Protective Effect And Mechanism Of Osthole On Hyperthyroidism-induced Myocardial Injury

BackgroundOsthole(Ost)is a coumarin compound extracted from the fruit of the Cnidium monnieri(L.)Cuss.Osthole has the effect of anti-inflammatory,anti-oxidation and lowering blood pressure.It was shown that osthole can reduce myocardial ischemia-reperfusion injury and inhibit mitochondria-mediated apoptosis.However,whether osthole has a regulation effect on hyperthyroidism-induced cardiac dysfunction is not clear.ObjectiveThe purpose of the present study was to explore the protective effect and mechanism of osthole on hyperthyroidism-induced myocardial injury.This study will provide theoretical basis for the prevention and treatment of hyperthyroidism-induced cardiac dysfunction.Methods1.Establishment of hyperthyroidism rat model: The rats were subcutaneously injected with L-Thyroxine(T4,0.025 mg/kg/d)for 2 weeks.2.Experimental groups: SD rats were randomly divided into control group(Control,CT),T4 group(T4),osthole+T4 group(Ost 20 mg/kg+T4,Ost 40 mg/kg+T4,Ost 60mg/kg+T4).Rats were gavaged with osthole for two weeks before T4 injection.3.Detection of hemodynamics: Carotid cannulation technology was used to detect the heart rate(HR),systolic pressure(SP),diastolic pressure(DP),mean arterial pressure(MAP),left ventricular systolic pressure(LVSP),left ventricular end diastolic pressure(LVEDP)and left ventricular pressure maximal rise/fall rate(±d P/dtmax)in hyperthyroidism rats.4.Detection of oxidative stress related indicators: The levels of lactate dehydrogenase(LDH),superoxide dismutase(SOD),malonaldehyde(MDA)and nitric oxide(NO)in plasma and cell culture medium were measured by ELIASA.5.Histomorphological observation: HE staining was used to observe the histomorphological change of osthole on hyperthyroidism-induced myocardial injury tissue.6.Detection of protein expression: Western Blot was used to observe the effect of osthole on the expression of Akt/e NOS and Bcl-2/Bax protein in T4-induced injured rat myocardial tissue and H9c2 cells Western Blot.7.Detection of cell survival rate: The effect of osthole on the survival rate of T4 injured H9c2 cells was detected by MTT assay.8.Detection of apoptosis: H9c2 cells were stained by Annexin V-FITC and PI,and then the apoptotic rates were detected by flow cytometry.Results1.Effect of osthole on hyperthyroidism-induced injured rat myocardiumCompared with the control group,the heart-to-body ratio was significantly increased and myocardial tissue damaged severely,the gap became lagged and myocardial cells disordered in the T4 group.The heart rate of T4 rats was faster than that of the control group and the maximum rate of rise and the maximum rate of decline were significantly higher than those of the control group are.In addition systolic pressure,mean arterial pressure,left ventricular systolic pressure,left ventricular end-diastolic pressure is higher than the control group.Compared with the T4 group,osthole intervention can reduce the heart-to-body ratio,tissue damage,lower blood pressure,and recover heart function.Rat plasma test showed that compared with the control group,plasma LDH activity and MDA concentration were significantly increased,SOD activity and NO concentration were significantly decreased in T4 group;Compared with T4 group,osthole intervention could reverse all results.The experimental results show that osthole can slow down heart rate,lower blood pressure,restore heart function and reduce oxidative stress to play its protective role.2.Effect of osthole on T4-injured H9c2 cellsThe results of MTT assay showed that compared with the control group,T4 could lead to a decrease of H9c2 cells survival rate,and the appearance was convex and dead cells number were increased,increased apoptotic rate also can be detected by fluorescent staining;Compared with the T4 group,osthole intervention can significantly increase the survival rate and decreased apoptotic rate of H9c2 cells.After detecting the supernatant of H9c2 cells,we found that compared with the control group,the LDH activity and MDA concentration were significantly increased,SOD activity and NO concentration were significantly decreased in T4 group;Compared with T4 group,osthole intervention can reverse all these indicators.This indicates that osthole protects H9c2 cells from T4 injury is associated with anti-oxidation and anti-apoptosis effects.3.Molecular mechanism of osthole in protecting hyperthyroidism-induced myocardial injuryThe protein detection results showed that compared with the control group,the expression levels of p-Akt and p-e NOS in the myocardial tissue or H9c2 cells in T4 group were significantly decreased;Compared with T4 group,osthole can increase the expression of these proteins.This indicates that osthole protects the myocardium of hyperthyroid rats by promoting the expression of p-Akt and p-e NOS.Compared with the control group,the expression of Bax protein was significantly increased and the protein expression of Bcl-2 was decreased in H9c2 cells of T4 group.Compared with T4 group,osthole intervention could decrease the expression of Bax and increase the expression of Bcl-2.This indicates that osthole exerts its anti-apoptotic effect by regulating the expression of Bax and Bcl-2.ConclusionsOsthole has the protective effect on hyperthyroidism-induced myocardial injury in rats,and its mechanism is closely related to the promotion of NO secretion via the Akt-e NOS signaling pathway,exerting anti-oxidation and anti-apoptosis activities.