Penehyclidine hydrochloride (PHC) can protect against myocardial ischemia/reperfusion (I/R) injury. However, the possible mechanisms of PHC in anoxia/reoxygenation (A/R)-induced injury in H9c2 cells remain unclear. In the present study, H9c2 cells were pretreated with PI3K/Akt inhibitor LY294002, ATP-sensitive K + (KATP) channel blocker 5-hydroxydecanoate (5-HD), PHC, or KATP channel opener diazoxide (DZ) before subjecting to A/R injury. Cell viability and cell apoptosis were determined by cell counting kit-8 assay and annexin V/PI assay, respectively. Myocardial injury was evaluated by measuring creatine kinase (CK) and lactate dehydrogenase (LDH) activities. Intracellular Ca 2+ levels, reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨ m ), and mitochondrial permeability transition pore (mPTP) were measured. The levels of cytoplasmic/mitochondrial cytochrome c (Cyt-C), Bax, Bcl-2, cleaved caspase-3, KATP channel subunits (Kir6.2 and SUR2A), and the members of the Akt/GSK-3β and Akt/mTOR signaling pathways were determined by western blotting. We found that PHC preconditioning alleviated A/R-induced cell injury by increasing cell viability, reducing CK and LDH activities, and inhibiting cell apoptosis. In addition, PHC preconditioning ameliorated intracellular Ca 2+ overload and ROS production, accompanied by inhibition of both mPTP opening and Cyt-C release into cytoplasm, and maintenance of ΔΨ m . Moreover, PHC preconditioning activated mitochondrial KATP channels, and modulated the Akt/GSK-3β and Akt/mTOR signaling pathways. Similar effects were observed upon treatment with DZ. Pretreatment with LY294002 or 5-HD blocked the beneficial effects of PHC. These results suggest that the protective effects of PHC preconditioning on A/R injury may be related to mitochondrial KATP channels, as well as the Akt/GSK-3β and Akt/mTOR signaling pathways.

Aims and Objective: The lack of effective treatments for myocardial ischemia-reperfusion (MI-R) injury severely restricts the effectiveness of the treatment of ischemic heart disease. In the present research, we aimed to investigate the protective effect and molecular mechanism of penehyclidine hydrochloride (PHC) on MI-R cells. Method: Cell viability was quantified using CCK8. Cell apoptosis was analyzed using flow cytometry. Western blot and Elisa assays were used for the detection of target proteins. Result: PHC pretreatment attenuated the inhibition of cell viability and decreased the percentage of apoptosis induced by simulated ischemia reperfusion (SIR). Platelet-derived growth factor B (PDGF-B) and its downstream AKT pathway were activated in PHC pretreated cells. After siRNA-PDGF-B transfection, cell viability was inhibited and the apoptosis was activated in PHC pretreated SIR cells, suggesting that PHC protected cells from SIR. PDGF-B knockdown also increased the levels of CK, LDH, IL-6 and TNF-α in PHC pretreated SIR cells. The effect of AKT inhibitor on H9C2 cells was consistent with that of PDGF-B knockdown. Conclusion: PHC pretreatment can protect cardiomyocytes from the decrease of cell activity and the increase of apoptosis caused by reperfusion through up-regulating PDGF-B to activate PI3K pathway. Our study indicates that PHC is a potential drug to protect cells from reperfusion injury and PDGF-B is a potential target for preventing MI-R injury.