Objective To observe the effect of tetramethypyrazine (TMP) on the expression of hypoxia-related factors in human umbilical vein endothelial cells (HUVECs). Methods The second to fifth passage cultured HUVECs were divided into five groups: control group, CoCl2induced hypoxic group and 50, 100, 200 mu;mol/L TMP treatment groups. HUVECs in control group were not treated. HUVECs inCoCl2induced hypoxic group were treated with 150 mu;mol/LCoCl2for four hours. HUVECs in 50, 100, 200 mu;mol/L TMP treated groups were pretreated with 150 mu;mol/LCoCl2 for four hours, followed by treatment with 50, 100, 200 mu;mol/L TMP for eight hours. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA levels of prolyl hydroxylase 2 (PHD2), hypoxia-induced factor-1alpha;(HIF-1alpha;) and vascular endothelial growth factor (VEGF). Protein levels of PHD2, HIF-1alpha;, and VEGF were detected using Western blot. Results Compared with the control group, theCoCl2 induced hypoxic group showed decreased mRNA and protein levels of PHD2 (t=3.734, 3.122;P＜0.05), while those of HIF-1alpha; and VEGF increased (HIF-1alpha; mRNA:t=4.589,P＜0.05; HIF-1alpha; protein:t=3.778,P＜0.05. VEGF mRNA:t=3.926,P＜0.05; VEGF protein:t=3.257,P＜0.05). Compared with theCoCl2 induced hypoxic group, 50, 100, 200 mu;mol/L TMP treated groups showed increased mRNA and protein levels of PHD2 (PHD2 mRNA: t=3.286, 3.617, 3.886;P＜0.05. PHD2 protein: t=2.813, 3.026, 3.078; P＜0.05); while those of VEGF decreased (VEGF mRNA: 50 mu;mol/L TMP: t=1.696,P＞0.05; 100 mu;mol/L TMP:t=2.974,P＜0.05; 200 mu;mol/L TMP: t=3.492,P＜0.05; VEGF protein: 50 mu;mol/L TMP: t=1.986,P＞0.05; 100 mu;mol/L TMP: t=2.976,P＜0.05; 200 mu;mol/L TMP:t=3.136,P＜0.05); although changes in HIF-1alpha;mRNA levels were not statistically significant (t=1.025, 0.726, -1.386;P＞0.05), showed a decrease in HIF-1alpha;protein levels (50 mu;mol/L TMP: t=2.056,P＞0.05; 100 mu;mol/L TMP:t=3.058,P＜0.05; 200 mu;mol/L TMP:t=3.828,P＜0.05). ConclusionIn HUVECs, TMP can upregulate the mRNA and protein expression of PHD2, while down regulating HIF-1alpha; protein expression and VEGF mRNA and protein expression under acute hypoxic conditions.
Objective To observe the influence of prolyl hydroxylase 2 (PHD2) expression on endothelial barrier dysfunction induced by high glucose in human retinal vascular endothelial cells (HRECs). Methods The HRECs were treated by different culture medium with various glucose concentrations (5 mmol/L glucose, 5 mmol/L glucose +25 mmol/L mannitol, 30 mmol/L glucose) as normal control group, mannitol control group and high glucose group, respectively. After the cells cultured for 24 and 48 hours, the protein levels of PHD2, hypoxia-inducible factor-1alpha; (HIF-1alpha;) and occludin was detected by Western blot; the expression of vascular endothelial growth factor (VEGF) in the supernatant was determined by enzymelinked immuno sorbent assay (ELISA); the transcription levels of PHD2, HIF-1alpha;, VEGF and occludin were determined by the reversetranscription polymerase chain reaction (RT-PCR); the paracellular permeability between endotheliums was detected by 7times;104 molecular weight FITCdextran. Results Compared with normal control group, the protein level of PHD2 in mannitol control group and high glucose group firstly decreased and then increased, the protein level of HIF-1alpha; increased while that of occludin decreased; the secretion of VEGF increased in high glucose group but not in mannitol control group (PHD2：F=7.618, 8.627;P＜0.05. HIF-1alpha;：chi;2=7.692, 7.652;P＜0.05. occludin：F=23.23, 7.317;P＜0.05. VEGF：F=10.768, 4.562; P＜0.05). Compared with normal control group, the mRNA levels of PHD2, HIF-1alpha;, VEGF and occludin in mannitol control group and high glucose group increased (PHD2：F=5.69, 14.27;P＜0.05. HIF-1alpha;：F=6.07, 10.47;P＜0.05. VEGF：F=12.31, 9.14;P＜0.05. occludin：F=8.77, 8.00;P＜0.05). Compared with normal control group, the paracellular permeability of mannitol control group and high glucose group increased (chi;2=20.57,F=56.09;P＜0.05). Conclusions High glucose induced altered expression of PHD2 which might play an important role in endothelial barrier dysfunction. The mechanism might be associated with HIF-1alpha; and VEGF.