Effect of Areca Nut on Helicobacter pylori-Induced Gastric Diseases in Mice

Abstract

Areca nut (AN) chewing is a habit in many countries in Central, Southern, and Southeast Asia. It is strongly associated with the occurrence of oral, pharyngeal, and esophageal cancer as well as systemic inflammation. However, the association between AN intake and the development of gastric lesions has not yet been identified. The aim of this study was to investigate the effect of AN on gastric diseases using a mouse model for Helicobacter pylori infection. We studied four groups of mice: those fed a normal diet (ND), those fed a diet containing 2.5% AN (AD), those fed ND and infected with H. pylori PMSS1 strain (ND/HP), and those fed AD and infected with H. pylori PMSS1 strain (AD/HP). Food intake and body weight were monitored weekly during the experiments. At 10 weeks, the mice were sacrificed, and the stomach weight, H. pylori colonization, and gastric inflammation were evaluated. The stomach weight had increased significantly in the ND/HP and AD/HP groups along with increases in H. pylori colonization; however, there was no significant difference between these two groups with respect to stomach weight and colonization. On histological grading, mononuclear cell infiltration was severer in the AD/HP group than in the ND/HP group. These data suggest that chronic gastric inflammation was aggravated by AN treatment in the mice with H. pylori-induced gastric lesions. Furthermore, as previously suggested, this animal model is useful to determine the effect of potential carcinogens on gastric lesions induced by H. pylori infection.

Introduction

Areca nut (AN) is the fruit seed of the palm tree Areca catechu, which is often mixed with betel quid and used as a masticatory substance by approximately 600 million people worldwide [14]. AN is consumed by people of all age groups, and chewing AN is a popular habit in Asian and Oceanic countries. It is considered to be commonly used as a psychoactive substance, such as tobacco, alcohol, and caffeine [24], and its constituents include carbohydrate, fat, protein, crude fiber, polyphenols (such as flavonol and tannin), alkaloid, and mineral matter [21]. Systemic effects of AN are mainly due to its principal alkaloid, arecoline [12]. Previous studies have shown that AN chewers have a higher prevalence of periodontal diseases and of oral, pharyngeal and esophageal cancers [16]. Recently, it has also been reported that chewing AN may increase the risk of systemic inflammation [28].

Helicobacter pylori is a microaerophilic gram-negative spiral-shaped bacterium found in the human stomach. H. pylori is the major causative agent in chronic gastritis and peptic ulcer disease as well as a cause of gastric cancers [17]. An association between H. pylori infection and the development of gastric disease is also found in studies using animal models [2,10,11,22]. Among these models, the mouse model using H. pylori strain PMSS1 is widely used [2,20,27]. Fernando et al. [8] suggested that chewing betel quid that contains AN and lime, with or without tobacco, may predispose persons infected with H. pylori to the development of severe gastric diseases, including gastric cancers, as a result of chewing and swallowing this substance. Thus, to identify an association between AN intake and H. pylori-induced gastric disease, we investigated the effects of AN on gastric lesions and on bacterial colonization in mice infected with the H. pylori PMSS1 strain.

 

Materials and Methods

Diet Preparation

AN was provided by the University of Peradeniya, Sri Lanka. Dried AN was finely powdered and mixed with a normal diet AIN76A (Research Diets, USA) at a concentration of 2.5% (w/w) in Gwangju Institute of Science and Technology, Korea. The diet containing AN was pelleted, sealed, and stored at 4℃ until use.

Animal Experiments

We purchased 5-week-old male C57BL/6 mice from Orient Bio Inc. (Korea). The animals were maintained in a temperature-controlled room at 22℃, with a 12 h light-dark cycle. Food and water were provided ad libitum. After 1 week of acclimation, 34 mice were divided into four groups: those fed a normal diet (the ND group) (n = 6), those fed a diet containing 2.5% AN (the AD group) (n = 8), those fed a normal diet and infected with H. pylori PMSS1 strain (the ND/HP group) (n = 10), and those fed a diet containing 2.5% AN and infected with H. pylori PMSS1 strain (the AD/HP group) (n = 10) (Fig. 1).

Fig. 1.Experimental design. Mice in the ND group were inoculated with Brucella broth (▽) and fed a normal diet (□). The AD group of mice was inoculated with Brucella broth and fed a diet containing 2.5% AN (■). The ND/HP group was inoculated with H. pylori strain PMSS1 (▼) and fed a normal diet. The AD/HP group was inoculated with H. pylori strain PMSS1 and fed a diet containing 2.5% AN.

At 6 weeks after intragastric inoculation with H. pylori PMSS1 strain, the mice in the ND/HP and AD/HP groups were fed the AIN76A diet and the AIN76A diet containing 2.5% AN, respectively, for 10 weeks. As a negative control, ND mice and AD mice that received Brucella broth were fed the AIN76A diet. The body weight and food intake were measured every week during the experimental period. At the end of this period, the mice were sacrificed, gastric mucosal tissues were weighed and examined histologically, and H. pylori colonization was determined, as described below. The animal experiments were performed in accordance with institutional guidelines, and protocols were approved by the animal ethics committee of the Yonsei University College of Dentistry in Seoul, Korea (2012-0076).

H. pylori Culture and Infection of Mice

The H. pylori PMSS1 strain was stored as frozen stock at -80℃ in brain–heart infusion medium supplemented with 20% glycerol and 10% fetal bovine serum (FBS; Gibco-BRL, USA). H. pylori was grown on horse blood agar plates containing 4% Columbia blood agar base (Oxoid, Basingstoke, UK), 5% defibrinated horse blood (Hemostat Laboratories, Dixon, CA, USA), 0.2% β-cyclodextrin, 10 μg/ml of vancomycin, 2.5 U/ml of polymyxin B, 5 μg/ml of trimethoprim, and 8 μg/ml of amphotericin B at 37℃ under microaerobic conditions. A microaerobic atmosphere was generated using a CampyGen sachet (Oxoid) in a GasPak jar (BD, USA). For liquid culture, H. pylori was grown in Brucella broth (Difco & BBL Diagnostics, USA) containing 10% FBS and 10 μg/ml of vancomycin. Cultures were shaken in a microaerobic environment. According to the growth curve, 2 × 107 bacteria were collected and resuspended in 250 μl of Brucella broth, which was introduced via intragastric gavage to produce infection after the mice had fasted for 24 h.

Determining the Number of Viable H. pylori in the Stomach

The number of viable H. pylori colonies in the stomachs of the mice was determined as previously described, with minor modifications [29]. After the mice had fasted for 18 h, they were euthanized, and their stomachs were excised. The stomach was dissected along the greater curvature, the forestomach was removed, and the stomachs were washed with 0.01 M of phosphate-buffered saline (pH 7.4) and then divided into two halves longitudinally. One half of each stomach was homogenized in 1 ml of Brucella broth using a homogenizer (Kinematica, Switzerland). The diluted homogenates were applied to horse blood agar plates used to culture H. pylori and were supplemented with 100 μg/ml of vancomycin, 100 μg/ml of bacitracin, and 10 μg/ml of nalidixic acid. The plates were incubated at 37℃ under microaerobic conditions for 5 days. The colonies were counted, and the number of viable H. pylori was expressed as colony-forming units (CFU) per gram of stomach tissue.

Histological Analysis

The remaining half of each stomach was fixed in 10% neutral buffered formalin and embedded in paraffin. The paraffin was cut into 4 μm sections and stained with hematoxylin and eosin for morphological observation. Two aspects of gastric lesions were recorded according to the updated Sydney system: the infiltration of polymorphonuclear leukocytes (PMNs) and chronic inflammation based on mononuclear cell infiltration. Morphological features of the gastric antrum and corpus were graded on a scale of 0 to 4: Grade 0, normal; Grade 1, slight; Grade 2, mild; Grade 3, moderate; and Grade 4, severe [7]. Microscopic images were obtained at magnifications of ×100 for the corpus and ×200 for the antrum.

Statistical Analysis

The SPSS statistics ver. 23 program (IBM, USA) was used for all statistical analyses. Analysis of variance was used to compare different groups, and multiple comparisons were performed using Scheffé’s method. A p-value of <0.05 was regarded as statistically significant.

 

Results

Gross Changes in Food Intake and Body Weight of Mice

We measured the food intake and body weight of the mice over 10 weeks to determine whether feeding AN caused any gross changes in these values. During this period, food intake was similar in all four groups of mice (Fig. 2A). The body weight increased in the AD group as compared with the ND group (Fig. 2B). Thus, importantly, the ND/HP and AD/HP groups showed no difference in either food intake or weight gain.

Fig. 2.Gross changes in food intake and body weight in mice during the experimental period. (A) Food intake change. (B) Body weight change. Data are presented as the means ± SD. * indicates a significant difference at p < 0.05.

Effect of AN on Viable H. pylori Colonization in the Mouse Stomach

To determine the effect of AN on H. pylori colonization in the gastric mucosa of mice, the number of H. pylori colonies was measured at 10 weeks after the mice had been fed the normal or AN-containing diet. Obviously, the ND/HP and AD/HP groups infected with H. pylori showed a significant increase in H. pylori colonization as compared with the ND and AD groups without H. pylori infection (Fig. 3A). However, there was no significant difference between the ND/HP and AD/HP groups infected with H. pylori, suggesting that AN treatment had no effect on H. pylori colonization.

Fig. 3.Viable number of H. pylori colonies in the gastric mucosa, and the ratio of stomach weight to body weight in the H. pylori-infected mice. (A) Viable H. pylori colonization was evaluated by means of a whole stomach culture system. (B) Stomachs were weighed at 10 weeks after the mice were fed a normal or AN-containing diet, and results were expressed as the ratio of stomach weight to body weight in the H. pylori-infected mice. * indicates a significant difference at p < 0.05.

Effect of AN on Stomach Weight of H. pylori-Infected Mice

To determine the effect of AN on the stomach weight of the mice infected with H. pylori, the stomachs were weighed at 10 weeks after the mice had been fed either the normal or the AN-containing diet, with results expressed as the ratio of stomach weight to body weight. The stomach weight in the ND/HP and AD/HP groups infected with H. pylori was significantly increased (Fig. 3B); however, there was no significant difference between these two groups, suggesting that AN treatment did not affect gross stomach weight.

Effect of AN on Gastric Lesions of H. pylori-Infected Mice

To determine whether AN affects H. pylori-induced gastric lesions, we observed the histopathological changes (i.e., infiltration of PMNs and mononuclear cells) in the corpus and antrum (Figs. 4A and 4B). Although PMN infiltration was similar in all four groups of mice, mononuclear cell infiltration was significantly increased in the ND/HP and AD/HP groups as compared with the mice in non-infected groups (Figs. 4C and 4D). More importantly, the mononuclear cell infiltration was more severe in the AD/HP group than in the ND/HP group (Fig. 4D). In addition, it is worth noting that no hyperplastic or metaplastic lesions were observed in any group throughout the experimental period.

Fig. 4.Effect of areca nut on H. pylori-induced gastric lesions in mice. (A) The corpus part of the gastric mucosa was stained with hematoxylin and eosin, and microscopic images were obtained (magnification of ×100). (B) The antrum part of the gastric mucosa was stained with hematoxylin and eosin, and microscopic images were obtained (magnification ×200). (C, D) Histological grading of the PMN infiltration (C) and of lymphocyte infiltration (D) in the gastric mucosa. Inflammatory responses of the gastric mucosa were graded according to morphological criteria. * indicates a significant difference at p < 0.05.

 

Discussion

The distinctive characteristic of H. pylori infection is chronic gastric inflammation [26]. Various animal models have been employed to investigate human H. pylori infection. Recently, it was reported that certain H. pylori strains were able to colonize the gastric mucosa of mice [23] and of Mongolian gerbils [30]. Of the several animal models infected with H. pylori, mice appear to have mild responses in terms of gastric inflammation [9]. The commonly used mouse-adapted strain is H. pylori SS1, which does not translocate cytotoxin-associated gene A (CagA), a virulence factor, into the host cells or induce interleukin (IL)-8 [6]. Since the presence of the functional CagA in H. pylori strains is associated with more severe gastric inflammation and the development of neoplastic gastric disease [4,26], the H. pylori PMSS1 strain with a functional CagA in vivo system has been employed for the mouse model [2].

In this mouse study, this PMSS1 strain induced moderate chronic inflammation in the animals’ gastric mucosa. This finding suggests that the mice infected with the H. pylori PMSS1 strain probably mimic the human’s asymptomatic response to CagA-positive H. pylori infection. Furthermore, the pre-neoplastic lesions such as glandular hyperplasia and metaplasia were not observed during the period of H. pylori infection. To develop pre-neoplastic lesions, it might be necessary for a more long-term infection or infection combined with exposure to carcinogens such as AN.

Among the mice without H. pylori infection, those fed AD showed no inflammatory changes as compared with the control ND group during the experimental period. In contrast, in the presence of H. pylori infection, the mice fed AD showed more severe lymphocyte infiltration than did the ND/HP group fed a normal diet in the presence of H. pylori infection. Previous studies have reported that AN extracts increase the expression of inflammatory cytokines such as tumor necrosis factor-α, IL-1β, IL-6, and IL-8 [5], and H. pylori induces the increased production of proinflammatory cytokines [13,15]. Combined with these findings, our data suggest that AN treatment aggravates H. pylori-induced gastritis by inducing the increased expression of inflammatory cytokines in the gastric mucosa.

Considering that fewer than 10% of patients with H. pylori infection have symptomatic gastric disease, it can be assumed that AN may affect disease progression from asymptomatic infection to severe gastric disease. Because H. pylori-induced gastritis has been reported to progress to gastric cancer [25], the intake of AN may affect gastric carcinogenesis induced by H. pylori infection. Since there was no significant difference in the extent of H. pylori bacterial colonization between groups ND/HP and AD/HP, the more severe chronic gastric inflammation with more lymphocyte infiltration in AD/HP was most likely aggravated by the effect of AN itself and not by the differential colonization of H. pylori.

Recently, Ji et al. [18] reported that AN induced DNA damage and expression of inflammatory cytokines such as IL-6 and IL-8 in oral cancer cells, and these effects were more prominent on cells cultured in low serum condition (1% FBS) or serum-free condition than cells cultured in 10% FBS condition. In addition, it was shown that H. pylori infection reduced the gastric mucosal blood flow in animal models [3,19] and humans [1], which may resemble the low serum condition. This would be a potential mechanism for the exacerbating gastric inflammation induced by H. pylori infection.

Further studies will be needed to determine the main pathway related to the progression of gastric inflammation with AN intake. In addition, this animal model is useful for the experiments to determine the effect of potential carcinogens on H. pylori-induced gastric lesions. In conclusion, AN induced severer chronic gastritis infection with the H. pylori PMSS1 strain, and the chewing of betel quid containing AN may predispose those infected with H. pylori to severe gastric diseases, including gastric cancers.