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QSKL, a clinical effective compound, plays cardio-protective roles in heart failure. But its underlying mechanism needs to be further elaborated. The purpose of this study was to identify the protective effects of QSKL on oxygen-glucose deprivation/recovery of oxygen glucose (OGD/R) induced-apoptosis in H9C2 cells and to clarify the underlying mechanism.
A model of apoptosis induced by OGD/R was established in vitro. The changes in cell viability were examined with CCK-8 assay to determine the available concentration of QSKL. 2′, 7′-Dichloroflurescin diacetate (DCFH-DA) and Hoechst 33342 were used to detect the effect of QSKL on ROS level and apoptotic rate, respectively. JC-1 was used to detect the mitochondrial membrane potential. We also used western blot to examine the effect of QSKL on the regulation of proteins related to apoptosis: p53, Mdm2, Bcl-2, Bax, caspase-8 and cleaved-caspase-3. LY294002, an inhibitor of PI3K, was used to detect the effect of QSKL on PI3K/Akt signaling pathway.
Compared with control group, cell survival rate in model group was 54.3±6.6%. Compared with model group, 400μg/ml, 600μg/ml, 800μg/ml and 1000μg/ml QSKL elevated cell viability (p ＜ 0.01) and cell survival rates were 73.7 ± 4.6%, 84.2 ± 2.9%, 87 ± 1.4%, 74.5 ± 3.7% respectively. In model group, ROS generation in cells increased, mitochondrial membrane potential and red/green fluorescence ratio decreased, apoptotic rate was 23.9 ± 5.8% (p < 0.01). Application of QSKL could make a reduction on intracellular ROS generation (p < 0.01) and increase mitochondrial membrane potential, which thus elevating the ratio of red/green fluorescence (p < 0.01). In addition, the apoptotic rate dropped to 8.9 ± 2.2%, which had a significant difference compared with the model group (p < 0.01). With the detection of western blot, Bcl-2 decreased slightly in model group, but had no difference compared with control group, while Bcl-2/Bax ratio was significantly lower (p < 0.01), caspase-8 and cleaved-caspase-3 was significantly increased (p < 0.01), Bax, p53 and Mdm2 slightly increased (p < 0.05). Compared with model group, QSKL significantly increased Bcl-2/Bax ratio (p <0.01), reduced the expression of p53, Bax, caspase-8 and cleaved-caspase-3 (p < 0.01), and increased the expression of Mdm2 (p < 0.05). After application of LY294002, the protective effect of QSKL was inhibited, and there were no difference on the expression of cleaved-caspase-3 and p-Akt compared with model group (p > 0.05).
QSKL protects H9C2 cells from OGD/R-induced apoptosis by reducing intracellular ROS level, elevating mitochondrial membrane potential and regulating apoptotic proteins. The protective and anti-apoptotic effects of QSKL could be mediated through modulating the p-Akt-P53/Mdm2 apoptotic pathway.