Role of Hcdc14A in the in vitro diabetes environment-induced human brain vascular endothelial cell injury

Abstract

Objective To investigate the effects of the in vitro diabetes environment such as high glucose, high free fatty acid(FFA), and hypoxia on human cell division cycle protein 14A(Hcdc14A)and related cyclins expressions, cell proliferation and apoptosis in human brain vascular endothelial cells(HBVEC), and to explore the new mechanism of HBVEC injury induced by diabetes. Methods HBVECs were stimulated respectively with high glucose, high FFA, and hypoxia under different conditions. The mRNA and protein levels of Hcdc14A were detected by RT-PCR and Western blot. At the same time, the protein levels of cyclin B, cyclin D, cyclin E, and p53 were detected by Western blot. The cell proliferation was tested by XTT. The cell apoptosis was detected by AnnexinV/PI. Results After HBVECs were incubated with various concentrations of glucose(10, 15, and 25 mmol/L)and FFA(50, 100, and 200 μmol/L)or with 25 mmol/L glucose and 200 μmol/L FFA for 24, 48, and 72 h, respectively, the mRNA and protein expressions of Hcdc14A were dose- and time-dependently decreased, along with decreased cyclin B, cyclin D, and cyclin E protein expressions and increased p53 protein expression(all P<0.05). Treatment with hypoxia showed a similar result. Compared with control, high glucose, FFA, and hypoxia, the combined intervention of high glucose+ FFA+ hypoxia significantly downregulated the mRNA and protein(all P<0.05)expressions of Hcdc14A, along with decreased cyclin B, cyclin D, and cyclin E protein expressions and increased p53 protein expression(all P<0.05). Compared with control, high glucose, FFA, and hypoxia, the combined intervention of above stimuli obviously reduced the proliferation of HBVECs(40.7%±12.1% vs 100.0%±10.2%, 67.5%±12.1%, 55.3%±11.2%, and 64.4%±11.3%, all P<0.05)and increased the cell apoptosis(22.5%±1.77% vs 5.7%±0.83%, 9.3%±0.95%, 16.3%±1.69%, and 16.3%±1.15%, all P<0.05). Conclusion The findings suggest that Hcdc14A may play an important role in diabetes-induced injury of brain vascular endothelial cells, and seems to be one of the major signaling pathways involved in cell injury. (Chin J Endocrinol Metab, 2015, 31: 59-65) Key words: Human cell division cycle protein 14A; Human brain vascular endothelial cells; High glucose; Free fatty acid; Hypoxia