| [Objective] To study the effects of glucose concentration on expreesion and transcriptive activity of hypoxia-inducible factor-1?under the hypoxia condition, and to explore the possible molecular mechanism of it as well as the Shexiang Baoxinwan intervention.[Methods] The HUVECs cultured in vitro were divided into four groups:normoxia and (21%O2) coupled with 5.5mM glucose, normoxia coupled with 25mM glucose, hypoxia (1%O2) coupled with 5.5mM glucose and hypoxia coupled with 25mM glucose. The cells were treated with 25mM glucose concentration for 48h and hypoxia for 6h. The expression levels of HIF-la mRNA and VEGF mRNA were measured by realtime PCR. HIF-la protein and its ubiquitin mediated degradation were detected by Western Blot. Luciferase reporter genes, containing the EPO enhancer region or full-length VEGF promoter construct, were used to identify the hypoxia induced HIF-la transactivation. On the basis of above studise, the gene and the protein level of the factor inhibiting HIF-1 (FIH), were detected in cells treated by gradient glucose concentration (5.5mM?15mM?20mM?25mM?45mM) for 48h, and normoxia or hypoxia for 6h. An alternative method, based on reported derivatization methods to give a fluorescent derivative, was used to detect the HIF enzymatic activity. The location of FIH in HUVECs was observed by using cell immunofluorescence technique. In the study of medicine intervation, HUVECs were treated for 48h with different concentrations of Shexiang Baoxinwan. The HUVECs'proliferation was detected by using CCK8 kit. The cellular migration ability was mesured by methods of Transwell plates and the scratch test, respectively, and the cell apoptosis was detect by Annexin/PI through flow cytometer. Then the expression levels of HIF-la and FIH genes and proteins were measured by realtime PCR and Western Blot, respectively.[Results] Compared with the normoxia groups (glucose concentrations of both 5.5mM and 25mM), HIF-la of both protein expression and transactivation were significantly increased, its ubiquitin mediated degradation was significantly decreased in hypoxia groups (all P<0.05). However, further normoxia and hypoxia conditions, the different glucose concentrations were not found to impact the relative gene and protein expressions of HIF-la. Exposure to high glucose (25mM) mainly impaired hypoxic induced HIF-1?transactivation and VEGF expression (P<0.05), moreover, deferoxamine (DFO) could turnover the depression of high glucose to hypoxia induced HIF-la transactivation. Both the relative gene expression and protein expressions of FIH were attenuated in HUVECs grown in hypoxia than in normoxia (P<0.05). In the hypoxia condition, with the gradient increased glucose concentration, FIH expressions (both mRNA and protein) in the groups of 20mM and 25mM glucose concentrations were significantly higher than in the group of 5.5mM glucose concentration (P<0.05), and its enzymatic activity in the group of 25mM glucose concentration was also increased compared with that in the group of 5.5mM glucose concentration (P<0.05). 0.1?g/ml Shexiang Baoxinwan could increase proliferation of HUVEC. The cellular migration ability was promoted when the concentration of Shexiang Baoxinwan is 0.1?g/ml and 1?g/ml, 1?g/ml Shexiang Baoxinwan could reduce cell apopsis. On the opposite,5?g/ml Shexiang Baoxinwan would inhibit the HUVECs proliferation and migration ability, but increase the apopsis. The relative gene expression and protein expressions of FIH were attenuated in the setting of Shexiang Baoxinwan, especially 2?g/ml concentration (P<0.05). However, Western blot analysis failed to demonstrate significantly change in HIF-la stability in the setting of Shexiang Baoxinwan?[Conclusion] High glucose concentration impaired the hypoxia induced transactivition of HIF-la, and the possible mechanism is that FIH expression and enzymatic activity increase in the condition of hypoxia and high glucose. Low concentration of Shexiang Baoxinwan could attenuate FIH expression. |
PDF Full Text Request