| Background:Acute myocardial infarction(AMI)refers to local myocardial ischemic necrosis induced by a sudden interruption of blood flow because of acute coronary artery occlusion,which leads to high morbidity and mortality in the world.The most common treatment for AMI is recanalization of occluded vessels to save the risk region of heart,such as thrombolysis therapy,percutaneous coronary intervention and coronary artery bypass surgery,etc.However,the reperfusion therapy itself can intensify the death of cardiomyocytes which is called ischemia reperfusion injury(IRI).Therefore,how to prevent and treat the reperfusion injury has become one of important tasks in the treatment of patients with AMI.Interleukin(IL)-23 and IL-17A,as potent pro-inflammatory cytokines,involved in pathogenesis of a variety of inflammatory diseases.Studies have found that there is a closely relationship between IL-23,IL-17A and JAK2/STAT3 signaling pathway.The expression of IL-17A could be regulated by IL-23,which may be related to the activation of JAK2/STAT3 signaling pathway.Previous studies have confirmed that IL-17A played an important role in myocardial I/R injury.Therefore,the research mainly focuses on the relationship among IL-23-JAK2/STAT3-IL-17A axis and its role in myocardial I/R injury in rats.This would be helpful to clarify the mechanism of myocardial I/R injury,and will give a new sight to prevent the myocardial I/R injury.Methods:1.In vitro:Sprague-Dawley neonatal rat cardiomyocytes were cultured and used to establish myocardial cell hypoxia and reoxygenation(H/R)model,then were treated with exogenous IL-23,anti-IL-23 and AG490(a selective inhibitor of JAK2/STAT3 signaling pathway).Myocardial damage indexes of LDH,CK,oxidative stress parameters SOD,MDA and inflammatory biomarkers TNF-a,IL-6 were measured,apoptosis rate of cardiomyocytes was assessed by flow cytometry.The expression of IL-23,IL-17A,apoptosis associated protein Bcl-2,Bax,signaling pathway associated protein JAK2,P-JAK2,STAT3,P-STAT3 were detected by Western Blot.2.In vivo:IL-23 and empty plasmid adenoviruses(named Ad-IL-23 and Ad-GFP)were constructed by cloning the IL-23 cDNA or empty plasmid.Then,the recombinant viruses were amplified in HEK 293 cells and finally purified by using Adeno-XTMPurification Kit in order to reach the titer of 1011 PFU/mL;90 male Sprague-Dawley rats(200-250g)were randomly assigned to the following six treatment groups:Group 1:sham-operated control(SO)(n=15):rats were subjected to surgical operation without myocardial ischemia.Group 2:ischemia and reperfusion(I/R)(n=15):rats were subjected to occlusion of the left anterior descending coronary artery(LAD)for 30 min,followed by reperfusion for 4 h.Group 3:Ad-GFP+I/R(n=15):rats were treated with Ad-GFP(100?L,1×1011 PFU/mL)72 h before LAD occlusion.Group 4:Ad-IL-23+I/R(n=15):rats were treated with Ad-IL-23(100?L,1×1011 PFU/mL)72 h before LAD occlusion.Group 5:anti-IL-23+I/R(n=15):rats were treated with anti-IL-23(200?L)neutralized monoclonal antibodies(mAbs)30 min before LAD occlusion.Group 6:Ad-IL-23+AG490+I/R(n=15):rats were treated with Ad-IL-23(100?L,1×1011 PFU/mL)72 h.And AG490(3 mg/kg)was injected 30 min before LAD occlusion.Myocardial infarct size were defined by TTC and Evans'Blue;apoptosis in heart was detected by TUNEL staining;the concentration of serum enzyme LDH,CK content in myocardial injury were measured;the SOD activity,MDA concentration,inflammatory factor of TNF-a,IL-6 were measured;The expression of IL-23,IL-17A,apoptosis associated protein Bcl-2,Bax,caspase-3,signaling pathway associated protein JAK2,P-JAK2,STAT3,P-STAT3 were detected by Western Blot.Results:1.In vitro experiments:Compared with control group,the concentration of LDH,CK,TNF-a,IL-6 and MDA were significantly increased and the SOD activity was decreased in H/R group.Western Blot analysis revealed that the expression of IL-23,IL-17A,Bax were significantly increased and the expression of Bcl-2 was decreased(all P<0.05);when compared with H/R group,the concentration of LDH,CK,TNF-a,IL-6 and MDA and the expression of IL-23,IL-17A,Bax were further increased and the SOD activity and the expression of Bcl-2 were further decreased in IL-23+H/R group,and the expression of signaling pathway activation associated protein P-JAK2,P-STAT3 were significantly increased(all P<0.05);On the contrary,the concentration of LDH,CK,TNF-?,IL-6 and MDA and the expression of IL-23,IL-17A,Bax were decreased and the SOD activity and the expression of Bcl-2 were increased,the expression of signaling pathway activation associated protein P-JAK2,P-STAT3 were significantly decreased in anti-IL-23+H/R group(all P<0.05);Compared with IL-23+H/R group,the concentration of LDH,CK,TNF-a,IL-6 and MDA and the expression of IL-17A,Bax were decreased and the SOD activity and the expression of Bcl-2 were increased,the expression of signaling pathway activation associated protein P-JAK2,P-STAT3 were significantly decreased in IL-23+AG490 +H/R group(all P<0.05).2.In vitro experiments:Recombiant Ad-IL-23 was constructed successfully with the titer of 1×1011 PFU/mL;Compared with SO group,the apoptisis rate and the serum concentration of LDH and CK were significantly increased in I/R group,the SOD activity was decreased and the concentration of MDA,TNF-a,IL-6 were increased in heart tissue,and the expression of IL-23,IL-17A and caspase-3 were significantly increased,Bcl-2/Bax rate was decreased(all P<0.05).However,the above indicators of Ad-GFP+I/R group had no significant difference with I/R group(all P>0.05).Compared with I/R group,the infarct size and apoptisis rate were significantly increased,the serum concentration of LDH and CK were further increased,the SOD activity was further decreased and the concentration of MDA,TNF-?,IL-6 were further increased the expression of IL-23,IL-17A,caspase-3 and P-JAK2,P-STAT3 were significantly increased,Bcl-2/Bax rate was decreased in Ad-IL-23+I/R(all P<0.05).On the contrary,the infarct size,apoptisis rate,the serum concentration of LDH,the concentration of MDA,TNF-a,IL-6,IL-23,IL-17A,caspase-3 and P-JAK2,P-STAT3 were decreased,the SOD activity and Bcl-2/Bax rate were increased in anti-IL-23+I/R group(all P<0.05);Compared with Ad-IL-23+I/R group,the infarct size,apoptisis rate,the serum concentration of LDH and CK,the concentration of MDA,TNF-a,IL-6,IL-17A,cleaved caspase-3 and P-JAK2,P-STAT3 were decreased,the SOD activity and Bcl-2/Bax rate were increased in Ad-IL-23+AG490+I/R group(all P<0.05).Conclusions:1.In vitro experiments we confirmed that IL-23 could promote myocardial H/R injury,which may be related to increased oxidative stress,inflammation and cell apoptosis;IL-23 may upregulate the expression of IL-17A by activation of JAK2/STAT3 signaling pathway;The IL-23-JAK2/STAT3-IL-17A axis mediated the aggravation of myocardial H/R injury;2.Construction of shuttle plasmid carrying IL-23 gene,homologous recombination in HEK293 cells after adenovirus packaging the formation of Ad-IL-23,amplified,purified virus titer up to 1×1011 PFU/mL;3.In vitro experiments we further confirmed that IL-23 could promote myocardial I/R injury,which may be related to increased oxidative stress,inflammation,and cell apoptosis;IL-23 may promote myocardial I/R injury by up-regulation of IL-17A,which may be related to activation of JAK2/STAT3 signaling pathway;The IL-23-JAK2/STAT3-IL-17A axis mediated the aggravation of myocardial I/R injury... |
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