Elsevier

Phytomedicine

Volume 21, Issue 4, 15 March 2014, Pages 470-478
Phytomedicine

Carthami Flos suppresses neutrophilic lung inflammation in mice, for which nuclear factor-erythroid 2-related factor-1 is required

https://doi.org/10.1016/j.phymed.2013.10.005Get rights and content

Abstract

Carthami Flos (CF) is used in traditional Asian medicine to treat blood stagnation and its associated diseases in patients. While the underlying mechanism for this effect remains unknown, CF has been reported to activate Nrf2, a transcription factor that is critical in protecting from various inflammatory lung diseases including acute lung injury (ALI). Here, we examined whether CF has a therapeutic effect on lung inflammation and assessed the impact of Nrf2 on the effect of CF using an ALI mouse model. Treatment of bone marrow derived macrophages with standardized aqueous extract of CF (AECF) activated Nrf2, resulting in the expression of Nrf2 dependent genes including GCLC, NQO-1 and HO-1. While intranasal LPS treatment of wild type mice resulted in neutrophilic infiltration and a concomitant expression of pro-inflammatory cytokine genes in the lung, the hallmarks of ALI, an intratracheal spraying of AECF to the lung 2 h after LPS treatment suppressed the inflammatory response. By contrast, similar treatment in nrf2−/− mice with AECF failed to attenuate the inflammatory response. Thus, our results show that AECF attenuated neutrophilic lung inflammation in mice, which required Nrf2. Since AECF administration abrogates lung inflammation after LPS treatment, we propose CF as a potential therapeutics in the management of ALI.

Introduction

Inflammation is part of protective process of innate immunity. However, it can lead to deleterious ramifications when uncontrolled, as evidenced in conditions such as acute lung injury (ALI) or chronic lung diseases, such as pulmonary fibrosis, asthma and chronic obstructive airways disease (Serhan et al. 2008). Uncontrolled, excessive inflammation is also a hallmark of other chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease psoriasis, and atherosclerosis (Nathan and Ding 2010). Despite extensive research in the last three decades, effective treatments for ALI and acute respiratory diseases remain elusive (Rubenfeld et al., 2005, Ware and Matthay, 2000).

Accumulating results show that macrophages play a pivotal role in regulating lung inflammation (Beutler and Rietschel 2003). Macrophages sense pathogen associated molecules such as lipopolysaccharide (LPS) in Gram negative bacteria, and initiate an inflammatory response. Toll-like receptor 4 (TLR4) on the surface of macrophages is known to bind LPS to initiate a signaling cascade that eventually activates key pro-inflammatory transcription factor NF-κB. Activation of NF-κB results in the expression of various pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6 and IL-8, recruiting neutrophils and accentuating inflammation (Beutler and Rietschel 2003). The responses that promote inflammation also activate nuclear factor-erythroid 2-related factor-1 (Nrf2), a member of the cap‘n’collar family of basic leucine zipper transcription factors (Johnson et al. 2008). Although Nrf2 is known to regulate the expression of detoxification and antioxidant enzymes such as glutamate-cysteine ligase catalytic subunit (GCLC), NAD(P)H:quinine oxidoreductases-1 (NQO-1) and heme oxygenase-1 (HO-1) (Itoh et al., 1997, Jaiswal, 2000), recent studies have indicated the Nrf2 plays an essential role in protecting from excessive inflammation in the lungs and airways (Blake et al., 2010, Boutten et al., 2011, Chan and Kan, 1999, Iizuka et al., 2005, Ishii et al., 2005, Rangasamy et al., 2005). Consistent with these results, polymorphisms in human nrf2 gene are known to be associated with an increased risk of acute lung injury (Marzec et al. 2007). Thus, it is becoming evident that, along with NF-κB, Nrf2 contributes to regulation of inflammation (Kim et al. 2011).

Carthami Flos (CF) has long been prescribed to patients who suffer from various symptoms associated with blood stagnation, and recent reports suggest that CF improves blood circulation by lowering the levels of blood lipids (Lin et al. 2012). While the underlying mechanisms for these effects remain unknown, accumulating reports suggest that CF can modulate inflammation. Methanol extract of CF activates Nrf2 and inhibits NF-κB activation in RAW 264.7 cells (Jun et al. 2011). Aqueous extract of CF suppresses LPS-induced expression of IL-1β, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) in RAW 264.7 (Wang et al. 2011). In addition, Carthamus red, a chemical constituent of CF, has antioxidant and hepatoprotective effects via activation of Nrf2 in rats (Wu et al. 2013). Thus, given the critical role of Nrf2 in protecting from various inflammatory lung diseases, we investigated the possibility that CF was effective in suppressing acute lung inflammation using an ALI mouse model. Furthermore, we sought to determine the extent to which Nrf2 affected the function of CF in suppressing lung inflammation. Our findings show that CF attenuates neutrophilic lung inflammation, through activation of Nrf2.

Section snippets

Plant material and fingerprinting analysis

Standardized aqueous extract of Carthami Flos (Carthamus tinctorius L.; catalog number: CW04-081) was obtained from the plant extract bank at KRIBB (Korea Research Institute of Bioscience and Biotechnology), Daejeon, Korea. HPLC was performed using an ACQUITY UPLCTM system (Waters Corporation, Milford, MA, USA) which was equipped with a photodiode array detector. Chromatographic separations were performed on a 2.1 mm × 100 mm, 1.7 μm ACQUITY BEH C18 (Waters Corporation) chromatography column. The

Carthami Flos activates Nrf2

Although methanol extract of CF activates Nrf2 (Jun et al. 2011), it is unclear whether aqueous extract of CF does the same. To determine whether an aqueous extract of CF activates Nrf2, we obtained a standardized aqueous extract of CF (AECF) from KRIBB, Korea (Fig. 1), and then tested the possibility that the cytotoxicity of AECF caused cellular toxicity, resulting in Nrf2 activation. RAW 264.7 cells were treated with various amounts of AECF. At 16 h after treatment, we performed MTT assay. As

Discussion

CF is a medicinal herb that has long been used to treat patients with diseases associated with poor blood circulation in traditional Asian medicine. It also serves as a key constituent of Dangkwisoo-San (DS), a traditional Asian herbal formula that is used to treat symptoms associated with blood stagnation and inflammation (Lyu et al. 2012). In this study, we sought to determine whether CF regulated inflammation in the lung. We found that aerosol administration of AECF suppressed neutrophilic

Conflict of interest

The authors do not have a commercial or other association that might have a conflict of interest.

Acknowledgement

This research was supported by KRIBB (KGM1221312 to M.J.) and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2012R1A1A2044346 to M.J. and NRF-2013R1A1A3013027 to K.H.K.).

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