Cadmium Exposure Promotes TMAO To Induce Senescence Of Vascular Endothelial Cells Through Oxidative Stress

BackgroundWith the rapid development of our country’s industrialization process,heavy metal pollution in the environment has caused great harm to human health and has received widespread attention.Cadmium is a heavy metal and an environmental toxicant.It can be gradually enriched to humans in the upper layers of the food chain through air,water,and crops.Today,diet and smoking are the main sources of cadmium exposure in the general population.Studies have shown that cadmium mainly accumulates reactive oxygen species in tissues and affects many organs during oxidative damage.Oxidative stress is usually related to cardiovascular diseases.When oxidative stress affects blood vessels,it can induce endothelial dysfunction,such as vascular endothelial cells.senescence.At the same time,the imbalance of the intestinal flora caused by cadmium will reduce the integrity of the intestinal barrier and induce an increase in intestinal permeability.In addition,due to changes in people’s diet(such as high-fat diets),the production of trimethylamine(TMA),a metabolite dependent on intestinal microbes,is increased.When the integrity of the intestinal barrier is reduced,more TMA passes through the liver.The enzyme FMO3 is oxidized to trimethylamine oxide(TMAO),which can cause various cardiovascular diseases,including senescence of vascular endothelial cells.Therefore,after long-term low-concentration cadmium enters the human body from the digestive tract,whether it will carry TMAO and affect it,thereby aggravating the senescence of vascular endothelial cells,the current research in this area is still insufficient.ObjectiveThis study mainly explores the effects of low concentration of cadmium and TMAO on the senescence of vascular endothelial cells,and explores the mechanism of vascular endothelial cell senescence from the perspective of oxidative stress.MethodsIn vitro experiments are conducted to study the effects of cadmium and TMAO on vascular endothelial cells,mainly starting from oxidative stress.First,prepare cell culture solutions of the following substances with different final concentrations.The experimental group is 300 μmol/L TMAO(TMAO group),and the concentration is 10 μmol/L,30 μmol/L,and 60 μmol/L cadmium chloride(Cadmium group),and add 300 μmol/L TMAO(cadmium+TMAO group)on the basis of each concentration of cadmium chloride,and set up a control group to interfere with human umbilical vein endothelial cells for 24 hours,48 hours,and 72 hours.hour.The relative survival rate of vascular endothelial cells was measured by the MTT method,and the cell growth was observed under an inverted microscope.The cell senescenceβ-galactosidase(SA-β-gal)positive cell ratio was observed by the staining method,and the cell’s growth was observed.Aging situation.Then,the level of telomerase and intercellular adhesion factor-1 were measured by enzyme-linked immunosorbent assay,the reactive oxygen species produced by endothelial cells under different interventions were detected by the fluorescent probe DCFH-DA,which is sensitive to reactive oxygen species,and the intracellular detection by colorimetry The activity of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)and the changes in the content of malondialdehyde(MDA)in cell culture fluid.Western blotting detects the expression of senescence-related proteins(P21 and P26).ResultsThe relative survival rate of endothelial cells:the relative survival rate of cells decreases with the increase of the concentration of cadmium chloride,and decreases with the extension of the intervention time.SA-β-gal:The human umbilical vein endothelial cells treated with 300 μM TMAO,different concentrations of cadmium chloride,and cadmium chloride plus TMAO appear cell senescence,such as the cell morphology becomes larger and flat,and the SA-β-GAL activity increases.Telomerase activity:The activity of telomerase decreased with the increase of cadmium concentration and the increase of interferents.The simple TMAO group,the different concentration of cadmium group and the addition of different concentrations of cadmium on the basis of TMAO were statistically different from the control group(P<0.05).In the 48h intervention group,it can be seen that the telomerase activity of the "cadmium+TMAO"group was lower than that of the "TMAO group"and "cadmium group",and there was a significant difference(P<0.05).Production of reactive oxygen species:The reactive oxygen sensitive fluorescent probe DCFH-DA can produce green fluorescent DCF after reacting with the reactive oxygen species in the cell.After different treatments of endothelial cells,the production of reactive oxygen species in the cell is significantly different.Compared with pure TMAO or pure cadmium chloride under the joint intervention of cadmium chloride+TMAO,stronger green fluorescence can be observed,indicating that strong oxidative stress occurs in the cells to produce more reactive oxygen species.SOD activity:The activity of superoxide dismutase enzyme decreased with the increase of cadmium concentration and the increase of interferents.The simple TMAO group,the different concentration of cadmium group and the addition of different concentrations of cadmium on the basis of TMAO were statistically different from the control group(P<0.05).GSH-Px activity:The activity of glutathione peroxidase decreases with the increase of cadmium concentration and the increase of interfering substances.The different concentration of cadmium group and the addition of different concentration of cadmium on the basis of TMAO were statistically different from the control group(P<0.05).Compared with the control group,the activity of glutathione peroxidase decreased in the TMAO group,But the difference is not obvious(P>0.05).MDA content:The content of MDA increases with the increase of cadmium concentration and interference.The different concentration of cadmium group and the addition of different concentration of cadmium on the basis of TMAO were statistically different from the control group(P<0.05).Compared with the control group,the MDA content of the TMAO group increased,but the difference was not significant(P>0.05).The expression of senescence-related proteins:Compared with the control group,the expression of senescence-related proteins p16 and p21 in the cells of the TMAO group,60μmol/L cadmium group,and 60 μmol/L cadmium+TMAO group increased.Conclusion1.Cadmium can induce the oxidative stress response of vascular endothelial cells alone,generate a large number of reactive oxygen species,and reduce the activity of antioxidant enzymes in cells,which may cause the aging of vascular endothelial cells.2.By giving TMAO and different concentrations of cadmium chloride on human umbilical vein endothelial cells to intervene,compared with TMAO or cadmium chloride group,TMAO exist together with cadmium chloride,under the condition of aging of vascular endothelial cells can be observed beta-galactose glucoside enzyme(SA-β-gal)increased proportion of positive cells,telomerase activity decline,show the presence of both.It makes telomerase lose the function of repairing telomere,promote telomere shortening,and accelerate the aging of vascular endothelial cells.3.The combination of low concentration of cadmium and TMAO can aggravate the senescence of vascular endothelial cells and increase the expression of senescence related proteins p16 and p21 in cells.4.The activities of superoxide dismutase(SOD)and glutathione peroxidase(GSH),the antioxidants used to scavenge ROS,decreased,while the malondialdehyde(MDA),the product of lipid peroxidation,increased,indicating that Cd and TMAO together enhanced oxidative stress of cells.