Journal of Ethnopharmacology 151 (2014) 1031–1039

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Apigenin has anti-atrophic gastritis and anti-gastric cancer progression effects in Helicobacter pylori-infected Mongolian gerbils Chao-Hung Kuo a,b, Bi-Chuang Weng a,c, Chun-Chieh Wu d, Sheau-Fang Yang d,e, Deng-Chang Wu a,b,f,n, Yuan-Chuen Wang c,nn a

Division of Gastroenterology, Department of Internal Medicine and Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, ROC Department of Medicine, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC c Department of Food Science and Biotechnology, National Chung Hsing University, Taichung, Taiwan, ROC d Department of Pathology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, ROC e Department of Pathology, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung, Taiwan, ROC f Division of Internal Medicine, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC b

art ic l e i nf o

a b s t r a c t

Article history: Received 6 September 2013 Received in revised form 19 November 2013 Accepted 20 November 2013 Available online 25 December 2013

Ethnopharmacological relevance: Apigenin, one of the most common flavonoids, is abundant in celery, parsley, chamomile, passionflower, and other vegetables and fruits. Celery is recognized as a medicinal vegetable in Oriental countries to traditionally treat inflammation, swelling, blood pressure, serum lipid, and toothache. In this study, we investigated apigenin treatment effects on Helicobacter pylori-induced atrophic gastritis and gastric cancer progression in Mongolian gerbils. Materials and methods: Five to eight-week-old Mongolian gerbils were inoculated with Helicobacter pylori for four weeks without (atrophic gastritis group) or with N0 -methyl-N0 -nitro-N-nitroso-guanidine (MNNG) (gastric cancer group) in drinking water, and were then rested for two weeks. During the 7th– 32th (atrophic gastritis group) or the 7th–52th (gastric cancer group) weeks, they were given various doses (0–60 mg/kgbw/day) of apigenin. At the end of the 32th (atrophic gastritis group) or the 52th (atrophic gastritis group) week, all Mongolian gerbils were sacrificed using the CO2 asphyxia method. The histological changes of Helicobacter pylori colonization, neutrophil and monocyte infiltrations, and atrophic gastritis in both atrophic gastritis and gastric cancer Mongolian gerbils were examined using immunohistochemistry stain and Sydney System scoring. Results: Apigenin treatments (30–60 mg/kgbw/day) effectively decreased atrophic gastritis (atrophic gastritis group) and dysplasia/gastric cancer (gastric cancer group) rates in Mongolian gerbils. Apigenin treatment (60 mg/kgbw/day) significantly decreased Helicobacter pylori colonization and Helicobacter pylori-induced histological changes of neutrophil and monocyte infiltrations and atrophic gastritis in both atrophic gastritis and gastric cancer Mongolian gerbils. Conclusions: Apigenin has the remarkable ability to inhibit Helicobacter pylori-induced atrophic gastritis and gastric cancer progression as well as possessing potent anti-gastric cancer activity. & 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: Histological observation Sydney system 40 ,5,7-trihydroxyflavone Anti-inflammation

1. Introduction More than 50% of the world population is infected with Helicobacter pylori. Ten to fifty percent of infected individuals

n Corresponding author at: Division of Gastroenterology, Department of Internal Medicine and Cancer Center, Division of Internal Medicine, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung Medical University Hospital; Department of Medicine, Faculty of Medicine, College of Medicine, Kaohsiung Medical University; Kaohsiung, Taiwan, ROC. Tel.: þ886 7 312 1101x7755. nn Corresponding author at: Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuo-Kuang Rd., Taichung 402, Taiwan, ROC. Tel: +886 4 2284 0385 x 4220; fax: +886 4 2285 4053. E-mail addresses: [email protected] ([email protected] (Y.-C. Wang).

0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.11.040

develop peptic ulcer disease, and 1–3% of them progress to gastric cancer (Taylor and Parsonnet, 1995). The WHO classified Helicobacter pylori as a group I carcinogen in 1994 (Anonymous, 1994). Gastric adenocarcinomas are classified as either well differentiated (known as intestinal-type) or undifferentiated (known as diffuse-type); the former is characterized by a corpusdominated gastritis with gastric atrophy and intestinal metaplasia, whereas the latter is characterized by gastritis throughout the stomach but no atrophy (Konturek et al., 2006; Fox and Wang, 2007). The Correa pathway (Correa, 1992) defined intestinal-type adenocarcinoma development as being a multistep and multifactor process. Helicobacter pylori infection induces chronic inflammation of the gastric mucosa, and then progresses to atrophic gastritis,

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intestinal metaplasia, dysplasia, and finally adenocarcinoma. Atrophic gastritis appears to be a critical initiating step in the progression toward gastric cancer (Correa, 1992; Fox and Wang, 2001; Fox and Wang, 2007). Interestingly, higher gastric pH, nitrates, and salts are stimulators, whereas ascorbic acid and β-carotene are inhibitors (Correa, 1992; Fox and Wang, 2007). Parietal, mucous neck, and chief cells are the major components of the oxyntic gland of the stomach body, which are responsible for gastric juice, mucus, and enzyme secretions, respectively (Fox and Wang, 2007). Once Helicobacter pylori attaches to host cells, the immune response immediately initiates through cellular signal transduction cascade. Specifically in inflammation defense, monocytes and polymorphonuclear neutrophils infiltrate into oxyntic glands extensively triggering a cytokine cascade that culminates in gland dilation and mineralization, progressing to focal fibrosis and finally complete loss of oxyntic parietal and chief cells (atrophic gastritis). (Fox and Wang, 2001; Houghton et al., 2002; Konturek et al., 2006; Fox and Wang, 2007; Konturek et al., 2009; Peek et al., 2010). Apigenin (Fig. 1) is one of the most common flavonoids. It is found in vegetables and fruits, and especially abundant in celery, parsley, guava, bilimbi fruit, garlic, bell pepper, onion, chamomile, and passionflower (Miean and Mohamed, 2001; Shukla and Gupta, 2010; Anonymous, 2013). Celery is recognized as a medicinal vegetable in Oriental countries to traditionally treat inflammation (pneumonia, cystitis, urethritis, pharyngolaryngitis, pharyngitis, hepatitis, and nephritis), swelling, blood pressure, serum lipid, and toothache (Lee, 1994). Chamomile prepared from the dried flowers of Matricaria chamomilla is traditionally consumed as single ingredient herbal tea in the America and European countries as a natural aid for both sleeplessness and anxiety. Infusion chamomile contains apigenin at 0.8–1.2% concentration (Shukla and Gupta, 2010). Passionflower (Passiflora incarnata) was traditionally used in the America and European countries for the treatment of boils, wounds, liver problems, anxiety, insomnia, asthma, shingles, and Parkinson0 s disease. Passionflower contains high levels of flavonoids (apigenin, luteolin, and scopoletin) and indole alkaloids (Anonymous, 2013). A few papers have characterized apigenin0 s anti-inflammatory activity both in vitro and in vivo. The relevant mechanisms include: (1) suppression of nuclear factor kappa B (NF-κB) activation; and (2) inhibitions of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and proinflammatory cytokine [interleukin IL-1β (IL-1β), IL-6, IL-8, and tumor necrosis factor-α (TNF-α)] production (Raso et al., 2001; Smolinski and Pestka, 2003; Ueda et al., 2004; Comalada et al., 2006; Lee et al., 2007; Nicholas et al., 2007; Ha et al., 2008). Anti-gastric carcinoma activity of apigenin was also reported being due to its anti-proliferation and apoptosis-inducing activities (Wu et al., 2005; Yuan et al., 2007; Li-Weber, 2013). Additionally, other bioactivities including anti-platelet aggregation, antioxidation, and anti-Helicobacter pylori activity have been also studied (Zhang et al., 2008; Han et al., 2009; Das et al., 2010; Wright et al., 2010). The bioactivity of apigenin is probably due to hydroxylation at A and B rings coupled with a double bond at C2 and C3 in the flavonoid skeleton (Kawaii et al., 1999; Raso et al., 2001; Ueda et al., 2004; Comalada et al., 2006), and its intrinsic scavenging property (Wang and Huang, 2013).

Fig. 1. Chemical structure of apigenin (40 ,5,7-Trihydroxyflavone).

In our previous paper (Wang and Huang, 2013) with an in vitro focus, remarkable anti-Helicobacter pylori induced inflammation was demonstrated in apigenin, in which the suppression of NF-κB pathway activation and inhibition of inflammatory factor [reactive oxygen species (ROS), COX-2, intercellular adhesion molecule-1 (ICAM-1), IL-6, and IL-8] expressions were involved. Consequently, in this study, we investigated apigenin treatment effects on Helicobacter pylori-induced atrophic gastritis and gastric cancer progression in Mongolian gerbils using immunohistochemistry stain and Sydney System scoring.

2. Materials and methods 2.1. Reagents Apigenin (98% purity) was purchased from Shaanxi Huike Botanical Development Co. (Shaanxi, China), with ethanol and dimethyl benzene from JT Baker (USA), fetal bovine serum (FBS) from SAFC Biosciences (Australia), Brucella broth from Becton Dickinson and Company (USA), basal granular diet from Altromin Spezialfutter GmbH & Co. (Germany), Embedding Medium from Leica Biosystems (Germany), N0 -methyl-N0 -nitro-N-nitroso-guanidine (MNNG) from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), methyl cellulose from Nacalai Tesque, Inc. (Kyoto, Japan), Helicobacter pylori Rapid Test Device from ABON Biopharm Co., Ltd. (China), normal saline from Nang Kuang Pharmaceutical Co., Ltd. (Tainan, Taiwan), neutral-buffered formalin from Hsiang Hsin (Taipei, Taiwan), and hematoxylin-eosin (H-E) from Surgipath (USA). 2.2. Helicobacter pylori strain Helicobacter pylori strain KMUH-G926 (cagA þ/vacAþ ) was isolated from a Helicobacter pylori-severely infected Mongolian gerbil. A volume of 0.1 mL of Helicobacter pylori suspension was added to 9 mL containing 10% (v/v) FBS of Brucella broth, and then was cultivated in an Anaerobic Workstation (Ruskinn Bugbox/ BB1103346, England) with 5% CO2 and 10% CO2-in-air at 37 1C for 72 h to produce 9  108 CFU/mL of the bacterial counts. 2.3. Mongolian gerbils and experimental designs Five to eight-week-old male and female Mongolian gerbils (50– 55 g weights) were obtained from the Laboratory Animal Center of Taiwan University (Taipei, ROC), and housed in an air-conditioned biohazard room with a 12:12-h light/dark cycle, 70–80% humidity, and 22–24 1C temperature. They were given water and basal granular diet ad libitum. Experiments were performed according to the experimental guidelines of the ethics committee of Kaohsiung Medical University Chung-Ho Memorial Hospital Laboratory Animal Center. Those guidelines were established according to the Guide for The Care and Use of Laboratory Animals by the American National Research Council (NRC) and were approved by the ethics committee constituted in accordance with the rules and guidelines stated by government of Taiwan (Article 16 of the Animal Protection Act announced on 03 Jun, 2010; Document no. 0990041068). The experiments were divided into atrophic gastritis and gastric cancer groups. For the atrophic gastritis group (Fig. 2), Mongolian gerbils were divided into six groups, with 10 Mongolian gerbils in each group. Group I-A was the vehicle. In the first four weeks, groups I-C–I-F were orally inoculated with Helicobacter pylori (9  108 CFU/ml, 2 mL) twice weekly and groups I-A–I-B were only given Brucella broth. During the 7th to the 32nd weeks, groups I-A I-F were intragastrically administered various doses (0–60 mg/ kgbw/day) of apigenin once daily in which apigenin was suspended

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groups II-B–II-G were given 50 μg/mL MNNG with drinking water, and consequently switched to distilled water. Additionally, at the 7th week, groups II-A–II-G were intragastrically administered various doses (0–60 mg/kgbw/day) of apigenin once daily until the end of the 52nd week in which apigenin was suspended in 0.5% methyl cellulose. At the end of the 52nd week, all Mongolian gerbils in the gastric cancer group were sacrificed using the CO2 asphyxia method. 2.4. Body and relative organ weights After sacrificing, the body and organs (stomach, liver, and kidneys) of each Mongolian gerbil were weighed. Relative weight (%) was calculated as the absolute weight of each organ compared to the individual body weight. Fig. 2. Experimental design for Mongolian gerbils with apigenin treatments against Helicobacter pylori-induced atrophic gastritis. Mongolian gerbils (males and females, 50–55 g weights) were divided into six groups with 10 Mongolian gerbils in each group. Group I-A was the vehicle. In the first four weeks, groups I-C–I-F were orally inoculated with Helicobacter pylori (9  108 CFU/mL, 2 mL) twice weekly and groups I-A–I-B were only given Brucella broth. During the 7th–32nd weeks, groups I-A–I-F were intragastrically administered various doses (0–60 mg/ kgbw/day) of apigenin once daily in which apigenin was suspended in 0.5% methyl cellulose. At the end of the 32nd week, all Mongolian gerbils were sacrificed using the CO2 asphyxia method.

2.5. Helicobacter pyloriHelicobacter pyloriinfection assay The fresh heart blood of the sacrificed Mongolian gerbils was collected and centrifugated at 1308g for 10 min. A volume of 30 μL of the supernatant was examined for Helicobacter pylori infection using an Helicobacter pylori Rapid Test Device. 2.6. Tissue preparation and histological examination The Mongolian gerbil stomachs were dissected along the greater curvature, washed with normal saline, fixed with 10% neutral-buffered formalin for 24 h, dehydrated with ethanol and dimethyl benzene; embedded in Embedding Medium, and then sliced into 4 μm-thick sections. The sections were stained with H-E, and then histologically examined for Helicobacter pylori density, neutrophil and monocyte infiltrations, intestinal metaplasia, and dysplasia under a light microscope (Olympus DP72, Tokyo, Japan) at 1000 times magnification, and at 40x for atrophic gastritis and gastric cancer. The degree of Helicobacter pylori colonization, infiltrations in neutrophils and monocytes, and atrophic gastritis were evaluated using the Sydney System (Dixon et al., 1996) on the basis of a four-point scale (0–3, 0¼ normal; 1¼ mild; 2, moderate; 3 ¼ marked). The dysplasia was identified as nuclear abnormalities including increased size, hyperchromatism, and an irregular shape of the glandular epithelial cells; whereas the gastric cancer was identified as cells forming irregular, invasive neoplastic glands with destruction of the normal gastric mucosa and submucosa architecture. 2.7. Statistical analysis

Fig. 3. Experimental design for Mongolian gerbils with apigenin treatments against Helicobacter pylori-induced gastric cancer. Mongolian gerbils (males and females, 50–55 g weights) were divided into seven groups with eight Mongolian gerbils in each group. Group II-A was the vehicle. In the first four weeks, groups II-D  II-G were orally inoculated with Helicobacter pylori (9  108 CFU/mL, 2 mL) twice weekly and groups II-A–II-C were only given Brucella broth. During the 2nd–20th weeks, groups II-B–II-G were given 50 μg/mL MNNG with drinking water, and then switched to distilled water. Additionally, at the 7th week, groups II-A–II-G were intragastrically administered various doses (0–60 mg/kgbw/day) of apigenin once daily until the end of the 52nd week in which apigenin was suspended in 0.5% methyl cellulose. At the end of the 52nd week, all Mongolian gerbils were sacrificed using the CO2 asphyxia method.

Data for body and relative organ weights, Sydney System scores for Helicobacter pylori colonization, infiltrations in neutrophils and monocytes, and atrophic gastritis were subjected to one-way analysis of variance (one-way ANOVA) with Dunnett0 s test by SPSS 10.0 software (SPSS Inc., USA) using least significant difference to identify significant differences of experimental groups from the control. Po 0.05 or P o0.01 was considered to be significant.

in 0.5% methyl cellulose. At the end of the 32nd week, all Mongolian gerbils in the atrophic gastritis group were sacrificed using the CO2 asphyxia method. For the gastric cancer group (Fig. 3), Mongolian gerbils were divided into seven groups, with eight Mongolian gerbils in each group. Group II-A was the vehicle. In the first four weeks, groups II-D–II-G were orally inoculated with Helicobacter pylori (9  108 CFU/mL, 2 mL) twice weekly and groups II-A–II-C were only given Brucella broth. During the 2nd to the 20th weeks,

3.1. Body and relative organ weights

3. Results

For the atrophic gastritis group (Table 1), neither the Helicobacter pylori infection (I-C) nor the 60 mg apigenin/kgbw/day treatment (I-B) resulted in significant differences of the body and relative organ (stomach, liver, and kidney) weights from the vehicle (I-A) (P o0.05). There were no significant differences in those weights between the 10–60 mg/kgbw/day of apigenin

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Table 1 Body weights and relative organ weights of Helicobacter pylori-induced atrophic gastritis and gastric cancer in Mongolian gerbils treated with various doses (0–60 mg/kgbw/day) of apigenin. Treatment

Body weight (g)

Atrophic gastritis group I-A 70.17 2.1 I-B 81.9 7 3.0 I-C 75.17 3.1 I-D 73.17 3.1 I-E 74.8 7 3.1 I-F 80.5 7 2.3 Gastric cancer group II-A 78.0 7 2.7 II-B 80.6 7 3.8 II-C 78.0 7 3.9 II-D 71.5 7 2.4 II-E 72.1 7 2.9 II-F 72.0 7 2.4 II-G 75.6 7 2.9

Relative organ weight (%) Stomach

Liver

Kidney

0.92 7 0.03 1.007 0.03 1.047 0.04 1.017 0.03 1.047 0.05 1.02 7 0.03

3.20 7 0.08 3.577 0.07 3.497 0.07 3.477 0.11 3.477 0.08 3.54 7 0.06

0.747 0.04 0.79 7 0.03 0.747 0.02 0.82 7 0.02 0.80 7 0.02 0.767 0.02

1.02 7 0.03 0.84 7 0.02 0.92 7 0.05 1.05 7 0.06 1.147 0.12 1.08 7 0.06 1.017 0.05

3.577 0.17 3.54 7 0.12 3.28 7 0.09 3.75 7 0.10 3.517 0.12 3.82 7 0.08 3.78 7 0.15

0.86 7 0.04 0.89 7 0.02 0.86 7 0.03 0.83 7 0.02 0.80 7 0.03 0.95 7 0.10 0.87 7 0.03

Relative organ weight (%)¼ [absolute organ weight (g)/body weight of Mongolian gerbils (g)]  100. Values presented are the mean 7 standard error, which derived from 10 and 8 Mongolian gerbils for groups atrophic gastritis and gastric cancer, respectively. There are no significant difference from I-C and II-D for atrophic gastritis and gastric cancer groups, respectively, at Po 0.05 (atrophic gastritis group) and P o 0.01 (gastric cancer group).

Table 2 Helicobacter pylori infection and histological change ratesa of Helicobacter pyloriinduced atrophic gastritis and gastric cancer in Mongolian gerbils treated with various doses (0–60 mg/kgbw/day) of apigenin. Treatment Helicobacter pylori infection Atrophic gastritis group I-A 0 (0/10) I-B 0 (0/10) I-C 100 (10/10) I-D 100 (10/10) I-E 100 (10/10) I-F 100 (10/10) Gastric cancer group II-A 0 (0/8) II-B 0 (0/8) II-C 0 (0/8) II-D 100 (8/8) II-E 100 (8/8) II-F 100 (8/8) II-G 100 (8/8) a

Atrophic gastritis

Intestinal metaplasia

0 0 100 100 80 40

(0/10) (0/10) (10/10) (10/10) (8/10) (4/10)

0 0 0 0 0 0

(0/10) (0/10) (0/10) (0/10) (0/10) (0/10)

0 0 0 100 100 100 100

(0/8) (0/8) (0/8) (8/8) (8/8) (8/8) (8/8)

0 0 0 0 0 0 0

(0/8) (0/8) (0/8) (0/8) (0/8) (0/8) (0/8)

Dysplasia

0 0 0 0 0 0 0 0 0 100 100 75 13

Gastric cancer

(0/10) (0/10) (0/10) (0/10) (0/10) (0/10)

0 0 0 0 0 0

(0/10) (0/10) (0/10) (0/10) (0/10) (0/10)

(0/8) (0/8) (0/8) (8/8) (8/8) (6/8) (1/8)

0 0 0 88 88 63 0

(0/8) (0/8) (0/8) (7/8) (7/8) (5/8) (0/8)

the percentage of positive numbers to total test numbers.

treatments (I-D-I–I-F) and the 0 mg/kgbw/day treatment (I-C) (P o0.05). For the gastric cancer group, the results were similar with the atrophic gastritis group. The Helicobacter pylori infection (II-D), 50 μg/mL MNNG given (II-B), and 60 mg apigenin/kgbw/day treatment (II-C) did not result in significant changes of the body and relative organ weights from the vehicle (II-A) (P o0.01). There were no significant differences in those weights between the 0–60 mg apigenin/kgbw/day treatments (II-D–II-G) (Po 0.01).

cancer (II-D–II-F treatments, 8/8) groups. 100% (10/10) of atrophic gastritis rates were found in both the I-C and II-D treatments (both being þHelicobacter pylori/–apigenin); 30 and 60 mg/kgbw/day of apigenin treatments (I-E and I-F) decreased those rates to 80% and 40%, respectively, in the atrophic gastritis group treatments, but not for the gastric cancer group. Intestinal metaplasia was not observed in either group. Both dysplasia and gastric cancer histological changes were not found in the atrophic gastritis group; however, they were discovered in the gastric cancer group (II-D–II-G treatments). With increasing treatment doses (30 and 60 mg/kgbw/day, II-F and II-G), both rates decreased from 100% and 88% (II-D) to 13–75% and 0–63%, respectively, for dysplasia and gastric cancer. Summarizing Table 2, Helicobacter pylori infection resulted in atrophic gastritis in 32 week-treated Mongolian gerbils (the atrophic gastritis group). Furthermore, Helicobacter pylori infection with MNNG resulted in gastric cancer in 52 week-treated Mongolian gerbils (the gastric cancer group). However, 30 and 60 mg/ kgbw/day of apigenin treatments effectively reduced those incidences in both groups. 3.3. Apigenin effects on histological changes of atrophic gastritis Mongolian gerbils Fig. 4(a–c) are histological changes under a light microscope, and Fig. 4(d–f) are Sydney System scores of those changes. As shown in Fig. 4(d–f), there were no histological changes (Helicobacter pylori density, neutrophil and monocyte infiltrations, and atrophic gastritis) for the vehicle (I-A) and apigenin-only (I-B) treatments; however, Helicobacter pylori infection (I-C) significantly increased those changes [2.7070.48 (Helicobacter pylori density) (F5, 54 ¼105.46, P o0.05; one-way ANOVA), 2.90 70.32 (neutrophil) (F5, 54¼ 153.95, Po0.05; one-way ANOVA), 2.90 7 0.32 (monocyte) (F5, 54¼ 85.35, P o0.05; one-way ANOVA), and 1.00 70.00 (atrophic gastritis) (F5, 54 ¼29.52, P o0.05; one-way ANOVA)]. In contrast, apigenin treatment (60 mg/kgbw/day) significantly decreased those scores to 2.00 70.47, 1.90 70.57, 1.80 7 0.63, and 0.40 70.52, respectively (F5, 54 ¼105.46, 153.95, 85.35, and 29.52, respectively; P o0.05; one-way ANOVA). 3.4. Apigenin effects on histological changes of gastric cancer Mongolian gerbils Fig. 5(a–c) are histological changes under a light microscope, and Fig. 5(d–f) are Sydney System scores of those changes. As shown in Fig. 5(d–f), there were no histological changes (Helicobacter pylori density, neutrophil and monocyte infiltrations; and atrophic gastritis) for the vehicle (II-A), MNNG-only (II-B), and apigenin-only (II-C) treatments; however, Helicobacter pylori infection (II-D) significantly increased those changes [2.8870.35 (Helicobacter pylori density) (F6, 49¼147.74, Po0.05; one-way ANOVA), 2.8870.35 (neutrophil) (F6, 49¼130.62, Po0.05; one-way ANOVA), 2.8870.35 (monocyte) (F6, 49¼ 117.66, Po0.05; one-way ANOVA), and 3.007 0.00 (atrophic gastritis)] (Po0.05) (F6, 49¼51.57, Po0.05; one-way ANOVA). Contrarily, apigenin treatment (60 mg/kgbw/day) significantly decreased those scores to 1.8870.35, 1.7570.46, 2.1370.64, and 1.7570.34, respectively (F6, 49¼147.74, 130.62, 117.66, 51.57, respectively; Po0.05; one-way ANOVA) (Po0.05).

3.2. Helicobacter pylori infection and histological change rates

4. Discussion

As shown in Table 2, in spite of apigenin treatments (10–60 mg apigenin/kgbw/day), 100% of Helicobacter pylori colonization rates were found in all Helicobacter pylori-infected Mongolian gerbils for both the atrophic gastritis (I-C–I-F treatments, 10/10) and gastric

In this study, Helicobacter pylori infection of Mongolian ger bils resulted in atrophic gastritis and gastric cancer after 32 and 52 week treatments, respectively, for which the histological changes (Helicobacter pylori colonization, neutrophil and monocyte

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I-A

I-C

3

2

1

Apigenin

neutrophil monocyte

3

2

1

0

0

H. pylori (mg/kgbw/day)

I-F 4

Sydney score of neutrophil/monocyte infiltration

Sydney score of H. pylori density

4

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0 I-A

60 I-B

0 I-C

10 I-D

30 I-E

60 I-F

0 I-A

60 I-B

0 I-C

10 I-D

30 I-E

60 I-F

H. pylori Apigenin 0 (mg/kgbw/day) I-A

60 I-B

0 I-C

10 I-D

30 I-E

60 I-F

Sydney score of atrophic gastritis

4

3

2

1

0

H. pylori Apigenin (mg/kgbw/day)

Fig. 4. Effect of apigenin treatments (0–60 mg/kgbw/day, I-A–F) on histological changes (a–c) and Sydney System scores (d–f) for Helicobacter pylori-induced atrophic gastritis in Mongolian gerbils in which (a) and (d) are Helicobacter pylori density, (b) and (e) are neutrophil/monocyte infiltrations, and (c) and (f) are atrophic gastritis. The black and red arrows in (b) indicate neutrophils and monocytes, respectively. The bars of Sydney scores (d–f) represented are the mean 7 standard error (n ¼10) with single star showing the significant difference from treatment I-C ( þ Helicobacter pylori/-apigenin) at P o 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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II-A

3

2

1

Sydney scores of neutrophil /monocyte infiltration

Sydney score of H. pylori density

*

*

3

*

2

1

0

0

H. pylori MNNG Apigenin 0

0

60

0

10

30

60

(mg/kgbw/day)

II-A II-B II-C II-D II-E II-F II-G

Sydney score of atrophic gastritis

neutrophil monocyte

4

4

f

II-G

II-D

H. pylori MNNG Apigenin 0

0

60

0

10

30

60

(mg/kgbw/day)

II-A II-B II-C II-D II-E II-F II-G

*

4

3

2

1

0

H. pylori MNNG Apigenin 0

0

60

0

10

30

60

(mg/kgbw/day)

II-A II-B II-C II-D II-E II-F II-G Fig. 5. Effect of apigenin treatments (0–60 mg/kgbw/day, II-A–G) on histological changes (a–c) and Sydney System scores (d–f) for Helicobacter pylori-induced cancer in Mongolian gerbils in which (a) and (d) are Helicobacter pylori density, (b) and (e) are neutrophil/monocyte infiltrations, and (c) and (f) are atrophic gastritis. The black and red arrows in (b) indicate neutrophils and monocytes, respectively. The bars of Sydney scores (d–f) represented are the mean 7standard error (n¼ 8) with single star showing the significant difference from treatment II-D ( þ Helicobacter pylori/-apigenin) at P o 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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the gastric cancer progression was inhibited. Apigenin has a gastric cancer prevention effect. Equally, those rate reductions were also probably due to apoptosis in gastric adenocarcinoma in Mongolian gerbils. In our previous study (Wang and Huang, 2013), 74 μM of apigenin significantly decreased IκBα degradation and COX-2/ROS production. The active NF-κB (dimer p50/p65) is a pro-apoptotic gene (Bad, Bak, and Bax) inhibitor, which inhibits apoptosis in gastric carcinoma cells (Noto and Peek, 2012; Li-Weber, 2013). COX-2 inhibitors are able to inhibit some cell cycle checkpoints and pro-apoptotic gene expressions (Sobolewski et al., 2010). Moreover, ROS is a stimulator for NF-κB activation (Handa and Yoshikawa, 2010; Li-Weber, 2013). Therefore, suppression of NF-κB activation, reduction of COX-2/ROS production, and the intrinsic scavenging property of apigenin observed in our previous study (Wang and Huang, 2013) would be the key effectors to activate pro-apoptotic gene expressions resulting in apoptosis in gastric adenocarcinoma in Mongolian gerbils, and finally leading to decreased gastric cancer rates (Table 2, Fig. 5). Apigenin has a potent anti-gastric cancer effect. Furthermore, a few studies have pointed out that the most significant anti-inflammatory effects of flavonoids were demonstrated in hydroxylations at positions 5, 7, 30 and 40 coupled with a double bond at C2–C3 in the flavonoid skeleton. Specifically, luteolin (30 ,40 ,5,7-tetrahydroxyflavone) and quercetin (30 ,40 ,5,7-tetrahydroxyflavonol) had the greatest antiinflammatory activity and anti-TNF-α production among the test flavonoids. Apigenin (40 ,5,7-trihydroxyflavone) is characterized by 3 hydroxylations at C-40 ,5,7 and a double bond at C2–C3 in the flavonoid skeleton; the anti-inflammatory activity was slightly lower than that of luteolin and quercetin but still remarkable (Kawaii et al., 1999; Raso et al., 2001; Ueda et al., 2004; Comalada et al., 2006). Those chemical structure features of apigenin can further explain the good anti-inflammatory and anti-gastric cancer progression/potent anti-gastric cancer activities demonstrated in our previous (Wang and Huang, 2013) and present studies. Herein, the role that apigenin played on the Correa pathway (Correa, 1992; Fox and Wang, 2001) in Mongolian gerbils is proposed in Fig. 6. Apigenin has the remarkable ability to inhibit the progression of Helicobacter pylori-induced superficial gastritis, atrophic gastritis, dysplasia, and gastric cancer in Mongolian gerbils. Few in vivo studies have been concerned with the antiHelicobacter pylori-inducing gastritis and gastric cancer of natural products or clinical medicines. The natural products quercetin (González-Segovia et al., 2008), caffeic acid phenethyl ester (Toyoda et al., 2009), Cladosipbon fucoidan (Shibata et al., 2003),

infiltrations, atrophic gastritis, dysplasia, or gastric cancer) were observed. Notably, apigenin treatment (60 mg/kgbw/day) effectively decreased such changes either in the atrophic gastritis or gastric cancer Mongolian gerbils. In our previous in vitro study (Wang and Huang, 2013), apigenin treatments significantly inhibited NF-κB activation in the Helicobacter pylori-infected adenocarcinoma cells. Thus, the downstream inflammatory factor [COX-2, ICAM-1, ROS, IL-6, IL-8, and reactive oxygen species (ROS)] expressions remarkably decreased. Additionally, the intrinsic radical scavenging property of apigenin may contribute to ROS deletion (Zhang et al., 2008; Han et al., 2009). In Table 2 and Fig. 4, the 60 mg/kgbw/day of apigenin treatment effectively decreased neutrophil and monocyte infiltrations and atrophic gastritis in the Helicobacter pyloriinfected Mongolian gerbils. Both in vitro and in vivo studies have showed consistent results. We strongly suggested that apigenin has an ability to inhibit extensive, Helicobacter pylori-induced inflammation (superficial gastritis) and atrophic gastritis in Mongolian gerbils through the suppression of NF-κB activation and the related inflammatory factor inhibition as well as the intrinsic free radical scavenging property of apigenin. Atrophic gastritis is a critical initiating step in the progression toward gastric cancer (Correa, 1992; Houghton et al., 2002; Fox and Wang, 2007). In Figs. 4 and 5, the Sydney scores of atrophic gastritis for the atrophic gastritis group (1.007 0.00, I-C) were much lower than that of the gastric cancer group (3.00 70.00, II-D) because of MNNG incorporation and more than 20 week treatment in gastric cancer group. Histological changes of dysplasia and gastric cancer for the gastric cancer group were observed at 100% and 88% rates, respectively (Table 2); importantly, dysplasia is a necessary step for gastric cancer development (Correa, 1992; Houghton et al., 2002; Fox and Wang, 2007). Notably, the 60 mg/kgbw/day of apigenin treatment significantly reduced dysplasia and gastric cancer rates by 87 and 100%, respectively (Table 2), and reduced the Sydney score of atrophic gastritis by 42% (Fig. 5). From this evidence, we can conclude that apigenin not only significantly decreased atrophic gastritis incidence, but also inhibited the progression of dysplasia and gastric cancer in the Helicobacter pylori-infected Mongolian gerbils. Notably, apigenin was also reported for its apoptosis-inducing activity in gastric carcinoma cells through Akt activity inhibition (Wu et al., 2005; Yuan et al., 2007). The gastric cancer reductions in Mongolian gerbils in this study (Table 2 and Fig. 5) were partially due to the suppression of Helicobacter pylori-induced inflammation, and thus

6 weeks 32 weeks

52 weeks

H. pylori Superficial gastritis

Atrophic gastritis

Dysplasia

Gastric adenocarcinoma

Infected Mongolian gerbil

Apigenin

Fig. 6. Inhibitory effects of apigenin on the Correa pathway in Helicobacter pylori-infected Mongolian gerbils. Five to eight-week-old Mongolian gerbils were inoculated with Helicobacter pylori for four weeks without (atrophic gastritis group) or with MNNG (gastric cancer group) in drinking water, and were then rested for two weeks. During the 7th–32th (atrophic gastritis group) or the 7th–52th (gastric cancer group) weeks, they were given various doses (0–60 mg/kgbw/day) of apigenin. At the end of the 32th (atrophic gastritis group) or the 52th (atrophic gastritis group) week, all Mongolian gerbils were sacrificed using the CO2 asphyxia method. From the results, apigenin treatment (60 mg/kgbw/day) significantly inhibited the progression of superficial gastritis, atrophic gastric, dysplasia, and gastric adenocarcinoma in Mongolian gerbils.

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apple peel fraction (Pastene et al., 2010), Aloe vera DMSO extract (Kumari et al., 2010), Japanese rice-fluid (Ishizone et al., 2007), 95% ethanol extracts of finger-root and turmeric rhizomes (Mahady et al., 2006), green tea extract (Matsubara et al., 2003), and garlic water–ethanol extract (Iimuro et al., 2002) were reported for their anti-inflammatory activity in Helicobacter pylori-infected Mongolian gerbils, guinea pigs, or albino rats. Significant reductions in the histological changes (neutrophil/monocyte infiltration, gastritis, intestinal plasia, chronic/acute inflammation, and gastric lesions) have been well discussed. However, none of these focused on the anti-gastric cancer activity induced by Helicobacter pylori. There were two clinical COX-2 inhibitors [etodolac (Magari et al., 2005) and celecoxib (Kuo et al., 2009)] reported to have both antigastritis and gastric cancer activities in Helicobacter pylori-infected Mongolian gerbils. Specifically, comparing our study0 s apigenin and quercetin (González-Segovia et al., 2008), both compounds have similar chemical structures as aforementioned, and both exhibited anti-Helicobacter pylori induced gastric inflammation in test animals. However, the treatment dosage for apigenin (60 mg/ kg) was much lower than quercetin (200 mg/kg). Moreover, antigastric inflammatory, anti-gastric cancer progression, and potent anti-gastric cancer activities were demonstrated in apigenin; contrastingly, only the anti-gastric inflammatory effect was reported in quercetin (González-Segovia et al., 2008). In conclusion, apigenin is the first natural product showing anti-gastritis, anti-gastric cancer progression, and potent anti-gastric cancer activities in Helicobacter pylori-infected test animals.

Acknowledgments This work was supported by Grants from the Excellence for Cancer Research Center, Department of Health, Executive Yuan, Taiwan, ROC. (DOH101-TD-C-111-002), and the National Science Council, Taiwan, ROC. (NSC 98-2313-B-005-017-MY3). We thank Prof. Chien-Hung Lee, Department of Public Health, Kaohsiung Medical University (Kaohsiung, Taiwan, ROC.) who kindly assisted in the statistical analysis of this study.

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Apigenin has anti-atrophic gastritis and anti-gastric cancer progression effects in Helicobacter pylori-infected Mongolian gerbils.

Apigenin, one of the most common flavonoids, is abundant in celery, parsley, chamomile, passionflower, and other vegetables and fruits. Celery is reco...
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