HO-1Mediates The Protective Effects Of Curcumin Against Oxidative Stress-induced Cell Injury In H9c2Cardiomyoblasts

With the development of cardiac surgery, the survival rate has increased for pediatric patients with complicated and critical congenital heart disease in recent years. Myocardial ischemia/reperfusion injury becomes one of the most important factors for clinical outcome. Cardiomyocytes apoptosis induced by oxidative stress plays a critical role in the development of myocardial ischemia reperfusion injury. Oxidative stress is a pathological state with abnormal accumulation of free radicals such as O2-, H2O2and their metabolites, which caused by the excessive production of ROS and/or the dysfunction of antioxidant defense system, leading to detrimental effects on cellular and tissue function. Many studies showed that therapeutic strategies aimed at preventing apoptosis from oxidative stress might be a reasonable choice for the treatment of myocardial ischemia/reperfusion injury.Heme oxygenase-1(HO-1) is the rate-limiting enzyme responsible for the degradation of heme into free ferrous iron, carbon monoxide (CO) and bilirubin. Pharmacological and genetic induction of HO-1has been shown to exert anti-apoptotic effect in various cardiovascular diseases including myocardial infarction, ischemia reperfusion injury and heart failure. Curcumin is the major component of turmeric powder extracted from the rhizomes of the plant Curcuma longa. This bioactive phytochemical is a potent inhibitor of tumor promotion, and possesses anti-inflammatory and antioxidant activities. Previous study showed that curcumin could upregulate HO-1protein expression in a dose-dependent manner in endothelial cells and skin fibroblasts in vitro, and HO-1mediates the protective effects of curcumin against cell damage. Thus we speculate that curcumin could protect H9c2cells from oxidative stress-induced cell injury, and this effect was critically associated with HO-1.Protein kinase B (PKB, Akt), one of the most important downstream target kinases of PI3K, is an important signaling molecule activated by anti-apoptotic agents. Several studies reported that HO-1protein expression, which was upregulated through activation of the PI3K/Akt signaling pathway, provides protection against various forms of stress. Nuclear factor κB (NF-κB) is an inducible transcription factor that tightly regulates the expression of a large cohort of genes. The relationship between NF-κB activity and apoptosis remains unclear, and in some cellular systems, it has proven to be detrimental, and in others, protective.In this study, we used the rat cardiac myoblast cell line (H9c2) stimulated with H2O2as a model to examine the role of HO-1in curcumin mediated protection against oxidative damage and the possible mechanism.Part Ⅰ Role of HO-1in the protection of H9c2cardiomyoblasts injury induced by oxidative stressObjective To investigate the protective effect of HO-1on the oxidative injury induced by H2O2in H9c2cardiomyoblasts and explore the possible mechanisms.Methods H9c2cells were pretreated for6h with50μM hemin in the absence or presence of10μM of ZnPP-Ⅸ, which was added1h before hemin, and then incubated for3h with400μM H2O2. HO-1activity was measured by dual-wavelength spectrophotometric determination. Cell viability was estimated using the MTT assay. Apoptosis was estimated by nuclear staining of Hoechst33342and caspase-3activity assay. The MDA content was measured by thio-barbituric acid (TBA) method. The total SOD activity was measured by nitroblue tetrazolium (NBT) assay. The NF-κB protein level was measured by western blot analysis.Results Compared with the control group, hemin increased the HO-1activity in a dose-dependent manner. Pretreatment of hemin significantly increased the HO-1activity, leading to a significant increase in cell survival rate, a decrease in apoptosis rate and caspase-3activity. Meanwhile, increase of HO-1activity induced by hemin could reduce the MDA content and increase the total SOD activity. In addition, the upregulation of the NF-κB protein lever induced by H2O2was inhibited by hemin pretreatment. These effects of hemin can be partly reversed by ZnPP-IX.Conclusion The increase of HO-1activity induced by hemin, protects H9c2cardiomyoblasts from H2O2-induced oxidant injury, probably through regulation of NF-κB nulear translocation and cellular redox banlance. HO-1mediates the protective effects of curcumin against oxidative stress-induced cell injury in H9c2cardiomyoblastsObjective To investigate the protective effects of curcumin against oxidative stress-induced cell injury in H9c2cardiomyoblasts and explore the role of HO-1in this effect.Methods H9c2cells were pretreated for12h with15μM curcumin in the absence or presence of10μM of ZnPP-IX, which was added1h before curcumin, and then incubated for3h with400μM of H2O2. HO-1activity was measured by dual-wavelength spectrophotometric determination. Cell viability was estimated using the MTT assay. Apoptosis was estimated by flow cytometry analysis and caspase-3activity assay. The MDA content was measured by thio-barbituric acid (TBA) method. The total SOD activity was measured by nitroblue tetrazolium (NBT) assay. The protein levels of HO-1, Bcl-2, Bax and cleaved caspase-3were measured by western blot analysis.Results Compared with the control group, curcumin increased the HO-1mRNA, protein expression and activity in a dose-dependent manner. Compared with the H2O2group, curcumin led to a significant increase in cell survival rate, a decrease in apoptosis rate and caspase-3activity. Meanwhile, curcumin could increase the bcl-2/bax ratio and decrease the protein expression level of cleaved caspase-3in H2O2treated-H9c2cells. In addition, curcumin could reduce the MDA content and increase the total SOD activity. These effects of curcumin were partly blocked by ZnPP-Ⅸ. Conclusion Curcumin protects H9c2cardiomyoblasts from H2O2-induced cell injury probably through the regulation of HO-1and cellular redox balance. The mechanism of HO-1mediates the protective effects of curcumin against oxidative stress-induced cell injury in H9c2cardiomyoblastsObjective To investigate the mechanism of HO-1mediates the protective effects of curcumin against oxidative stress-induced cell injury in H9c2cardiomyoblasts.Methods H9c2cells were treated with5-15μM curcumin for12h or15μM curcumin for0.5-12h. The protein expression levels of p-Akt and t-Akt were measured by western blot analysis. H9c2cells were pretreated for12h with15μM curcumin in the absence or presence of50μM of LY294002(or10uM of ZnPP-IX), which was added1h before curcumin, and then incubated for3h with400uM of H2O2. The expression levels of HO-1, cleaved caspase-3or NF-κB were measured by western blot analysis.Results Curcumin increased the levels of p-Akt/t-Akt in a dose-dependent manner. The levels of p-Akt/t-Akt increased remarkably in the first30min and then began to decrease continuously in the following hours. The inhibition of curcumin on the protein expression of cleaved caspase-3in H2O2-treated cells was partly reversed by LY294002. Meanwhile, the upregulation of HO-1protein expression induced by curcumin was also partly reversed by LY294002. In addition, the activation of NF-κB induced by H2O2was inhibited by curcumin pretreatment. ZnPP-IX partly reversed the effect of curcumin on NF-κB suppression, leading to the attenuation of curcumin mediated apoptosis resistance.Conclusion The protective effects of curcumin against oxidative stress-induced cell injury in H9c2cardiomyoblasts requires the upregulation of HO-1expression through the PI3K/Akt signaling pathway and HO-1mediated NF-κB suppression.