Andrographolide acts as an anti-inflammatory agent in LPS-stimulated RAW264.7 macrophages by inhibiting STAT3-mediated suppression of the NF-κB pathway

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Abstract

Ethnopharmacological significance

Inflammation is involved in numerous diseases, such as chronic inflammatory disease and cancer. Many plant products exhibit useful biological activities, including antifungal, antibacterial, and anti-inflammatory actions.

Aim of study

However, our understanding of the anti-inflammatory effects of andrographolide is limited.

Materials and methods

We use lipopolysaccharide (LPS)-stimulated macrophages as a model of inflammation to investigate the anti-inflammatory effects of andrographolide, which contains polyphenolic structures.

Results

We found that andrographolide exhibited a potent anti-inflammatory effect. In this study, we investigated the inhibitory effects of andrographolide on the induction of nitric oxide synthase (NOS) and cyclooxygenase-2 (COX-2) as well as their respective downstream products, NO and PGE2, in RAW264.7 cells treated with LPS. Treatment with andrographolide also reduced nuclear factor-κB (NF-κB) and activation protein-1 (AP-1) DNA-binding activity. Western blot analysis showed that andrographolide significantly inhibited the phosphorylation of signal transducer and activator of transcription-3 (STAT3) and the protein expression of CCAAT/enhancer-binding protein δ (C/EBPδ). We also found that andrographolide suppressed LPS-induced suppressor of cytokine signalling 1 and 3 (SOCS1 and 3) mRNA expression, which, in turn, inhibited apoptosis signalling and mitochondria membrane potential activation. Our results demonstrate that andrographolide downregulates inflammatory iNOS and COX-2 gene expression by inhibiting the activation of NF-κB and STAT3 by interfering with the expression of SOCS1 and SOCS3 signalling.

Conclusion

Therefore, andrographolide exerts a potent anti-inflammatory effect and could potentially be developed as a useful agent for the chemoprevention of cancer or inflammatory diseases.

Introduction

Macrophages play a key role in inflammatory and immune reactions by releasing a variety of inflammatory mediators such as cytokines, chemokines, growth factors, iNOS, COX-2 (Ramana et al., 2006) and increased circulating levels of lipopolysaccharide (LPS) lead to increased mitochondrial activity and the formation of reactive oxygen species (ROS), resulting in disturbed redox homeostasis in macrophages that activate redox-sensitive transcription factors, such as NF-κB and AP-1 (Woo et al., 2004, Liu and Malik, 2006), and lead to apoptotic cell death of macrophages (Woo et al., 2004, Asehnoune et al., 2004, Liu and Malik, 2006). Therefore, interfering with signalling pathways that lead to LPS-mediated apoptosis may represent an important therapeutic target for suppressing inflammatory responses.

Inflammatory stimuli, such as LPS activation of macrophages, lead to the activation of the transcription factors NF-κB and AP-1, which promote the expression of several pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6 (Nunez Miguel et al., 2007), as well as other inflammatory mediators, including nitric oxide (NO) and PGE2, which are synthesised by inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX), respectively. Another important transcription factor involved in pro-inflammatory cytokine-induced inflammatory gene expression is signal transducer and activator of transcription-3 (STAT3). Previous studies show that a STAT-binding gamma activated site is necessary for expression of iNOS in LPS-induced RAW 264.7 macrophages (Marrero et al., 1998). The STAT3 pathway is activated in response to several cytokines, including IL-1β, IL-4, and IL-10 (Yu et al., 2002). Additionally, STAT3 has a dual role in IL-6 mediated signalling; its activation may result in increased IL-6, but also IL-6 itself may lead to phosphorylation of STAT3, resulting in diverse biological responses (Fielding et al., 2008).

Andrographis paniculata (Burm.f.) Nees (Acanthaceae) is a plant indigenous to Southeast Asian countries that has been used as an official herbal medicine in China for many years. Extracts from the plant and their constituents exhibit a wide spectrum of biological activities, including antitumour, antiviral and anti-inflammatory properties (Calabrese et al., 2000, Gabrielian et al., 2002, Abu-Ghefreh et al., 2009, Parichatikanond et al., 2010). Andrographolide (AP), a bicyclic diterpenoid lactone, is the major constituent of Andrographis paniculata. It is particularly efficient at regulating immune responses and works as an anti-inflammatory agent by reducing the generation of ROS in human neutrophils (Calabrese et al., 2000, Shen et al., 2002). It has also been shown to have hepatoprotective effects in animal studies (Singha et al., 2007, Lee et al., 2010).

The relationship between AP and inflammatory reactions has been investigated in macrophage cell systems, whereas there is limited understanding of inhibitory STAT3 mechanisms by AP during the inflammation responses. Therefore, the objective of this study was to determine if AP causes resistance to apoptosis in LPS-stimulated macrophage. To address our hypothesis, we analysed the following: (i) whether the molecular mechanisms of the anti-inflammatory action of AP prevent LPS-induced iNOS, COX-2 and NF-κB activation and expression of apoptotic proteins; (ii) the role of STAT3 phosphorylation and that it was down-regulated by the AP in this model; and (iii) whether the downstream signalling events in the LPS-induced activation of the TNF-α, IL-1β and IL-6 pathways are regulated by AP.

Section snippets

Cell culture and reagents

The RAW 264.7 cell line was obtained from the American Type Culture Collection (Manassas, VA). The cells were cultured in Dulbecco Modified Eagle's Medium (DMEM) (Gibco BRL Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (Gibco BRL Life Technologies Inc.) and maintained at 37 °C in a humidified incubator containing 5% CO2. Andrographolide, lipopolysaccharide, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from

AP inhibited LPS-induced iNOS and COX-2 proteins expression and the production of nitrite and PGE2 in RAW 264.7 cells

The protein levels of iNOS and COX-2 were undetectable in RAW264.7 cells without LPS-stimulation (Fig. 1A, B). Treatment with LPS alone markedly increased iNOS and COX-2 protein levels, whereas co-treatment with AP significantly and dose-dependently suppressed the expression of iNOS and COX-2. The reduced protein expression of iNOS and COX-2 was consistent with the reductions in total nitrite and PGE2 in culture media (Fig. 1C, D). Interestingly, LPS (500 ng/ml) or co-culture of LPS and AP for 12

Discussion

This study shows that AP blocks LPS-induced inflammatory signals that lead to apoptotic cell death of RAW264.7 macrophage. We demonstrated that in RAW264.7 macrophage, AP significantly reduced iNOS and COX-2 protein expression induced by LPS and subsequent NO and PGE2 synthesis. Furthermore, our data show that the underlying anti-inflammatory mechanisms of AP are due, at least in part, to inhibition of LPS-induced, STAT3 phosphorylation-mediated NF-κB activation.

LPS induces iNOS gene

Acknowledgments

This work was supported by grant CMRPG380811 (KC, Lee) and CMRPD170111 (TY, Lee) from Chang Gung Memorial Hospital, Taoyuan, Taiwan.

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