Protective Effects And Mechanisms Of Danshen Yin’s Extracts On Myocardial Ischemia Injury Model Of Rats

Coronary heart disease(CHD),is a disease resulting from myocardial ischemia and ischemia caused by coronary vascular stenosis or occlusion related with coronary atherosclerosis or(and)functional changes(narrow),which is also known as the ischemic heart disease.Dans hen Yin,a Chinese decoction recorded in the book of Shi Fang Miao Yong written by Chen Xiuyuan in Qing Dynasty,now is still commonly used for clinical treatment of CHD and the effect is remarkable.In the experimental researches of recent years,it was found that Danshen Yin had several pharmacological effects such as the functions of anti-myocardial fibrosis,anti-apoptosis,and anti-coagulation,affecting the myocardial enzymes,reducing the levels of inflammation factors and improving the damage of myocardial ischemia.However,there are still a lot of unknowns about the specific mechanisms of myocardial protection of Danshen Yin and it needs constant experiments to explore.Therefore,in this topic we are trying to observe the protection on the myocardial ischemic injury and to explore the possible mechanisms of Danshen Yin’s extracts(DSYE)from different angles and levels.Objective1.Build the myocardial ischemic injury rat model to observe the influences on the serum myocardial enzymology level,myocardial infarction area,and pathological changes of the ischemic myocardium in rats,in order to investigate the protective effects of DSYE on the ischemic myocardium.2.Observe the influences on the oxidative stress and the PI3K-Akt signal pathway in the ischemic myocardium,so that to explore the mechanisms of DSYE in protecting the ischemic myocardium.MethodsThe rats were induced to the myocardial ischemia model by coronary artery ligation,and ten of which just by threading without ligation as the sham operation group.The successful model rats were randomly divided into six groups:untreated model group,group treated by Compound Danshen Dripping Pills(CSDP),group treated by Nicorandil Tablets,group treated by DSYE with a low dose,group treated by DSYE with a midium dose,and group treated by DSYE with a high dose,with ten rats in each group.The drugs were administered correspondingly via intragastric administration once a day for a month,while the 10 rats of the sham operation group were given the same volume of purified water per day.Two hours after the last administration,the rats were anesthetized for electrocardiogram monitoring(ECG),and then collect the blood samples of rats via abdominal aorta,and quickly remove the hearts of rats by thoracotomy at last.Automatic biochemical analyzer was used to detect the levels of serum creatine kinase isoenzyme(CK-MB)and lactate dehydrogenase(LDH).Optical and electron microscope were used to observe the pathological changes of myocardial tissue and myocardial cell ultrastructure respectively,and TUNEL assay was applied to determine the myocardial apoptosis in rats.Biochemical methods was adopted to detect the levels of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and malondialdehyde(MDA).Immunohistochemistry was used to detect the expression levels of Bax/Bcl-2 protein in myocardial tissue.And the expression levels of Akt,p-Akt,GSK-3β,p-GSK-3β and Caspase-3 proteins were examined by western blot(WB).Results1.Levels of the serum myocardial enzymology:The CK-MB and LDH levels of rats in the model group were significantly higher than the sham operation group(P<0.01).The CK-MB and LDH levels of rats in the three DSYE groups were all lower than the model group,and the differences in the midium and high dose group were statistically significant(P<0.05).2.The infarction area of ischemic myocardial:Compared with the model group,the infarction areas of ischemic myocardial in rats treated by DSYE with a midium or high dose were reduced significantly(P<0.05);low dose of DSYE could partly diminish the infarction area,but the difference was not significant(P>0.05).3.The pathological changes of myocardial tissues:Compared with the sham operation rats,the myocardial tissues in the model group were found with fibrosis,swelling and necrosis,and telangiectasia and severe inflammatory cell infiltration,and even some myocardial were found with coagulation necrosis,vacuolation and inflammatory foci;Compared with the model group,the midium and high dose of DSYE relieved the fibrosis,swelling and necrosis of the myocardial tissues,as well as the inflammatory cell infiltration,with the inflammatory foci and vacuolation disappearing.4.Changes of myocardial ultrastructure:In the sham operation rats,the myocardial cells shaped well with intact mitochondrias and the myocardial fibers arranged orderly.In the model rats,the myocardial mitochondrias were swelling like vacuolizations or even dissolved,the myocardial fibers arranged disorderly,and the mesenchymal cells appeared collagen deposition,showing that the myocardial structural damages were serious.Rats in the low dose of DSYE group showed no obvious mitigation in myocardial cell damage.Myocardial cell damages of rats treated by the midium and high dose of DSYE were improved with the oval nucleus substantially intact and the mitochondria swelling reduced.5.Apoptosis index of cardiomyocytes:Compared with the sham operation rats,the apoptosis index of cardiomyocytes in the model rats was increased significantly(P<0.01).Compared with the model group(P<0.01),DSYE reduced the apoptosis index,the difference in low dose group was not significant(P>0.05),while the differences in the midium and high dose groups were significant(P<0.01 or P<0.05).6.Oxidative stress in myocardial tissue:Compared with sham operation group,the SOD and GSH-Px activities in myocardial were both decreased(P<0.01),while the MDA level was increased(P<0.01)significantlyin the model rats.Compared with the model group,the GSH-Px activities in the low and high dose DSYE groups were increased significantly(P<0.05 or P<0.01);the SOD activities in the midium and high dose DSYE groups were increased markedly(P<0.05 or P<0.01);and the MDA levels were significantly reduced by the three DSYE groups(P<0.01).7.Influence on PI3K-Akt signal pathway:The immunohistochemical results showed that the expression levels of the apoptotic proteins Bax and Caspase-3 were higher and the antiapoptotic protein Bcl-2 was lower in the model rats than that in the sham operation rats,significantly(P<0.01);Compared with the model group,DSYE with different doses reduced the Bax and Caspase-3 expression levels while enhanced the Bcl-2 levels(P<0.01).The WB detection results showed that the differences of the levels of total Akt and GSK-3β in each group were not statistically significant(P>0.05);the levels of P-Akt and P-GSK-3β were significantly decreased in the model group,compared with the sham operation group(P<0.05 or P<0.01);and DSYE enhanced the levels of P-Akt protein significantly with a midium and high dose,as well as the P-GSK-3β protein,contrasted to the model group(P<0.05).Conclusion1.DSYE could play a protective role on the myocardial ischemic injury rat models by reducing the serum enzyme levels and myocardial infarct size in a dose dependent manner.2.DSYE was able to improve the changes of the myocardial pathological morphology and the myocardial cell ultrastructure,reduce the ischemic myocardial apoptosis index,hinting that DSYE had an effect on protecting the damaged myocardium and inhibiting the cardiomyocyte apoptosis.3.DSYE could increase the vitality of SOD and GSH-Px(oxygen free radical scavenging enzymes)and reduce the content of MDA(a peroxidation product)in myocardial,suggesting that it was possible for DSYE to suppress the oxidative stress state,which might be a reason for reducing the myocardial cell injury.4.DSYE might prompt the expression of apoptosis inhibitory protein Bcl-2,cut down the expression of pro-apoptotic protein Bax and Caspase-3,which was probably one of the reasons for the inhibition of myocardial apoptosis.5.DSYE could increase the phosphorylation levels of Akt and GSK-3β protein.P-Akt accelerates the Bad phosphorylated and separated from the Bcl-XL so that the Bcl-XL restores the inhibition effect on pro-apoptotic protein Bax/Bak;P-Akt can also boost the activity of p-GSK-3β to bring the reductions of the Ca2+ entering mitochondria and the ROS generating.Therefore,the results suggested that DSYE might play a role in maintaining the integrity of mitochondrial structure and function,and regulating the expressions of apoptosis-related protein genes by affecting the PI3K-Akt signaling pathway,which might be a molecular mechanism of the protective effect on ischemic myocardium.In summary,our results indicated that DSYE had several protective effects on the myocardial ischemic model rats,including reducing the serum enzyme levels and infarct size,improving the changes of the myocardial pathological morphology and the myocardial cell ultrastructure,and inhibiting the cardiomyocyte apoptosis.The possible mechanisms of DSYE might be related to the mitigation of mitochondrial damage and cell apoptosis by inhibiting the oxidative stress and influencing the PI3K-Akt signaling pathway in cardiomyocytes.