose in each group was measured after 3 days,5days,1week and 2weeks of STZ injection.Wound healing rates were calculated at 3rd day,7th day,11 th day and 14 th day after trauma.The granulation tissue of wounds in rats of each group at 5 days,8days,11 days and 14 days after trauma was analyzed.HE staining was used to observe the morphological changes of the wounds.Ki67 and CD31 staning was used to observe angiogenesis in wounds.2.UPLC-Q-TOF/MS was applied to analyze the components in the simplified formula of TLXDS.Molecular docking technology was used to predict the targets of these components.The function and pathway annotations of target protein was performed through relevant databases such as Uniprot and KEGG.The drug components-targetfunction diagram was constructed using Cytoscape software.3.Human umbilical vein endothelial cells were cultured in vitro.CCK-8 assay was used to detecte cell activity.Western blotting was used to detect the signals related to angiogenesis protein expression of the drug components.Results 一、Animal experiment results 1.Wound healing rate of rats in each group: After the rats were modeled,the skin of DM group was slightly thinner than that of the NC group.Compared with the NC group,the wound healing of DM group was significantly delayed.The wound healing rate was lower than that in the NC group(P<0.05).The wound healing rate in the DM+TLXDS group was significantly higher than that in the DM group 2 days after the administration.There was no significant difference in blood glucose between the diabetic rats during the drug intervention period.2.immunohistochemical staining showed that Ki67 and vascular endothelial cell proliferation marker CD31 in DM group were decreased compared with those in the NC group.The expression of Ki67 and CD31 in wounds of DM+TLXDS group was significantly higher than that in the DM group.二、Network pharmacological analysis results 1.UPLC-Q-TOF/MS drug components analysis: A total of 28 compounds were identified from the simplified formula of TLXDS in positive and negative ion modes.These compounds contain flavonoids,saponins,coumarin,steroids and other ingredients.2.Molecular docking and network pharmacology analysis: The results of molecular docking showed that the 17 compounds contained in the assay interacted with 17 target proteins,with a total of 210 pairs of component-target relationships.Among them,5 targets were related to immune regulation,6 targets were related to antibacterial and anti-inflammatory effects,6 targets were related to cell differentiation,10 targets were related to cell migration,6 targets were related to angiogenesis,2 targets were related to stimulation of epithelial growth factor,6 targets were related to vasodilation and 2 targets were related to estrogen.Further network pharmacological analysis showed that the flavonoids,saponins,coumarins,steroids and anthraquinones contained in this formula could regulate the related signal pathways through the regulation of related target proteins to jointly produce antibacterial and anti-inflammatory,immune regulation and angiogenesis and other biological effects.三、The results of tissue and cell experiments The effects of astragaloside,flavonoids and nodakenin on the expression of PI3K/Akt/e NOS signaling pathway proteins: The results of Western Blotting showed that astragaloside IV could affect the expression of PI3 K protein in endothelial cells.Astragaloside II and nodakenin could affect the expression of downstream Akt phosphorylation protein,Calycosin could further affect the expression of e NOS phosphorylation protein.The drug components showed a synergistic effect.Conclusions 1.The simplified formula of Tuo-Li-Xiao-Du powder could effectively improve the wound healing by promoting angiogenesis in wounds of diabetic rats.2.The flavonoids,saponins,coumarins,steroids and anthraquinones contained in this formula could be the main material basis for the drug’s efficacy.Some signal pathways regulated by these components might be the potential molecular mechanisms to the TLDXS treatment in diabetic wound healing.3.The simplified formula of Tuo-Li-Xiao-Du powder could effectively promote the vascularization of the wounds.Its molecular mechanism of promoting angiogenesis may be related to the regulation of the PI3K/AKt/e NOS pathway by the drug components contained in Astragalus membranaceus and Angelica. |
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