The Study Of Effect And Mechanisms Of Hydroxysafflor Yellow A On Anoxia/reoxygenation-induced Apoptosis Of H9c2 Cardiomyocytes

Background:Since heart transplantation,coronary artery bypass grafting and percutaneous coronary intervention are widely utilized, acute ischemia-reperfusion (I/R) injury of the heart is a frequently encountered pathophysiology phenomenon in clinical practice. Apoptosis is initiated shortly after the onset of myocardial ischemia and becomes markedly enhanced during reperfusion. Thus, inhibiting the cardiomyocyte apoptosis induced by I/R will result in keeping more myocardium and improving reperfusion therapeutic effectiveness of acute myocardial ischemia.Heme oxygenase-1 (HO-1) is the induction type of heme oxygenase, it was reported that could be induced to be overexpressed to anti-apoptosis.Many kinds of agents can upregulate HO-1 expression through activation of signaling pathways such as ERK1/2 and Akt, enhancing translocation of nuclear factor erythroid-2-related factor 2 (Nrf2) to the nucleus, where it binds to antioxidant response element (ARE) sequences, leading to the transcriptional activation of HO-1 to provide protection to the cells against various forms of stress.Hydroxysafflor yellow A (HSYA) is the overriding effective chemical component isolated from the flower of the medicinal plants safflower. Previous studies reported that HSYA can alleviate I/R injury and inhibite hypoxia-induced apoptosis of endothelial cells. However, there is still no report on the effect of HSYA on cardiomyocyte apoptosis induced by I/R, and the mechanisms of its effect are unclear. This study includes two experimental parts to investigate the effect and mechanisms of HSYA on A/R-induced apoptosis of the H9c2 cardiomyocytes.Part 1:Upregulation of heme oxygenase-1 expression by hydroxysafflor yellow A inhibiting apoptosis induced by anoxia/reoxygenation of H9c2 cardiomyocytesObjective:We utilized the H9c2 cardiomyocytes subjected to anoxia-reoxygenation (A/R) in a serum-free medium in vitro simulating ischemia/reperfusion in vivo, which were treated by HSYA, to study the effect and mechanisms of HSYA on A/R-induced apoptosis of H9c2 cardiomyocytes.Methods:The cultured H9c2 cardiomyocytes were randomly divided into control (Con) groups. A/R groups, different dosage (1.5.20.80μM) HSYA treated groups, cell viability was measured by MTT, determination of cardiac troponin I concentrations in the supernatant was executed by enzyme linked immuno assay and Western blot analysis was used to measure the expression of HO-1 in the H9c2 cardiomyocytes of different groups. Then the H9c2 cardiomyocytes were randomly divided into Con groups. A/R groups,20μM HSYA treated groups and HO-1 inhibiter (ZnPP-Ⅸ) groups, which were gone ahead with additional experiment. Assay for HO-1 enzyme activity was executed, flow cytometric detection of apoptosis rate was used and Western blot analysis was used to measure the expression of HO-1,CC3 in H9c2 cardiomyocytes and the Bcl-2/Bax ratio in the mitochondria of the H9c2 cardiomyocytes of different groups.results:1. HSYA enhanced cell viability of the H9c2 cardiomyocytes subjected to A/R.2. HSYA alleviated A/R injury of the H9c2 cardiomyocytes subjected to A/R.3. HSYA upregulated HO-1 expresssion in the H9c2 cardiomyocytes subjected to A/R.4. ZnPP-Ⅸhad no noticeable influence on HO-1 expression upregulated by HSYA in the H9c2 cardiomyocytes subjected to A/R.5. HO-1 enzymatic activity upregulated by HSYA in the H9c2 cardiomyocytes subjected to A/R was completely suppressed by ZnPP-Ⅸ.6. The effect of HSYA providing protection to the H9c2 cardiomyocytes against apoptosis induced by A/R was notably negated by ZnPP-Ⅸ.7. The effect of HSYA increased the Bcl-2/Bax ratio in the mitochondria of the H9c2 cardiomyocytes subjected to A/R was partly negated by ZnPP-Ⅸ.8. The effect of HSYA reduced caspase-3 activation of the H9c2 cardiomyocytes subjected to A/R was negated notably by ZnPP-Ⅸ.Conclusions:HSYA can provide protection to the H9c2 cardiomyocytes against apoptosis induced by A/R, and its antiapoptotic effect depends on the upregulation expression and enzymatic activity of HO-1. Part 2:The signal transduction pathway of HSYA upregulating HO-1 expression in the H9c2 cardiomyocytes subjected to A/RObjective:Close behind the first experimental part, We utilized the H9c2 cardiomyocytes subjected to A/R in a serum-free medium in vitro simulating I/R in vivo, which were treated by HSYA, to study the signal transduction pathway of HSYA upregulating HO-1 expression in the H9c2 cardiomyocytes subjected to A/R.Methods:The cultured H9c2 cardiomyocytes were randomly divided into Con groups,A/R groups,HSYA(20μM) treated groups and HO-1 inhibiter groups, Western blot analysis was used to measure the expression of p-ERK1/2,p-Akt in the H9c2 cardiomyocytes of different groups. Then the H9c2 cardiomyocytes were randomly divided into Con groups,A/R groups,HSYA (20μM) treated groups and PI3K inhibiter (LY294002) groups, which were gone ahead with additional experiment. Western blot analysis was used to measure the expression of p-Akt,HO-1,CC3 in the H9c2 cardiomyocytes and Nrf2 levels in the nucleus of the H9c2 cardiomyocytes of different groups.results:1. HSYA or ZnPP-Ⅸhad no striking influence on ERK1/2 phosphorylation in the H9c2 cardiomyocytes subjected to A/R.2. HSYA activated Akt phosphorylation in the H9c2 cardiomyocytes subjected to A/R, and ZnPP-Ⅸhad no influence on Akt phosphorylation activated by HSYA.3. Akt phosphorylation activated by HSYA in the H9c2 cardiomyocytes subjected to A/R was completely blocked by LY294002. 4.The CC3 levels supressed by HSYA in the H9c2 cardiomyocytes subjected to A/R was notably negated by LY294002, and HO-1 expression upregulated by HSYA in the H9c2 cardiomyocytes subjected to A/R was partly blocked by LY294002.5. HSYA enhanced the translocation of Nrf2 to the nucleus in the H9c2 cardiomyocytes subjected to A/R, which was partly blocked by LY294002.Conclusions:HSYA can provide protection to the H9c2 cardiomyocytes against apoptosis induced by A/R. Its antiapoptotic effect largely depends on the upregulation expression and enzymatic activity of HO-1 via the PI3K/Akt pathway, enhancing the translocation of Nrf2 to the nucleus and binding to ARE sequences, leading to the transcriptional activation of HO-1.