| Ischemic heart disease is a life-threatening disorder with high morbidity and mortality worldwide,and recovering the blood of the coronary artery in time is considered to be the most effective treatment.However,when blood flows back into the myocardial tissue,the injury induced by ischemia further aggravates.This phenomenon is called myocardial ischemia reperfusion(I/R)injury.Oxidative stress caused by ROS overproduction is one of the important pathogenesis of I/R injury.It was reported that myocardial I/R injury is associated with the activation of ROS/JNK signal pathway.Our previous study found that cytoplasmic JNK is abnormally activated by excessive ROS by hypoxia/reoxygenation(H/R)in the I/R model in vitro,and translocates to the outer membrane of mitochondria to combine with SH3 domain binding protein 5,then initiating downstream signaling pathway to promote ROS production.And then aggravates mitochondrial oxidative stress and and whole cell H/R injury.Mito-Tempo(MT)is a novel mitochondria-targeted antioxidant that can easily cross lipid bilayers and accumulate massively in the mitochondria,which can effectively eliminate mitochondrial ROS.Although some studies have shown that MT have good therapeutic effects on diseases closely related to oxidative stress,and one study has shown that MT can improve the H/R injury of H9C2 cells,the effect and mechanism of MT on the model of myocardial I/R in vivo that is closer to the clinical patients are still unclear.In this study,we established the I/R model of mice by the left anterior descending ligation to investigate whether MT could improve myocardial I/R injury and protect cardiac function.On this basis,we investigated whether the protective effect of MT is related to the elimination of ROS and inhibition of cytoplasmic JNK activation and mitochondrial translocation.Methods1.The myocardial I/R model was established by the left anterior descending ligation of C57BL/6JNifdc mouse.2.The experimental groups were as follows:(1)Sham;(2)Sham+MT;(3)Model;(4)Model+0.5mg/kg MT(0.5 mg/kg MT);(5)Model+1.0 mg/kg MT(1.0 mg/kg MT);(6)Model+2.0 mg/kg MT(2.0 mg/kg MT).3.Method and time of administration: different doses of MT were administered by intravenous injection 15 min before ligation of left anterior descending.4.The myocardial tissues of mice were homogenized by the grinding bead homogenization method,emulsification disperser method,and glass homogenizer method,and mitochondria were separated by differential centrifugation method.5.The isolated mitochondrial membrane potential was detected by JC-1.6.The contents of myocardial enzymes such as creatine kinase(CK),creatine kinase MB(CKMB),lactate dehydrogenase(LDH)and cardiac troponin I(c Tn-I)in serum of mice were detected by biochemical methods.7.The area of myocardial infarction and ischemia in mice were detected by Evans Blue /TTC staining.8.The morphologies and structures of myocardial tissues of mice were observed by HE staining on paraffin sections9.Ultrastructures of myocardial fibers,mitochondria and microvessels were observed by transmission electron microscopy.10.The content of Malondialdehyde(MDA)in myocardial tissue of mice was detected by thiobarbituric acid assay.11.Cardiac functions of mice were detected by echocardiography,including Left ventricular ejection fraction(LVEF),Left ventricular short axis shortening rate(LVFS),Left ventricular end-systolic volume(LVESV),Left ventricular end-diastole volume(LVEDV),Left ventricular inner diameter at end-diastole(LVIDd)and Left ventricular inner diameter at endsystole(LVIDs).12.The levels of total ROS and mitochondrial ROS in myocardial tissue were detected by DHE and Mito-Sox staining.13.The levels of cytoplasmic and mitochondrial p-JNK/total JNK were detected by Western blot.Results1.The level of p-JNK in myocardial tissue showed a trend of increasing at first and then decreasing with the prolongation of reperfusion time in the range of 15-120 min after 45 min’s ischemia.The level of p-JNK reached the peak at 15 min after reperfusion,and began to decrease gradually after 60 min after reperfusion.2.Compared with Sham group,the contents of various myocardial enzymes in serum of mice in Model group increased in early reperfusion,and obvious myocardial infarction appeared in late stage.MT inhibited the above damage changes in a dose-dependent manner.3.Compared with the Sham group,the morphology and structure of the myocardial tissue of mice in the Model group was significantly changed,which showed that the myocardial fiber was broken and dissolved,interstitium was edematous,inflammatory cells infiltrated,and a large number of red cells were exudated in the space between the muscle fibers.MT improved the damage of the above morphology and structure in a dose-dependent manner.At the same time,with transmission electron microscopy,the disorganization in the myofibrils and mitochondrial cristae,and the swollen microvascular endothelial cells were observed.MT improved the untrastructural damage of the above myocardial tissue in the Model group.4.Compared with Sham group,the level of MDA in myocardium of Model group was significantly increased,and the difference was statistically significant(P<0.05).With the increase of MT dose,the level of MDA in myocardium of I/R mice decreased gradually,and the effect of high dose of MT(2.0 mg/kg)was the most obvious in all groups.5.Compared with Sham group,LVEF and LVFS in Model group were significantly decreased,while LVEDV,LVESV,LVEDd,and LVIDs were significantly increased.MT dosedependently increased LVEF and LVFS,and decreased LVEDV,LVESV,LVIDd and LVIDs.6.Compared with Sham group,total ROS and mitochondrial ROS production increased in Model group,and cytoplasmic JNK was abnormally activated and transferred to mitochondria.Compared with the Model group,the contents of total ROS and mitochondrial ROS,as well as the protein expression levels of cytoplasm and mitochondrial p-JNK in MT group were significantly decreased,and the differences were statistically significant(P<0.05).7.Compared with Sham group,the expression level of PGC-1α in myocardium in Model group was decreased,and the difference was statistically significant(P<0.05).The expression level of PGC-1α was significantly increased after MT administration.8.Mitochondria with high purity were obtained by three homogenating methods,and the differences in extraction efficiency were not statistically significant.Compared with the bead homogenizer method,the mitochondrial membrane potential obtained by the emulsion disperser method and the glass homogenizer method decreased,and the differences were statistically significant(P<0.05).The morphology and structure of mitochondria obtained by grinding bead homogenizer were complete,while the morphologies and structures of mitochondria obtained by emulsification disperser and glass homogenizer were incomplete or broken.Conclusions1.MT can dose-dependently improve the morphological and structural damage of myocardial tissue after I/R,decrease the leakages of myocardial enzymes,reduce the infarct area,and protect cardiac function.2.MT can clear cytoplasmic and mitochondrial ROS,inhibit cytoplasmic JNK activation and mitochondrial translocation,and protect mitochondria in myocardial tissue,which might be important mechanisms for its protective effects on myocardial I/R injury.3.The combination of the grinding bead homogenation method and differential centrifugation method can ensure the integrity of the structure and good function of the isolated mitochondria,and it is a method worthy of recommendation to mitochondrial extract effectively. |
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