Anti-inflammatory effect of glucose-lysine Maillard reaction products on intestinal inflammation model in vivo

International Immunopharmacology 52 (2017) 324–332

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Anti-inflammatory effect of glucose-lysine Maillard reaction products on intestinal inflammation model in vivo

MARK

Chung-Oui Honga,1,2, Chae Hong Rheea,b,2, Min Cheol Pyoa, Kwang-Won Leea,⁎ a b

Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 1, 5-ga, Anam-dong, Sungbuk-gu, Seoul 02841, Republic of Korea Quality Assurance Team, Gyeongsan Plant, Maeil Dairies Co., Ltd., Gyeongsan-si, Gyeongsangbuk-do 38451, Republic of Korea

A R T I C L E I N F O

A B S T R A C T

Keywords: Inflammatory bowel diseases Dextran sulfate sodium Colitis Maillard reaction products Cytokines

Inflammatory bowel diseases (IBDs) are chronic disorders that are characterized by intestinal epithelial inflammation and injury. Currently, the most employed therapies are antibiotics and anti-inflammatory drugs; however, the side effects limit long-term effectiveness. We evaluated the impact of glucose-lysine Maillard reaction products (Glc-Lys MRPs) on colitis, induced in rats by an administration of 5% dextran sulfate sodium (DSS) in drinking water. Glc-Lys MRPs ameliorate DSS-induced colitis, as determined by a decrease in disease index activity, colon weight/length ratio, nitric oxide levels in serum, recovery of body weight loss, colon length and serum lysozyme levels. Furthermore, Glc-Lys MRPs increase the glutathione content and the activity of glutathione peroxidase, superoxide dismutase and catalase, and inhibit lipid peroxidation and myeloperoxidase activity in colon tissues. In particular, Glc-Lys MRPs suppress the mRNA level of the inflammatory cytokines and nuclear factor-κB in colon tissues. This study suggests the potential of Glc-Lys MRPs in preventing or treating IBDs.

1. Introduction Inflammatory bowel diseases (IBDs), such as Crohn's disease and ulcerative colitis, are chronic relapsing disorders of the gastrointestinal tract that are characterized pathologically by intestinal epithelial inflammation and injury [1]. The representative symptoms of IBDs are diarrhea, occult bleeding, abdominal pain, body weight loss and anemia [2]. Ulcerative colitis is histologically characterized by a continuous inflammation of colonic lamina propria with crypt abscesses, distortion and loss, ulceration, and infiltration of neutrophils, monocytes and lymphocytes [2]. Crohn's disease is a relapsing inflammatory disease, mainly affecting the gastrointestinal tract, and frequently presenting with abdominal pain, fever and clinical signs of bowel obstruction or diarrhea with passage of blood and/or mucus [1]. IBDs are considered an autoimmune disease, and its pathogenesis depends on complex interactions between genetic and environmental factors and innate and adaptive immune mechanisms, which are still not entirely clear [3]. Dextran sulfate sodium (DSS) has been used to induce colitis in animals, and the mouse model of DSS-induced colitis mimics the morphological and pathological features of human disease [4]. The DSSinduced colitis model exhibits high reproducibility for colitis lesions,

which are induced primarily in the left colon in a similar fashion to human colitis [5]. DSS is a heparin-like sulfated polysaccharide containing 17% sulfur with up to three sulfate groups in a glucose molecule [6]. Depending on the concentration and the duration and frequency of DSS administration, animals develop acute or chronic colitis [7]. The current treatments for IBDs focus on ameliorating disease symptoms and are associated with several disadvantages. Although antibiotic therapy is commonly used, it may adversely affect the balance of gut microflora and cause antibiotic resistance. Moreover, immunosuppressant and anti-inflammatory drugs such as corticosteroids have many undesirable side effects [1,8]. Finding new, and effective treatments for IBDs are thus becoming an increased focus of research. The Maillard reaction, which is well known as a non-enzymatic browning reaction, mainly occurs from a reaction between the amino group of proteins and the carbonyl group of sugars during food processing and storage. This reaction forms Maillard reaction products (MRPs) with many different physical and chemical properties [9,10]. Many studies have reported that MRPs have anti-oxidant, anti-mutagenic, carcinogenic and anti-bacterial activities [11]. However, there are few studies on the anti-inflammatory effect of MRPs. Fructose-tyrosine MRPs exhibited anti-inflammatory activity in astrocytes and BV-2

⁎

Corresponding author. E-mail address: [email protected] (K.-W. Lee). Current address: Life Science Research Institute, Novarex Co., Ltd., Ochang-eup, Cheongwon-gu, Cheongju-si, Chungcheongbuk-do 28126, Republic of Korea. 2 These authors contributed equally to this work. 1

http://dx.doi.org/10.1016/j.intimp.2017.09.009 Received 17 July 2017; Received in revised form 12 September 2017; Accepted 12 September 2017 1567-5769/ © 2017 Elsevier B.V. All rights reserved.

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Rats were randomly allocated into 5 groups: control (saline) (n = 6); 5% DSS, 5% DSS + saline group (n = 6); 5% DSS + MRPL, 5% DSS + 42.1 mg of Glc-Lys MRPs (210 mg/kg B.W.) (n = 6); 5% DSS + MRPH, 5% DSS + 421 mg of Glc-Lys MRPs (2100 mg/kg B.W.) (n = 6); and, 5% DSS + GL, and 5% DSS + 421 mg of Glc-Lys without MRPs (2100 mg/kg B.W.) (n = 6). During the experimental period, food and water consumption were not significantly different in each group (data not shown). Rats were sacrificed 18 h after the last day of the induction period. A blood sample was taken from the vena cava caudalis for nitric oxide (NO) and lysozyme assays in serum. Immediately after blood collection, the colon was excised and flushed by gentle washing with ice-cold PBS 3 times before measuring its length and weight. The distal segments of the colon were collected for histopathological studies. Adjacent segments were treated with Trizol reagent and immediately frozen in liquid nitrogen and kept at −80 °C for cytokine genes expression analysis. A section of the remaining colon was taken for the measurement of myeloperoxidase activity (MPO), glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) content. A histopathological section was fixed immediately in 10% formalin. The other sections were frozen immediately in liquid nitrogen and stored at − 80 °C for biochemical analysis.

cells [11]. Kitts, Chen and Jing [12] studied the anti-inflammatory effect of sugar- (glucose, fructose or ribose) amino acid (lysine or glycine) MRPs using Caco-2 cells as an intestinal epithelial cell model. The effect of glucose-lysine MRPs (Glc-Lys MRPs) in the regulation of the intestinal barrier and its anti-oxidant and anti-inflammatory response in DSS-induced colitis is unknown. The objective of this study is to evaluate the impact of Glc-Lys MRPs on DSS-induced colitis in rats. We hypothesized that Glc-Lys MRPs have an important role in maintaining the integrity of the intestinal mucosal barrier and enhancing the anti-inflammatory response in colitis, which implies a novel means of preventing or treating IBDs. We evaluated the disease activity index (DAI), biochemical inflammatory markers, histopathological status, anti-oxidant enzyme activities and levels of inflammatory cytokines in a DSS-induced animal model with the treatment of Glc-Lys MRPs. 2. Materials and methods 2.1. Chemicals Catalase (CAT), 5, 5-dithiobis-(2-nitrobenzoic acid) (DTNB), hexadecyltrimethylammonium bromide (HTAB), hydrogen peroxide (H2O2), glucose, lipopolysaccharide (LPS), lysine, lysozyme hydrogen peroxide (H2O2), glutathione (GSH), glutathione peroxidase (GPx), Micrococcus lysodeikticus, myeloperoxidase, nitric oxide (NO), o-dianisidine, phosphate buffered saline (PBS) and superoxide dismutase (SOD) were purchased from Sigma (St. Louis, MO, USA). Dextran sulfate sodium (DSS) was purchased from MP Biomedicals, LLC (Santa Ana, California, USA). All chemicals were of the highest possible grade.

2.5. Disease activity index (DAI) The consistency and condition of the stools of the rats were measured on the last day of the 7 consecutive days of DSS treatment using the DAI scoring system described by Nagib, Tadros, ELSayed and Khalifa [8]. The scores were determined as follows: stool consistency (0 and 1: normal, 2 and 3: loose stool, 4: diarrhea) and stool bleeding (0: negative, 1: ± , 2: +, 3: ++, 4: gross). The DAI score was calculated using the following formula:

2.2. Preparation of Glc-Lys MRPs Glucose (1 mol/L) was mixed in a 1:1 M ratio with lysine (1 mol/L) in distilled deionized water. The mixture was immediately placed in a screw-capped glass tube and boiled at 100 °C for 30 min in a shaking bath. After cooling for 30 min, glucose-lysine (Glc-Lys) MRPs were lyophilized and then stored at −80 °C until further use. A positive control of fresh Glc-Lys was made with a mixture of Glc and Lys, immediately lypolized, and then stored at −70 °C until further use.

DAI = (stool consistency score + stool bleeding score) 2.

2.6. Measurement of NO and lysozyme levels in serum Blood was collected from the vena cava and left in an ice box for 10 min. After centrifugation at 12,000 RPM for 10 min, the supernatant was collected. Serum was stored at −80 °C for further study. The analysis of NO levels in serum was determined by measuring nitrite (NO2−) levels using a colorimetric method based on the Griess reaction with some modification [14]. A serum lysozyme assay was done by measuring the lysis of suspension of Micrococcus lysodeikticus according to a slightly modified method of Nudo and Catap [15]. Lysozyme activity was expressed as mg lysozyme/g protein. Protein content was measured with a BCA protein kit using BSA as the standard.

2.3. Animals Six- to seven-week-old male Wistar rats with a body weight of 150 ± 10 g were purchased from Samtako Bio Korea Co. (Gyeonggido, Korea). They were allowed free access to a standard diet and tap water. Rats were housed in stainless steel cages in a room kept at 22 ± 1 °C with a 12-h light/12-h dark cycle (lights on from 07:00 to 19:00). Animals were acclimatized to the laboratory setting for at least 1 week before the start of the experimental protocols. All animal handling was performed according to the guidance of the Committee for Ethical Usage of Experimental Animals of Korea University.

2.7. Measurement of glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation (MDA) assay in colon tissue

2.4. In vivo colitis model The in vivo colitis model was designed with slight modifications according to Hayashi, Narumi, Tsuji, Tsubokawa, Nakaya, Wakayama, Zuka, Ohshima, Yamagishi and Okada [2], Liu, Beaumont, Walker, Chaumontet, Andriamihaja, Matsumoto, Khodorova, Lan, Gaudichon and Benamouzig [13] and Nagib, Tadros, ELSayed and Khalifa [8]. Before colitis induction, rats were treated daily with oral administration of saline alone or Glc-Lys MRPs (210 or 2100 mg/kg body weight (B.W.)) or Glc-Lys without MRPs (2100 mg/kg B.W.) for 7 d. Then, the experimental colitis model was induced by an ad libitum administration of 5% (wt/vol) DSS (molecular weight 36,000 to 50,000 kDa) in drinking water for 7 d. During this colitis induction period, rats also received daily oral administrations of Glc-Lys MRPs or Glc-Lys without MRPs.

Colon tissue was homogenized in ice-cold PBS, divided into aliquots, and frozen at − 80 °C until analysis. Spectrophotometric analysis of GSH content was done with a slightly modified method according to Owens and Belcher [16] and Nagib, Tadros, ELSayed and Khalifa [8] based on the reaction of DTNB with GSH. The GSH content was expressed as nM/mg protein. GPx and CAT and lipid peroxidation content were determined by our previous methods [17,18]. SOD activity was determined with a slightly modified method according to Kovaceva, Platenik, Vejrazka and Stipek [19]. Lipid peroxidation was measured by estimating the level of thiobarbituric acid reactive substances (TBARS) as determined by MDA, and the MDA content was expressed as nM/mg protein. 325

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A

B 3.5

240

a b

c

2.5

c

c c

200

b

3.0

DAI (point)

Body weight (g)

220

Control 5% DSS 5% DSS+MRPL 5% DSS+MRPH 5% DSS+LG

d 180

2.0

d 1.5

1.0

160 0.5

140

a

0.0

1

7

11

Control

14

day

5% DSS

5% DSS + MRPL

5% DSS + MRPH

5% DSS + GL

Fig. 1. Effect of Glc-Lys MRPs on relative body weight and disease activity index (DAI) in dextran sulfate sodium (DSS)-induced colitis in rats. Data are expressed as means ± S.D. (n = 6). Means within rows with different letters are significantly different (A, p < 0.01; B, p < 0.001) according to a one-way analysis of variance. Control, only saline group; 5% DSS, 5% DSS + saline group; 5% DSS + MRPL, 5% DSS + 42.1 mg of Glc-Lys MRPs (210 mg/kg BW); 5% DSS + MRPH, 5% DSS + 421 mg of Glc-Lys MRPs (2100 mg/kg BW); 5% DSS + GL, 5% DSS + 421 mg of fresh Glc-Lys (2100 mg/kg BW).

(TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL10) and nuclear factor-κB (NF-κB) mRNAs were calculated by normalization to β-actin mRNA.

2.8. Histopathological examination Cross sections of colonic tissue were processed for histopathological observation by hematoxylin and eosin (H & E) staining and observed at different magnifications (100 ×, 200 × and 400×). The prepared paraffin wax tissue blocks were prepared for sectioning at 4 μm thickness by a rotary microtome (Leica Microsystems Ltd., Melbourne, Australia). The obtained tissue sections were collected on glass slides, deparaffinized, stained with H & E, and then examined using a light microscope (Olympus, Tokyo, Japan) [8]. The main histologic findings for experimentally-induced colitis are 1) acute ulcer, 2) mucosal erosion, 3) inflammatory cell infiltration in the mucosa, 4) inflammatory cell in the submucosa, and 5) submucosal edema. The former four were graded and scored as follows: None, score 0; mild grade, score 1 (1–2 foci/ × 100); moderate grade, score 2 (3–4 foci/ × 100); severe grade, score 3 (5 ≥ foci/ × 100). Submucosal edema was scored as present (score 1) or absent (score 0).

2.11. Data analysis All experiments were carried out in 6 replicates, and the results are presented as means ± standard deviations. A one-way analysis of variance (ANOVA) was used to assess significant differences among the treatment groups. A p-value of < 0.05 was considered statistically significant. 3. Results 3.1. Oral administration of Glc-Lys MRPs improves DAI and colonic damage in DSS-induced colitis It is known that rats and mice treated with 5% or 3% DSS develop a severe illness characterized by bloody diarrhea and sustained body weight loss [8,21]. In our study, rats were treated orally by gavage with Glc-Lys MRPs at two different doses (42.1 and 421 mg/200 g B.W. of the rat) from day 1 to 14, and colitis was induced beginning on day 8 with DSS treatment. Our results showed that B.W. gain in the DSS-induced colitis group (5% DSS, 190.7 ± 3.1 g) was significantly less than in the control (228.5 ± 5.4 g) and treatment groups: low-dose Glc-Lys MRP (MRPL, 42.1 mg/200 g B.W.; 204.2 ± 7.0 g), high dose Glc-Lys MRP (MRPH, 421 mg/200 g B.W.; 215.7 ± 3.6 g) and fresh Glc-Lys (GL, 421 mg/200 g B.W.; 200.3 ± 5.0 g) (Fig. 1A). Interestingly, the MRPH-treated group had the highest B.W. gain among all of the groups except the control group, followed by the MRPL- and GL-treated groups. DAI is a representative index of the degree of colitis [8]. As shown in Fig. 1B, the DSS-induced colitis group (2.9 ± 0.4 points) exhibited a significant increase in DAI compared to the control group (0.1 ± 0.2 points). However, MRPL- (1.9 ± 0.2 points), MRPH- (1.1 ± 0.5 points) and GL treatment (2.0 ± 0.4 points) significantly decreased the level of DAI. While the MRPH-treated group displayed the lowest DAI level, the MRPL-treated group and the GL-treated group did not exhibit statistically significant differences. Colon shortening is a representative symptom of colitis. As for the morphology of the colon (Fig. 2A and B), the colon length in the DSSinduced colitis group (12.5 ± 0.6 cm) was significantly shorter by

2.9. Determination of myeloperoxidase (MPO) activity In this study, we measured MPO activity as a marker of immune cell infiltration into the colon tissue of rats with DSS-induced colitis. We measured the activity of MPO to assess neutrophil infiltration in the colon with a slightly modified protocol according to Hayashi, Narumi, Tsuji, Tsubokawa, Nakaya, Wakayama, Zuka, Ohshima, Yamagishi and Okada [2]. The activity of MPO is expressed as U/mg protein. 2.10. Quantitative real-time RT-PCR analysis Cytokine mRNA expression in colon tissue was assessed with a previously published method [6,20]. Levels were measured by quantitative real-time reverse transcriptase-polymerase chain reaction (qRTPCR). The primers were purchased from Cosmo Genetech (Seoul, Korea), and were used at a final concentration of 20 μM. The primer sequences are shown in Supplementary Table 1. qRT-PCR was performed using the real-time SYBR Green method on a BioRad iQ-5 thermal cycler (Hercules, CA, USA), and PCR was conducted in a 20 μL mixture of a solution containing the template (2 μL), water (6 μL), primers (2 μL) and SYBR green PCR Master Mix (10 μL). The PCR conditions were: 94 °C for 1 min, followed by 40 cycles of 94 °C for 10 s, 60 °C for 30 s and 72 °C for 30 s. The levels of tumor necrosis factor-α 326

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A

C

B

0.5

20

a

a

18

c

16

C olon length (cm)

C olon weight/length (g/cm)

b

14

b

0.4

d

12 10 8 6

e

c d a

0.3

0.2

0.1

4 2

0.0

0

Control

5% DSS

5% DSS + MRPL

5% DSS + MRPH

Control

5% DSS + GL

5% DSS

5% DSS + MRPL

5% DSS + MRPH

Control

5% DSS + GL

5% DSS

5% DSS + MRPL

5% DSS + MRPH

5% DSS + GL

Fig. 2. Effect of Glc-Lys MRPs on colonic length and relative colonic weight/length ratio in DSS-induced colitis in rats. A Macroscopic appearance, B colon length, and C colon weight/ length (grams per centimeter). Data are expressed as means ± S.D. (n = 6). Means within rows with different letters are significantly different (B, p < 0.001; C, p < 0.05) according to a one-way analysis of variance.

MRPs (MRPH), NO generation in serum significantly decreased to the control level. As shown in Fig. 3B, the mean serum lysozyme levels (mg/g protein) in the DSS-induced colitis group (17.5 ± 7.7 mg/g protein) were significantly lower than in the normal control group (57.2 ± 5.5 mg/g protein), which indicates that DSS induced colitis and then lowered lysosomal contents and defense activity. The MRPL- (30.6 ± 4.8 mg/g protein), MRPH- (35.9 ± 5.1 mg/g protein) and GL-treated groups (27.3 ± 5.2 mg/g protein) had significantly increased serum lysozyme levels, similar to that in the DSS-induced colitis group. The highest lysozyme level was observed in the MRPH-treated group.

nearly 26% compared to the control group (17.4 ± 0.6 cm). On the other hand, administration of MRPL (15.5 ± 0.4 cm), MRPH (16.8 ± 0.6 cm) or GL (14.0 ± 0.5 cm) in DSS-induced colitis significantly prevented shortening of the colon in comparison with the colitis group. The colon weight/length ratio changed in a similar manner as the DAI level. The colon/weight length ratios of the MRPL(0.34 ± 0.02 g/cm), MRPH- (0.31 ± 0.02 g/cm) and GL-treated groups (0.36 ± 0.01 g/cm) were lower than that of the DSS-induced colitis group (0.39 ± 0.01 g/cm). The control value was 0.28 ± 0.01 g/cm.

3.2. Glc-Lys MRPs treatment reduces NO level and improves lysozyme activity in serum in DSS-induced colitis

3.3. Histopathological evaluation of Glc-Lys MRPs in DSS-induced colitis

Glc-Lys MRP treatment plays an important role as an immune marker of NO generation and lysozyme activity associated with inflammation. As shown in Fig. 3A, NO generation in the DSS-induced colitis group (8.0 ± 1.1 μM) significantly increased (3.0-fold) over that in the control group (2.7 ± 0.4 μM). However, NO generation in the MRPL- (3.9 ± 0.6 μM), MRPH- (3.1 ± 0.2 μM) and GL-treated groups (5.0 ± 0.7 μM) was reduced much more than in the DSS-induced colitis group. With the administration of a high-dose of Glc-Lys

As seen in Fig. 4, each rat in the control group displayed normal, intact mucosae with little infiltration of mononuclear cells in the lamina propria and muscular layer; whereas, the DSS-induced colitis group exhibited various severe degrees of colitis. On the other hand, these DSS-induced colonic damages were ameliorated by treatment with GlcLys MRPs. Next, we performed a histopathological evaluation of the DSS-induced colitis group, MRPL-, MRPH- and GL-treated groups compared with the control group, which is summarized in Table 1. The Fig. 3. Effect of Glc-Lys MRPs on NO levels and lysozyme activity in the serum of DSS-induced colitis in rats. Data are expressed as means ± S.D. (n = 6). Means within rows with different letters are significantly different (p < 0.05) according to one-way analysis of variance.

B

A 10

70

b

a 60

d

6

c 4

a

a

Lysozyme (mg/g protein)

NaNO 2 (μ M )

8

50

d 40

cd

30

c

b

20

2

10

0

0

Control

5% DSS

5% DSS + MRPL

5% DSS + MRPH

5% DSS + GL

Control

5% DSS

327

5% DSS + MRPL

5% DSS + MRPH

5% DSS + GL

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Control

5% DSS + MRPL

5% DSS

5% DSS + MRPH

5% DSS + GL

100×

200×

400×

Fig. 4. Effect of Glc-Lys MRPs on histopathological evaluation in DSS-induced colitis in rats. Colon tissues were stained with H & E. , mucosal erosion; , inflammatory cell infiltration in the mucosa; , loss of cryptal glands; , submucosal edema; , inflammatory cell infiltration in the submucosa; mucosal irregularity.

,

submucosa and submucosal edema. Only one case displayed mucosal ulceration. On the other hand, the MRPL- and MRPH-treated groups exhibited attenuation of colitis as assessed by four histologic parameters. Of 5 rats, one or two had mild mucosal erosion, and four displayed mild inflammatory cell infiltration in the mucosa; however, the sum of the scores was not statistically significant. The MRPL-treated group had 4 and 2 cases of mild submucosal edema and mild inflammatory cell infiltration in the submucosa, respectively, which were not observed in the MRPH-treated group. The GL-treated group included 1 case of severe acute ulcer, as in the DSS-induced group, 2 cases of mild submucosal edema and 1 case of inflammatory cell infiltration in the submucosa. Also, 4 cases of mild or severe mucosal erosion and mild, moderate, or severe inflammatory cell infiltration in the mucosa in all 5 cases were observed. A comprehensive evaluation of the MRPLand MRPH-treated groups demonstrated an improvement in moderate or mild colitis as compared with the DSS-induced colitis group.

DSS-induced colitis group had acute ulcers (0.60 ± 1.34), mucosal erosion (1.80 ± 0.84), inflammatory cell infiltration in the mucosa (2.00 ± 1.00) or submucosa (0.60 ± 0.55), and submucosal edema (0.80 ± 0.45) in the microscopic tissue damage compared to the control group. On the other hand, the mucosal erosion scores in the MRPL- and MRPH-treated groups were significantly lower (0.20 ± 0.45 and 0.40 ± 0.55) than in the DSS-induced colitis group and the GL-treated group (1.80 ± 0.84 and 1.60 ± 1.34). Inflammatory cell infiltration in the mucosa decreased in the MRPL- and MRPH-treated groups (1.00 ± 0.71 and 0.80 ± 0.45) compared with the DSS-induced colitis group (2.00 ± 1.00). The GL-treated group exhibited a lower score than did the DSS-induced colitis group, but the difference was not statistically significant. The submucosal edema score in the MRPH-treated group was significantly lower (0.0 ± 0.00) than in the DSS-induced colitis group and the MRPL-treated group (0.80 ± 0.45 and 0.80 ± 0.45). The GL-treated group had a lower score than the DSS-induced colitis or MRPL-treated groups, but the difference was not statistically significant. The score of inflammatory cell infiltration in the submucosa was similar with acute ulcers. The DSS-induced colitis (0.60 ± 0.55), MRPL-treated (0.40 ± 0.55) and GL-treated (0.20 ± 0.45) groups displayed mild inflammatory cell infiltration in the submucosa. However, the MRPH-treated group exhibited no evidence of inflammatory cell infiltration in the submucosa. All cases of DSS-induced colitis displayed a moderate to severe degree of mucosal erosion and inflammatory cell infiltration in the mucosa. Three or four cases had mild inflammatory cell infiltration in the

3.4. Glc-Lys MRPs treatment induces GSH, GPx, SOD and CAT and reduces lipid peroxidation in colon tissue in a DSS-induced colitis model We measured GSH and MDA levels and the enzymatic activities of GPx, SOD and CAT in the colon tissue (Table 2). While administration of DSS significantly reduced the levels of GSH, administration of GlcLys MRPs significantly ameliorated the GSH depletion produced by DSS. The GSH levels of the control, DSS-induced colitis, MRPL-treated, MRPH-treated, and GL-treated groups were 48.3 ± 3.2 nM/mg

Table 1 Effect of glucose-lysine Maillard reaction products (Glc-Lys MRPs) on the histological score in DSS-induced colitis in rats. Group Control 5% DSS 5% DSS + MRPL 5% DSS + MRPH 5% DSS + GL

Acute ulcer 0.00 0.60 0.00 0.00 0.60

± ± ± ± ±

0.00 1.34 0.00 0.00 1.34

Mucosal erosion 0.00 1.80 0.20 0.40 1.60

± ± ± ± ±

a

0.00 0.84b 0.45a 0.55a 1.34b

Inflammatory cell infiltration in the mucosa 0.00 2.00 1.00 0.80 1.60

± ± ± ± ±

a

0.00 1.00b 0.71c 0.45ac 0.89bc

Inflammatory cell infiltration in the submucosa

Submucosal edema

0.00 0.60 0.40 0.00 0.20

0.00 0.80 0.80 0.00 0.40

± ± ± ± ±

0.00 0.55 0.55 0.00 0.45

± ± ± ± ±

0.00a 0.45b 0.45b 0.00a 0.55ab

Values are presented as means ± S.D. (n = 6). Means within rows with different letters are significantly different (p < 0.05), according to a one-way analysis of variance. Control, only saline group; 5% dextran sulfate sodium (DSS), 5% DSS + saline group; 5% DSS + MRPL, 5% DSS + 42.1 mg of Glc-Lys MRPs (210 mg/kg BW); 5% DSS + MRPH, 5% DSS + 421 mg of Glc-Lys MRPs (2100 mg/kg BW); 5% DSS + GL, 5% DSS + 421 mg of fresh Glc-Lys (2100 mg/kg BW). Means within columns followed by different letters are significantly different (p < 0.05) according to a one-way analysis of variance.

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Table 2 Effect of Glc-Lys MRPs on glutathione (GSH), lipid peroxidation (MDA), glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase(CAT) in DSS-induced colitis in rats. Group

GSH (nM/mg protein)

Control 5% DSS 5% DSS + MRPL 5% DSS + MRPH 5% DSS + GL

48.3 32.8 37.7 41.1 35.7

± ± ± ± ±

3.2a 2.2b 2.2c 3.5d 1.8bc

MDA (nM/mg protein) 259.8 387.0 285.6 259.3 321.2

± ± ± ± ±

GPx (U/mg protein)

22.0a 24.7b 15.7c 10.3a 26.7d

1.88 0.32 0.62 0.92 0.59

± ± ± ± ±

0.40a 0.08b 0.10c 0.24d 0.08c

SOD (U/mg protein) 2.44 0.67 1.36 2.07 1.31

± ± ± ± ±

0.48a 0.39b 0.27c 0.37a 0.35c

CAT (U/mg protein) 0.28 0.17 0.21 0.24 0.20

± ± ± ± ±

0.02a 0.01b 0.01c 0.01d 0.01c

Values are presented as means ± S.D. (n = 6). Means within rows with different letters are significantly different (p < 0.05), according to a one-way analysis of variance. Control, only saline group; 5% dextran sulfate sodium (DSS), 5% DSS + saline group; 5% DSS + MRPL, 5% DSS + 42.1 mg of Glc-Lys MRPs (210 mg/kg BW); 5% DSS + MRPH, 5% DSS + 421 mg of Glc-Lys MRPs (2100 mg/kg BW); 5% DSS + GL, 5% DSS + 421 mg of fresh Glc-Lys (2100 mg/kg BW). Means within columns followed by different letters are significantly different (p < 0.05) according to a one-way analysis of variance.

(Fig. 5). The mRNA expression levels of all inflammatory cytokines that were tested significantly increased in the DSS-induced colitis group than in the control group. However, the MRPL-, MRPH- and GL-treated groups showed significantly lower levels of inflammatory cytokines compared with the DSS-induced colitis group. There was no statistically significant difference between the MRPL- and MRPH-treated groups, except in the level of NF-κB mRNA. The GL-treated group had significantly reduced mRNA levels of all inflammatory cytokines than did the DSS-induced colitis group; but, there was no significant difference compared with the MRPL-treated group, except in TNF-α and IL-1β mRNA levels. However, the MRPH-treated group showed significantly lower mRNA levels of all inflammatory cytokines as compared with the GL-treated group. The greatest reduction in the mRNA levels of all inflammatory cytokines was induced by MRPH treatment. This study demonstrates that the TNF-α cytokine level in the control group (2.10 ± 0.71 relative quantification) was significantly lower than in the DSS-induced colitis group (27.81 ± 5.55 relative quantification). However, the TNF-α cytokine level in the MRPL- (6.10 ± 3.25 relative quantification), MRPH- (4.50 ± 1.48 relative quantification) and GLtreated groups (12.51 ± 3.52 relative quantification) were significantly lower than in the DSS-induced colitis group. The mRNA level of IL-1β in the control group (3.46 ± 1.39 relative quantification) and MRPL(134.86 ± 67.24 relative quantification), MRPH(87.89 ± 49.26 relative quantification) and GL-treated groups (219.03 ± 53.89 relative quantification) were significantly lower than in the DSS-induced colitis group. The level of IL-6 in the DSS-induced group (69.87 ± 29.19 relative quantification) was higher than that in the control group (4.24 ± 1.58 relative quantification) and in the MRPL- (12.32 ± 8.06 relative quantification), MRPH- (8.63 ± 3.26 relative quantification) and GL-treated groups (23.76 ± 4.82 relative quantification). The mRNA level of IL-10 displayed a similar pattern to that of the TNF-α, IL-1β and IL-6 cytokines. Rats that were given MRPL (25.78 ± 5.21 relative quantification), MRPH (9.91 ± 5.30 relative quantification), and fresh GL (29.67 ± 12.59 relative quantification) had reduced mRNA levels of IL-10 when compared to that in the colitis group (66.03 ± 23.89 relative quantification). The relative value in the control group was 4.86 ± 2.63. The results also demonstrated that the mRNA level of the inflammatory cytokine NF-κB in the control group (2.07 ± 0.73 relative quantification) was significantly lower than that in the DSS-induced colitis group (11.48 ± 1.13 relative quantification); however, the NF-κB level in the MRPL- (6.55 ± 0.84 relative quantification), MRPH- (4.82 ± 0.64 relative quantification) and GL-treated groups (6.66 ± 0.81 relative quantification) were significantly lower than in the DSS-induced colitis group. There was no significant difference between the control group and the MRPH-treated group in the mRNA levels of TNF-α, IL-6 and IL-10 cytokines. Together, these data suggest that MRPL, MRPH and fresh-GL ameliorate inflammation in the rat colitis model.

protein, 32.8 ± 2.2 nM/mg protein, 37.7 ± 2.2 nM/mg protein, 41.1 ± 3.5 nM/mg protein and 35.7 ± 1.8 nM/mg protein, respectively. There was no significant difference between the DSS-induced colitis group and the GL-treated group. Significantly, the highest GSH level was seen in the MRPH-treated group. Administration of DSS alone increased the MDA content (387.0 ± 24.7 nM/mg protein) compared with the control group (259.8 ± 22.0 nM/mg protein). The MRPL(285.6 ± 15.7 nM/mg protein), MRPH- (259.3 ± 10.3 nM/mg protein) and GL-treated groups (321.2 ± 26.7 nM/mg protein) had reduced MDA levels in comparison with the DSS-induced colitis group. The MRPH-treated group showed no significant difference with the control group. We also investigated the activities of antioxidant enzymes on colitis. The GPx activity was significantly lower in the DSSinduced colitis group (0.32 ± 0.08 U/g tissue) than in the control group (1.88 ± 0.40 U/g tissue). This result suggests that antioxidantassociated enzymes inhibit colitis. Interestingly, GPx activity in the MRPL- (0.62 ± 0.10 U/g tissue), MRPH- (0.92 ± 0.24 U/g tissue) and GL-treated groups (0.59 ± 0.08 U/g tissue) were dramatically increased compared with that in the DSS-induced colitis group. The DSS-induced colitis group (0.67 ± 0.39 U/g tissue) also showed significantly decreased SOD activity compared with the control group (2.44 ± 0.48 U/g tissue). The MRPL- (1.36 ± 0.27 U/g tissue), MRPH(2.07 ± 0.37 U/g tissue) and GL-treated groups (1.31 ± 0.35 U/g tissue) exhibited recovery of enzymatic activity compared with the colitis group. In addition, the MRPH-treated group had the greatest enhancement in enzymatic activity and was not significantly different from the control group. While the group treated with only DSS showed a decrease in CAT activity (0.17 ± 0.01 U/g tissue) compared with the control group (0.28 ± 0.02 U/g tissue), the groups treated with MRPL, MRPH and GL significantly increased CAT activity (by 0.21 ± 0.01 U/g tissue, 0.24 ± 0.01 U/g tissue and 0.20 ± 0.01 U/g tissue, respectively) compared with the DSS-induced group. The MRPL-treated group and the GL-treated group had higher CAT activity than did the DSS-induced colitis group; although, the difference was not statistically significant. 3.5. Glc-Lys MRPs treatment inhibits immune cell infiltration into colon tissue and inflammatory cytokine mRNA expression in colonic tissue in DSSinduced colitis As shown in Fig. 5, MPO activity was significantly higher in the DSSinduced colitis group (5.04 ± 0.97 U/g tissue) than in the control group (0.31 ± 0.13 U/g tissue). This result implied excessive infiltration of neutrophil cells into colon tissue following the development of colitis. Interestingly, MPO activity in the MRPL- (1.58 ± 0.26 U/g tissue), MRPH- (0.81 ± 0.20 U/g tissue) and GL-treated groups (3.02 ± 0.96 U/g tissue) were dramatically decreased compared with that in the DSS-induced colitis group. The most substantial decrease in the level of MPO activity was found in the MRPH-treated group. To determine the anti-inflammatory effect of MRPL, MRPH and fresh GL on DSS-induced colitis, inflammatory markers were used for qRT-PCR analysis, including TNF-α, IL-1β, IL-6, IL-10 and NF-κB

4. Discussion To our knowledge, this is the first study demonstrating the anti329

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Fig. 5. Effect of Glc-Lys MRPs on myeloperoxidase (MPO) activity in DSS-induced colitis in rats. Data are expressed as means ± S.D. (n = 6). Means within rows with different letters are significantly different (p < 0.05) according to a one-way analysis of variance.

in disease activity [8,25] Glc-Lys MRPs administration consistently lower all of these parameters, recovering the healthy appearance of the colon, as compared with animals exposed to DSS alone. Intestinal barrier dysfunction is one of the major contributing factors in multiple pathological conditions of the gastrointestinal tract [26]. Glc-Lys MRPs promote the prevention of intestinal mucosa damage and protect against DSS-induced colitis damage. This study proposes that the anti-inflammatory mechanism of GlcLys MRPs in DSS-induced colitis is partially associated with its influence on antioxidant systems. Inflammation induces neutrophil infiltration and the production of neutrophil-derived free radicals, such as hydrogen peroxide, superoxide and hydroxyl radicals, as well as the release of other neutrophil-derived mediators [27]. NO is an important intracellular and extracellular signal substance. Nitrite is a product of the oxidative metabolism of NO [12]. Lysozymes are especially active against gram-positive bacteria. Lysozymes break down carbohydrate chains on bacterial surfaces, destroying the structural integrity of the cell wall. The bacteria then burst under their own internal pressure

inflammatory effects of Glc-Lys MRPs on DSS-induced colitis in rats, as a model of human IBDs. The etiology of IBDs varies, and the administration of anti-TNF-α antibody, 5-aminosalicylates, corticosteroids, azathioprine and 6-mercaptopurine display therapeutic efficacy with limited toxicity and side effects for all types of IBDs [22,23]. Thus, it is necessary to search for substances that prevent or treat the symptoms of IBDs. MR, first introduced by Maillard [24], has been studied by many researchers who have focused on its antioxidants activities. MRPs have many structural forms [9]. Hence, we prepared Glc-Lys MRPs, and investigated the effects of Glc-Lys MRPs against IBDs using a DSS-induced in vivo model of colitis, which is widely used as a representative model of human colitis [2]. Kitts, Chen and Jing [12] demonstrated in vitro anti-inflammatory bioactivities of sugar- (glucose, fructose, ribose) amino acid (lysine, glycine) compounds using Caco-2 cells as an intestinal epithelial cell model. Oral administration of DSS to rats and mice for several days causes intense, bloody diarrhea, significant body weight loss and massive granulocyte infiltration that leads to colon damage and an increase 330

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attenuation of colon epithelial cell necrosis, edema, ulcers and neutrophil infiltration, especially in the MRPH-treated group, because during the process of colitis, pro-inflammatory cytokines such as TNF-α and IL-1β are released in the colon and exacerbate damage. The proinflammatory mediator NF-κB significantly decreased following the administration of Glc-Lys MRPs compared with the DSS-induced colitis group. NF-κB causes induction of inflammatory cytokines and plays an important role in immune modulatory disorders; it induces an inflammatory response in the intestine, leading to inflammatory bowel diseases [41]. The inflammatory cytokines IL-6 and IL-10 were significantly downregulated by treatment with Glc-Lys MRPs. IL-6 is produced by Th2 cells and acts as a mediator of the acute-phase response. IL-10 is an immunoregulatory cytokine produced by monocytes and macrophages [42]. The inhibition of inflammatory mediators and/ or cytokines could lead to reduced infiltration of inflammatory cells into the colonic mucosa following administration of Glc-Lys MRPs. In summary, many studies on substances to treat or prevent IBDs have been done using the DSS-colitis model. This study was the first to reveal that Glc-Lys MRPs may ameliorate DSS-induced colitis, as determined by a reduction in clinical symptoms, DAI, macroscopic properties of the disease, biochemical inflammatory markers and histological inflammation, as well as the enhancement of enzymatic activity that leads to resistance to inflammation and suppression of pro-inflammatory cytokines and inflammatory mediators. This result suggests that MR enhances the anti-inflammatory activity of Glc-Lys MRPs. However, the effect of Glc-Lys MRPs in IBDs must be elucidated by studies into the mechanisms by which it controls anti-inflammatory responses. This study suggests the potential of Glc-Lys MRPs in preventing or treating IBDs. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.intimp.2017.09.009.

[28]. The modulating effect of Glc-Lys MRPs on NO and lysozyme expression levels in serum were improved in the treatment group compared with the DSS-induced colitis group. The reaction of NO with the superoxide anion forms peroxynitrite, a potent cytotoxic oxidant eliciting lipid peroxidation and cellular damage [29,30]. As an indicator of lipid peroxidation, MDA was measured in the current study. Furthermore, the GSH level, GPx, SOD and CAT activity, which are known as antioxidants and antioxidant enzymes, were also measured in colon tissues. As a consequence, Glc-Lys MRPs reduced MDA formation and increased GPx, SOD and CAT activities compared with those in DSStreated rats, suggesting that Glc-Lys MRPs not only inhibit lipid peroxidation, but also encourage radical scavenging by enhancing the activity of antioxidant enzymes. An anti-inflammatory effect is associated with antioxidant activity via phenolics from Laggera alata in acute and chronic models of inflammation [21], and olmesartan medoxomil ameliorates colitis in rats by increasing MDA and GSH [8]. MPO has emerged as a specific marker of neutrophil activity as it provides an index of poly-morphonuclear infiltration [31,32]. Many researchers have described MPO activity in immune cell infiltration in DSS-induced colitis models and demonstrated an anti-inflammatory aspect of many substances [2,8]. MPO activity was significantly higher in DSS-induced colitis, which indicated neutrophil infiltration, and was reduced following Glc-Lys MRPs administration. This finding suggests that Glc-Lys MRPs administration not only reduces inflammatory infiltration, but also deactivates the inflammatory response. Glc-Lys MRPs ameliorated neutrophil infiltration, as evidenced by the suppression of colonic MPO activity. Interestingly, Kitts et al. reported that Glu-Lys MRP mixture, which was prepared at 121 °C for up to 90 min, exhibited significant intracellular antioxidant activity and NO and IL-8 inhibitory activities in an intestinal epithelial cell model [12]. Therefore, it is plausible that Glu-Lys MRPs reacted at 100 °C for 30 min, having antioxidant and anti-inflammatory bioactivities in our study, may reduce MPO activity, which is an indicator of neutrophil infiltration resulting in less recruitment of neutrophils in colon tissues. Its etiology of IBDs is not fully understood, but seems to result from a dysregulation in the immune response [33]. Our histological results demonstrated that control rats exhibited normal mucosa. However, the DSS-induced colitis group showed clear evidence of colitis with significant acute ulcer, mucosal erosion, inflammatory cell infiltration in the mucosa and/or submucosa and submucosal edema. The integrity of the intestinal mucosa is a physical and metabolic barrier against toxins and pathogens in the lumen. In particular, the MRPH-treated group showed mild colitis, and the MRPLtreated group had moderate colitis compared to the DSS-induced colitis group. In contrast, the GL-treated group showed less colitis than did the DSS-induced colitis group, but was not significantly different in all measurements of colitis. Histopathological results revealed that Glc-Lys MRPs had both preventive and therapeutic effects on IBDs. Together with MPO activity and histological evaluation, our results demonstrated that Glc-Lys MRPs dramatically affected recovery from colitis through the protection of the mucosal barrier. Inflammation can activate monocytes and macrophages, and promote neutrophil infiltration [34]. Activated macrophages produce substantial amounts of pro-inflammatory cytokines [35]. NF-κB is activated rapidly in response to a wide range of stimuli, including pathogens, stress signals and pro-inflammatory cytokines, such as TNF-α and IL-1β [36]. The pro-inflammatory cytokines TNF-α and IL-1β trigger a systemic inflammatory response [37]. These cytokines have profound effects on numerous cell types that, in turn, secrete a variety of inflammatory mediators that create complex networks of interactions and lead to multiple inflammatory cascades [31,38]. NF-κB, a key transcription factor, is responsible for macrophage activation and inflammation in mammals, including human beings [39]. Cytokines seem to have a crucial role in controlling intestinal inflammation and the associated clinical symptoms of IBDs [40]. These effects were clearly reflected on the histopathological evaluation in this study. It revealed

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