Study Of The Anti-Apoptotic Effect And Mechanism Of Panax Notoginseng Saponin On Rat Cardiomyocytes Induced By Ischemia

Objective It had been demonstrated that PNS could protect cardiomyocytes injury induced by ischemia, but the underlying molecular mechanism of this protective effect was still unclear. This study aimed at investigating the anti-apoptotic effect of PNS on rat cardiomyocytes induced by ischemia in vitro and in vivo, and at investigating the potential mechanism involved in the anti-apoptotic effect of PNS. Methods H9c2 cells were divided into control group, model group and different concentrations of PNS (0.01g/L,0.05g/L,0.25g/L,0.5g/L,0.75g/L,2.25g/L and 3.75g/L) groups. Cells of control group were cultured in DMEM/F12 supplemented with 10%FBS in a humidified atmosphere of 5% CO2. Cells of model group were subjected to serum, glucose and oxygen deprivation (SGOD). Cells of PNS groups were subjected to SGOD and treated with different concentrations of PNS at the same time. Apoptotic cells were detected by flow cytometer (FCM) after stained with Annexin V/PI. Mitochondrial membrane potential (Δψm) and intracellular reactive oxygen species (ROS) were detected by JC-1 and DCFH-DA, respectively. Adult male Sprague-Dawley rats were subjected to left anterior descending (LAD) coronary artery ligation and were divided into ischemia group, PNS-L (30mg/kg/d) group and PNS-H (60mg/kg/d) group. Rats of sham operation group (sham) were subjected to thoracotomy without LAD ligation. PNS were administered by mouth from the next day. Rats of sham operation group and ischemia group were given isovolemic physiological saline. Rats cardiac function were examined by echocardiograph six weeks later. Then the hearts were snipped, weighed, and made the frozen section for TUNEL staining which can be used to detect the apoptotic cells of cardiac tissue. Western blot assay was used to detect the expression of proteins caspase-3, cleaved caspase-3, bcl-2, Akt, MIF, AMPK, phosphorylated Akt (p-Akt) and phosphorylated AMPK (p-AMPK).Results It was shown that PNS could protect H9c2 cell from apoptosis induced by SGOD in dose-dependent manner by FCM analysis. The apoptotic ratio of normal control group was 7.15%±1.56%, that of model group was 67.79%±3.74%(vs. control P=0.000).And the apoptotic ratios of PNS groups were 65.58%±1.90%,66.99%±2.86%,53.22%±3.62%(vs. model P=0.006), 37.34%±2.16%(vs. model P=0.000),31.34%±2.52%(vs. model P=0.000),24.42%±0.81%(vs. model P=0.000),19.76%±5.21%(vs. model P=0.000), respectively. While all of LY294002 (a specific inhibitor of PI3K), ISO-1(a MIF antagonist) and Compound C (an AMPK inhibitor) could significantly reverse the anti-apoptotic effect of PNS (P=0.000, P=0.003, P=0.000 vs. PNS2.25g/L). Besides, PNS could decrease the protein expression of cleaved caspase-3, thus inhibited the activation of caspase-3. However, LY294002 reversed the inhibitory effect of PNS on the activation of caspase-3. It was indicated that PNS could protect mitochondrial membrane potential from depolarization by JC-1 staining. DCFH-DA staining demonstrated that PNS could significantly decrease intracellular ROS. In vivo, data of echocardiograph demonstrated that PNS-H could significantly improve cardiac function of rat ischemic heart since PNS-H could significantly increase the'EF (P=0.010 vs. ischemia) and FS (P=0.004 vs. ischemia), and significantly decrease the LVDd (P=0.001 vs. ischemia) and LVDs (P=0.000 vs. ischemia} in the meanwhile. PNS-L could also improve the ischemic rats cardiac function, but the difference between groups had no statistical significance compared with ischemia group. The anti-apoptotic effect of PNS was confirmed by TUNEL staining in vivo since PNS could significantly decrease the percentage of TUNEL positive cells and decrease the protein expression of cleaved caspase-3, meanwhile, increase the protein expression of bcl-2 in cardiac tissue. Western blot assay shew that expression of p-Akt protein was significantly downregulated when the cells were subjected to SGOD (P=0.000 vs. control), while PNS could significantly increase the expression of p-Akt protein (P=0.004 vs. model), but this effect was significantly reversed by LY294002 (P=0.002 vs. PNS 2.25g/L). The MIF and p-AMPK protein expression were upregulated in SGOD alone group (P=0.047, P=0.006 vs. control). PNS was able to further increase the MIF and p-AMPK protein expression (P=0.043,P=0.003 vs. model). Western blot also shew that PNS-H could significantly increase the p-Akt, MIF, p-AMPK protein expression in cardiac tissue (P=0.000, P=0.020,P=0.010 vs. ischemia). PNS-L could also increase the p-Akt, MIF, p-AMPK protein expression in cardiac tissue, but the difference between groups had no statistical significance compared with ischemia group.Conclusion These results indicated that PNS could inhibit rat cardiomyocytes apoptosis induced by ischemia through the mitochondrion-dependent pathway, which was involved with PI3K/Akt pathway. Besides, the upregulation of MIF and p-AMPK protein expression also involved in the anti-apoptotic effect of PNS. The results were meaningful for further illuminating the molecular mechanism of cardiac protection of PNS. And they will provide a meaningful experimental basis for better exploring the clinical usage of PNS in the future.