Study On The Injury Mechanism Of HUVECs Induced By Homocysteine And Explore The Protective Effect Of Pae

Nowadays experimental and clinical studies provide convincing evidence that homocysteine is an independent risk factor for atherosclerosis, and it plays an important role in the pathogenesis of atherosclerosis and cardiovascular diseases. Despite several cellular mechanisms have been proposed and confirmed, how do hyperhomocysteine contribute to atherosclerosis remain unclear. So exploring pathogenesis of atherosclerosis has important academic value and social significance.Paeonol is the primary active component of a traditional Chinese medicine Moutan Cortex. It has a wide range of pharmacological activities, and the cardiovascular benefits are anti-atherosclerosis, anti-thrombus,anti-arhythmia. Up to now, some reports have indicated that Paeonol has beneficial effects on endothelial cells and it can decrease the progression of atherosclerosis in vivo. However, there are no experimental researches on cellular models injuried by homocysteine which study the protective effect of Paeonol. In this article, we developed human umbilical endothelial cellular models exposed to homocysteine. We observed the potentially beneficial effects of Paeonol, and we explored the molecular mechanisms of Paeonol. Paeonol may be used to prevent and remedy atherosclerosis in clinical in the future, and our study will provide valuable experimental and theoretical basis.Part I Study on the injury mechanism of the human umbilical veins endothelial cells (HUVECs) induced by homocysteine(Hcy)Objective:To investigate the effects of homocysteine on the NF-kB pathway, eNOS and the content of NO in the human umbilical veins endothelial cells and explore the possible molecular mechanisms in which Hcy induce endothelial cells injury.This study will provide new targets for the prevention and therapy of atherosclerosis.Methods:1. Isolation and cultivation of HUVECsHUVECs were obtained using the complex enzymes of0.1%I collagenase and0.25%trypsin-0.02%EDTA by the technique of irrigative digestion, and were cultured with DMEM supplemented with20%fetal bovine serum20μg/L bFGF in an atmosphere of5%CO2at37℃in the culture incubator, and then were passaged using0.125%trypsin-0.01%EDTA digestion.2. Experimental group:We used one or two passages cells in our study. HUVECs were divided into normal control group (HO)and different dose groups of Hcy. Normal control group cells were cultured with DMEM containing20%fetal bovine serum,20μg/L bFGF. Different dose groups of Hcy were randomly divided into4groups (H1,H2,H3,H4),and each group was treated with2.5mmol/L Hcy,5mmol/L Hcy,10mmol/L Hcy,15mmol/L Hcy except DMEM containing20%fetal bovine serum,20μg/L bFGF. Each group was continued to culture for24hours.3. The morphological changes of HUVECs were observed in each group respectively with inverted microscope.4. Cell viability was detected in HUVECs in each group by the method of MTT.5. RT-PCR was used to measure the expression of NF-kB p65, eNOS mRNA in HUVECs in each group.6. Western blotting was used to detect the expression of IkB-α, ICAM-1protein in HUVECs in each group.7. Immunohistochemistry was used to observe the change of NF-κB p65nuclear translocation8. NO was detected in the cultured supernate of HUVECs in each group by the method of nitric acid reductase.Results: 1. Isolation and cultivation of HUVECs:Primary cultured HUVECs showed a typical cobblestone monolayer or cobblestone mosaic arrangement. The morphology and growth patterns of passage cells were similar to primary cultured cells.2. The morphological changes of HUVECs observed were as follows in each group respectively with inverted microscope.Compared with the normal control group, H1group had no significant changes in cell morphology, but H2group cells became round and the gap widened. H3, H4group cells clustered, and the cell lining was selectively lost. There were more cells shedding.3. Cell viability was detected by the method of MTT.The cell viability in H1group had no significant difference compared with nomal control group (P>0.05). The cell viability in the other groups (H2, H3, H4) decreased obviously compared with nomal control group (P<0.01).4. The expression of NF-kB p65, eNOS mRNA in HUVECs in each group detected by RT-PCR.4.1The expression of NF-kB p65mRNA:Compared with nomal control group, The expression of NF-kB p65mRNA in Hcy dose groups increased significantly (P<0.05,P<0.01) in a dose-dependent manner (P<0.05).4.2The expression of eNOS mRNA:The eNOS mRNA expression in H1group had no significant difference compared with nomal control group (P>0.05). The eNOS mRNA expression in the other groups was remarkably lower than that of nomal control group in a dose-dependent manner (P<0.05).5. The expression of IkB-α, ICAM-1protein in HUVECs in each group detected by Western blotting.5.1The expression of IkB-α protein:The expression of IkB-α protein in Hcy dose groups reduced significantly compared with nomal control group (P<0.05, P<0.01) in a dose-dependent manner (P<0.05).5.2The expression of ICAM-1protein:The expression of ICAM-1protein in Hey dose groups increased significantly compared with nomal control group (P<0.05, P<0.01).6. Immunohistochemistry was used to observe the change of NF-κB p65nuclear translocation.NF-κB p65positive expression products were buffy particles. In the normal control group, NF-κB p65almost did not have the positive particles in the cells. The buffy particles in H1group were few. The buffy particles in the other groups increased obviously. Buffy particles in the nucleus increased significantly with the increasing concentrations of Hey indicating a clear tendency of nuclear translocation. Compared with the normal control group, the positive NF-kB p65expression particles increased significantly in Hey dose groups (P<0.01).7. NO was detected in the cultured supernate of HUVECs in each group by the method of nitric acid reductase.Compared with the normal control group, the content of NO in the cultured supernate of Hey dose groups decreased gradully in a concentration-dependent manner (P<0.05, P<0.01).Conclusions:1. Hey can increase the expression of NF-kB p65mRNA obviously, and increase the degradation of IκB-α, allowing the translocation of active NF-kB into the nucleus.Then, NF-kB up-regulates the expression of intercellular adhesion molecule-1. Taken together, Hey can lead to atherosclerosis in the end.2. Hey can inhibit the expression of eNOS mRNA obviously, so it can reduce the synthesis of eNOS and decrease the activation of eNOS. Therefore, NO production was reduced. So Hcy can accelerate the pathogenesis and development of atherosclerosis. Part II Explore the protective effect of Pae on HUVECs injured by homocysteine(Hcy)Objective:We used human umbilical vein endothelial cells (HUVECs) as the research objects, and we set up damaged endothelial cells models induced by homocysteine. We investigated the effects of Paeonol on the NF-kB pathway, eNOS and the content of NO in the endothelial cells injured by homocysteine. We explored the protective effects of Paeonol and its anti-atherosclerotic molecular mechanisms.Methods:1. Experimental group:We divided the HUVECs into5groups randomly: normal control group, Hcy injured model group (10mmol/L Hcy), the low-dose group of Pae (10mmol/L Hcy+0.15mmol/L Pae), the middle-dose group of Pae (10mmol/L Hcy+0.3mmol/L Pae), the high-dose group of Pae (10mmol/L Hcy+0.6mmol/L Pae).2. The morphological changes of HUVECs were observed in each group respectively with inverted microscope.3. Cell viability was detected in HUVECs in each group by the method of MTT.4. RT-PCR was used to measure the expression of NF-kB p65, eNOS mRNA in HUVECs in each group.5. Western blotting was used to detect the expression of IkB-α, ICAM-1protein in HUVECs in each group.6. Immunohistochemistry was used to observe the change of NF-kB p65nuclear translocation.7. NO was detected in the cultured supernate of HUVECs in each group by the method of nitric acid reductase.Results:1. The morphological changes of HUVECs were observed in each group respectively with inverted microscope.Compared with normal control group, Hcy injured model group showed flake separation and shedding phenomenon. In Pae dose groups the intercellular space became narrow, and the cell shape tended to be normal.2. Cell viability was detected in HUVECs in each group by the method of MTT.The cell viability in Hcy injured model group decreased obviously compared with nomal control group (P<0.01). The cell viability in the low-dose、the middle-dose and the high-dose group of Pae was remarkably higher than that of Hcy injured model group (P<0.05).3. The expression of NF-kB p65, eNOS mRNA in HUVECs in each group detected by RT-PCR.3.1The expression of NF-kB p65mRNA:Compared with nomal control group, NF-kB p65mRNA expression in Hcy injured model group increased significantly (P<0.01). NF-kB p65mRNA expression in the low-dose, the middle-dose and the high-dose group of Pae was remarkably lower than that of Hcy model group (P<0.05, P<0.01) and they were in a dose-dependent manner (P<0.05).3.2The expression of eNOS mRNA:Compared with nomal control group, eNOS mRNA expression in Hcy injured model group decreased significantly (P<0.01). The eNOS mRNA expression in the low-dose, the middle-dose and the high-dose group of Pae was remarkably increased than that of Hcy injured model group (P<0.01) and they were in a concentration-dependent manner (P<0.05).4. Western blotting was used to detect the expression of IkB-α, ICAM-1protein in HUVECs in each group.4.1The expression of IkB-α protein:Compared with nomal control group, IkB-α protein expression in Hcy injured model group decreased significantly (P<0.01). IkB-α protein expression in the low-dose, the middle-dose and the high-dose group of Pae was remarkably increased than that of Hcy model group (P<0.01).4.2The expression of ICAM-1protein:Compared with nomal control group, ICAM-1protein expression in Hcy injured model group increased significantly (P<0.01). ICAM-1protein expression in the low-dose, the middle-dose and the high-dose group of Pae was remarkably lower than that of Hcy injured model group (P<0.05, P<0.01).5. Immunohistochemistry was used to observe the change of NF-kB p65nuclear translocation.The buffy particles located in nucleus and cytoplasm in Hcy injured model group. Compared with the normal control group, the difference was significant in Hcy injured model group (P<0.01). In Pae low-dose group, there were still many positive particles in the nucleus, and the positive particles in the nucleus in Pae middle-dose group decreased significantly. Furthermore, there were almost no buffy particles in the nucleus in Pae high-dose group. Compared with Hcy injured model group, the positive NF-kB p65expression particles in the nucleus was significantly reduced in Pae dose groups (P<0.01).6. NO was detected in the cultured supernate of HUVECs in each group by the method of nitric acid reductase.Compared with nomal control group, the content of NO in the cultured supernate of Hcy injured model group decreased significantly (P<0.01). The content of NO in Pae dose groups was remarkably higher than that of model group (P<0.01) and they were in a concentration-dependent manner (P<0.05).Conclusions:1. Pae played a protective role on endothelial cells injured by Hcy.2. Pae inhibitd the expression of NF-kB mRNA in endothelial cells injured by Hcy, and then Pae suppressed the activation of NF-kB. Pae down-regulated the expression of ICAM-1protein in a result.3. Pae improved the gene expression of eNOS, and then Pae increased the content of NO in endothelial cells injured by Hcy.