Anti-apoptotic Potency Of RP105on Myocardial Ischemia Reperfusion Injury Via Suppression Of TLR4Signaling Pathways And The Mechanism Research In Rats

Objective The major aim of the present work was to provide molecular mechanisticby which RP105protects cardiomyocytes from cell apoptosis during ischemic and ensuingreperfusion conditions via suppression TLR4-triggerred signaling pathways in rats.Methods40adult male Sprague-Dawley rats (SPF grade, weighting220-250g) wererandomly allocated into four equal groups(n=10):(1)normal non-ischemic group (Shamgroup);(2) myocardial I/R group(I/R group);(3) myocardial I/R with Ad-EGFPgroup(Ad-E group);(4) myocardial I/R with Ad-EGFP-RP105group(Ad-E-R group). Onthe forth day after adenovirus or normal saline delivery, all of the rats underwent30min ofcoronary occlusion followed by reperfusion for4h with the exception of shamed group.Then, immunohistochemistry and fluorescence microscopy were approached for theexpression of EGFP and RP105; Evans Blue/triphenyltetrazolium chloride (TTC)double-staining was performed to determinate the infarct area of myocardium; the in vivoterminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL) assaywas used to detect the apoptotic cardiomyocytes; Real-time PCR was performed for thelevel of EGFP and RP105mRNA. Moreover, we preferred to western blot analysis toassess the expression level of objective proteins in dissected myocardial tissue, such asBax、cytochrome c、Bcl-2、cleaved caspase-3、caspase-9、RP105、TLR4、P38MAPK、p-P38MAPK、c-Jun/AP-1and p-c-Jun/AP-1.Results (1) Adenoviral transduction using both Ad-EGFP-RP105and Ad-EGFP wassufficient to induce the expression of EGFP and RP105in myocardium.(2) Relative tonon-ischemic sham control, myocardium from I/R、Ad-E and Ad-E-R groups showedenhanced levels of infracted area and apoptotic index. Furthermore, compared with shamgroup, increased levels of pro-apoptotic molecules, including Bax, cytochrome c,caspase-9(procaspase-9) and cleaved caspase-3, and diminished anti-apoptotic proteinBcl-2were measured in I/R、Ad-E and Ad-E-R groups, which were concomitant withelevated levels of TLR4, P38MAPK, p-P38MAPK, c-Jun and p-c-Jun proteins.(3)Relative to I/R and Ad-E groups, the myocardium previously transduced withAd-EGFP-RP105had less infarct tissue and apoptotic index, and decreased levels ofpro-apoptotic protein and increased level of anti-apoptotic protein. In addition,over-expression of RP105(Ad-E-R group) also significantly reduced the levels of TLR4,p-P38MAPK and p-c-Jun in I/R myocardium.(4) Transduction of Ad-EGFP (Ad-E group) had no significant effect on the infract area and apoptotic index in comparison with I/Rgroup. No significantly statistic differences were found in I/R and Ad-E groups on thelevels of mitochondrial-associated molecular and TLR4-triggerred signaling pathways.(5)Adenoviral transduction of RP105shared no significant effect on total cellular level ofP38MAPK and c-Jun. In addition, low levels of p-P38MAPK/P38MAPK ratio andp-c-Jun/c-Jun ratio were also calculated in sham group, while myocardium from I/Rinjury(I/R) governed increased levels of the ratios. I/R injury followed by adenoviraltransduction using Ad-EGFP had no significant effect on the levels of the ratios. Of note,overexpression of RP105significantly lessened the levels of p-P38MAPK/P38MAPK ratioand p-c-Jun/c-Jun ratio in I/R myocardium.Conclusion (1) Intramyocardial injection of adenoviral vectors Ad-EGFP andAd-EGFP-RP105with subsequent ischemia and revascularization-treatment contributed tothe overexpression of RP105and EGFP in the administrated heart;(2) Prophylacticoverexpression of RP105with Ad-EGFP-RP105prior to MIRI markedly suppressed infarctarea and apoptotic cardiomyocytes;(3) The activity of TLR4signaling pathways, whichwere concomitant with the phosphorylation of P38MAPK and c-Jun/AP-1in ischemiareperfusion injury, participated in the pathophysiology of cardiomyocytes apoptosis;(4)Over-expression of RP105contributed to anti-apoptotic activity on myocardium damageinduced by IR suffering through causally ameliorating mitochondrial apoptosis pathway,characterized by the decreased release of cytochrome c into cytosol and down-regulatedexpression of caspase-9and cleaved caspase-3with a concomitant repression of Bax andenhancement of Bcl-2.(5) Selectively intramyocardial overexpression of RP105viaadenoviral vectors led to the significant repression of TLR4, P38MAPK, and AP-1signaling pathways and causally resulted in the amelioration of mitochondrial apoptosis,thereby predisposing the cardioprotective effect against the apoptosis of cardiomyocytesand myocardial injury...