ABSTRACT
Objective: To study the effect and mechanism of Angelica dahurica extract (AD) on neovascularization maturation in db/db mice. Methods: Forty-eight 8-week-old male db/db mice were randomly divided into model group and A. dahurica extract-treated group, and 24 littermate male db/m mice were set as control group. A mouse model of diabetic ulcer was prepared by punching the back. The A. dahuricae extract-treated group was administered with AD 1.8 g/kg ig, and control group and model group were ig administered with the same amount of normal saline for 21 d. The healing condition of mouse wounds was recorded at 2, 4, 7, 11, 14 and 21 d after trauma. The number and maturation of new blood vessels in wound tissues was observed by HE, immunohistochemistry and immunofluorescence staining at 11 d after trauma. Expression of fork head transcription factor (FOXO1) and angiopoietin-2 (Ang-2) in wound tissues were detected by Western blotting and immunohistochemistry. According to different intervention conditions in vitro, endothelial cells were divided into normal glucose and hypoxia group, high glucose and hypoxia group, hypertonic and hypoxic group, high glucose and hypoxia + A. dahurica extract-treated group. Endothelial cells were co-cultured with pericytes under different intervention conditions, and the effects of A. dahurica intervention on endothelial cells' chemotaxis and tubular structure formation in vitro under high glucose and hypoxia were observed. Western blotting was used to detect the levels of FOXO1, p-FOXO1 and Ang-2 proteins and their upstream protein kinase B (Akt) and phosphorylation protein changes in wound tissues and endothelial cells. Results: The wound healing rate of the A. dahurica extract-treated group, the general recovery of the wound, the number of blood vessels and the maturation of new blood vessels in the wound tissue were significantly better than the model group (P < 0.05). FOXO1 and Ang-2 protein expression levels in the A. dahurica extract-treated group were significantly higher than the model group (P < 0.05); p-Akt and p-FOXO1 protein expression in the A. dahurica extract-treated group were lower than model group (P < 0.05). After A. dahurica intervention in endothelial cells under high glucose and hypoxia in vitro, the expression levels of FOXO1 and Ang-2 protein in the cells were significantly reduced (P < 0.05), p-Akt and p-FOXO1 protein expression were significantly increased (P < 0.05); In addition, A. dahurica extract could increase the chemotaxis of endothelial cells to pericytes under high glucose and hypoxia and promote the formation of tubular structures of endothelial cells and pericytes in vitro under high glucose and hypoxia. Conclusion: A. dahurica extract can promote the formation and maturation of neovascularization, so as to improve the rate and quality of wound healing in db/db mice. The mechanism may be related to the down-regulation of FOXO1/Ang-2 pathway, the recruitment of endothelial cells to pericytes, and the promotion of the maturation of wound new blood vessels.
ABSTRACT
OBJECTIVE: To study the transport properties of puerarin across Caco-2 cell membrane and determine the influence of Radix Angelicae Dahuricae extract on the transport of puerarin using the well-characterized, human-based intestinal Caco-2 cell model as a platform. METHODS: The characteristics of the bidirectional transport of puerarin was investigated. The effects of time, drug concentration, pH, P-gp inhibitor and MRP inhibitor on the absorption of puerarin were observed. Then the influence of the extract of Radix Angelicae Dahuricae on the transport of puerarin was studied. Drug concentration was measured by HPLC and the apparent permeability coefficients (Papp) and apparent permeability ratio (PDR) were calculated. RESULTS: Puerarin was better absorbed in weak acid of pH5.5. The Ka and Papp of puerarin was of concentration-dependent, and the transport showed saturation with increasing time and concentration. When P-gp/MRP inhibitors were added to the model, the Papp AP-BL of puerarin in Caco-2 cell increased and the PappBL-AP/PappAP-BL puerarin reduced. The absorption of puerarin was improved when combined with Radix Angelicae Dahuricae. CONCLUSION The intestinal absorption of puerarin in Caco-2 cell monolayer model is mainly a passive diffusion process, and active transportation mediated by P-gp and MRP transporter is also involved. Radix Angelicae Dahuricae can enhance the intestinal absorption of puerarin.