Activation Of Nrf2/HO-1 Signaling Pathway Protects Myocardial Apoptosis From Coronary Microembolism

Research BackgroundCME is a refractory complication of PCI in the treatment of acute coronary syndrome.It often occurs when the atherosclerotic plaque ruptures and blocks the distal microvessels,resulting in no reflow or slow reflow phenomenon,which may be accompanied by myocardial contractile function.Disorders,arrhythmias,inflammations,and microinfarctions occur.Nuclear factor 2?Nrf2?is an important transcription factor.Nf2 is activated by various stimuli and is a major player in maintaining and restoring homeostasis.At the same time,Nrf2plays an important role in the regulation of cardiomyocyte apoptosis.Important role.HO-1 is a defensive enzyme and also has strong anti-apoptotic and anti-oxidative stress effects.More and more studies have focused on the role of Nrf2/HO-1 signaling pathway in myocardial apoptosis.However,it is unclear whether Nrf2/HO-1 signaling pathway can improve CME-induced myocardial apoptosis.Research purposesThe aim of this experiment was to establish a rat CME model to observe the effects of Nrf2 and HO-1 on cardiomyocyte apoptosis,cardiac function,and myocardial enzymes,and to explore possible molecular pathways of myocardial cell apoptosis induced by CME in rats.Treatment of CME-induced myocardial apoptosis provides a potential new site of action.Research methodsEighteen SD rats were randomly divided into sham?N?group,AAV-Nrf2?N?group,and AAV-control?N?group.The rats were injected with 0.5 ml normal saline through the tail vein 14 days before surgery.1×10¹¹U AAV-Nrf2 and AAV-control virus,and the expression level of Nrf2 was detected by western blot.Forty Sprague-Dawley rats were randomly divided into 4 groups of 10 in each group:sham operation group,CME group,AAV-Nrf2 group?CME+AAV-Nrf2?and AAV-control?CME+AAV-control?.Each rat in the AAV-Nrf2 group was injected with a total volume of 1×10¹¹ UAAV-Nrf2through the tail vein.The AAV-control was injected in the same way with the same amount of AAV-control,and the sham group was injected with an equal volume of saline,and then the 42?m plastic microspheres were injected through the syringe.In the left ventricle injected into the CME group and the Nrf2 group,0.1 ml physiological saline was additionally injected into the left ventricle of the sham-operated group rats in the same manner as the experimental control instead of the plastic microspheres.Six hours after CME,serum cTnI was detected by enzyme labeling,cardiac function?LVEF,LVEDd,FS,and CO?was assessed by echocardiography;HE staining was used to observe the microspheres entering the myocardium;HBFP Staining was used to assess the degree of early myocardial necrosis;TUNEL staining was used to detect myocardial apoptosis.The mRNA expression levels of Nrf2,HO-1,bax,bcl-2,caspase-9,and caspase-3 were detected by qPT-PCR,and Nrf2,HO-1,bax,and bcl-2 were detected by Western blotting.Caspase-9 and cleaved-caspase-3protein expression levels.Research resultThe Nrf2 gene expression level test showed that the transfection of AAV-Nrf2 could increase the expression of Nrf2 protein in the myocardium of AAV-Nrf2?N?rats compared with Sham?N?and AAV-control?N?groups.?P<0.05?.The results of echocardiography showed that 6 hours after coronary microembolization in rats,compared with the sham group,the myocardial contractility was significantly reduced in the CME group and the AAV-control group,and the left ventricular ejection fraction?LVEF?,The fractional shortening?FS?and cardiac output?CO?were significantly decreased?P<0.05?,while the left ventricular end diastolic diameter?LEVDd?was significantly increased?P<0.05?.In contrast,in the AAV-Nrf2 group,left ventricular ejection fraction?LVEF?,shortened fraction?FS?,and cardiac output?CO?increased significantly compared with the CME group,whereas left ventricular end diastolic diameter?LEVDd?was significant.Decrease?P<0.05?.Six hours after coronary microembolization in rats,the CME group?0.32±0.015?g/L vs 0.05±0.002?g/L,P<0.05?and AAV-control?0.33±0.012?g/L vs 0.05±0.002?g/L,P<0.05?were compared with the sham operation group.The serum c TnI levels in the serum of the rats in the group were significantly higher than those in the control group?P<0.05?.In addition,compared with the CME group,the cTnI level in the AAV-Nrf2 group was significantly decreased?0.19±0.014?g/L??P<0.05?.The results of HBFP staining showed that there was no obvious microinfarction in the ventricular anterior myocardium in the sham-operated group.Obvious early ischemic myocardial cells,necrotic cardiomyocytes,and red blood cells were stained in the CME group,AAV-Nrf2 and AAV-control groups.Redness;AAV-Nrf2 group was significantly smaller than the CME group,the difference was statistically significant?P<0.05?.TUNEL staining showed that there was no obvious cardiomyocyte apoptosis in the sham-operated group,but the CME group,the AAV-control group,and the AAV-Nrf2 group showed a brown-brown apoptotic cardiomyocytes.The normal nucleus of the cardiomyocytes was pale.Compared with CME group,the apoptosis of cardiomyocytes in AAV-Nrf2 group was significantly reduced,and the apoptosis rate was also significantly lower in the Nrf2 group than in the CME group?P<0.05?.The results of RT-PCR showed that the expression levels of Nrf2,HO-1and bcl-2 mRNA in sham group and AAV-Nrf2 group were significantly higher than those in CME group and AAV-control group?P<0.05?.At the same time,the expression levels of bax,caspase-9,and caspase-3 mRNA in CME group and AAV-control group were significantly higher than those in sham-operated group and AAV-Nrf2 group,but compared with CME group,bax,capspase-9 in AAV-Nrf2 group.The mRNA expression of caspase-3 and caspase-3 were significantly decreased,and the difference was statistically significant?P<0.05?.Western blot results showed that the expression levels of Nrf2,HO-1 and bcl-2 protein in the sham operation group and Nrf2 group were significantly higher than those in the CME group?P<0.05?.Meanwhile,the CME group and AAV-control The expression levels of bax,caspase-9,and cleaved-caspase-3proteins in the AAV-Nrf2 group were significantly lower than those in the sham-operated group and the AAV-Nrf2 group.Compared with the CME group,the expression levels of bax,capsase-9,and cleaved-caspase-3 proteins were significantly decreased in the AAV-Nrf2 group.The difference was statistically significant?P<0.05?.Analysis conclusionThis study shows that Nrf2 activates HO-1 to reduce cardiomyocyte apoptosis induced by CME,and it can improve cardiac function,reduce myocardial infarct size,and reduce myocardial enzyme levels.The data generated in this study provides a potential therapeutic target for reducing CME-induced cardiomyocyte apoptosis.