Oxidative stress suppression by luteolin-induced heme oxygenase-1 expression

Abstract

Luteolin, a flavonoid that exhibits antioxidative properties, exerts myocardial protection effects. However, the underlying molecular mechanisms are not yet fully understood. To investigate the effects of luteolin on myocardial injury protection and its possible mechanisms, a myocardial injury model was established with intragastric administration of 4 mg/kg isoproterenol (ISO) to male Sprague–Dawley rats (200–220 g) daily for 2 days. We found that pretreatment of luteolin (160, 80 and 40 mg/kg, i.g., respectively) daily for 15 days can prevent ISO-induced myocardial damage, including decrease of serum cardiac enzymes, improvement electrocardiography and heart vacuolation. Luteolin also improved the free radical scavenging and antioxidant potential, suggesting one possible mechanism of luteolin-induced cardio-protection is mediated by blocking the oxidative stress. To clarify the mechanisms, we performed the in vitro study by hydrogen peroxide (H₂O₂)-induced cytotoxicty model in H9c2 cells. We found that luteolin pretreatment prevented apoptosis, increased the expression of heme oxygenase-1 (HO-1), and enhanced the binding of Nrf2 to the antioxidant response element, providing an adaptive survival response against H₂O₂-derived oxidative cytotoxicity. The addition of Znpp, a selective HO-1 competitive inhibitor, reduced the cytoprotective ability of luteolin, indicating the vital role of HO-1 on these effects. Luteolin also activated Akt and ERK, whereas the addition of LY294002 and U0126, the pharmacologic inhibitors of PI3K and ERK, attenuated luteolin-induced HO-1 expression and cytoprotective effect. Taken together, the above findings suggest that luteolin protects against myocardial injury and enhances cellular antioxidant defense capacity through the activation of Akt and ERK signal pathways that leads to Nrf2 activation, and subsequently HO-1 induction.

Introduction

Myocyte apoptosis plays an important role in pathological cardiac damage in several cardiovascular diseases, such as myocardial infarction and ischemia/reperfusion injury (van Empel et al., 2005). Evidence suggests that oxidative stress, which is caused by the over-accumulation of intracellular reactive oxygen species (ROS), is one of the leading factors triggering myocyte apoptosis (Takano et al., 2003). Excessive ROS production causes the detrimental modification of vital intracellular macromolecules, such as DNA, proteins, and lipids, resulting in cellular apoptotic death (Ryter et al., 2007). Hence, the modulation of intracellular ROS levels and regulation of apoptotic cascade are considered crucial therapeutic strategies for treating cardiovascular diseases.

Luteolin exists mainly in glycosylated form in many types of plants, including fruits, vegetables, and medicinal herbs. Our laboratory recently demonstrated that cynaroside, the glycosylated form of luteolin, prevents oxidative injury to cardiac myocytes. Cynaroside is first hydrolyzed into luteolin in the intestinal tract, and then subsequently absorbed and metabolized (Ying et al., 2008). Moreover, luteolin shows various beneficial activities, including cardiovascular protection, anti-inflammatory activity, anticancer activity, and other biological activities (Lopez-Lazaro, 2009, Park et al., 2007b, Seelinger et al., 2008), most of these effects are connected with its antioxidative function. Data from in vitro studies also demonstrate that luteolin exerts prominent antioxidative effects (Rice-Evans et al., 1996). Luteolin has significant protective effects against myocardial ischemia injury (Liao et al., 2011, Rump et al., 1995). Moreover, epidemiological studies have revealed that a high dietary intake of luteolin can significantly reduce the risk of acute myocardial infarction and decrease mortality from cardiovascular diseases (Marniemi et al., 2005, Mink et al., 2007). These studies highlighted the potential function of luteolin in the prevention and treatment of cardiovascular diseases. However, the cellular and molecular mechanisms of luteolin-mediated cardiovascular protection still need to be defined.

The expression of heme oxygenase-1 (HO-1), one of the major antioxidative/cytoprotective enzymes, is induced by various stimuli, such as oxidative stress, heavy metals, pro-inflammatory cytokines, UV irradiation, and thiol-reactive substances, suggesting that HO-1 may play a critical role in cytoprotection (Park et al., 2007a). Enhanced HO-1 expression can reduce the incidence of oxidant-induced cell damage (Ryter et al., 2002). NF-E2-related factor (Nrf2), a redox-sensitive transcription factor capable of interacting with antioxidant response elements (AREs), plays a key role in the transcriptional activation of HO-1 gene expression (Choi and Alam, 1996). Recently, emerging evidence has revealed that the induction of HO-1 through Nrf2 activation confers protection against oxidative stress in the heart (Zhu et al., 2005, Zhu et al., 2008). In particular, luteolin has been shown to induce neurite outgrowth and augment cellular antioxidant defense capacity through the ERK-dependent induction of Nrf2-driven HO-1 expression (Lin et al., 2010). Therefore, an intriguing issue is whether or not luteolin activates Nrf2 and the subsequent up-regulation of HO-1 expression to suppress oxidative stress in myocytes.

In the present study, we examined the protective effects of luteolin against isoproterenol (ISO)-mediated myocardial injury in vivo. We demonstrated that luteolin administration can protect against H₂O₂-induced apoptosis in H9c2 cardiomyoblasts. This function relies on activating Nrf2 through the Akt/protein kinase B- and extracellular signal-regulated kinase (ERK)-dependent signal transduction pathways to up-regulate HO-1 expression.