Induction of HO-1 through p38 MAPK/Nrf2 signaling pathway by ethanol extract of Inula helenium L. reduces inflammation in LPS-activated RAW 264.7 cells and CLP-induced septic mice

doi:10.1016/j.fct.2012.12.027

Food and Chemical Toxicology

Volume 55, May 2013, Pages 386-395

https://doi.org/10.1016/j.fct.2012.12.027 Get rights and content

Abstract

High mobility group box 1 (HMGB1) plays a crucial mediator in the pathogenesis of many inflammatory diseases. We recently proposed that heme oxygenase-1 (HO-1) negatively regulates HMGB1 in inflammatory conditions. We investigated whether ethanol extract of Inula helenium L. (EIH) activates p38 MAPK/Nrf2/HO-1 pathways in RAW264.7 cells and reduces inflammation in CLP-induced septic mice. EIH induced expression of HO-1 protein in a time- and concentration-dependent manner. EIH significantly diminished HO-1 expression in siNrf2 RNA-transfected cells. As expected, the inhibited expression of iNOS/NO, COX-2/PGE2, HMGB1 release by EIH in LPS-activated RAW264.7 cells was significantly reversed by siHO-1RNA transfection. Furthermore, EIH not only inhibited NF-κB luciferase activity, phosphorylation of IκBα in LPS-activated cells but also significantly suppressed expression of adhesion molecules (ICAM-1 and VCAM-1) in TNF-α activated human umbilical vein endothelial cells. The induction of HO-1 by EIH was inhibited by SB203580 but not by SP600125, PD98059, nor LY294002. Most importantly, administration of EIH significantly reduced not only increase in blood HMGB1, ALT, AST, BUN, creatinine levels but also decrease macrophage infiltrate in the liver of septic mice, which were reversed by ZnPPIX, a HO-1 inhibitor. We concluded that EIH has anti-inflammatory effect via the induction of p38 MAPK-dependent HO-1 signaling pathway.

Graphical abstract

Highlights

► Heme oxygenase-1 negatively regulated high mobility group box 1 in inflammation. ► Demonstration of a role for p38 MAPK/Nrf2 signal for induction of heme oxygenase-1. ► Inula helenium L. maybe useful for treatment of sepsis..

Introduction

It has been shown that heme oxygenase-1 (HO-1) and its by-products play important roles in the resolution of inflammation (Otterbein et al., 1999, Ryter and Choi, 2006). High mobility group box-1 (HMGB1), a non-histone DNA-binding molecule, has been implicated as a multifunctional ubiquitous protein in many eukaryotic cells. HMGB1 was firstly found to be expressed in the nucleus, which regulates the cell cycle, cellular differentiation, and gene transcription (Bustin, 1999). In addition to its nuclear role, the pro-inflammatory cytokine-stimulating activity of extracellular HMGB1 has been recognized. Indeed, HMGB1 has been proposed to be a crucial mediator in the pathogenesis of many diseases, including sepsis, arthritis, cancer, autoimmunity diseases, and diabetes (Lotze and Tracey, 2005, Andersson and Erlandsson-Harris, 2004, Ardoin and Pisetsky, 2008, Zhang et al., 2009). Moreover, HMGB1 is expressed in atherosclerotic lesions in humans and animals (Kalinina et al., 2004). Recently, we and others reported that HO-1 activity negatively regulates HMGB1 under systemic inflammatory conditions, such as sepsis (Jang et al., 2012, Tsoyi et al., 2009, Tsoyi et al., 2011a, Takamiya et al., 2009).

Although Inula helenium L. (Compositae) and its active components have been widely used as anti-inflammatory, anti-microbial, and anti-cancer agents (Qiu et al., 2011, Dorn et al., 2006, Seo et al., 2008, Konishi et al., 2002), no report has found that the anti-inflammatory effect of I. helenium is related with HO-1 activity. Therefore, the present study tested the hypothesis that ethanol extract of I. helenium (EIH) induces HO-1 expression, which is responsible for the reduction of both pro-inflammatory mediators and HMGB1 release in LPS-activated macrophages. Since HO-1 couples the activation of mitochondrial biogenesis to anti-inflammatory cytokine expression, it might significantly reduce inflammatory cytokines. In addition, we explored the signal pathway(s) by which EIH up-regulates HO-1 induction. Here, we report that EIH inhibited expression of iNOS, COX-2, and HMGB1 release in LPS-activated macrophages as well as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in TNF-α-activated human umbilical vein endothelial cells (HUVECs). Most importantly, administration of EIH significantly reduced the blood concentration of HMGB1 and attenuated multiple organ injury in cecal ligation and puncture (CLP)-induced mice, which were restored with zinc-protophorphyrin IX (ZnPPIX), a competitive inhibitor of HO-1, suggesting the importance of HO-1 induction by EIH for anti-inflammatory action.

Section snippets

Materials

Dulbecco’s modified Eagle’s medium (DMEM), fatal bovine serum (FBS), and other tissue culture reagents were purchased from Gibco BRL Co. Primary antibodies for HO-1, cyclooxygenase-2 (COX-2), Nuclear factor erythroid 2 related factor 2 (Nrf2), PCNA, ICAM-1, VCAM-1, and responsible secondary antibodies used for Western blot analysis were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for β-actin were from Sigma–Aldrich (St. Louis, MO), and inducible nitric oxide synthase

Effect of EIH on cell viability

Cells were treated with different concentrations of EIH, and MTT assay was performed after 12 h of incubation as described in Section 2. As shown in Fig. 1a, an approximately 80% survival rate was observed at a dose of 50 μg/ml. However, when incubated for 24 h, significant toxic signs were observed at above 50 μg/ml of EIH (Fig. 1b). Therefore, through the entire experiments, the maximum concentration of EIH was limited to 50 μg/ml.

EIH increases HO-1 expression

Fig. 2 shows that EIH significantly increased HO-1 protein

Discussion

This study, for the first time, provides evidence for the mechanism by which EIH mediates its anti-inflammatory responses by inducing HO-1 in vitro and in vivo. HO-1 and its by-products (biliverdin, CO) are known to have cyto-protective properties, including anti-oxidant, anti-inflammatory, and anti-apoptotic activities (Otterbein et al., 1999, Ryter and Choi, 2006). There is increasing evidence demonstrating that the induction of HO-1 can prevent or mitigate the symptoms associated with

Conflict of Interest

The authors declare that there are no conflicts of interest.

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Induction of HO-1 through p38 MAPK/Nrf2 signaling pathway by ethanol extract of Inula helenium L. reduces inflammation in LPS-activated RAW 264.7 cells and CLP-induced septic mice

Abstract

Graphical abstract

Highlights

Introduction

Section snippets

Materials

Effect of EIH on cell viability

EIH increases HO-1 expression

Discussion

Conflict of Interest

FEBS Lett.

Free Rad. Biol. Med.

Chest

Curr. Opin. Pharmacol.

Neurochem. Int.

J Ethnopharmacol.

HMGB1 is a potent trigger of arthritis

J. Int. Med.

The role of cell death in the pathogenesis of autoimmune disease: HMGB1 and microparticles as intercellular mediators of inflammation

Mod. Rheumatol.

Regulation of DNA-dependent activities by the functional motifs of the high-mobility-group chromosomal proteins

Mol. Cell. Biol.

Tumor cell specific toxicity of Inula helenium extracts

Phytother. Res.

Higenamine reduces HMGB1 during hypoxia-induced brain injury by induction of heme oxygenase-1 through PI3K/Akt/Nrf-2 signal pathways

Apoptosis

Hemeoxygenase-1inhibits atherosclerotic lesion formation in ldl-receptor knockout mice

Circ. Res.

Ethyl pyruvate induces heme oxygenase-1 through p38 mitogen-activated protein kinase activation by depletion of glutathione in RAW 264.7 cells and improves survival in septic animals

Antioxid. Redox. Signal

Salvianolic acid B exerts vasoprotective effects through the modulation of heme oxygenase-1 and arginase activities

J. Pharmacol. Exp. Ther.

Ethanol extract of Prunella vulgaris var. lilacina inhibits HMGB1 release by induction of heme oxygenase-1 in LPS-activated RAW 264.7 cells and CLP induced septic mice

Phytother. Res.

Increased expression of the DNA-binding cytokine HMGB1 in human atherosclerotic lesions: role of activated macrophages and cytokines

Arterioscler. Thromb. Vasc. Biol.

Inhibition of activation of nuclear factor kappaB is responsible for inhibition of inducible nitric oxide synthase expression by higenamine, an active component of aconite root

J. Pharmacol. Exp. Ther.