| Cyclooxygenase 2(COX-2),as one of the isoenzymes COX prostaglandin endoperoxidase,can rapidly respond to a range of proinflammatory mediators and cytokines that mediate inflammation production.According to relevant reports,during the development of myocardial ischemia-reperfusion(I/R)injury,COX-2 is involved in the pathological inflammatory development of I/R injury,and plays an important role in the pathological process of myocardial injury.JunB,a member of the transcription factor-activating protein-1(AP-1)family,acts as a transcriptional regulator,which mainly includes the two large families of Jun and Fos.JunB has a wide range of effects,including promoting embryo development,promoting cell differentiation and proliferation,participating in inflammatory response,participating in oxidative stress response,etc.The literature describes the significant changes of JunB in the case of myocardial I/R or cell H/R,and then brings about an increased response with inflammatory factors,which is closely related to the inflammatory response.Calcium channel blockers(CCB)mainly block the L-type voltage-dependent calcium channel(L-VDCC)on the cell membrane to play its drug role.It is a kind of drug that can selectively block Ca2+flowing into the cell through voltage-dependent calcium channel and reduce the concentration of Ca2+in the cell.Generally,it can be divided into three categories according to chemical structure:dihydropyridines(DHPs)with nifedipine(Nif)as the representative drug,btzs with diltiazem(Dil)as the representative drug,and phenylalkanes(PAAS)with verapamil(Ver)as the representative drug.N-n-butyl haloperidol iodide(F2)is a CCB with cardiovascular activity.The traditional view is that CCB can protect L-VDCC on the membrane of myocardial and vascular smooth muscle cells,but in recent years,there are also studies that CCB has a non-L-VDCC-dependent cardioprotection.Previous studies in our laboratory showed that CCB can establish H/R model experiments on H9c2 cells knocked out of L-VDCC channel(Cacnalc-/-).CCB can block H/R injury through non-L-VDCC,and its mechanism can be It can be related to the regulation of COX-2 and junB,which proves that CCB may have a non-L-VDCC dependent protection mechanism against I/R injury.In order to explain the above problems more accurately and comprehensively,this study adopts the cardiac micro vascular endothelial cells(CMEC),which are the first affected by myocardial ischemia-reperfusion injury,to observe the effect of CCB on H/R injury of cells,we used COX-2 and junB as targets to explore CCB’s antagonism on H/R injury of cells through the regulation of the above two genes,because CMECs has no L-VDCC,so CCB’s improvement on H/R injury of CMECs has nothing to do with L-VDCC.Methods1.To establish a CMECs H/R model,the model time points of COX-2 and JunB in H/R damage were determined by detecting the protein expression of COX-2 and JunB after H/R and the leakage of lactate dehydrogenase(LDH).2.To explore the relationship between COX-2 and H/R by detecting LDH,SOD,MDA,inflammatory factors(IL-1β;,IL-6,TNF-α and ICAM-1),apoptosis,product,and activity using COX-2-specific inhibitor NS398.3.Using the JunB-siRNA transfection method to interfere with the expression of JunB,the LDH,SOD,MDA,inflammatory factors(IL-1β;,IL-6,TNF-α and ICAM-1,and apoptosis were detected,respectively,to explore the relationship between JunB and H/R.4.CCB with different concentrations(10μmol/L,1μmol/L,0.1μmol/L)was administered to detect the protein expression of COX-2 and JunB on CMECs and observe the drug effect.ResultsPart Ⅰ CCB regulates COX-2 against H/R in CMECs1.Compared with the Ctrl group,the protein expression of COX-2 was the highest at 3 h single deficiency,and the protein expression of COX-2 was high at CMECs H3h/R2h(p<0.05)Compared with Ctrl group,and the leakage of LDH increased at COX-2,so H3h/R2h was chosen as the time point of H/R damage model(p<0.05).2.Compared with the Ctrl group,LDH leakage increased,MDA concentration increased,TNF-α,IL-1β,IL-6 and ICAM-1 increased,apoptosis increased,SOD activity decreased,COX-2 activity increased,PGE2 and PGI2 production increased(p<0.05).Compared with H/R+DMSO group,LDH leakage,MDA concentration,TNF-α,IL-1β,IL-6 and ICAM-1 production decreased,apoptosis improved,SOD activity increased,COX-2 activity decreased,PGE2 and PGI2 production decreased(p<0.05).3.Compared with the Ctrl group,LDH leakage increased,MDA concentration increased,TNF-α,IL-1β,IL-6 and ICAM-1 increased,apoptosis increased,SOD activity decreased(p<0.05).Compared with H/R+NC group,LDH leakage decreased,MDA concentration decreased,TNF-α,IL-1β,IL-6 and ICAM-1 production decreased,apoptosis improved and SOD activity increased in H/R+CCX-2-siRNA group(p<0.05).4.After different concentrations(10μmol/L,1μmol/L,0.1μmol/L)of Ver,Nif,Dil and F2 were given respectively,the protein expression of COX-2 after H/R was effectively reduced by Nif and Dil(p<0.05),and the activity of COX-2 and the production of PGE2 were decreased by Nif and Dil in a dose-dependent manner(p<0.05).Part Ⅱ CCB regulates JunB against H/R in CMECs1.Compared with Ctrl group,the protein expression of JunB was the highest at 3 h,and the protein expression of JunB was higher at Ctrl group,CMECs H3h/R2h,and the leakage of LDH increased(p<0.05).2.Compared with Ctrl group,LDH leakage increased,MDA concentration increased,TNF-α,IL-1β,IL-6 production increased,apoptosis increased,SOD activity decreased in H/R group.Compared with H/R+NC group,LDH leakage decreased,MDA concentration decreased,TNF-α,IL-1β,IL-6 and ICAM-1 production decreased,apoptosis improved and SOD activity increased in H/R+JunB-siRNA group(p<0.05).3.After different concentrations(10μmol/L,1μmol/L,0.1 μmol/L)of Ver,Nif,Dil and F2,respectively,Nif was able to reduce the protein expression of JunB after H/R in a dose-dependent manner(p<0.05).Conclusions1.COX-2 and JunB are involved in the development of H/R injury in CMECs.2.Nif,and Dil against CMECs H/R damage by regulating COX-2.3.Nif antagonizes CMECs H/R damage by modulating JunB.4.In CMECs,CCB may regulate COX-2 and JunB through non-L-VDCC dependent pathway to against H/R injury. |
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