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The aerial part of Taraxacum coreanum extract has an anti-inflammatory effect on peritoneal macrophages in vitro and increases survival in a mouse model of septic shock

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Abstract

Ethnopharmacological relevance

Taraxacum coreanum Nakaiis a dandelion native to Korea and is widely consumed as an edible and medicinal herb. The aerial part of Taraxacum coreanum (TC) has been used therapeutically as a diuretic and anti-inflammatory agent, but its mechanism of action has not yet been evaluated.

Aim of the study

To investigate the anti-inflammatory potential of a Taraxacum coreanum chloroform fraction(TCC) and its mechanisms of action in vitro and in vivo.

Materials and methods

Isolated mouse peritoneal macrophages were stimulated in vitro with interferon-γ (IFN-γ) and lipopolysaccharide (LPS) in the presence or absence of TCC. The anti-inflammatory effects of TCC were assessed by measuring nitric oxide (NO) and prostaglandin E2 (PGE2) production, as well as expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), IκBα, phospho-IKK, mitogen-activated protein kinases (MAPKs), and signal transducer and activator of transcription (STAT1). The effects of TCC were tested in vivo by measuring cytokine production and survival in a mouse model of lethal septic shock. And the standard compounds of Taraxacum coreanum were analyzed by HPLC using a C18 column.

Results

Treatment of primary macrophages with TCC in vitro significantly inhibited all of the inflammatory parameters measured, including LPS-induced NO and PGE2 production, iNOS and COX-2 expression, IκBα degradation, IKK phosphorylation, and MAPK and STAT1 activation. In a mouse model of LPS-induced septic shock, TCC inhibited the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, and increased survival by 83%.Standard compounds (gallic acid, syringic acid) of Taraxacum coreanum were qualified by HPLC analysis.

Conclusions

TCC possesses potent anti-inflammatory activity in vitro and in vivo, which occurs at least partly through inhibition of proinflammatory signaling and mediator release. These results strongly support the therapeutic potential of TCC as an anti-inflammatory agent in vivo.

Introduction

Inflammation is a complex pathophysiological process mediated by a variety of signaling molecules produced by leukocytes, macrophages, and mast cells (Aoki et al., 2008). The symptoms result from increased blood flow to the damaged or infected tissue causing fever, redness, swelling, and pain. Macrophages are major inflammatory cells and immune effector cells. One major function of macrophages is phagocytosis of cellular and acellular debris during inflammation and healing (Scull et al., 2010). Activated macrophages are present in inflamed tissues and play an important role in inflammatory disease through the release of inflammatory mediators. Macrophages can be stimulated by a variety of agents such as interferon-γ (IFN-γ) and microbial products (Gautam and Deodhar, 1989, Paulnock and Lambert, 1990), some of which have also been shown to trigger the release of nitric oxide (NO) (Tripathi et al., 2007), prostaglandin (PG)E2, and proinflammatory cytokines (Guastadisegni et al., 2002). Lipopolysaccharide (LPS) is a constituent of the outer membrane of Gram-negative bacteria and is a potent activator of macrophages. In addition, expression of inducible nitric oxide synthase (iNOS) and production of NO is induced in macrophages by binding of LPS and IFN-γ to toll-like receptor-4 (TLR4) and IFN-γ receptor (IFNγR), respectively (Cohen, 2002).

NF-κB is a transcription factor that plays pivotal roles in the immediate-early stages of immune, acute phase, and inflammatory responses, as well as in cell survival (Makarov, 2001). In resting cells, NF-κB is located in the cytoplasm as an inactive complex bound to the inhibitory protein IκBα. Activation of NF-κB typically requires phosphorylation of IκBα by the IκBα kinase (IKK) complex, which results in IκBα degradation and allows NF-κB to translocate to the nucleus (Perkins, 2007). Activation of NF-κB is also regulated by upstream kinases such as mitogen-activated protein kinases (MAPKs) (Guha and Mackman, 2001). The NF-κB and MAPK pathways induce the expression of various inflammation mediators, including NO, prostaglandins (PGs), and inflammatory cytokines such as TNF-α, IL-1β, and IL-6 (O’Neill and Bowie, 2007, Medzhitov, 2008, Hajishengallis and Lambris, 2011). Members of the MAPK family, including extracellular signal-regulated kinase1/2 (Erk1/2), p38 MAPK, and c-Jun NH2-terminal kinase (JNK), have been shown to play a significant role in the mediation of signals triggered by cytokines, growth factors, and environmental stress (Reibman et al., 2000). During this process, the signal transducer and activator of transcription (STAT) family of proteins act as regulatory transcription factors that are initially cytosolic but translocate to the nucleus upon cytokine-mediated signaling (Rawlings et al., 2004).

The genus Taraxacum is a member of the family Compositae. About 2000 species of Taraxacum have been identified in the northern hemisphere, among which Taraxacum platycarpum, Taraxacum ohwianum, Taraxacum hallaisanense, Taraxacum coreanum, and Taraxacum officinale are found in South Korea. Taraxaci Herba have long been used as medicinal herbs to treat inflammatory diseases such as hepatitis, arthritis, rheumatism, breast abscess, lung abscess, intestinal abscess, scrofula, sore and swollen throat (Xu and Wang, 2002, Schutz et al., 2006, Park et al., 2011). In addition, various studies on Taraxacum extracts and their constituents have demonstrated anti-inflammatory, antinociceptive, anti-oxidant, and anti-cancer activity (Jeon et al., 2008, Choi et al., 2010, You et al., 2010). In South Korea, Taraxacum platycarpum H. Dahlstedt, Taraxacum officinaleWeber, Taraxacum mongolicum Handel-Mazzetti and Taraxacum coreanum Nakai have been used as traditional medicine Taraxaci Herb (Korea Food and Drug Administration, 2011).Among them, Taraxacum coreanum (TC), known as ‘white dandelion’, grows chiefly in the South Korea. Thus, Taraxacum coreanum is well used herbal medicine to treat inflammatory diseases in Korean traditional clinic. However, several studies have reported the anti-inflammatory effects of Taraxaci Herba, there are no studies of anti-inflammatory effects of Taraxacum coreanum. Recent research has identified several metabolites of Taraxacum coreanum, including carnitine, mannose, heptadecanoic acid, 9,12-octadecadienoic acid, octadecadienoic acid, hexacosanoic acid, stigmasterol, β-amyrin, β-sitosterol, and lanosterol. Of these, β-amyrin (Brendolise et al., 2011) and 9,12-octadecadienoic acid (Leung and Foster, 1995) have been shown to possess anti-inflammatory activities.

In the present study, to further our understanding of the anti-inflammatory properties of Taraxacum coreanum, we investigated the effect of a Taraxacum coreanum extract on macrophage production of inflammatory mediators in vitro, as well as its effects in an in vivo model of lethal bacterial sepsis. Also, we conducted a quantitative analysis of contained components of Taraxacum coreanum by analyzing gallic acid and syringic acid with high performance liquid chromatography (HPLC).

Section snippets

Sample preparation

Fresh aerial parts of Taraxacum coreanum were collected from Jeongseon (Kangwon province, Republic of Korea) in May 2009. A voucher specimen (s004h) was deposited in the laboratory of Herbology, College of Korean Medicine, Kyung Hee University. After drying, the aerial part (50 g) was extracted with 100% methanol under reflux at 85 °C for 2 h. This process was repeated 3 times and the final extract was filtered, evaporated under vacuum, and lyophilized. The yield of extract was 29.0%. Five grams

Effect of TCC on viability of peritoneal macrophages

We examined the effect of TCC on viability of peritoneal macrophages in vitro by incubating cells with 10, 25, 50, 100, 200, or 400 μg/ml of TCC for 24 h. The results of the MTT assay showed that TCC was not cytotoxic to primary macrophages at concentrations up to 200 μg/ml, but viability was significantly reduced at 400 μg/ml (Fig. 1A). Therefore, we selected concentrations of 50, 100, and 200 μg/ml for further study.

Effect of TCC on NO production and iNOS expression in stimulated macrophages

iNOS is expressed in macrophages in response to a variety of inflammatory stimuli

Discussion

The purpose of this study was to investigate the anti-inflammatory effects of TCC by evaluating activation of proinflammatory signaling pathways and production of inflammatory mediators in LPS+IFN-γ-stimulated primary macrophages. To this end, we examined the effects of TCC on NO and PGE2 production and on expression of iNOS, COX-2, IκBα, IKK, p38, ERK1/2, JNK, and STAT1, all of which play crucial roles in regulating the macrophages response to inflammation.

NO is a key inflammatory mediator and

Acknowledgments

This study was supported by the Korean Medical Science Center, Kyung Hee University.

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