| Homocysteine(Hcy)is an independent risk factor for atherosclerotic cardiovascular disease and is associated with the incidence of cardiovascular events.Studies have confirmed that Hcy oxidation process is catalyzed by metal-containing enzyme proteins to produce reactive oxygen species,which leads to endothelial dysfunction.In addition,clinical studies have shown that elevated Hcy in patients with hyperhomocysteinemia(HHcy)is accompanied by elevated plasma Cu2+,and there is a positive correlation between the two,which is especially true in people with cardiovascular disease.common.This indicates that Cu2+ plays an important role in Hcy damage to endothelial cells.And with the new understanding of Hcy-Cu complexes,it was found that Hcy-Cu2+ complex also plays a very important role in the formation of atherosclerosis.ZnMoS4 is a novel nanoparticle ion exchanger with extremely high selectivity for copper ions.It not only penetrates the cell membrane and acts as a copper antidote in the cell,but also maintains the homeostasis of other essential biometal ions.Our previous studies have confirmed that Hcy combined with Cu2+ can significantly aggravate cell damage and reduce cell survival rate compared with Hcy and Cu2+-treated umbilical vein endothelial cells.The mechanism may be that Hcy relies on Cu2+ to activate the glycolytic pathway and NADPH oxidase,inhibit the glutathione(GSH)antioxidant system,and generate too much ONOO-causing endothelial oxidative damage.This experiment will further demonstrate the possible mechanism of Hcy combined with Cu2+ to cause more serious damage and damage to blood vessels in vivo,and to explore the protective effect of ZnMoS4 on the aorta of rats with Hcy-Cu complex injury.This will provide a new target for the treatment of hyperhomocysteinemia.Part 1 Hcy damage to rat aortic endotheliumObjective:To investigate the effect of Hcy on the rat aorta.Methods:A HHcy rat model was established.Ten 6-week-old male Sprague-Dawley rats were randomly divided into 2 groups,5 in each group:①normal methionine group(4.6 g/kg Met);②high methionine group(7.5 g/kg Met);Each group of feeds was fed for 8 weeks,and the body weight of the rats was monitored.After the experiment,the blood vessels of the eyeballs were used to detect the Hcy solubility in the serum.The aorta was dissected and used for the measurement of vascular ring tension,HE staining,transmission electron microscopy,and observation of the rat aorta.Degree of damage;Results:Compared with the normal group,the endothelium-dependent diastolic function of the aorta in the high methionine group was weakened,and the aortic endothelial cells were significantly damaged with mitochondrial damage.Conclusion:Increased concentration of Hey in vivo can damage aortic endothelial cells.Part 2 Mechanism of Hcy Combined with Cu2+ Injury of AortaObjective:To investigate the mechanism of Hcy combined with Cu2+ in the injury of aorta.Methods:Experiment1:30 male Sprague-Dawley rats aged 6 weeks were randomly divided into 6 groups:5 in each group:①low copper control group(1.6 mg/kg Cu);②normal copper control group(10 mg/kg Cu);③ high copper control group(300mg/kg Cu);④low copper + high methionine group(1.6mg/kg Cu + 7.5g/kg Met);⑤ normal copper + high methionine group(10mg/kg Cu + 7.5g/kg Met);⑥high copper + high methionine group(300mg/Kg Cu+7.5g/kg Met);each group of feed for 8 weeks,monitor the body weight of each group,after the end of the experiment,dissect all model rats,take the aorta under the living body for The degree of injury of the rat aorta was observed by vascular ring tension measurement,HE staining and transmission electron microscopy.Experiment 2:20 male Sprague-Dawley rats aged 6 weeks were randomly divided into 4 groups:5 in each group:①normal copper control group(10 mg/kg Cu);②low copper + high methionine group(1.6 mg/kg Cu+7.5)g/kgMet);3③normal copper + high methionine group(10 mg/kg Cu + 7.5 g/kg Met);④high copper + high methionine group(300 mg/kg Cu + 7.5 g/kg Met);each group of feed for 8 weeks,The body weight of each group was monitored.After the experiment,the blood samples were taken from the ocular arteries to measure the serum lactic acid content.All the rats in the model were dissected and the aorta was removed for HE staining.Immunohistochemistry and fluorescence staining were used to detect ROS and NOX activities.Results:Experiment 1:The aortic vasodilation function of rats in normal copper,low copper and high copper group was similar.The pathological results showed no aortic endothelial injury;normal copper + high methionine group,low copper + high methionine group,high copper + high The vasodilation function of the methionine group was significantly weaker than that of the 123 group,and the high copper +high methionine group<normal copper + high methionine group<low copper +high methionine group;endothelial cell destruction,mitochondrial damage increased significantly compared with the first three groups,high copper + high methionine group>normal copper + high methionine group>low copper + high methionine group.Experiment 2:Compared with the normal control group,the expression of aortic hexokinase Ⅱ,gultl,and NOX subunits in the normal copper + high methionine group,high copper + high methionine group increased,ROS and NOX activities were significantly enhanced,and endothelial cells Destruction and mitochondrial damage were severe and proportional to the concentration of copper;the expression 3-NT protein decreased with increasing copper concentration.Conclusion:The content of Cu2+ in serum does not affect the effect of normal Hcy on blood vessels.However,in HHcy,the increase of copper content may aggravate the damage of Hcy on aorta.The mechanism may be through activation of NADPH oxidase and glycolysis.Pathway,producing ROS;generating too much ONOO"causing endothelial cell oxidative damage.Part 3 Protective effect of nano-copper exchanger(ZnMoS4)on Hey injured aortaObjective:To investigate the protective effect of ZnMoS4 on Hcy injured aortaMethods:Experiment 1:Extraction of primary human umbilical vein endothelial cells,grouping of cells:①normal group,②Hcy+Cu2+ group,③Hcy+Cu2+tetrathiomolybdate(TM)group(2,5,10,50 uM/L),④Hcy+Cu2++ nano-copper ion exchanger(ZnMoS4)group(2,10,50,100 uM/L),after 24 hours of dosing,cell viability was detected by flow cytometry;At the same time,the acute drug toxicity test compares the toxicity of both.Experiment 2:15 male Sprague-Dawley rats aged 6 weeks were randomly divided into 3 groups:5 in each group:①normal control group;②high methionine group(7.5 g/kg Met);③high methionine group + nano-copper chelating agent group(7.5 g/kg Met+ZnMoS4):After 1 week of feeding,ZnMoS4(40 ml of ZnMoS4 was added to 2 kg of rat diet)was added.Each group of feeds was fed for 8 weeks.The body weight of the rats was monitored.All the model rats were dissected after anesthesia.The aorta was taken out under the living body for fluorescence detector to test NOX activity,HE staining and transmission electron microscopy.Results:Experiment 1:Hcy+Cu2+ addition of TM and ZnMoS4 with different solubility can improve cell survival rate,but ZnMoS4 is much less toxic than TM.Experiment 2:Compared with the high methionine group,the degree of NOX activity in the vascular ring of the high methionine + ZnMoS" group was significantly reduced,and the degree of endothelial cell destruction and mitochondrial damage was significantly reduced.Conclusion:ZnMoS4 has protective effect on Hcy injured aorta.Part 4 Identification of Hcy and Cu2+ ComplexesObjective:To identify Hcy-Cu2+ complexesMethods:Matrix-assisted laser desorption ionization mass spectrometry(MALDI-TOF)and high performance liquid chromatography-linear ion trap mass spectrometry(ESI-MS)and nuclear magnetic resonance spectrum(NMR).Results:Peak spectra of three mass-nuclear ratios of 365.9m/z,367.9m/z,and 369.98m/z appeared in the chromatogram of high performance liquid chromatography-linear ion trap mass spectrometry.In the nuclear magnetic resonance spectrum,the peak of Hcy-Cu2+ complexes becomes less,widens and shifts.Conclusion:Hcy and Cu2+ can form complexes. |
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