| Myocardial infarction (MI) is a major cause of death in the United States and many developed countries. Short periods of ischemia are leading to some functional impairment initially, but the hemodynamic recovery will be detected certain period after blood reflow. Therefore, it is necessary to reestablish the early reperfusion after MI in order to salvage the ischemic tissue and prevent the myocardial damage. Nevertheless, reperfusion of the myocardium after ischemia is associated with ultrastructural changes, which finally lead to irreversible injury. Cellular calcium overload, oxygen radical production, and inflammatory hypothesis have been proposed as the major causes of ischemia-reperfusion (I/R) injury.There is increasing evidence that opening of the mitochondrial permeability transition pore (mPTP) plays a key role in triggering apoptosis of cardiomyocytes following I/R. mPTP is a large, nonselective conductance pore located in the inner mitochondrial membrane. Opening of mPTP is induced, during the early stages of reperfusion, by Ca2+ overload, a massive production of reactive oxygen species (ROS) and abrupt restoration of pH. This in turn causes the dissipation of the mitochondrial membrane potential (ATm), uncoupling of oxidative phosphorylation, inhibition of ATP production, and mitochondrial swelling. In addition, mPTP opening leads to the release of pro-apoptotic proteins including cytochrome c (Cyt c) from the mitochondria into the cytoplasm. The released cytochrome c in turn activates the mitochondrial apoptotic pathway and aggravates myocardial injury. Hence, inhibition of mPTP opening and the subsequent activation of the mitochondrial apoptotic pathway is a promising therapeutic strategy to protect the heart from I/R injury.Panax quinquefolium saponin (PQS) is extracted from the stems and leaves of Radix panacisquinquefolii (American ginseng). Previous studies have shown that PQS can increase ATP content and energy charge (EC), upregulate the expression of the anti-apoptotic protein Bcl-2, and downregulate Fas expression in the myocardium of AMI rats. These studies, however, did not explore its association with the regulation of mPTP opening. In addition, in the cerebral cortex of ischemic rats, PQS was shown to upregulate the anti-apoptotic protein Bcl-2, and reduce the levels of cleaved-caspase-9 and cleaved-caspase-3. Although these studies demonstrated the anti-apoptotic effects of PQS, the underlying mechanism was not explored. Recently, our group showed that PQS can reduce the extent of myocardial I/R injury and apoptosis of cardiomyocytes both in vivo as well as in vitro, which mechanistically, is associated with a reduction in the level of endoplasmic reticulum stress (ERS). Furthermore, we demonstrated that PQS can also alleviate cardiac ventricular remodeling following myocardial infarction by suppressing ERS-related apoptosis. However, whether the anti-apoptotic property of PQS is related to the regulation of mPTP opening and inhibition of the mitochondrial apoptotic pathway during I/R, remains unclear.In the current study, we hypothesized that PQS induces cardioprotection by inhibiting the opening of mPTP and consequently, the mitochondrial apoptotic pathway in I/R injured myocardium. To address this question, we investigated the effect of PQS on mPTP and the expression of pro- and anti- apoptotic factors in the myocardium in rat models of I/R injury. Furthermore, we hypothesized that PQS inhibits the opening of mPTP during I/R via PI3K/Akt/GSK-3β pathway. To address this question, the cardiomyocytes of H/R injury were used to investigate whether the effects of PQS are associated with inhibition of mPTP and the activation of PI3K/Akt/GSK-3β pathway.Part I Panax quinquefolium saponin attenuates cardiomyocyte apoptosis and opening of the mitochondrial permeability transition pore in a rat model of ischemia/reperfusionObjective:Opening of the mitochondrial permeability transition pore (mPTP) is a critical event during ischemia/reperfusion (I/R) injury. Recently, we showed that Panax quinquefolium saponin (PQS) alleviates apoptosis of cardiomyocytes by suppressing excessive endoplasmic reticulum stress (ERS) during I/R injury. Here, we hypothesized that this anti-apoptotic effect might be mediated through inhibition of mPTP and the mitochondrial apoptotic pathway.Methods:Ninety healthy male Sprague-Dawley rats were randomly divided into sham, I/R, PQS+I/R (200 mg/kg/d), Cyclosporine A (CsA,10 mg/kg), CsA+I/R (10 mg/kg), and PQS+CsA+I/R. I/R was modeled in rats by ligating the left anterior descending artery (LAD) for 30 min followed by 120 min of reperfusion. To evaluate the cardioprotective function of PQS, we measured hemodynamics, serum content of lactate dehydrogenase (LDH), serum content of creatine kinase-MB (CK-MB), myocardial infarct size, and myocardial apoptotic index (AI). We investigated the underlying mechanism by examining changes in the mitochondrial ultrastructure and membrane potential (ATm), dynamics of mPTP opening, expression of cleaved caspase-3, cleaved caspase-9 in the myocardium, Bcl-2 and Bax in the mitochondria versus cytosol, and translocation of cytochrome c.Results:Administration of PQS to I/R rats significantly reduced serum LDH level, serum CK-MB level, infarct size and AI. In addition, PQS protected the mitochondrial structure, markedly inhibited mPTP opening and Δψm depolarization, led to upregulation of Bcl-2 and downregulation of Bax in the mitochondria compared to the cytosol, and suppressed the expression of cleaved-caspase-9 and cleaved-caspase-3, as well as I/R induced translocation of cytochrome c to the cytoplasm.Conclusion:Our results show that PQS can alleviate apoptosis of cardiomyocytes during I/R injury, possibly due to repressed mitochondrial apoptotic pathway associated with the opening of mPTP induced by myocardial I/R injury.Part Ⅱ Panax quinquefolium saponin protects cardiomyocytes from hypoxia/reoxygenation injury by inhibition of mitochondrial permeability transition pore opening via PI3K/Akt/GSK-3β pathway.Objective:The activation of phosphatidylinositol 3-kinase/serine threonine kinase (PI3K/Akt) pathway and the phosphorylation of glycogen synthase kinase 3β (GSK-3β) are crucial in suppressing mPTP opening during hypoxia/reoxygenation (H/R). The aim of this study was to investigate whether the cardioprotective effects of PQS during H/R are associated with PI3K/Akt/GSK-3p pathway.Methods:The cardiomyocyte injury of neonatal rats was induced by hypoxia/reoxygenation (H/R). PQS (160 μg/mL) pretreatment was administered at 24h before H/R. CCK-8 assay was used to detect the cell viability; annexin V/PI analysis and TUNEL assay were used to evaluate the apoptotic rate of cardiomyocytes; cytosolic Ca2+ concentrations, mPTP opening and mitochondrial membrane potential (Δψm) were analyzed by fluo-4, calcein release and JC-1, respectively. The ATP content was measured to detect the effect of PQS on mitochondrial function with firefly luciferase.Results:PQS pretreatment attenuated the H/R-induced decreased cell viability and increased cardiomyocyte apoptosis, elevated the cytosolic Ca2+ concentrations, inhibited mPTP opening and Δψm depolarization, and increased the cellular ATP content. PQS pretreatment at 24-hour before H/R significantly suppressed H/R-induced translocation of cytochrome c from mitochondria to cytoplasm, down-regulate the expression of cleaved-caspase-3, and up-regulate the expression of p-Akt and p-GSK-3β. All these effects of PQS were abolished by LY294002, a specific PI3K/Akt inhibitor.Conclusion:PI3K/Akt/GSK-3β pathway partially mediates the cardioprotection of PQS via inhibiting the mPTP opening, mitochondrial dysfunction and the apoptosis of cardiomyocytes during H/R injury.
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