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
Graphical abstract
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.
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