Elsevier

Biochemical Pharmacology

Volume 91, Issue 1, 1 September 2014, Pages 40-50
Biochemical Pharmacology

Andrographolide inhibits TNFα-induced ICAM-1 expression via suppression of NADPH oxidase activation and induction of HO-1 and GCLM expression through the PI3K/Akt/Nrf2 and PI3K/Akt/AP-1 pathways in human endothelial cells

https://doi.org/10.1016/j.bcp.2014.06.024Get rights and content

Abstract

Andrographolide, the major bioactive component of Andrographis paniculata, has been demonstrated to have various biological properties including anti-inflammation, antioxidation, and anti-hepatotoxicity. Oxidative stress is considered a major risk factor in aging, inflammation, cancer, atherosclerosis, and diabetes mellitus. NADPH oxidase is a major source of endogenous reactive oxygen species (ROS). In this study, we used EA.hy926 endothelial-like cells to explore the anti-inflammatory activity of andrographolide. Andrographolide attenuated TNFα-induced ROS generation, Src phosphorylation, membrane translocation of the NADPH oxidase subunits p47phox and p67phox, and ICAM-1 gene expression. In the small hairpin RNA interference assay, shp47phox abolished TNFα-induced p65 nuclear translocation, ICAM-1 gene expression, and adhesion of HL-60 cells. Andrographolide induced the gene expression of heme oxygenase 1 (HO-1) and glutamate cysteine ligase modifier subunit (GCLM) in a time-dependent manner. Cellular glutathione (GSH) content was increased by andrographolide. shGCLM attenuated the andrographolide-induced increase in GSH content and reversed the andrographolide inhibition of HL-60 adhesion. shHO-1 showed a similar effect on andrographolide inhibition of HL-60 adhesion to shGCLM. The mechanism underlying the up-regulation of HO-1 and GCLM by andrographolide was dependent on the PI3K/Akt pathway, and both the Nrf2 and AP-1 transcriptional factors were involved. Our results suggest that andrographolide attenuates TNFα-induced ICAM-1 expression at least partially through suppression of NADPH oxidase activation and induction of HO-1 and GCLM expression, which is PI3K/Akt pathway-dependent.

Introduction

Reactive oxygen species (ROS) are recognized as a major cause of inflammation and result in injury to and dysfunction of the cardiovascular system. Proinflammatory cytokines such as tumor necrosis factor-alpha (TNFα) can trigger the production of ROS [1]. Excess production of ROS, such as superoxide anions, hydrogen peroxide, peroxynitrite, nitric oxide, and hydroxyl radicals, not only results in endothelial dysfunction [2] but also activates signaling pathways involved in increased inflammatory cytokine gene expression [3]. In vascular cells, ROS are produced by NADPH oxidase, xanthine oxidase, NO synthase, mitochondrial respiration, and lipoxygenase as well as by cyclooxygenase [4].

NADPH oxidase is a major source of endogenous ROS in endothelial cells and macrophages [5], [6]. The NADPH oxidase is composed of gp91phox, p22phox, p40phox, p47phox, p67phox, and Rac subunits. The cytosolic subunits, p40phox, p47phox, p67phox, and Rac, mediate the activation of NADPH oxidase by phosphorylation and membrane translocation [7]. NADPH oxidase is activated by various cytokines and growth factors such as TNFα and vascular endothelial growth factor [8], [9]. Moreover, NADPH oxidase-derived ROS serve as signal intermediates in selected downstream pathways such as Src, PI3K, and p38 in endothelial cells [9].

The Src family of protein tyrosine kinases includes c-Src, Fyn, Yrk, and Yes, which are widely expressed in different tissues, whereas the other members including Lyn, Hck, Fgr, and Blk have a more restrictive distribution [10]. Because Src can activate signaling molecules such as PI3K and phospholipase C, Src is considered to be an important signaling transmitter in many cellular processes including growth, gene transcription, adhesion, and apoptosis [11]. A previous study showed that Src participates in the activation of TNFα-induced p47phox in human airway smooth muscle cells [12]. Thus, taken together, these data suggest that proinflammatory cytokine-mediated vascular inflammation might be highly related to NADPH oxidase-derived ROS in endothelial cells.

Glutathione (GSH), a member of the cellular antioxidant defense system, assists in the clearance of excessive ROS and maintains the redox homeostasis in cells [13]. The cellular GSH level is influenced by multiple factors, and a primary determinant of the intracellular GSH level is the rate of de novo synthesis. Glutamate cysteine ligase catalyzes the rate-limiting step in GSH synthesis. The mammalian glutamate cysteine ligase holoenzyme is a heterodimer that is composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM) [14]. Previous studies found that mice deficient in the Gclm or Gclc gene have a markedly reduced GSH content in aortas and liver [15], [16]. Thus, the expression of GCLC and GCLM was considered to determine the cellular GSH level.

Heme oxygenase 1 (HO-1) is considered to be a phase II enzyme and antioxidant [17]. HO-1 catalyzes the rate-limiting step in heme catabolism and produces carbon monoxide, free iron, and biliverdin, which is further catabolized into bilirubin by biliverdin reductase [18], [19]. Previous studies suggested that dietary phytochemicals provide chemoprevention and therapeutic potential because they induce HO-1 and thereby enhance cellular antioxidant capacity [20], [21]. Several signaling molecules, such as mitogen-activated protein kinases (JNK, ERK, and p38) and PI3K/Akt, and transcriptional factors, such as activator protein 1 (AP-1) and NF-E2-related factor-2 (Nrf2), participate in the regulation of GCLC, GCLM, and HO-1 gene expression [20], [22], [23].

Andrographis paniculata (Burm. F.) Nees is a traditional herb in China, Korea, and other regions in Southeast Asia. Andrographolide is one of the major bioactive components and is the most abundant diterpenoid in the leaves of A. paniculata [24]. Andrographolide has been studied for its beneficial properties such as anti-inflammation, anti-cancer, and antioxidation [25], [26], [27]. Antioxidation and anti-inflammation are considered to be therapeutic strategies for prevention or treatment of atherosclerosis. The present study was undertaken to clarify the role of NADPH oxidase, GCLM, and HO-1 in the andrographolide inhibition of TNFα-induced ICAM-1 expression and the underlying mechanisms involved.

Section snippets

Reagents

Cell culture medium (RPMI-1640), RPMI-1640 without phenol red, Dulbecco's modified Eagle's medium (DMEM), penicillin–streptomycin solution, and 0.25% trypsin–EDTA were obtained from GIBCO (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from HyClone (Logan, UT, USA). Andrographolide was purchased from Calbiochem (Darmstadt, Germany). Human TNFα, sodium bicarbonate, HEPES, 5,5′-dithiobis(2-nitro-benzoic acid) (DTNB), LY294002, DMSO, and all other chemicals were purchased from

Effect of andrographolide on TNFα-induced ROS generation

To determine whether andrographolide attenuates TNFα-induced ROS generation, we pretreated cells with 7.5 μM andrographolide for 16 h and then challenged cells with 1 ng/ml TNFα for an additional 20 min. As shown in Fig. 1, TNFα induced ROS generation at 20 min, and pretreatment with 7.5 μM andrographolide significantly inhibited this ROS generation. N-Acetylcysteine (NAC) was used as a positive control.

TNFα-induced NADPH oxidase activation and the essentiality of NADPH oxidase in TNFα-induced ICAM-1 expression and monocyte adhesion in EA.hy926 cells

NADPH oxidase is a major source of ROS. Activation of NADPH oxidase is initiated by the membrane

Discussion

Enhancement of cellular antioxidant capacity is believed to reduce the risk of oxidative stress-mediated diseases. Andrographolide is one of the major bioactive components and the most abundant diterpenoid in the leaves of A. paniculata [37]. Our laboratory has shown that andrographolide has anti-inflammatory [22] and chemopreventive [27] activities and that these activities are associated with HO-1 induction. In this study, we demonstrated that andrographolide suppressed TNFα-induced ROS

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was supported by grants NSC-100-2313-B-039-002-MY3 and CMU101-ASIA-11 from the National Science Council, China Medical University, and Asia University, Taiwan.

References (62)

  • T. Li et al.

    Heme oxygenase-1 inhibits progression and destabilization of vulnerable plaques in a rabbit model of atherosclerosis

    Eur J Pharmacol

    (2011)
  • M. Fujii et al.

    Bilirubin and biliverdin protect rodents against diabetic nephropathy by downregulating NAD(P)H oxidase

    Kidney Int

    (2010)
  • C.C. Franklin et al.

    Structure, function, and post-translational regulation of the catalytic and modifier subunits of glutamate cysteine ligase

    Mol Aspects Med

    (2009)
  • C. Chen et al.

    Dietary cancer-chemopreventive compounds: from signaling and gene expression to pharmacological effects

    Trends Pharmacol Sci

    (2005)
  • S.K. Biswas et al.

    Depressed glutathione synthesis precedes oxidative stress and atherogenesis in Apo-E(−/−) mice

    Biochem Biophys Res Commun

    (2005)
  • B.C. Liao et al.

    Cinnamaldehyde inhibits the tumor necrosis factor-α-induced expression of cell adhesion molecules in endothelial cells by suppressing NF-κB activation: Effects upon IκB and Nrf2

    Toxicol Appl Pharmacol

    (2008)
  • F. Nadori et al.

    Presence of distinct AP-1 dimers in normal and transformed rat hepatocytes under basal conditions and after epidermal growth factor stimulation

    Hepatology

    (1997)
  • S.M. Yeligar et al.

    Ethanol-induced HO-1 and NQO1 are differentially regulated by HIF-1alpha and Nrf2 to attenuate inflammatory cytokine expression

    J Biol Chem

    (2010)
  • Y.S. Kim et al.

    Curcumin attenuates inflammatory responses of TNF-alpha-stimulated human endothelial cells

    J Cardiovasc Pharmacol

    (2007)
  • Y. Shi et al.

    Reactive oxygen-derived free radicals are key to the endothelial dysfunction of diabetes

    J Diabetes

    (2009)
  • H. Cai et al.

    Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress

    Circ Res

    (2000)
  • X.L. Chen et al.

    Superoxide, H2O2, and iron are required for TNF-alpha-induced MCP-1 gene expression in endothelial cells: role of Rac1 and NADPH oxidase

    Am J Physiol Heart Circ Physiol

    (2004)
  • Y.S. Bae et al.

    Macrophages generate reactive oxygen species in response to minimally oxidized low-density lipoprotein: toll-like receptor 4- and spleen tyrosine kinase-dependent activation of NADPH oxidase 2

    Circ Res

    (2009)
  • H. Raad et al.

    Regulation of the phagocyte NADPH oxidase activity: phosphorylation of gp91phox/NOX2 by protein kinase C enhances its diaphorase activity and binding to Rac2, p67phox, and p47phox

    FASEB J

    (2009)
  • J.M. Li et al.

    Acute tumor necrosis factor alpha signaling via NADPH oxidase in microvascular endothelial cells: role of p47phox phosphorylation and binding to TRAF4

    Mol Cell Biol

    (2005)
  • S.J. Parsons et al.

    Src family kinases, key regulators of signal transduction

    Oncogene

    (2004)
  • S.M. Thomas et al.

    Cellular functions regulated by Src family kinases

    Annu Rev Cell Dev Biol

    (1997)
  • Y. Chen et al.

    Hepatocyte-specific Gclc deletion leads to rapid onset of steatosis with mitochondrial injury and liver failure

    Hepatology

    (2007)
  • A.M. Choi et al.

    Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein in oxidant-induced lung injury

    Am J Respir Cell Mol Biol

    (1996)
  • C.S. Huang et al.

    Protection by chrysin, apigenin, and luteolin against oxidative stress is mediated by the Nrf2-dependent up-regulation of heme oxygenase 1 and glutamate cysteine ligase in rat primary hepatocytes

    Arch Toxicol

    (2013)
  • Y. Zhong et al.

    Heme oxygenase-1-mediated reactive oxygen species reduction is involved in the inhibitory effect of curcumin on lipopolysaccharide-induced monocyte chemoattractant protein-1 production in RAW264.7 macrophages

    Mol Med Rep

    (2013)
  • Cited by (0)

    View full text