Fisetin protectes H9c2 cardiomyoblast cells against H2O2induced apoptosis through Akt and ERK1/2 signaling pathways : Evaluation of cardiac muscle cell therapy efficacy of fisetin = H2O2 유도 된 흰쥐 심장 H9c2 유래세포 사멸에 대해 Akt 및 ERK1 / 2 신호 전달 경로를 통한 Fisetin의 심장보호 작용의 기전연구

다국어 초록 (Multilingual Abstract)

Fisetin (tetrahydroflavone), a nutral component of plants, is known to have direct radical scavenging, anti-inflammation and cell survival properties. This study investigated whether fisetin can protect myocardial cells against cell death induced by H2O2, which is associated with cardiovascular risk.
The effects of fisetin on the expression of antioxidant proteins such as superoxide dismutase (SOD, Cu / Zn-SOD and Mn-SOD), catalase (CAT), and heme oxygenase (HO-1) in H9c2 cells exposed to H2O2 were analyzed by western blot Regulation of the enzyme involved in the removal of fisetin during to H2O2-induced oxidative stress resulted in an increase in Cu / Zn-SOD expression compared to that in cells treated with H2O2 alone at a concentration of 5 and 10 μM . However, HO-1, Mn-SOD and CAT expression was not increased after treatment with fisetin.
These data indicate that fisetin reduces oxidative damage by enhancing the antioxidant capacity associated with Cu / Zn-SOD in cell. The enzyme can be rapidly a first line of defense when cell are exposed to oxygen-derived free radicals or organisms are under oxidative stress. These results indicate that fisetin protects H9c2 cells treated with H2O2 from reactive oxygen species (ROS) produced during oxidative stress and enhances antioxidant enzyme activity.
Fisetin reduced H2O2-induced apoptosis in H9c2 cells, Further fisetin clearly showed cardioprotective effects and enhanced cell survival, suggesting that recovery of mitochondrial energy played a role in its protection. We therefore assessed the effect of fisetin on cell death by FACS analysis after double staining with annexin-V and propidium iodide. Survival increased in a concentration-dependent manner. relative to that in the control group, where H2O2 treated H9c2 cells were not exposed to fisetin, The effect of fisetin on the expression of apoptotic regulatory proteins including caspase-3, (Bax, and Bcl-2) in H2O2-stimulated cells was examined to confirm the anti-apoptotic effect of fisetin in H2O2-treated cells. Bcl-2 expression decreased after H2O2 treatment, whereas H2O2 treatment significantly increased the expression of cleaved caspase-3 and Bax compared to that in the control cells (without H2O2 treatment). Fisetin pretreatment significantly reduced both H2O2-induced Bax levels in cleaved caspase-3 and H9c2 cells. In addition, H2O2 treatment reduced the ratio of Bcl-2 to Bax compared with the normal control, whereas fisetin treatment induced recovery of the Bcl-2-/-Bax ratio in a dose-dependent manner. Fisetin increased protein kinase B (Akt) and extracellular regulated kinase (ERK) 1/2 phosphorylation.
We examined signaling pathways that might, support the protective effect of fisetin against cell damage induced by H2O2. Activation of the PI3K-/-Akt pathway in H9c2 cells inhibits apoptosis and promotes cell survival, and mitogen-activated protein kinases (ERK1 / 2), including MAPKs, are involved in the regulation of apoptosis. Therefore, we examined whether the P13K/AKT pathway could be altered by fisetin in H2O2-exposed cell. H9c2 cells were treated with H2O2 for 30 minutes and then the expression and phosphorylation of Akt, ERK1-/-2 and glycogen synthase kinase (GSK) -3β were measured. Phosphorylation of Akt and ERK 1/2 were dramatically increased in a concentration-dependent manner after H9c2 cells exposed to H2O2 were treted with fisetin. However, no significant change in GSK-3 b type phosphorylation was observed after fisetin treatment of H2O2-exposed H9c2 cells.
These results indicate that the Akt and ERK1-/-2 signaling pathways may be involved in the anti-apoptotic effects of fisetin in H9c2 cells exposed to H2O2 in particular. Inhibition of the Akt and ERK1-/-2 signaling pathways plays an important role in the regulation of apoptosis in H2O2-exposed H9c2 cells. H9c2 cells were pretreated with LY294002 (a PI3K / Akt pathway inhibitor, 20 μM) and PD98059 (an MEK inhibitor, 20 μM) and fisetin (10 μM to determine whether increased Akt and ERK1-/-2 phosphorylation contributeds to the cardioprotective effects of fisetin).
In the absence or presence of H2O2, neither LY294002 nor PD98059 did affected cell viability. However, pre-treatment with LY294002 and PD98059 reduced the protective effect of fisetin in cell exposed to H2O2. When cells were pretreated with LY294002, PD98059 and fisetin the viability of the cells exposed to H2O2 decreased compared to that of cells treated with fisetin alone. It was This was further analyzed by Western blotting with. The effects of LY294002 and PD98059 decrease Akt and ERK1-/-2 phosphorylation, respectively by western blot analysis. Fisetin pre-treatment decreased both Akt and ERK1-/-2 the LY294002 and PD98059. treatment group. Also, LY294002 및 PD98059는 caspase-3, Bax, In addition, LY294002 and PD98059 reversed the protective effect of fisetin in H2O2 exposed cell and increased antioxidant enzyme activity as determined by the expression of apoptosis-related proteins such as cleaved caspase-3, Bax and Bcl. The, he data suggest that the protective action of against H2O2-induced death of H9c2 cells is at least in part mediated by the Akt and ERK1 /-2 signaling pathwasys. Fisetin increased the phosphorylation of apoptotic cells and intracellular ROS, while reducing the expression of Cu-/-Zn-superoxide dismutase (SOD and Akt cell kinase (ERK) 1/2 Let. In particular, fisetin significant reduction apoptosis by inhibiting cleaved caspase-3 and Bax-, and increased the anti-apoptotic activity of Bcl-2 the enzymes in H9c2 cells. However, heme oxygenase, Mn-SOD and catalase were not changed by fisetin under these conditions. Taken together, these data suggest that, the potential cardioprotective effects of fisetin may be dependent on Akt and Cu-/-Zn-SOD-mediated activation of Bcl-2 via the ERK1 / 2 pathway. Therefore fisetin could be useful in the treatment of myocardial cells caused by ischemia / reperfusion.

목차 (Table of Contents)

• ABSTRACT = i
• TABLE OF CONTENTS = v
• LIST OF FIGURES = vii
• ABBREVIATION = xii
• 1. INTROUDCTION = 1
• 2. MATERIALS AND METHODS = 6
• 2.1. Materials and reagents = 6
• 2.2. Cell culture = 6
• 2.3. Cell viability = 7
• 2.4. Assessment of apoptotic cell death = 8
• 2.5. Measurement of intracellular ROS = 9
• 2.6. Western blotting = 10
• 2.7. Statistical analysis = 11
• 3. Results = 12
• 3.1. Fisetin protected H9c2 cells against H2O2-induced cytotoxicity = 12
• 3.2. Fisetin reduced intracellular ROS generation and enhanced anti-oxidant enzyme activity in H2O2-treated H9c2 cells = 18
• 3.3. Fisetin decreased H2O2-induced apoptosis in H9c2 cells = 30
• 3.4. Fisetin increased protein kinase B(Akt) and extracellular regulated kinase (ERK) 1/2 phosphorylation = 45
• 3.5. Suppression of the Akt and ERK1/2 signaling pathway plays an important role in apoptosis regulation in H2O2-exposed H9c2 cells = 52
• 4. DISCUSSION = 67
• 5. CONCLUSION = 73
• References = 74
• 국문요지 = 87