| Objective:As the natural umbrella of the blood vessels, the endothelial cells can synthesize andsecrete a variety of biological active substances, to regulate blood pressure and bloodcoagulation and anticoagulation equilibrium.Homocysteine can increase the risk ofcardiovascular disease, which induces apoptosis of endothelial cells.Metformin is anantidiabetic drugs, a study found that metformin can reduce the risk of breast cancerand cardioascular disease.Our study was designed to investigate whether metforminreduces endothelial cell injury caused by homocysteine and its possible mechanism.Methods:(1) The study observed the different solubility of homocysteine (Hcy) effect onvascular endothelial cell activity.The experiment was divided into normal group, Hcy group, vascular endothelialcells inoculated drug treatment after24hours. Hcy group were using250μM,500μM,1000μM and2000μM solubility of Hcy treated24hours;With CCK8kitsdetect each cell activity after drug treatment.(2) The study observed the effect of different concentrations of Metformin(Metformin) effects on vascular endothelial cell activityThe experiment was divided into normal group, the Merformin group, vascularendothelial cell vaccination after24hours of drug treatment. Merformin groups wereusing250μM,500μM,1000μM and2000μM solubility of Merformin treated24hours With CCK8kits each cell detect activity after drug treatment.(3) Our study explored the role and mechanism of Merformin intervention bybuilding the Hcy model of vascular endothelial cell injury.The experiment was divided into normal group, Hcy group, Merformin group,Hcy+Merformin group.Cells inoculated medication after24hours.Hcy group to join1000μM solubility Hcy, Merformin group to join1000μM solubility Merformin, Hcy+Merformin group at the same time to join1000μM solubility of Hcy and1000μMMerformin solubility, and work together for24hours.To detect the following aspects: groups of CCK8test cell activity, the determination of endothelial in lactatedehydrogenase (LDH), total superoxide dismutase (SOD) and glutathione peroxidase(gsh-px) activity and trace malondialdehyde (MDA) level, NADPHmRNA qRT-PCRtest, SODmRNA and CATmRNA expression, Western Blot test p-AMPK alpha and t-AMPK alpha protein expression.Results:①the vascular endothelial cell activity(1) compared with normal the group, the cell activity of low solubility Hcygroup (P <0.05), and with the increase of Hcy concentration, cell activity decreased.(2) compared with normal group, each group of solubility Merformin cellactivity has no obvious change (P>0.05), and endothelial cell activity has nothing todo with Merformin concentration.(3) compared with normal group, Hcy group cell activity were lower (P<0.05),Merformin group had no obvious effect on cell activity (P>0.05), and cell activity inthe Hcy group high than the Hcy+Merformin group (P <0.05).②the activity of lactate dehydrogenase (LDH)Compared with normal group, the cells of LDH activity high Hcy group(P<0.05), LDH activity Merformin group of cells have no obvious difference(P>0.05), Hcy+low Merformin LDH activity than Hcy group (P<0.05), Merformincan reduce Hcy leads to the rise of the LDH activity.③the total SOD activity in the cellCompared with the normal group, the total SOD activity of cells Hcy grouplower (P<0.05), group Merformin cell total SOD activity has no obvious difference(P>0.05).Hcy+Merformin group total SOD activity is higher than Hcy group (P<0.05), Merformin ease total Hcy leads to the decrease of SOD activity.④malondialdehyde (MDA) levelCompared with normal group, the generation of cell MDA level high Hcy group(P <0.05), Merformin group was no obvious difference was found between thegeneration of cell MDA level (P>0.05).Hcy+Merformin group of cells to theproduction of MDA level level is lower than Hcy group (P <0.05), Merformin canreduce the cell of Hcy leads to the increase of MDA. ⑤glutathione peroxidase (gsh-px) activitiesGroup compared with normal group, Hcy group of glutathione peroxidaseactivity lower (P <0.05), group Merformin cellular glutathione peroxidase activity hasno obvious difference (P>0.05).Hcy+Merformin group of glutathione peroxidaseactivity is higher than Hcy group (P<0.05), Merformin ease Hcy caused by theactivity of glutathione peroxidase reduced.⑥NADPHmRNAexpression quantityCompared with normal group, the expression of cell NADPHmRNA of Hcygroup higher (P<0.05), the expression of Hcy+cells NADPHmRNA Merformingroup amount is lower than Hcy group (P<0.05), Merformin can reduce the amountof the expression of cell NADPHmRNA Hcy leads to the rise.⑦SODmRNAexpression quantityCompared with normal group, the expression of cell SODmRNA of Hcy groupare lower (P<0.05), the expression of SODmRNA Hcy+cells Merformin group highthan Hcy group (P<0.05), Merformin ease Hcy lead to reduce the amount of theexpression of cell SODmRNA.⑧CATmRNAexpression quantityCompared with normal group, the expression of cell CATmRNA ofHcy groupare lower(P<0.05), the expression of Hcy+cells CATmRNA Merformin group higherthan Hcy group (P<0.05), Merformin ease Hcy lead to reduce the amount of theexpression of cell CATmRNA.⑨p-AMPKα and t-AMPKα protein expression.Compared with normal group, the expression of t-AMPKα protein in eachexperimental group had no significant difference (P>0.05);Compared with normalgroup, the expression levels of Hcy group of p-AMPKα protein was no significantdifference e (p>0.05), Merformin and Hcy+Merformin group of p-AMPKα proteinexpression quantity increased (p<0.05).Merformin make fine.Intracellular p-AMPKalpha/t-the ratio of AMPKα increases, so that the phosphorylation of AMPKα levels.Conclusion:①Homocysteine can reduce the activity of endothelial cells, and with theincrease of homocysteine, the greater the effect on the activity of vascular endothelial cell, its influence induced endothelial cell activity and intracellular oxygen freeradicals, MDA, LDH and NADPH level or activity rise, and make the intracellularantioxidant enzymes such as SOD, CAT and gsh-px activity.②Merformin had no obvious effect on vascular endothelial cell activity (P>0.05), and endothelial cell activity unrelated to Merformin concentration.③Metformin can reduce homocysteine on endothelial cell injury and possiblemechanism is through the excited AMPK pathway, increase the phosphorylation ofAMPK, which relieves the oxygen free radicals in cells induced by homocysteine,MDA, LDH and NADPH level or activity rise, antioxidant enzymes such as SOD,CAT and gsh-px activity. |
