Chemico-Biological Interactions 210 (2014) 26–33

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Celastrol ameliorates murine colitis via modulating oxidative stress, inflammatory cytokines and intestinal homeostasis Mohamed E. Shaker a,⇑, Sylvia A. Ashamallah b, Maha E. Houssen c a

Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt Department of Pathology, Faculty of Medicine, Mansoura University, Mansoura 35516, Egypt c Department of Biochemistry, Faculty of Pharmacy, Damanhour University, Damanhour, Egypt b

a r t i c l e

i n f o

Article history: Received 5 October 2013 Received in revised form 10 December 2013 Accepted 17 December 2013 Available online 30 December 2013 Keywords: Colitis Intestinal homeostasis Celastrol Inflammasome

a b s t r a c t Therapeutic agents that block the nuclear factor-kappa B (NF-jB) pathway might be beneficial for incurable inflammatory diseases, such as ulcerative colitis. Here, we investigated the effect of the novel NF-jB inhibitor celastrol on murine colitis. Colitis was induced in male mice by administration of 5% (w/ v) dextran sulfate sodium (DSS) in drinking water for a period of 5 days, followed by a 2 day recovery period. Celastrol (2 mg/kg, oral) was administered daily over the 1 week of the study. Our results indicated that treatment with celastrol attenuated DSS-induced colon shortening and neutrophil infiltration. Besides, celastrol ameliorated DSS-induced colon injury and inflammatory signs as visualized by histopathology. The mechanisms behind these beneficial effects of celastrol were also elucidated. These include (i) counteracting DSS-induced oxidative stress in the colon via decreasing lipid peroxidation products (malondialdehyde and 4-hydroxynonenal) and increasing the antioxidant levels (reduced glutathione, glutathione-S-transferase and superoxide dismutase); (ii) inhibiting DSS-induced activation of the NLRP3-inflammasome, as evidenced by decreased production of IL-1b and IFN-c as indirect measure of IL-18 in the colon; (iii) targeting DSS-induced activation of the IL-23/IL-17 pathway by abating the elevation of IL-23 and IL-17A levels in the colon; (iv) augmenting the anti-inflammatory defense mechanisms via increasing IL-10 and TNF-a levels in the colon; (v) and more importantly, maintaining intestinal epithelial reconstitution and homeostasis via attenuating the overexpression of CD98 in colonic epithelial cells. In conclusion, our study provides novel insights into the beneficial effects of celastrol as a promising candidate for the treatment of ulcerative colitis in humans. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Ulcerative colitis is one of the inflammatory bowel diseases affecting the distal colon and rectum that is generally recognized as a dysregulated immune response to the intestinal microbiota [1,2]. Of the various signaling pathways involved in the colonic inflammation, the Toll-like receptor 4 (TLR4) pathway is considered the most important. TLR4 recognizes lipopolysaccharide (LPS), which is present in the cell wall of gram-negative bacteria. Interaction of LPS with TLR4 triggers signaling cascades that lead to the activation of the transcription factor nuclear factor-kappa B (NF-jB) and the production of proinflammatory cytokines and proteins, such as IL-1b, IL-6, IL-8, IL-12, TNF-a and IFN-c [3]. Therefore, understanding the molecular mechanisms involved in this pathway is an important step in countering the damaging effects of these proinflammatory mediators in ulcerative colitis.

⇑ Corresponding author. Tel.: +20 502246253; fax: +20 502247496. E-mail address: [email protected] (M.E. Shaker). 0009-2797/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbi.2013.12.007

Unfortunately, current treatments for ulcerative colitis, like corticosteroids, sulfasalazine, classical immunosuppressives and antibiotics, focus only on the suppression of inflammation, and their use is associated with numerous and serious side effects. Emerging therapies that block the TNF-a pathway, such as infliximab and adalimumab, might be effective in the treatment of ulcerative colitis [4]. However, a large proportion of patients failed to respond to the therapy, and others were subjected to anaphylactic reactions and infections [5]. Hence, the use of active compounds from medicinal plants represents an attractive approach for ulcerative colitis treatment. Celastrol, a triterpene extracted from the root bark of the Chinese medicinal plant Tripterygium wilfordii (also known as Thunder of God Vine), has recently gained much interest as a natural remedy for inflammation, cancer and autoimmune diseases [6,7]. Molecular studies have identified several targets for celastrol which are mostly dependent on the inhibition of NF-jB signaling [8,9]. In the present study, we hypothesized that the natural NF-jB inhibitor celastrol might be a promising candidate for the treatment of ulcerative colitis in humans. To evaluate this idea,

M.E. Shaker et al. / Chemico-Biological Interactions 210 (2014) 26–33

the effect of celastrol was investigated on the dextran sulfate sodium (DSS)-induced colitis model in mice. This model has the advantage of mimicking human inflammatory bowel disease and is considered the most suitable for investigating potential therapeutics [10]. Here, we report for the first time that the natural triterpene celastrol ameliorates DSS-induced colitis in mice via modulating colonic oxidative stress, inflammatory cytokines and intestinal epithelial homeostasis. Thus, our study provides novel insights regarding the potential usefulness of celastrol in the treatment of ulcerative colitis in humans.

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centrifuged at 2000g for 10 min at 4 °C for serum preparation, followed by storage at 80 °C. After measuring the length with digital caliber, and several washings with normal saline, portions of colon tissues were stored at 80 °C for the enzyme-linked immunosorbent assay (ELISA) and oxidative stress/antioxidant assays. Portions of colons were also fixed in 10% (v/v) neutral-buffered formalin solutions for 24 h for histopathological and immunohistochemical analysis.

2.5. Colonic histopathological and immunohistochemical evaluations 2. Materials and methods 2.1. Drugs and chemicals Celastrol (purity 98%) was purchased from Cayman (Ann Arbor, MI, USA). DSS (molecular weight 40 kDa) was purchased from TdB Consultancy (Uppsala, Sweden). 1-Methyl-2-phenylindole, 1,1,3, 3-tetramethoxypropane, 5,50 -dithiobis(2-nitrobenzoic acid), L-glutathione reduced and vanadium III chloride (VCl3) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Methanesulfonic acid was purchased from Merck (Darmstadt, Germany). 3,30 ,5,50 -Tetramethylbenzidine and hexadecyltrimethylammonium bromide were purchased from MP Biomedicals (Irvine, CA, USA). 1-Chloro2,4-dinitrobenzene (CDNB) and AEBSF was purchased from Acros Organics (Morris Plains, NJ, USA). Pyrogallol was purchased from Fluka (Buchs SG, Switzerland). 2.2. Animals Adult male BALB/c mice (28–32 g) were purchased from Nile Pharmaceuticals (Cairo, Egypt). The animals were allowed access to food and tap water ad libitum throughout the acclimatization and experimental periods. All animals received humane care in compliance with the National Institutes of Health and the Research Ethics Committee Criteria for Care of Laboratory Animals at Mansoura University. 2.3. Induction of colitis by dextran sulfate sodium (DSS) and treatment protocol Acute colitis in mice was induced by adding DSS to the drinking water at a concentration of 5% (w/v) for a period of 5 days. Thereafter, the DSS solution was replaced with drinking water for additional 2 days to allow some colonic epithelial recovery. From the first day of adding DSS to the drinking water, celastrol treatment was administered to mice daily over the 1 week. Celastrol was freshly prepared by predissolving 2 mg in 0.05 ml dimethylsulfoxide due its limited aqueous solubility, followed by dilution with normal saline to 10 ml. 2.4. Experimental design The mice were divided into four groups as follows: (1) Control: allowed to drink tap water and received only the vehicle (0.3 ml/ 30 g/day, oral); (2) DSS: allowed to drink tap water containing 5% (w/v) of DSS over 5 days and received only the vehicle (0.3 ml/ 30 g/day, oral) over the 1 week; (3) DSS + Celastrol (2 mg/kg): allowed to drink tap water containing 5% (w/v) of DSS over 5 days and received celastrol at a dose of 2 mg/kg/day (0.3 ml/30 g, oral) over the 1 week; (4) Celastrol (2 mg/kg): allowed to drink tap water and received celastrol at a dose of 2 mg/kg/day (0.3 ml/ 30 g, oral) over the 1 week. At the end of the week, blood samples were withdrawn from thiopental-anesthetized animals via cardiac puncture and

Standard histopathological techniques were followed for processing portions of distal colon, preparation of paraffin blocks and staining of the slides with hematoxylin–eosin to evaluate DSS-induced colitis. Histological scoring was based on 3 parameters as previously described [11]. Severity of inflammation was scored as follows: 0, rare inflammatory cells in the lamina propria; 1, increased numbers of granulocytes in the lamina propria; 2, confluence of inflammatory cells extending into the submucosa; 3, transmural extension of the inflammatory infiltrate. Crypt damage was scored as follows: 0, intact crypts; 1, loss of the basal onethird; 2, loss of the basal two-thirds; 3, entire crypt loss; 4, change of epithelial surface with erosion; 5, confluent erosion. Ulceration was scored as follows: 0, absence of ulcer; 1, 1 or 2 foci of ulcerations; 2, 3 or 4 foci of ulcerations; 3, confluent or extensive ulceration. Values were added to give a maximal histological score of 11. For immunohistochemical analysis, anti-CD68 and anti-CD98 (Santa Cruz, CA, USA) were used as primary antibodies to detect their targeted proteins using standard immunohistochemical methods.

2.6. Enzyme-linked immunosorbent assay (ELISA) for cytokines Portions of colons were homogenized (10% w/v) in ice-cold lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 2 mM AEBSF). The lysates were centrifuged at 8000g for 10 min at 4 °C, and the supernatants were transferred for ELISA. Mouse IL-1b, IFN-c, IL-17A, IL-23(p19/40), IL-10 and TNF-a concentrations were determined using the ELISA MAX™ Deluxe set (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions. The protein content was assayed by the Bradford method [12]. IL-17A and TNF-a concentrations were also determined in serum.

2.7. Measurement of colonic myeloperoxidase (MPO) activity The extent of neutrophil accumulation in the colon was measured by assaying myeloperoxidase (MPO) activity as previously described [13] with a slight modification. After processing the supernatant of colon homogenate, a part of the corresponding pellet (50 mg) was weighed, homogenized in 1 ml of the buffer (0.1 M NaCl, 0.02 M NaH2PO4, 0.015 M EDTA, pH 4.7) and centrifuged at 6000g for 20 min at 4 °C. The pellets were then resuspended in 0.5 ml of 0.05 M sodium phosphate buffer (pH 5.4) containing 0.5% (w/v) hexadecyltrimethylammonium bromide. The suspensions were freeze–thawed three times, heated for 2 h at 60 °C to increase myeloperoxidase recovery, and finally centrifuged at 6000g for 20 min at 4 °C to separate the supernatants for MPO assay. The reaction was started by mixing 0.2 ml of 1.6 mM 3,30 ,5,50 -tetramethylbenzidine in dimethylsulfoxide with 0.8 ml of 0.05 M sodium phosphate buffer (pH 5.4) containing 0.006% (v/v) H2O2 and 0.2 ml of the 6000g supernatant from the colon tissue sample. MPO activity was assayed by measuring the change in optical density (OD) for 5 min at 650 nm. Results were expressed as change in OD per g of wet tissue.

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2.8. Preparation of colon homogenates for assessment of oxidative stress and antioxidant parameters Portions of colons were first homogenized (10% w/v) in 20 mM Tris–HCl (containing 1 mM EDTA, pH 7.4) and centrifuged at 3000g for 20 min at 4 °C. Protein contents of supernatants from colon homogenates were determined as previously described [14]. 2.8.1. Measurement of colonic reduced glutathione (GSH) content Colonic content of non-protein sulfhydryl as an indicator of GSH was measured as previously described [15] with a slight modification. Briefly, the protein in 0.225 ml of colon homogenate was precipitated with 0.025 ml of 50% (w/v) trichloroacetic acid and then centrifuged at 1000g for 5 min. The reaction mixture containing 0.125 ml of supernatant, 1 ml of 0.2 M Tris–HCl (containing 1 mM EDTA, pH 8.9) and 0.05 ml of 10 mM 5,50 -dithiobis-(2-nitrobenzoic acid) in absolute methanol was kept at room temperature for 5 min, and the yellow color developed was measured spectrophotometrically at 412 nm. Results were calculated from a standard GSH curve (0–500 nmol/ml) and expressed as nmol/mg protein. 2.8.2. Measurement of colonic glutathione-S-transferase (GSH transferase) activity GSH transferase activity was measured as previously described [16] with a slight modification. Briefly, 0.01 ml of 100 mM GSH in water and 0.01 ml of 100 mM CDNB in ethanol were added to 0.975 ml of the enzyme assay buffer (0.8% w/v of NaCl, 0.02% w/v of KCl, 0.144% w/v of Na2HPO4, 0.024% w/v of KH2PO4, pH 6.5). The increase in absorbance over 1 min was monitored at 340 nm after adding 0.05 ml of colon homogenate. A blank consisting of the homogenization buffer was also monitored in the same manner. GSH transferase activity was expressed as U/mg protein, where one unit of the enzyme was defined as the amount that conjugated with 1 lM CDNB in the presence of GSH per min at 25 °C. The extinction coefficient for CDNB conjugate at 340 nm for 1 cm path length is 0.0096 lM1. 2.8.3. Measurement of colonic malondialdehyde (MDA) and 4hydroxynonenal (4-HNE) contents Malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) concentrations were determined as previously described [17]. Briefly, 0.8 ml of 10.3 mM 1-methyl-2-phenylindole in acetonitrile diluted with methanol containing 32 lM FeCl3 (3:1) was added to 0.4 ml of colon homogenate sample with vortexing. After adding 0.2 ml of 99% (v/v) methanesulfonic acid, samples were mixed well, closed with a tight stopper and incubated at 45 °C for 40 min. The samples were then cooled on ice, centrifuged at 4000g for 10 min, and the absorbance was measured spectrophotometrically at 586 nm. A standard curve comprised of 1,1,3,3-tetramethoxypropane (0–20 nmol/ml) was also run for quantitation. 2.8.4. Measurement of colonic superoxide dismutase (SOD) activity Superoxide dismutase (SOD) activity was measured as previously described [18] with a slight modification. This method is based on the ability of SOD to inhibit the autoxidation of pyrogallol. Briefly, 0.1 ml of colon homogenate was mixed with 1.5 ml of 20 mM Tris–HCl (containing 1 mM EDTA, pH 8.2), then 0.1 ml of 15 mM pyrogallol was added. Thereafter, the change in OD per min was determined by monitoring the increase in OD at 420 nm for 3 min for the samples. The percentage of inhibition for the samples was calculated running a control with no sample under the same conditions. SOD enzyme activity was expressed as U/mg protein, where one unit was defined as the amount of the enzyme that inhibited the rate of pyrogallol autoxidation by 50%.

2.8.5. Measurement of colonic nitric oxide (NOx) content Total nitrate/nitrite (NOx) products were estimated as an index of nitric oxide synthesis as previously described [19]. Colon homogenate (0.5 ml) was added to 0.25 ml of 0.3 N NaOH. After incubation for 5 min at room temperature, 0.25 ml of 5% (w/v) ZnSO4 was added for deproteinization. This mixture was then centrifuged at 3000g for 20 min at 4 °C, and 0.5 ml of the resultant supernatant was added to 0.3 ml VCl3 (8 mg/ml) in 1 M HCl and 0.3 ml Griess reagent [0.15 ml of 2% (w/v) sulfanilamide in 5% (v/ v) HCl and 0.15 ml of 0.1% (w/v) N-(1-naphthyl)ethylendiamine dihydrochloride in distilled water]. After incubation for 45 min at 37 °C, samples were measured spectrophotometrically at 540 nm. Concentration of NOx in samples was determined from a standard curve of NaNO3 (0–100 nmol/ml). 2.9. Statistical analysis Data are expressed as means ± SEM in each experimental group. Statistical evaluations of the results, except those of histopathology scoring, were carried out by means of one way analysis of variance, followed by the Tukey–Kramer multiple comparison test. The results of histopathology scoring were analyzed by the nonparametric Kruskal–Wallis test with Dunn’s multiple comparison post-test. Statistical tests were performed with GraphPad Instat V 3.05 (GraphPad Software Inc., San Diego, CA, USA). 3. Results 3.1. Celastrol attenuates DSS-induced colon shortening and neutrophil infiltration The administration of DSS in drinking water to mice resulted in signs of acute colitis including diarrhea and bloody stools starting from day 3 and reaching maximum at day 5. After 7 days, the colon length was found to be significantly decreased in DSS-treated mice by 51%, compared with control mice (Fig. 1A). The administration of celastrol (2 mg/kg) to DSS-treated mice attenuated significantly (P < 0.001) the decrease in colon length, as compared with the DSS group. To ascertain whether celastrol might decrease neutrophil infiltration during DSS colitis, colonic MPO activity was determined. Colonic MPO activity was markedly increased in the DSS group (2.25-fold), but this increase was significantly (P < 0.05) reduced in the DSS + celastrol group (Fig. 1B). 3.2. Celastrol ameliorates DSS-induced colon injury and inflammatory signs By evaluating hematoxylin-eosin stained slides, marked histopathological changes in the distal colons of DSS-treated mice were observed (Fig. 2A and B). These included ulceration, crypt loss, erosion and inflammation of mucosa and submucosa. Semiquantitative scoring of these histological parameters confirmed that colitis severity in DSS-treated mice was significantly (P < 0.001) higher than in normal mice, 9.6 ± 0.31 versus 1.5 ± 0.17, respectively. Remarkably, the severity of ulceration, crypt loss and inflammation was significantly (P < 0.05) less in mice that received DSS + celastrol (4.2 ± 0.2), as compared with the DSS group. 3.3. Celastrol counteracts DSS-induced oxidative stress in the colon To investigate whether celastrol ameliorates DSS-colitis via inhibiting oxidative stress, we assessed the colonic levels of MDA+4-HNE, SOD, GSH transferase and GSH (Table 1). DSS-administration to mice resulted in an increase by approximately 110% in colonic MDA+4-HNE content, which was blunted by celastrol

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Fig. 1. Effect of celastrol (2 mg/kg/day) on DSS-induced changes of colon length (A) and MPO activity (B). Bars are means ± SEM (n = 8–10). Statistically significant differences are indicated as: ⁄⁄⁄P < 0.001 versus Control; #P < 0.05 and ###P < 0.001 versus DSS.

(2 mg/kg) treatment to DSS mice (40% only increase), as compared with the control group. Colonic SOD and GSH transferase activities, as well as GSH content were significantly reduced in DSS-treated mice by 82%, 75% and 70%, respectively, compared with the control group, but these reductions were restored by celastrol treatment to levels near to the normal control values. 3.4. Celastrol inhibits DSS-induced activation of the NLRP3inflammasome in the colon The activation of the NOD-like receptor protein 3 (NLRP3)inflammasome results in the secretion of IL-1b and the IFN-cinducing factor IL-18, which are two central players in colitis [20]. To test whether celastrol could also inhibit the NLRP3inflammasome, we determined the colonic protein levels of IL-1b, and also IFN-c as indirect measure of IL-18 (Table 2). Treatment with celastrol (2 mg/kg) to DSS-intoxicated mice resulted in a significant decrease in the protein levels of these mediators.

Fig. 2. Effect of celastrol (2 mg/kg/day) on DSS-induced changes of distal colonic histopathology (hematoxylin–eosin staining, 40, (A)) and the corresponding histolopathological score (B). Bars are means ± SEM (n = 8–10). Statistically significant differences are indicated as: ⁄P < 0.05 and ⁄⁄⁄P < 0.001 versus Control; # P < 0.05 versus DSS.

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Table 1 Effect of celastrol (2 mg/kg/day) on DSS-induced changes of colonic MDA+4-HNE, SOD, GSH transferase and GSH levels. Parameters Colon Colon Colon Colon

MDA+4-HNE (nmol/mg Protein) SOD (U/mg Protein) GSH Transferase (U/mg Protein) GSH (nmol/mg Protein)

Control

DSS ⁄

0.378 ± 0.035 130.4 ± 20.25 26.69 ± 4.59 0.77 ± 0.16

0.794 ± 0.07 23.18 ± 7.38⁄⁄⁄ 6.71 ± 1.29⁄⁄ 0.234 ± 0.056⁄

Data are means ± SEM (n = 8–10). Statistically significant differences are indicated as: ⁄P < 0.05,

⁄⁄

P < 0.01 and

DSS + Celastrol (2 mg/kg)

Celastrol (2 mg/kg)

0.529 ± 0.114 113.2 ± 17.22## 18.63 ± 5.06 0.568 ± 0.114

0.467 ± 0.1 120.1 ± 16.09 22.87 ± 2.61 0.754 ± 0.118

⁄⁄⁄

P < 0.001 versus Control;

##

P < 0.01 versus DSS.

Table 2 Effect of celastrol (2 mg/kg/day) on DSS-induced changes of colonic IL-1b, IFN-c, IL-23 and IL-17A proteins. Parameters

Control

DSS

DSS + Celastrol (2 mg/kg)

Celastrol (2 mg/kg)

Colon Colon Colon Colon

26.36 ± 3.5 8.35 ± 0.75 3.15 ± 0.6 2.38 ± 0.22

92.43 ± 17.52⁄⁄ 15.96 ± 2.83⁄ 15.45 ± 1.45⁄⁄⁄ 6.11 ± 0.72⁄⁄⁄

42.07 ± 5.56## 9.03 ± 0.85# 8.67 ± 1.55⁄,## 2.53 ± 0.42###

28.91 ± 4.86 6.23 ± 1.29 3.48 ± 0.87 2.63 ± 0.45

IL-1b (pg/mg Protein) IFN-c (pg/mg Protein) IL-23 (pg/mg Protein) IL-17A (pg/mg Protein)

Data are means ± SEM (n = 8–10). Statistically significant differences are indicated as: P < 0.001 versus DSS.

⁄⁄

P < 0.05,

⁄⁄

P < 0.01 and

⁄⁄⁄

P < 0.001 versus Control;

#

P < 0.05,

##

P < 0.01 and

###

3.5. Celastrol targets DSS-induced activation of the IL-23/IL-17 pathway in the colon

3.8. Celastrol-mediated inhibition of inflammatory cytokines is via modulating macrophages activation, but not migration

Recent studies have also shown that IL-1b and IL-18 can act in synergy with the IL-23/IL-17 pathway during colitis [21]. Therefore, we next investigated the effects of celastrol on the IL-23/IL-17 pathway by determining the colonic protein levels of IL-23 and IL-17. Interestingly, DSS-administration resulted in a pronounced elevation in the colonic IL-23 and IL-17A protein levels (Table 2). Celastrol (2 mg/kg) treatment to DSS-mice significantly decreased this elevation in the colonic in the protein levels of IL23 (P < 0.01) and IL-17A (P < 0.001).

The decreased levels of colonic cytokines in mice treated with celastrol might be simply a consequence of the reduction of macrophage migration rather than inhibition of activation. To clarify this hypothesis, we assessed the colonic expression for the macrophage marker CD68 immunohistochemically (Fig. 3A) by counting CD68-positive cells per field (X400). DSS-administration resulted in a pronounced increase in the number of CD68-postive cells per field (13.8 ± 1.17), compared with the control group (3.38 ± 0.42). Of note, we observed that celastrol treatment of DSS-mice did not decrease the number of CD68-postive cells per field (17.25 ± 3.21), compared with the DSS group.

3.6. Celastrol accrues DSS-induced activation of TNF-a in the colon 3.9. Celastrol prevents DSS-induced loss of intestinal epithelial homeostasis

TNF-a production is known to be increased during DSS-induced colitis, and its inhibition ameliorates the disease progression [22]. As expected, DSS-administration increased the levels of TNF-a in both colon and serum. Instead of decreasing the elevated TNF-a in DSS-treated mice, celastrol treatment increased TNF-a levels in both colon and serum, which correlated with colonic NOx content, but not serum IL-17A level (Table 3).

CD98 is a type II transmembrane glycoprotein whose expression increases in intestinal epithelial cells during inflammation, resulting in impairment of intestinal epithelial reconstitution and homeostasis, as well as tumorigenesis [23]. Next, we sought to investigate the effect of celastrol on the intestinal epithelial glycoprotein CD98 immunohistochemically (Fig. 3B). DSS-administration resulted in a pronounced increase in the colonic expression of CD98 in comparison to normal mice. Interestingly, treatment with celastrol to DSS-mice decreased the colonic expression of CD98.

3.7. Celastrol restores DSS-induced inhibition of IL-10 in the colon Next, we investigated whether celastrol can modulate the colonic anti-inflammatory IL-10. A significant reduction (P < 0.01) was observed in the colonic protein level of DSS-treated, compared with control mice. Interestingly, celastrol treatment to DSS-mice restored the colonic IL-10 levels near to the normal control values (Table 3).

4. Discussion The NF-jB pathway plays a central role in the production of inflammatory cytokines in various inflammatory diseases. Thus,

Table 3 Effect of celastrol (2 mg/kg/day) on DSS-induced changes of colonic TNF-a protein and NOx products, serum TNF-a and IL-17A, and Colonic IL-10 protein. Parameters

Control

DSS

DSS + Celastrol (2 mg/kg)

Celastrol (2 mg/kg)

Colon TNF-a (pg/mg Protein) Colon NOx (nmol/mg Protein) Serum TNF-a (pg/ml) Serum IL-17A (pg/ml) Colon IL-10 (pg/mg Protein)

3.76 ± 0.24 0.318 ± 0.053 14.58 ± 2.83 400.3 ± 46.39 22.56 ± 4.81

9.27 ± 0.91⁄⁄ 0.794 ± 0.144⁄ 129.3 ± 49.3 788.7 ± 86.2⁄⁄ 4.54 ± 0.42⁄⁄

12.95 ± 2.02⁄⁄⁄ 1.65 ± 0.088⁄⁄⁄,### 430.7 ± 133.9⁄⁄,# 654.1 ± 63.38⁄⁄ 16.77 ± 2.94#

5.15 ± 0.73 0.944 ± 0.152⁄⁄ 28.04 ± 4.9 478.2 ± 67.76 18.54 ± 3.05

Data are means ± SEM (n = 8–10). Statistically significant differences are indicated as: DSS.

⁄⁄

P < 0.05,

⁄⁄

P < 0.01 and

⁄⁄⁄

P < 0.001 versus Control; #P < 0.05 and

###

P < 0.001 versus

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Fig. 3. Representive captured pictures showing effect of celastrol (2 mg/kg/day) on DSS-induced changes of CD68-positive cells (400, (A)) and CD98 protein (200, (B)) expressions in epithelial cells of colon as assessed by immunohistochemistry. (A) DSS administration resulted in a pronounced increase in CD68-positive cells, and celastrol treatment did not decrease the number of DSS-induced CD68-positive cells. (B) DSS administration resulted in a pronounced increase in colonic expression of CD98, which was reversed by celastrol treatment.

therapeutic agents that block this pathway might be useful for the treatment of incurable inflammatory diseases, such as ulcerative colitis. Here, we report the first investigation on the effect of the novel NF-jB inhibitor celastrol on DSS-induced colitis in mice. Our data demonstrate that celastrol ameliorated the acute intestinal injury and inflammatory signs associated with DSS-administration, such as decreased colon length, bleeding, ulceration and increased colonic tissue damage scoring.

The mechanism behind DSS-mediated colon damage appears to be directly injuring the intestinal epithelia, resulting in increased uptake of bacterial endotoxins that stimulate secretion of proinflammatory cytokines from neutrophils and macrophages via TLR4 [24,25]. The increase in oxidative stress due to DSS-administration can be attributed mainly to the neutrophils, which accumulate upon colon damage within the epithelial crypts and intestinal mucosa and produce reactive oxygen and nitrogen species [26].

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Interestingly, celastrol treatment notably reduced DSS-induced colonic neutrophil infiltration (MPO) and oxidative stress (MDA+4-HNE, SOD, GSH transferase, GSH). Recent studies have revealed a critical role for the TLR4 and NLRP3-inflammasome in the pathogenesis of colitis. Recognition of bacterial LPS by TLR4 induces synthesis of the IL-1b and IL-18 precursors through activation of NF-jB [27]. In addition, NF-jBdependent signals have been also shown to regulate NLRP3 expression [28]. On the other hand, bacteria and bacterial products enter the cytosol via pore-forming toxins, type III or IV secretion systems, resulting in assembly and activation of the NLRP3-inflammasome components (NLRP3, Asc, and caspase-1) [29]. As a result, caspase-1 is activated to promote the processing and secretion of IL-1b and IL-18. Additionally, in vitro studies have shown that macrophages secreted high levels of IL-1b in a caspase-1-dependent manner when incubated with DSS, even without LPS [20]. Moreover, this IL-1b secretion was dependent on phagocytosis, lysosomal maturation and reactive oxygen species. Accordingly, our results indicate that celastrol could also inhibit the NLRP3-inflammasome, as indicated by reducing the elevated colonic protein levels of IL-1b, and IFN-c as indirect measure of IL-18. Another novel mechanism revealed by this study is the inhibition of IL-23/IL-17 pathway by celastrol. IL-23 induces the differentiation of naive CD4+ T cells into highly pathogenic helper Th17 T cells that produce IL-17, IL-6 and TNF-a, but not IFN-c [30]. The IL-23/IL-17 pathway has been shown to augment neutrophil recruitment during colitis through granulocyte-colony stimulating factor (G-CSF) and monocyte chemotactic protein-1(MCP-1) [31]. Moreover, deletion of the IL-17 receptor gene has been shown to protect against 2,4,6-trinitrobenzene sulfonic acid-induced colitis [32]. Most recently, mice deficient in IL-17A have been shown to have reduced colonic IL-6, IFN-c and TNF-a protein levels in colitis-associated cancer model [33]. Furthermore, IL-1b has been shown to act synergistically with IL-23 to promote Th17 responses from CD4+ T cells during the adaptive inflammatory response of the gut [21]. Thus, celastrol-mediated inhibition for both IL-23 and IL-17A provides another mechanism for preventing the amplification of the intestinal inflammatory cascade. Notably, the inhibitory effect of celastrol on the induction of IL-23 was lower than that of IL-17, and this can be linked to NF-jB-independent signals that induce IL-23, such as mitogen-activated protein kinases [34]. Despite its proinflammatory role, TNF-a has been recently shown to ameliorate DSS-induced colitis by promoting local glucocorticoid synthesis through directly inducing steroidogenic enzymes in the intestinal epithelial cells [35]. Besides, DSS-induced colitis was significantly exacerbated in mice deficient in TNF-a and its receptors, compared to wild type [36,37]. In this context, celastrol induced-increase of TNF-a may have an anti-inflammatory role in alleviating DSS-induced colitis, which is in agreement with other studies. Similarly, celastrol-mediated increase in NOx content in the colon of DSS-treated mice suggests an increase of inducible NO synthase activity that promotes intestinal blood flow and ulceration healing [38]. TNF-a production is usually increased during DSS-induced colitis and TLR4/NF-jB appears to be the major contributor to TNF-a expression. Nevertheless, upon inhibiting the downstream signaling of NF-jB by celastrol, there was activation of TNF-a in the colon by another transcription factor. LPS-induced TNF-a factor (LITAF) has been recently shown to mediate the increase of TNFa secretion in response to LPS stimulation from inflamed colonic lamina propria macrophages independent of NF-jB [39]. Hence, celastrol induced-increase of TNF-a and the associated increase NOx can be attributed to LITAF that may accrue upon inhibition of NF-jB. Unlike neutrophils, macrophage numbers did not decrease by celastrol treatment in the colon of DSS mice as visualized

Fig. 4. Proposed mechanism of action for celastrol in ameliorating colitis.

immunohistochemically by the macrophage marker CD68. This indicates that celastrol-mediated inhibition of inflammatory cytokines is via modulating macrophages activation, but not migration. Apart from their inflammatory role, macrophages may have an anti-inflammatory role by inhibiting mediators release, scavenging cellular debris and promoting wound healing [40]. The activation of macrophages of the gut into a counterinflammatory phenotype is critically dependent on the presence of IL-10 [41,42]. In line with this, celastrol-induced increase of colonic IL-10 in DSS-treated mice explained why celastrol possessed anti-inflammatory properties. Paradoxically, recent studies have revealed that mice lacking the NLRP3-inflammasome components exhibited greater loss of epithelial integrity and impairment of intestinal homeostasis in DSS-induced colitis than the wild-type mice [43–45]. Accordingly, innate signals are clearly protective and promote epithelial integrity. Therefore, we investigated whether the inhibition of inflammatory mediators by celastrol can result in impairment of intestinal homeostasis. Interestingly, celastrol supported intestinal homeostasis as indicated by decreasing DSS-induced overexpression of CD98. CD98 overexpression in intestinal epithelial cells has been recently shown to enhance the production of proinflammatory cytokines and chemokines by impairing intestinal epithelial reconstitution and homeostasis [23,46]. The upregulation of CD98 in intestinal inflammation and colorectal cancer has been shown to be mediated by the proinflammatory cytokine IFN-c [47]. Thus, celastrol via its prominent inhibitory effects on the colonic overexpression of IFN-c and CD98 might be a promising candidate for chemoprevention, and more significantly, treatment of inflammation-associated tumorigenesis in the colon. 5. Conclusion In summary, our study provides novel insight into the mechanisms involved in alleviating DSS-induced colitis in mice by celastrol as proposed (Fig. 4). These mechanisms compromise inhibition of colonic oxidative stress, inflammatory cytokines and intestinal homeostasis impairment. Based on our results in experimental animals, application of celastrol might be a promising approach in the treatment of ulcerative colitis in humans. Conflict of interest The authors have no conflict of interest to disclose. References [1] B. Khor, A. Gardet, R.J. Xavier, Genetics and pathogenesis of inflammatory bowel disease, Nature 474 (2011) 307–317. [2] M. Asquith, F. Powrie, An innately dangerous balancing act: intestinal homeostasis, inflammation, and colitis-associated cancer, J. Exp. Med. 207 (2010) 1573–1577.

M.E. Shaker et al. / Chemico-Biological Interactions 210 (2014) 26–33 [3] E.M. Palsson-McDermott, L.A. O’Neill, Signal transduction by the lipopolysaccharide receptor, Toll-like receptor-4, Immunology 113 (2004) 153–162. [4] G.Y. Melmed, S.R. Targan, Future biologic targets for IBD: potentials and pitfalls, Nat. Rev. Gastroenterol. Hepatol. 7 (2010) 110–117. [5] O.H. Nielsen, M.A. Ainsworth, Tumor necrosis factor inhibitors for inflammatory bowel disease, N. Engl. J. Med. 369 (2013) 754–762. [6] X. Pang, Z. Yi, J. Zhang, B. Lu, B. Sung, W. Qu, et al., Celastrol suppresses angiogenesis-mediated tumor growth through inhibition of AKT/mammalian target of rapamycin pathway, Cancer Res. 70 (2010) 1951–1959. [7] K.F. Wong, Y. Yuan, J.M. Luk, Tripterygium wilfordii bioactive compounds as anticancer and anti-inflammatory agents, Clin. Exp. Pharmacol. Physiol. 39 (2012) 311–320. [8] A. Salminen, M. Lehtonen, T. Paimela, K. Kaarniranta, Celastrol: molecular targets of thunder god vine, Biochem. Biophys. Res. Commun. 394 (2010) 439–442. [9] R. Kannaiyan, M.K. Shanmugam, G. Sethi, Molecular targets of celastrol derived from thunder of god vine: potential role in the treatment of inflammatory disorders and cancer, Cancer Lett. 303 (2011) 9–20. [10] M. Perse, A. Cerar, Dextran sodium sulphate colitis mouse model: traps and tricks, J. Biomed. Biotechnol. 2012 (2012) 718617. [11] H.S. Cooper, S.N. Murthy, R.S. Shah, D.J. Sedergran, Clinicopathologic study of dextran sulfate sodium experimental murine colitis, Lab. Invest. 69 (1993) 238–249. [12] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [13] C. Schierwagen, A.C. Bylund-Fellenius, C. Lundberg, Improved method for quantification of tissue PMN accumulation measured by myeloperoxidase activity, J. Pharmacol. Methods 23 (1990) 179–186. [14] R.E. Beyer, A rapid biuret assay for protein of whole fatty tissues, Anal. Biochem. 129 (1983) 483–485. [15] M.S. Moron, J.W. Depierre, B. Mannervik, Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver, Biochim. Biophys. Acta 582 (1979) 67–78. [16] W.H. Habig, M.J. Pabst, W.B. Jakoby, Glutathione S-transferases. The first enzymatic step in mercapturic acid formation, J. Biol. Chem. 249 (1974) 7130– 7139. [17] D. Gerard-Monnier, I. Erdelmeier, K. Regnard, N. Moze-Henry, J.C. Yadan, J. Chaudiere, Reactions of 1-methyl-2-phenylindole with malondialdehyde and 4-hydroxyalkenals. Analytical applications to a colorimetric assay of lipid peroxidation, Chem. Res. Toxicol. 11 (1998) 1176–1183. [18] S. Marklund, G. Marklund, Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase, Eur. J. Biochem. 47 (1974) 469–474. [19] K.M. Miranda, M.G. Espey, D.A. Wink, A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite, Nitric Oxide 5 (2001) 62–71. [20] C. Bauer, P. Duewell, C. Mayer, H.A. Lehr, K.A. Fitzgerald, M. Dauer, et al., Colitis induced in mice with dextran sulfate sodium (DSS) is mediated by the NLRP3 inflammasome, Gut 59 (2010) 1192–1199. [21] M. Coccia, O.J. Harrison, C. Schiering, M.J. Asquith, B. Becher, F. Powrie, et al., IL1beta mediates chronic intestinal inflammation by promoting the accumulation of IL-17A secreting innate lymphoid cells and CD4(+) Th17 cells, J. Exp. Med. 209 (2012) 1595–1609. [22] M. Onizawa, T. Nagaishi, T. Kanai, K. Nagano, S. Oshima, Y. Nemoto, et al., Signaling pathway via TNF-alpha/NF-kappaB in intestinal epithelial cells may be directly involved in colitis-associated carcinogenesis, Am. J. Physiol. Gastrointest. Liver Physiol. 296 (2009) G850–859. [23] H.T. Nguyen, G. Dalmasso, L. Torkvist, J. Halfvarson, Y. Yan, H. Laroui, et al., CD98 expression modulates intestinal homeostasis, inflammation, and colitisassociated cancer in mice, J. Clin. Invest. 121 (2011) 1733–1747. [24] I. Okayasu, S. Hatakeyama, M. Yamada, T. Ohkusa, Y. Inagaki, R. Nakaya, A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice, Gastroenterology 98 (1990) 694–702. [25] L. Stevceva, P. Pavli, A.J. Husband, W.F. Doe, The inflammatory infiltrate in the acute stage of the dextran sulphate sodium induced colitis: B cell response

[26] [27]

[28]

[29] [30] [31]

[32]

[33] [34]

[35]

[36]

[37] [38]

[39]

[40] [41] [42]

[43]

[44]

[45]

[46]

[47]

33

differs depending on the percentage of DSS used to induce it, BMC Clin. Pathol. 1 (2001) 3. Y. Naito, T. Takagi, T. Yoshikawa, Neutrophil-dependent oxidative stress in ulcerative colitis, J. Clin. Biochem. Nutr. 41 (2007) 18–26. B.K. Davis, H. Wen, J.P. Ting, The inflammasome NLRs in immunity, inflammation, and associated diseases, Annu. Rev. Immunol. 29 (2011) 707– 735. F.G. Bauernfeind, G. Horvath, A. Stutz, E.S. Alnemri, K. MacDonald, D. Speert, et al., Cutting edge: NF-kappaB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression, J. Immunol. 183 (2009) 787–791. L. Franchi, R. Munoz-Planillo, G. Nunez, Sensing and reacting to microbes through the inflammasomes, Nat. Immunol. 13 (2012) 325–332. Y. Iwakura, H. Ishigame, The IL-23/IL-17 axis in inflammation, J. Clin. Invest. 116 (2006) 1218–1222. R. Ito, M. Kita, M. Shin-Ya, T. Kishida, A. Urano, R. Takada, et al., Involvement of IL-17A in the pathogenesis of DSS-induced colitis in mice, Biochem. Biophys. Res. Commun. 377 (2008) 12–16. Z. Zhang, M. Zheng, J. Bindas, P. Schwarzenberger, J.K. Kolls, Critical role of IL17 receptor signaling in acute TNBS-induced colitis, Inflamm. Bowel Dis. 12 (2006) 382–388. Y.S. Hyun, D.S. Han, A.R. Lee, C.S. Eun, J. Youn, H.Y. Kim, Role of IL-17A in the development of colitis-associated cancer, Carcinogenesis 33 (2012) 931–936. W. Liu, X. Ouyang, J. Yang, J. Liu, Q. Li, Y. Gu, et al., AP-1 activated by toll-like receptors regulates expression of IL-23 p19, J. Biol. Chem. 284 (2009) 24006– 24016. M. Noti, N. Corazza, C. Mueller, B. Berger, T. Brunner, TNF suppresses acute intestinal inflammation by inducing local glucocorticoid synthesis, J. Exp. Med. 207 (2010) 1057–1066. Y. Naito, T. Takagi, O. Handa, T. Ishikawa, S. Nakagawa, T. Yamaguchi, et al., Enhanced intestinal inflammation induced by dextran sulfate sodium in tumor necrosis factor-alpha deficient mice, J. Gastroenterol. Hepatol. 18 (2003) 560– 569. R. Stillie, A.W. Stadnyk, Role of TNF receptors, TNFR1 and TNFR2, in dextran sodium sulfate-induced colitis, Inflamm. Bowel Dis. 15 (2009) 1515–1525. Y. Aoi, S. Terashima, M. Ogura, H. Nishio, S. Kato, K. Takeuchi, Roles of nitric oxide (NO) and NO synthases in healing of dextran sulfate sodium-induced rat colitis, J. Physiol. Pharmacol. 59 (2008) 315–336. K.N. Bushell, S.E. Leeman, E. Gillespie, A.C. Gower, K.L. Reed, A.F. Stucchi, et al., LITAF mediation of increased TNF-alpha secretion from inflamed colonic lamina propria macrophages, PLoS One 6 (2011) e25849. D.M. Mosser, The many faces of macrophage activation, J. Leukoc. Biol. 73 (2003) 209–212. S. Gordon, Alternative activation of macrophages, Nat. Rev. Immunol. 3 (2003) 23–35. J.E. Ghia, F. Galeazzi, D.C. Ford, C.M. Hogaboam, B.A. Vallance, S. Collins, Role of M-CSF-dependent macrophages in colitis is driven by the nature of the inflammatory stimulus, Am. J. Physiol. Gastrointest. Liver Physiol. 294 (2008) G770–777. I.C. Allen, E.M. TeKippe, R.M. Woodford, J.M. Uronis, E.K. Holl, A.B. Rogers, et al., The NLRP3 inflammasome functions as a negative regulator of tumorigenesis during colitis-associated cancer, J. Exp. Med. 207 (2010) 1045–1056. J. Dupaul-Chicoine, G. Yeretssian, K. Doiron, K.S. Bergstrom, C.R. McIntire, P.M. LeBlanc, et al., Control of intestinal homeostasis, colitis, and colitis-associated colorectal cancer by the inflammatory caspases, Immunity 32 (2010) 367–378. M.H. Zaki, K.L. Boyd, P. Vogel, M.B. Kastan, M. Lamkanfi, T.D. Kanneganti, The NLRP3 inflammasome protects against loss of epithelial integrity and mortality during experimental colitis, Immunity 32 (2010) 379–391. H.T. Nguyen, G. Dalmasso, Y. Yan, H. Laroui, M. Charania, S. Ingersoll, et al., Intestinal epithelial cell-specific CD98 expression regulates tumorigenesis in Apc(Min/+) mice, Lab. Invest. 92 (2012) 1203–1212. T. Kucharzik, A. Lugering, Y. Yan, A. Driss, L. Charrier, S. Sitaraman, et al., Activation of epithelial CD98 glycoprotein perpetuates colonic inflammation, Lab. Invest. 85 (2005) 932–941.