| Objective The major aim of the present work was to study the cardioprotection of RP105 on myocardial ischemia reperfusion injury(MIRI) and explore its molecular mechanism.Methods Forty male Sprague-Dawley(SD) rats with specific pathogen free(SPF)grade(Weight of 220-250g) were used for the current study and randomly divided into four equal groups(n=10).(1) Normal saline infection with sham operation(Sham group).(2)Normal saline infection with myocardial IR(IR group).(3) Ad-EGFP-RP105 infection with myocardial IR(Ad-RP105 group).(4) Ad-EGFP infection with myocardial IR(Ad-EGFP group). RP105 recombinant adenovirus vector(Ad-EGFP-RP105), no-load virus(Ad-EGFP) or saline were pre-infected at the apex of heart respectively. Rat model of MIRI were established by ligaturing the left anterior descending coronary artery for 30 min and followed by 2 h reperfusion. The expression of RP105 after adenovirus transfection in cardiac muscle tissue was observed by immunofluorescence. The levels of serum myocardial enzyme LDH and CK-MB were measured by blood biochemical analysis.Myocardial infarct area was measured by the Evans Blue/triphenyltetrazolium chloride(TTC) double-staining. HE dyeing was performed to observe rat myocardial tissue morphology. The levels of IFN-β and TNF-α in myocardial tissue were detected by Enzyme-linked immuno sorbent assay(ELISA). Western blot analysis and qRT-PCR were used to examine the protein and gene levels of TRL4/TRIF signaling pathway respectively.EMSA for the DNA-binding activity of IRF3 in cardiomyocytes was performed.Results(1) RP105 recombinant adenovirus vector was delivered into rat cardiac muscle tissue successfully.(2) IR group performed a significant increase in the activities of serum myocardial enzyme CK-MB and LDH compared to the sham group(P<0.05). After RP105 transduction, the CK-MB and LDH level decreased significantly compared to IR group or Ad-EGFP group(P<0.05).(3) Compared with the Sham group, the myocardial infarction area of IR group increased significantly(P<0.05). The myocardium transduced with Ad-EGFP-RP105 exerted cardioprotective effect and had less infarct tissue compared to IR group or Ad-EGFP group(P<0.05).(4) IR group showed obvious myocardium cell degeneration and necrosis, while delivery of RP105 partially rescued cell degeneration and necrosis compared to IR group or Ad-EGFP group.(5) Rats subjected to MIRI exhibited a remarkable increasing in inflammatory cytokines IFN-β and TNF-α in IR group, compared to sham group(P both<0.05). Notably, up-regulation of RP105 in cardiac muscle cell mitigated IFN-βand TNF-α(P both<0.05).(6) Comparing with sham group, the expression of relative protein TRIF, TBK-1, IRF3 and p-IRF3 were up-regulated obviously in IR group(P<0.05). Interestingly, transduction of RP105 into myocardium markedly decreased above protein expression(P<0.05).(7)The mRNA levels of TRIF, TBK-1, IRF3 and p-IRF3 were notably higher in IR group than that in sham group(P<0.05). Similarly,up-regulation of RP105 could reduce these mRNA expressions after the delivering of RP105(P<0.05). Myocardial ischemia reperfusion markedly increased the DNA-binding and transcriptional activity of IRF3(P<0.05), yet RP105 could attenuate it in some degree.Conclusion(1) TLR4/TRIF/IRF3 signaling pathway played an important role in the development of MIRI by increasing the inflammatory reaction.(2)RP105 over-expression in cardiocytes with adenovirus vectors could mitigate inflammatory factors, decrease myocardial infarct size and reduce serum myocardial enzyme via inhibiting TLR4/TRIF/IRF3 signaling pathway.(3) RP105, as an effective target for intervention in MIRI, combined with gene therapy was expected to become the effective measures of MIRI treatment and provided a theoretical foundation to clinical CHD patients subjected to revascularization with reperfusion injury. |
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