| Objective:All current cardiovascular disease as the first cause of death,and the incidence of cardiovascular disease,the development is closely associated with endothelial dysfunction.Through the study of vascular endothelial function to reveal the pathogenesis of cardiovascular disease has become an important research method.The mitochondrial autophagy is a research hotspot,homocysteine has been proved to be risk factors for cardiovascular disease,the same homocysteine can induce endothelial cells autophagy of mitochondria is worth exploring.It is now found that patients with high homocysteine are often accompanied by high copper(Cu),so the effect of the combined effects of Hcy and Cu on endothelial cells is worth exploring.However,many laboratories with vascular endothelial cell differentiation serious,too much,the experimental results of the said service effect.Through the experiments of extraction of primary human umbilical vein endothelial cells,to explore the mechanism of homocysteine combined with copper damage on vascular endothelial cell line,whether there is the involvement of mitochondrial autophagy,autophagy is beneficial or harmful,and the intervention effect of selenium on the possible role.Metholds:(1)Extraction of primary human umbilical vein endothelial cells.Umbilical vein endothelial cells extracted from newborn umbilical cord.(2)This study we use MTT method to find the effects of different lelvels of Hcy,Cu,Se,Hcy+Cu and Hcy+Cu+Se on cell proliferation.Sulfate solution and selenite sodium solution for copper ion and Se sources.We divide the experiment like the following groups:(1)simple Hcy on cell proliferation effects of Hcy concentrations were 10 ?M,20?M,40?M,100?M,200?M;(2)pure copper and pure selenium on cell proliferation and Cu concentrations were 10 ?M,20?M and 30?M,40?M,50?M,Se concentrations were 0.4?M,1?M,2?M,5?M,10?M;(3)plus copper is increased Hcy inhibited proliferation.The combination of concentration of Hcy 40?M +Cu 10?M,Hcy 40?M +Cu 20?M Hcy40?M+Cu 30?M,Hcy40?M+Cu40?M,Hcy40?M+Cu50?M,Hcy and Cu treatment time for 48 hours.(3)MTT method was used to explore the optimum Se concentration.Through the methods,we found that Hcy have obvious inhibition on cell proliferation from 40?M,in the limited concentration range of the experimental Cu and Se,only with copper or pure selenium had no obvious effect on cell proliferation,while the Hcy concentration is 40?M,with different concentrations of Cu were increased the inhibition of cell proliferation.This study selected Hcy 40?M +Cu 40?M as a model of human umbilical vein endothelial cell damage,to investigate the effects of selenium by adding different concentrations of selenium.The experiment was divided into normal control group,Hcy 40?M +Cu 40?M +Se 0.4?M group,Hcy 40?M +Cu 40?M +Se 1?M group,Hcy 40?M +Cu 40?M +Se 2?M group,Hcy 40?M +Cu 40?M +Se 5?M group,Hcy 40?M +Cu 40?M +Se 10?M group.The same treatment method.The cell type Hcy+Cu flow detection technology,Hcy+Cu+Se group ROS content,to explore the effects of Se content on ROS formation.DCFH-DA fluorescent probes were used to detect reactive oxygen species,which were divided into normal control group,Hcy 40?M +Cu 40?M group,Hcy 40?M+Cu 40? M+Se 2?M group.The cell type Hcy+Cu flow detection technology,Hcy+Cu+Se group of mitochondrial membrane potentialJC-1 dye was used to detect mitochondrial membrane potential.The experiment was divided into two groups: normal control group,Hcy 40?M+Cu 40?M group,Hcy 40? M+Cu 40?M+Se 2?M.The cell type Hcy+Cu flow detection technology,Hcy+Cu+Se group apoptosis rateV Annexin method was used to detect the cell apoptosis rate.The experiment was divided into normal control group,Hcy 40?M+Cu 40 ?M group,Hcy 40?M+Cu 40?M+Se 2?M group.Seventhly,Western blot method was used to detect the expression of LC3 and PINK1 protein expression,study the Hcy+Cu increase the possibility of human umbilical vein endothelial cells,mitochondrial autophagy,and Se intervention effect.The experiment was divided into normal control group,Hcy 40 ?M+Cu 40 ?M group,Hcy 40?M+Cu 40?M+Se 2?M group,Western-blot detection of LC3 PINK1 protein expression,to explore whether Hcu+Cu can lead to increased autophagy.result:(1)the primary human umbilical vein endothelial cells were extracted successfully.(2)40?M and above Hcy inhibited cell proliferation,P<0.05,and increased with the increase of Hcy concentration,the combined action of P<0.05.Hcy and Cu could aggravate the inhibition of cell proliferation.(3)0.4?M to 5?M Se for Hcy+Cu induced cell proliferation inhibition in,P<0.05,and the concentration of selenium was 2?M,the most obvious inhibition effect was,P<0.05.(4)the ROS content in Hcy+Cu+Se group was lower than that in Hcy+Cu group(P<0.05),but higher than that in Ctrl group(P<0.05).(5)the membrane potential of Hcy+Cu+Se group was higher than that of Hcy+Cu group(P<0.05),but lower than that of Ctrl group(P<0.05).(6)the apoptosis rate of Hcy+Cu+Se group was lower than that of Hcy+Cu group(P<0.05),but higher than that of Ctrl group(P<0.05).(7)the LC3II/LC3 I gray value of Hcy+Cu+Se group was lower than that of Hcy+Cu group(P<0.05),but higher than that of Ctrl group(P<0.05);the gray value of PINK1 group was lower than that of Hcy+Cu group(P<0.05),but higher than that of Ctrl group(P<0.05)(Hcy+Cu+Se).Conclusions:1.Homocysteine could inhibit the proliferation of human umbilical vein endothelial cells at 40?M concentration,the higher the homocysteine concentration,the more significant the cell proliferation inhibition was.2.Cu increased homocysteine inhibited proliferation of human umbilical vein endothelial cells.3.Selenium can reduce the effect of Cu and homocysteine on human umbilical vein endothelial cell proliferation,and is more suitable for the human umbilical vein endothelial cells of the selenium concentration is 2 M.4.Mitochondrial autophagy is involved in the mechanism of Cu and homocysteine damage in endothelial cells,which may be excessive and harmful,while selenium can mitigate this effect.5.The Cu and homocysteine may be through increased intracellular ROS and the PINK1/Parkin pathway by mitochondrial autophagy excessive damage and selenium may by reducing ROS inhibit the excessive mitochondrial autophagy. |
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