Anti-Inflammatory activity of chrysophanol through the suppression of NF-kappaB/caspase-1 activation in vitro and in vivo.

Abstract

Chrysophanol is a member of the anthraquinone family and has multiple pharmacological effects, but the exact mechanism of the anti-inflammatory effects of chrysophanol has yet to be thoroughly elucidated. In this study, we attempted to determine the effects of chrysophanol on dextran sulfate sodium (DSS)-induced colitis and lipopolysaccharide (LPS)-induced inflammatory responses in mouse peritoneal macrophages. The findings of this study demonstrated that chrysophanol effectively attenuated overall clinical scores as well as various pathological markers of colitis. Additionally, chrysophanol inhibited the production of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and the expression of cyclooxygenase (COX)-2 levels induced by LPS. We showed that this anti-inflammatory effect of chrysophanol is through suppression of the activation of NF-kappaB and caspase-1 in LPS-stimulated macrophages. These results provide novel insights into the pharmacological actions of chrysophanol as a potential molecule for use in the treatment of inflammatory diseases.

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Molecules 2010, 15, 6436-6451; doi:10.3390/molecules15096436

molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Anti-Inflammatory Activity of Chrysophanol through the
Suppression of NF-κ κB/Caspase-1 Activation in Vitro and in Vivo
Su-Jin Kim 1, Min-Cheol Kim 2, Byong-Joo Lee 1, Dae-Hee Park 1, Seung-Heon Hong 2
and Jae-Young Um 1,*
1 Department of Pharmacology, College of Oriental Medicine, Institute of Oriental Medicine, Kyung
Hee University, 1 Hoegi-Dong, Dongdaemun-Gu, Seoul, 130-701, Korea;
E-Mails: ksj1009@khu.ac.kr (S-J.K.); nlbo92@hanmail.net (B.-J.L.); neorufa@nate.com (D-H.P.)
2 Department of Oriental Pharmacy, College of Pharmacy, VestibuloCochlear Research Center of
Wonkwang University, Iksan, Jeonbuk, Korea; E-Mails: mchol@naver.com (M.-C.K.);
jooklim@wku.ac.kr (S.-H.H.)
* Author to whom correspondence should be addressed; E-Mail: jyum@khu.ac.kr;
Tel.: +82-2-961-9262; Fax: +82-2-967-7707.
Received: 6 August 2010; in revised form: 9 September 2010 / Accepted: 14 September 2010/
Published: 16 September 2010

Abstract: Chrysophanol is a member of the anthraquinone family and has multiple
pharmacological effects, but the exact mechanism of the anti-inflammatory effects of
chrysophanol has yet to be thoroughly elucidated. In this study, we attempted to determine
the effects of chrysophanol on dextran sulfate sodium (DSS)-induced colitis and
lipopolysaccharide (LPS)-induced inflammatory responses in mouse peritoneal
macrophages. The findings of this study demonstrated that chrysophanol effectively
attenuated overall clinical scores as well as various pathological markers of colitis.
Additionally, chrysophanol inhibited the production of tumor necrosis factor (TNF)-α,
interleukin (IL)-6 and the expression of cyclooxygenase (COX)-2 levels induced by LPS.
We showed that this anti-inflammatory effect of chrysophanol is through suppression of
the activation of NF-κB and caspase-1 in LPS-stimulated macrophages. These results
provide novel insights into the pharmacological actions of chrysophanol as a potential
molecule for use in the treatment of inflammatory diseases.
Keywords: chrysophanol; inflammation; colitis; macrophage

OPEN ACCESS

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1. Introduction
Ulcerative colitis (UC) is a typical inflammatory intestinal disease belonging to the inflammatory
bowel diseases (IBD). Unfortunately, despite many years of extensive research implicating immune
dysfunction, genetic susceptibility, and bacterial flora within the intestinal environment as possible
factors associated with development of the disease, its pathogenesis is still poorly understood.
Inflammation is a process that involves the action of multiple factors within a complex network.
The ingress of leukocytes into the inflammation site is important in the pathogenesis of inflammatory
conditions. Macrophage activation has been determined to perform an important role in the
inflammatory process [1,2] and to generate potent pro-inflammatory cytokines such as tumor necrosis
factor (TNF)-α, and interleukin (IL)-6, which induce inflammation and recruit other immune cells,
including neutrophils [1]. Although TNF-α and IL-6 are beneficial to host defenses, they can also
trigger pathological conditions when expressed in excess quantities [3]. For example, massive
stimulation of macrophages after a severe Gram-negative bacterial infection can result in excessive
production of pro-inflammatory cytokines and the development of fatal septic shock syndrome, as well
as multiple organ failure [4]. It was reported that mucosa from patients with UC indeed have shown
increased expression of IL-1 and IL-6 and proposed to play an integral role in its pathogenesis [5-7].
Hence, there is currently a strong interest in agents that can block the generation or activities of
inflammatory cytokines.
Cyclooxygenases (COX) have been implicated in a number of physiological events, including the
progression of inflammation, immunomodulation, and transmission of pain. Two COX isoenzymes
were identified. COX-1, the constitutive enzyme, makes prostaglandins which protects the stomach
and kidney from damage. Whereas COX-2 normally is expressed at very low levels, it is rapidly
induced by a variety of stimuli, such as cytokines, growth factors, hormones and carcinogens, and is
believed to be responsible for the production of prostaglandins associated with mediation of
inflammation. It is reported that COX-2 expression is increased in inflamed mucosa of patients with
UC [8,9].
Nuclear factor-kappa B (NF-κB) performs a crucial function in the expression of many genes
involved in immune and inflammatory responses. Inactive NF-κB complexes are sequestered in the
cytoplasm via binding to their inhibitory subunit, IκB. After a variety of stimuli, the IκB proteins are
phosphorylated, ubiquinated, and degraded, allowing for NF-κB to translocate into the nucleus where
it can bind specific DNA sequences located in the promoter regions of target genes and activate gene
transcription, thereby indicating its pivotal function in the regulation of immune and inflammatory
responses, via the control of the transcription of inflammatory cytokine genes [10,11]. Increased
activation of NF-κB has been found in macrophages and epithelial cells of patients with UC [12]. In
addition, increased DNA binding activity of NF-κB that is associated with secretion of high levels of
IL-1 and IL-6 is observed in macrophages from patients with UC [12,13]. These studies suggested that
the activation of NF-κB plays a critical role in the initiation of intestinal inflammation of UC. From
this, inhibition of NF-κB activation has been suggested as an anti-inflammatory strategy in UC.
Caspase-1 is a member of a family of caspases with large prodomains [14], and its activation is
involved in apoptosis and inflammation [15]. Caspase-1 activation induces inflammation via the
production of pro-inflammatory cytokines and the recruitment of neutrophils [16]. It was reported that

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caspase-1−/− mice reduced the production of IL-6 after stimulation with lipopolysaccharide (LPS).
Caspase-1 deficiency also reduced the clinical score including weight loss, diarrhea, rectal bleeding,
and colon length in colitis experimental model [17]. The results of these studies demonstrated that the
activation of caspase-1 is an attractive target for the treatment of inflammatory diseases.
Chrysophanol is a member of the anthraquinone family. The results of previous pharmaceutical
studies have shown that derivatives of anthraquinones exert a number of biological effects, including
anticancer [18,19], hepatoprotective [20], and antimicrobial [21]. However, the exact mechanisms
underlying the anti-inflammatory effects of chrysophanol remain to be thoroughly elucidated.
Dextran sulfate sodium (DSS)-induced colitis is characterized by mucosal infiltration of inflammatory
cells, epithelial injury, and ulceration [22]. The pathological mechanism of DSS-induced colitis includes
the production of inflammatory cytokines and mediators by macrophages that are activated after
phagocytosis of DSS. The principal objective of this study was to determine whether or not chrysophanol
modulates inflammatory reactions in DSS-induced colitis. Additionally, we elucidated the effect of
chrysophanol in LPS-phenotype similar to human acute and chronic UC [23]. The inhibitory induced
inflammatory responses in mouse peritoneal macrophages were evaluated.
2. Results and Discussion
2.1. Effect of Chrysophanol on Clinical Signs in DSS-Induced Colitis
It was reported that in mice chrysophanol has an effect on the intestines in DSS-induced
experimental colitis. As shown in Figures 1A and B, all mice treated with DSS showed the significant
weight loss and colon shortening, compared to control group. However, we observed that groups
administrated with chrysophanol showed the significant attenuation of body weight loss at seven days
and colon shortening caused by DSS. Relative colon lengths were represented in Figure 1C. In addition,
disease activity index (DAI) was determined by scoring changes in body weight, diarrhea score, and
rectal bleeding score in accordance with the method described by Murthy et al. [24]. DAI was
inhibited in groups administrated with chrysophanol compared to group of DSS (Figure 1D).
2.2. Effect of Chrysophanol on Levels of Inflammatory Mediators (IL-6 and COX-2) in DSS-Induced
Colitis
We investigated the effect of chrysophanol on IL-6 production in colitis tissues. At the end of
experiment, the colon tissues were homogenized, and ELISA was performed. As shown in Figure 2A,
the levels of IL-6 were significantly increased in the colon tissues of DSS-treated mice compared to
that of control. However, administration of chrysophanol reduced these induction induced by DSS.
The inhibition rates of IL-6 production by chrysophanol were 32.11 ± 5.8%. Also, the inhibition rates
of IL-6 production by SFZ were 30.01 ± 6.1%.
The effect of chrysophanol on the expression levels of COX-2 were evaluated by Western blot
analysis. The expressions of COX-2 were significantly increased in the colon tissues of DSS-treated
mice compared to that of control. However, administration of chrysophanol reduced the expression of
COX-2 induced by DSS (Figure 2B). The relative level of COX-2 was measured using an image
analyzer (Figure 2C).

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Figure 1. Effect of chrysophanol on clinical signs in DSS-induced colitis. Experimental
colitis in mice was induced by a 5% DSS dissolved in the drinking water for 7 days.
Chrysophanol was administered at doses of 5 mg/kg once a day for 7 days prior to 5% DSS
supplement. (A) Body weight of mice was measured; (B) The colons were removed at day
7 after DSS treatment, and the colon lengths were measured; (C) Relative colon lengths
were represented; (D) DAI was calculated as described in the Experimental. Sulfasalazine
(150 mg/kg) was used as a positive control. (1) Control group; (2) DSS alone-treated group;
(3) chrysophanol (5 mg/kg) + DSS treated group; (4) sulfasalazine (150 mg/kg) + DSS
treated group. Values were represented in the mean ± S.E.M. (n = 5) of duplicate
determinations from triplicate separate experiments (# p < 0.05 vs. control, * p < 0.05 vs.
DSS alone).

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Figure 2. Effects of chrysophanol on the level of IL-6 and COX-2 in DSS-treated colon
tissue. Experimental colitis in mice was induced by a 5% DSS dissolved in the drinking
water for 7 days. Chrysophanol was administered at doses of 5 mg/kg once a day for 7 days
prior to 5% DSS supplement. At the end of experiment, the colon tissues were cut out and
homogenized. (A) The levels of IL-6 in the indicated groups were measured by ELISA; (B)
The levels of COX-2 were evaluated by Western blot analysis; (C) The relative expression
level of COX-2 was measured using an image analyzer.1) Control group; 2) DSS alone-
treated group; 3) chrysophanol (5 mg/kg) + DSS treated group; 4) sulfasalazine
(150 mg/kg) + DSS treated group. Values were represented in the mean ± S.E.M. (n = 5) of
duplicate determinations from triplicate separate experiments (#p < 0.05 vs. control, *p <
0.05 vs. DSS alone).

2.3. Effect of Chrysophanol on Activation of NF-κB (p65) and Caspase-1 in DSS-Induced Colitis
Activation of NF-κB p65 is involved in colitis, thus inhibition of NF-κB activation has been
suggested as an anti-inflammatory strategy in colitis [12]. We examined whether chrysophanol
regulates the activation of NF-κB p65 in colitis tissues. The activation of NF-κB p65 were
significantly increased in the colon tissues (epidermis) of DSS-treated mice compared to that of control.
However, administration of chrysophanol reduced significantly the activation of NF-κB (p65) induced
in DSS-treated colon tissues (Figure 3A). The relative level of NF-κB was measured using an image
analyzer (Figure 3B). Histology study showed that DSS dramatically induced NF-κB p65 activation,
but chrysophanol diminished these increase (Figure 3C).