| Objective:It is known that there is a clear relationship between exposure to air particulate matters and cardiovascular disease morbidity and mortality.Epidemiological studies of human populations and numerous studies in experimental animals have shown that particulate matters is as a risk factor for cardiovascular disease,which are the leading cause of both mortality and disability in the world.In our study,the myocardial ischemic injury model was established in vivo and ex vivo,which were exposed by Standard Reference Material 1650b:Diesel Particulate Matter?DPM?to explore the protective effects of Shengmaisan?SM?on DPM-induced cardiovascular system damage in rat and investigate the possible protective molecular mechanism against the effects.Methods:1.The myocardial ischemic injury model was established by DPM-induced,the rats were administrated by SM intragastric for a week,the DPM suspension was instilled by tracheal infusion,and the left anterior descending coronary artery was ligated.2.To establish ischemia-reperfusion model,SM injection and DPM suspension were administered by Langendorff perfusion system,and the protective effect of SM on isolated rat heart was observed.3.For in vitro experment,H9c2 cardiomyocytes were exposed DPM for 24 h to investigate the possible effects of SM by restoring mitochondrial function through Nrf2pathway.In vivo experiments:SD rats were randomly divided into a sham operation group,a model group,a SM low dose group,a SM medium dose group,and a SM high dose group.SM was intragastrically administered for a week.After tracheal intubation,DPM suspension was instilled and the rats were anesthetized and undergoing ligation left anterior descending coronary artery surgery.Serum LDH,CK-MB contents were detected using biochemical kits;antioxidant index SOD,MDA,CAT,GSH-Px,NOX contents were detected;level of cTnT in serum was detected by immunoassay?ELISA?.The myocardial infarction area was evaluated by TTC staining.The pathological changes were observed by HE staining.The myocardial tissue and mitochondrial ultrastructure were observed by transmission electron microscope.The mitochondrial oxidation was detected by qPCR array.And expression of Nrf2 and downstream proteins was detected by qPCR.Ex vivo experiments:SD rats were randomly divided into a normal control group,a verapamil positive drug group,a model group,a SM 0.14 mg/mL group and a SM 0.28mg/mL group.After anesthetized,the heart was isolated and hung on the Langendorff aortic cannula.The K-H solution was continuously oxygenated at 37°C,95%O2-5%CO2.The self-made balloon was inserted into left ventricle,and pressure sensor was connected to a multi-channel physiological recorder,meanwhile,perfusion pressure,electrocardiogram and ventricular pressure curve were recorded.After equilibration,10 min of perfusion was administered,30 mL of 10?g/mL DPM suspension was perfused,40 min of total ischemia and 40 min for reperfusion.Lastly,HR,LVEDP,LVSP were measured and LVDP andądp/dtmax were calculated,and level of cTnT was measured by cardiac perfusion liquid at the end of reperfusion.In vitro experiments:H9c2 cardiomyocytes were treated with DPM 100?g/mL for 24 h as a cell injury model,and CCK8 was used to detect the effect of SM on DPM-induced injury.DCFH-DA fluorescent probe was used to detect ROS production,JC-1 fluorescent probe was used to detect mitochondrial membrane potential,and XFe energy metabolism instrument was used to quantitatively detect mitochondrial respiration to evaluate the effect of SM on DPM exposure for mitochondrial function.Results:1.Vivo experiments:?1?Compared with the sham operation group,DPM induced a significant increase in myocardial infarct size?P<0.001?,and LDH?CK-MB and cTnT levels were significantly increased;an increasing vascular and myocardial fiber gaps,many infiltrated inflammatory cells,local myocardial fiber edema were observed in myocardial histopathology;TEM showed that cells were edema,sarcomer light and dark bands were blurred,mitochondria appeared swelling and sputum dissolution in myocardial ultrastructure;compared with model group,significantly infarct size reduction was observed in SM group?P<0.001?;LDH?CK-MB?cTnT contents decreased;infiltrated inflammatory cells decreased,myocardial interfiber space decreased,myofibrils arranged neatly,sarcomere structure was clear;mitochondrial structure occasionally swollen,and had aligned cristae structure;?2?Compared with sham operation group,CAT and GSH-Px contents in model group were significantly decreased?P<0.05?,and MDA and NOX contents were significantly increased?P<0.01?.Compared with model group,CAT contents in SM low dose group increased significantly?P<0.05?,GSH-Px contents in SM middle dose group were increased significantly?P<0.05?.MDA and NOX levels in the SM group decreased significantly?P<0.05?;?3?Compared with sham operation group,DPM caused significant down-regulation of Cat and Gstk1,down-regulation of Ucp3 and up-regulation of Cyba.Compared with model group,Cat?Gstk1 and Ucp3 were significantly up-regulated after SM treatment,Cyba was down-regulated;?4?Compared with sham operation group,the relative expression levels of Nrf2,HO-1,CAT,SOD1,GPX1 and NQO1 mRNA in DPM were significantly decreased?P<0.05?,NOX mRNA in DPM were significantly increased?P<0.001?,and the relative expression levels of CAT and GPX1 in SM low dose group were significantly increased?P<0.05?,the relative expression levels of CAT,SOD1 and GPX1 in SM medium group were significantly increased?P<0.05?.2.Ex vivo experiments:?1?Compared with normal control group,ST-segment was elevated,HR was abnormalitied,and PP were significantly increased in model group,and LVDP,dp/dtmax and RPP were significantly decreased?P<0.05?;?2?Compared with model group,SM had no significant effect on isolated cardiac function,PP decreased,LVDP?dp/dtmax?RPP increased but no significantly difference;PP decreased significantly in verapamil group?P<0.05?,dp/dtmax?LVDP?RPP were significantly increased?P<0.001?;?3?Compared with normal control group,cTnT content in composite pattern model group was significantly increased?P<0.05?;compared with model group,SM 0.28 mg/mL group decreased cTnT content significantly?P<0.05?.3.Vitro experiments:?1?CCK8 detection of 100?g/mL DPM on cells,which had 57%cell survival,after SM treatment,cell survival rate increased,then selected SM 0.28 mg/mL?SM 0.56 mg/mL?SM 1.12 mg/mL completed the follow-up experiment;?2?After 24 h of DPM exposure,ROS was increased,which was significantly different from the normal control group?P<0.05?.Pretreatment of SM could reduced ROS production,SM 0.28 mg/mL and SM 0.56 mg/mL group showed significantly ROS reduction compared with the DPM group?P<0.01?;?3?After treatment with DPM,the mitochondrial membrane potential were increased;after SM pretreatment,the mitochondrial membrane potential of the cells were decreased,and there was no significantly difference;?4?After DPM exposure,mitochondrial basal respiration,ATP production,and non-mitochondrial respiration consumption were significantly increased compared with the normal control group,the above values were decreased?P<0.05?.After SM pre-treatment,compared with DPM group,basic respiratory value,ATP production and respiratory reserve decreased?P<0.05?.Conclusion:This study confirmed that DPM had aggravated myocardial ischemic injury and damaged mitochondrial function in myocardial cells,DPM acts an external stimulus to cause oxidative stress in cell,causing nuclear translocation of Nrf2,which in turn affects downstream phase II detoxification enzymes and antioxidant enzymes.The expression of the defense ability declines,at the same time,oxidative stress leads to excessive ROS production,which in turn affects mitochondrial function,and the mitochondrial respiration affects more ROS damaged cardiomyocytes;SM may have a protective effect on ischemia injury by regulating Nrf2 pathway to against oxidative stress. |
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