ABSTRACT
Objective To investigate the effect of ginkgo diterpene lactones on hypoxia-induced apoptosis and angiogenesis of human umbilical vein endothelial cells (HUVECs) and the related molecular mechanisms. Methods HUVECs were cultured under hypoxia for 24 h, and then treated with ginkgo diterpene lactones (low-dose: 6.25 mg/L and high-dose: 25.00 mg/L). MTT assay was used to detect the cell activity. Flow cytometry was used to detect the apoptosis of HUVECs. Transwell assay was employed to detect the migration of HUVECs. Quantitative real-time polymerase chain reaction and Western blotting were used to test the expression levels of mRNA and protein of hypoxia-inducible factor 1α (HIF-1α), apoptosis-related genes (B-cell lymphoma 2［Bcl-2］and B-cell lymphoma 2-related X protein［Bax］), and angiogenesis-related genes (vascular endothelial growth factor［VEGF］and transforming growth factor β［TGF-β］). Results Compared with the normal group, the HUVEC activity was significantly decreased after exposed to hypoxia for 24 h (P＜0.05), and the apoptosis rate of HUVECs and mRNA and protein expression levels of HIF-1α were significantly higher (all P＜0.05). The cell activity and migration ability of HUVECs were significantly higher in the low- and high-dose ginkgo diterpene lactones groups than those in the hypoxia group (all P＜0.05), and the apoptosis rates of HUVECs were significantly lower than those in the hypoxia group (both P＜0.05). Meanwhile, the cell activity and migration ability were significantly higher in the high-dose group than those in the low-dose group (both P＜0.05), and the apoptosis rate was significantly lower than that in the low-dose group (P＜0.05). The mRNA and protein expression levels of HIF-1α and Bax were lower in the low- and high-dose ginkgo diterpene lactones groups than those in the hypoxia group, and the mRNA and protein expression levels of Bcl-2, VEGF and TGF-β were higher than those in the hypoxia group; and the expression changes of the above genes were more significant in the high-dose group. Conclusion Ginkgo diterpene lactones can improve hypoxia-induced apoptosis and angiogenesis of HUVECs by regulating the expression levels of HIF-1α and apoptosis- and angiogenesis-related genes, and it might be used as a new agent to treat anoxic vascular diseases.
ABSTRACT
Ginkgo diterpene lactones meglumine injection (GDLI) is a commercially available product used for neuroprotection. However, the pharmacokinetic properties of the prototypes and hydrolyzed carboxylic forms of the primary components in GDLI, i.e., ginkgolide A (GA), ginkgolide B (GB), and ginkgolide K (GK), have never been fully evaluated in beagle dogs. In this work, a simple, sensitive, and reliable method based on ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) was developed, and the prototypes and total amounts of GA, GB, and GK were determined in beagle dog plasma. The plasma concentrations of the hydrolyzed carboxylic forms were calculated by subtracting the prototype concentrations from the total lactone concentrations. For the first time, the pharmacokinetics of GA, GB, and GK were fully assessed in three forms, i.e., the prototypes, the hydrolyzed carboxylic forms, and the total amounts, after intravenous administration of GDLI in beagle dogs. It was shown that ginkgolides primarily existed in the hydrolyzed form in plasma, and the ratio of hydrolysates to prototype forms of GA and GB decreased gradually to a homeostatic ratio. All of the three forms of the three ginkgolides showed linear exposure of AUC to the dosages. GA, GB, and GK showed a constant half-life approximately 2.7, 3.4, and 1.2 h, respectively, which were consistent for the forms at three dose levels (0.3, 1.0, and 3.0 mg·kg) and after a consecutive injection of GDLI for 7 days (1.0 mg·kg).
Subject(s)
Animals , Dogs , Ginkgo biloba , Chemistry , Ginkgolides , Pharmacokinetics , Lactones , Pharmacokinetics , Plant Extracts , Pharmacokinetics , Tandem Mass SpectrometryABSTRACT
Objective: To obtain the monoclonal antibodies against the protein of ginkgo diterpene lactones meglumine injection. Methods: After total proteins of ginkgo leaf running two-dimensional electrophoresis, the protein spots were digged and analyzed by mass spectrometry (MS). According to the result of MS, the polypeptide was synthesized and used to immunize the BALB/c mice. The indirect ELISA was utilized to develop monoclonal antibody after cell fusion between SP2/0 cells and spleen cells from the immunized BALB/c mice. Results: The immunoglobulin subtypes of five monoclonal antibodies were IgM and light chain was Kappa identified with a commercial capture-ELISA kit. Western blotting analysis also indicated that the monoclonal antibodies were specific to the protein of ginkgo leaf. Conclusion: The experiment would be helpful for the establishment of detection method specific to the proteins of ginkgo diterpene lactones meglumine injection in the future study.