Protective Effects Of Quercetin On Hyperthyroidism-induced Cardiovascular Dysfunction Via ANP/NO Signaling Pathyway

BackgroundQuercetin(QUE)is a monomeric active ingredient found in various Chinese herbal medicines and belongs to the class of polyhydroxylated flavonoids.It has been demonstrated that QUE is involved in the regulation of cardiac hypertrophy and atherosclerosis through anti-inflammatory and anti-oxidative effects,but its protective effect on the cardiovascular damage caused by hyperthyroidism is not yet clear.PurposeTo explore the protective effect and mechanism of QUE on cardiovascular injury caused by hyperthyroidism,and to provide theoretical basis for the prevention and treatment of hyperthyroidism cardiovascular disease.Method(1)Establishment of a hyperthyroid rat model: Subcutaneous injection of L-thyroxine(0.05 mg/kg)was given subcutaneously for 14 days.(2)Experimental groups: Control group,T4 group,QUE-L+T4 group,QUE-M+T4group,QUE-H+T4 group.L,M,and H were 10,30,and 60 mg/kg,gavage 2 weeks in advance.(3)Heart rate and blood pressure detection: Non-invasive caudal artery detection technique for detecting rats(4)Detection of Plasma Active Components: Detection of atrial natriuretic peptide(ANP)by radioimmunoassay,detection of nitric oxide(NO)using nitrate reductase method,and determination of lactate by pyruvate dinitrophenylhydrazine Lactate dehydrogenase(LDH),detection of superoxide dismutase(SOD)by WST-1.(5)Histomorphological observation: HE staining was used to observe heart tissue andaorta.(6)Atrial pressure and ANP secretion level detection: Atrial pressure was measured in isolated rat atrial perfusion and ANP secretion was measured by radioimmunoassay.(7)Protein detection: Western blot detection of NPR-A,Akt,eNOS and P-Akt,P-eNOS content in heart tissue and aorta.(8)Cell viability assay: MTT assay was used to observe cell viability.(9)Cell NO secretion assay: The cell culture medium was assayed for NO levels by nitrate reductase assay.(10)Apoptosis detection: Apoptosis of cell was detected by Annexin V-FITC and PI double staining.Result1.Effect of QUE on plasma active components and hemodynamics in hyperthyroid rats(1)Compared with the control group,heart and body ratio,heart rate and blood pressure of 1T4 group were significantly higher.QUE had an improvement effect on the abnormal increase of heart-body ratio,heart rate,and blood pressure of hyperthyroid rat.(2)Plasma active ingredients: Plasma SOD levels in the T4 group were significantly lower than those in the control group.QUE treatment increased hyperthermia in the hyperthyroid rat plasma;NO levels in the T4 group were significantly lower than those in the control group.QUE intervention in hyperthyroidism rat plasma The level of NO was increased;the level of plasma LDH was significantly higher in the T4 group than in the control group.The plasma LDH level in the HG group was decreased after the QUE intervention;the level of plasma ANP in the T4 group was significantly lower than that in the control group.QUE intervention in hyperthyroidism The level of ANP in the plasma of rats increased.2.Protective effect of QUE on cardiac damage caused by hyperthyroidism and its mechanism(1)HE staining of cardiac tissue showed that hyperthyroid rats had severe myocardial hypertrophy,severely impaired myocardial interstitial space,and disordered myocardial cells.QUE had protective effects on myocardial tissue injury in hyperthyroid rats.(2)Atrial pressure : When QUE 60 mg/kg was used,there was a decrease in atrial pressure in normal rats;when QUE 10,30,and 60 mg/kg were used,the effect of ACh on reducing atrial pressure in the control group was enhanced;T4 group was larger.Rat ACh inducedatrial pressure change was significantly lower than the control group,using QUE 60 mg/kg intervention ACh promoted atrial pressure changes significantly;T4 rats ACh secretion of atrial secretion of ANP significantly reduced compared with the control group,using QUE60 The effect of ACh on the secretion of ANP in the atria was significantly increased by mg/kg intervention.The above results suggest that QUE improves the ACh-induced atrial pressure and atrial ANP secretion in hyperthyroid rats.(3)Expression of protein in myocardial tissue: The expression of NPR-A in myocardial tissue of T4 group was significantly lower than that of control group.The expression of NPR-A in myocardial tissue of QUE intervention group was significantly increased;Akt,p-Akt in myocardial tissue of T4 group rats Compared with the control group,the expression of Akt and p-Akt was significantly increased in the QUE intervention group;the expression of eNOS and p-eNOS in the myocardial tissue of the T4 group was significantly lower than that in the control group.The eNOS,myocardial tissue in the QUE intervention group was significantly decreased.The expression of p-eNOS was significantly increased,suggesting that QUE increased the expression of eNOS-NO signaling pathway in myocardial tissue of hyperthyroid rats.(4)H9C2 cell viability assay: 0-20 ?M QUE had no effect on the survival rate of H9C2,while 10 ?M T4 significantly inhibited cell survival of H9C2.QUE intervention could improve the survival of H9C2 induced by 10 ?M T4.Detection of NO in 5H9C2 cell culture fluid: The level of NO in the T4 group was significantly lower than that in the control group.QUE increased the NO level in the H9C2 culture medium stimulated by T4.3.Protective effect of QUE on vascular endothelial cell damage caused by T4 and its mechanism(1)Aortic HE staining: The thoracic aorta of the hyperthyroidism was severely deformed and varied in thickness,and the intact endothelial layer could not be seen.The elasticity of the fibrous layer disappeared and the nucleus was arranged disorderly.For a large degree of recovery,the endovascular layer shows intact endothelium.(2)Protein detection in thoracic aorta: The expression of NPR-A in thoracic aorta of T4 group was significantly lower than that in control group,and the expression of NPR-A in thoracic aorta of QUE intervention group was significantly increased;in thoracic aorta of T4 group rats The expression of Akt and p-Akt was significantly lower than that of the control group.The expression of Akt and p-Akt in the thoracic aorta of the QUE intervention group was significantly increased.The expression of eNOS and p-eNOS in the thoracic aorta of theT4 group was significantly lower than that of the control group.The expression of eNOS and p-eNOS in thoracic aorta was significantly increased in QUE intervention group,which indicated that QUE increased the expression of eNOS-NO signaling pathway in thoracic aorta of hyperthyroid rats.The results suggest that QUE increases the expression of NPR-A in hyperthyroid rats and may increase the expression of eNOS-NO signaling pathway.(3)HUVECs cell viability assay: 0-10 ?M QUE had no effect on the survival rate of HUVECs,5 ?M T4 significantly inhibited the cell viability of HUVECs,and QUE improved the survival rate of HUVECs induced by 5 ?M T4.(4)Detection of NO in HUVECs cell culture medium: The level of NO in the T4 group was significantly lower than that in the control group.QUE increased the NO level in the culture medium of HUVECs stimulated by T4.(5)HUVECs apoptosis detection: QUE reduced the apoptosis rate of HUVECs induced by 5 ?M T4.ConclusionQUE prevents hyperthyroidism-induced cardiovascular injury through ANP and NO signaling pathways.