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Insect Tea Attenuates Hydrochloric Acid and Ethanol‑Induced Mice Acute Gastric Injury

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Insect Tea Attenuates Hydrochloric Acid and Ethanol‑Induced Mice Acute Gastric Injury EXPERIMENTAL AND THERAPEUTIC MEDICINE 14: 5135-5142, 2017 Insect tea attenuates hydrochloric acid and ethanol‑induced mice acute gastric injury XIN ZHAO1-4*, QIANG CHENG5,6*, YU QIAN3-6, RUOKUN YI3-6, LIANJIE GU1, SHANSHAN WANG1 and JIA-LE SONG1,5,6 1Chongqing Collaborative Innovation Center for Functional Food; 2Chongqing Engineering Research Center of Functional Food; 3Chongqing Engineering Laboratory for Research and Development of Functional Food; 4Department of Food Quality and Safety, Chongqing University of Education, Chongqing 400067; 5Department of Nutrition and Food Hygiene, School of Public Health; 6Institute of Preventive Medicine, Guilin Medical University, Guilin, Guangxi 541004, P.R. China Received October 10, 2016; Accepted June 22, 2017 DOI: 10.3892/etm.2017.5181 Abstract. The aim of the present study was to investigate the in ICR mice. This effect may be achieved via modulating protective effect of insect tea on HCl/ethanol-induced gastric serum neuropeptide levels, reducing gastric juice secretion, ulcers in ICR mice. The serum levels of vasoactive intestinal and modulating the inflammation‑ and antioxidant‑associated peptide, substance P, somatostatin, motilin and endothelin-1 protein expressions in gastric tissue. in mice were assessed using commercial kits and gastric tissues of superoxide dismutase (SOD, nitric oxide (NO) and Introduction malondialdehyde (MDA) were determined using western blot analysis Insect tea significantly reduced HCl/ethanol-induced Imbalances in offensive factors (including acid, pepsin, and gastric juice secretion and increased the pH of gastric juice Helicobacter pylori infection) and defensive factors (including (P<0.05). Insect tea treatment signfiicantly increased vasoac- mucin, prostaglandin, bicarbonate, nitric oxide, and growth tive intestinal peptide and somatostatin, and significantly factors) serve an important role in the pathology of chronic decreased motilin, substance P and endothelin levels in the gastric ulcers, which are associated with the development of serum (P<0.05). Treatment with insect tea was demonstrated gastric cancer (1). The prevalence of chronic gastric disease, to significantly increase levels of gastric SOD and NO and in particular gastric ulcers, is increasing (2); however, the to reduce levels of MDA in the gastric ulcer mouse model etiology is currently unclear. Lifestyle factors, including (P<0.05). The gastric expression of inhibitor of nuclear alcohol consumption, tobacco use and stress, are widely factor-κB (NF-κB), epidermal growth factor (EGF), EGF accepted as causative factors in the pathogenesis of chronic receptor, neuronal nitric oxide synthase (nNOS), endothelial gastric ulcers (2). NOS, Mn-SOD, Cu/Zn‑SOD and catalase was significantly Neuropeptides are a type of small protein-like molecule increased in mice treated with inset tea compared with that are associated with a wide range of brain functions and untreated model mice (P<0.05). Levels of NF-κB, and induc- have also been demonstrated to regulate gastric juice secre- ible NOS were demonstrated to be decreased in mice treated tion, motility and contractions (3). These neuropeptides, with insect tea compared with untreated model mice (P<0.05). including vasoactive intestinal peptide (VIP), substance P The results of the present study suggest that insect tea has a (SP), somatostatin (SS), motilin (MOT) and endothelin-1 protective effect against HCl/ethanol-induced gastric ulcers (ET-1), are widely distributed in the gastrointestinal tract and modulate the immune reaction in the intestinal mucosa (3,4). Abnormal levels of these neuropeptides have previously been observed in patients with gastrointestinal mucosal lesions and Correspondence to: Professor Jia-Le Song, Department of hemorrhage (5). Nutrition and Food Hygiene, School of Public Health, Guilin Medical At present, the primary clinical treatment for gastric ulcers University, 109 Huancheng North Road, Guilin, Guangxi 541004, is the administration of histamine-2 receptors antagonist P. R. Ch i na (including cimetidine and ranitidine) or proton-pump inhibitors E-mail: [email protected] (including lansoprazole) to reduce gastric juice secretion (6). However, long-term use of these agents may cause serious side *Contributed equally effects, including hypergastrinemia, nausea, abdominal pain, Abbreviations: VIP, vasoactive intestinal peptide; SP, substance P; constipation, diarrhea and increased risk of hip fracture (7). SS, somatostatin; MOT, motilin; ET-1, endothelin-1 Therefore, the development of more effective pharmacological agents to treat gastric ulcers with fewer side effects is of great Key words: insect tea, mice, acute gastric injury, expression importance. Insect tea, which is produced using insect feces, is a traditional drink that has long been used by ethnic minorities 5136 ZHAO et al: INSECT TEA ATTENUATES HCl/ETHANOL INDUCED GASTRIC INJURY in southwest China (8). Insect tea contains no tea leaves, (50 mg/kg/day for 28 days, Sigma-Aldrich; Merck KGaA, rather the name is derived from the color of the drink (9). A Darmstadt, Germany). All treatments were administered via number of previous studies have reported that insect tea has oral gavage. On experimental day 29, all mice had developed health benefits, including anticancer (8,10), antioxidant (11), jejunitis and were denied food but allowed to drink water hypoglycemic and antihypertensive effects (8). Insect tea has ad libitum. Mice in groups 3, 4 and 5 were gavaged with a also been reported to reduce inflammation‑induced colonic stomach injury inducer (0.1 ml HCl or ethanol per 10 g; 60% mucosa injury and promote duodenal motility activity (8). in 150 mM HCl) on day 30 and sacrificed after 1 h (12). Blood The aim of the present study was to investigate the potential samples were harvested from the inferior vena cava, centri- protective effect of insect tea on the gastrointestinal tract and fuged at 3,000 x g for 10 min at 4˚C and stored at -80˚C until to elucidate the mechanisms underlying these effects using required for further study. The animal protocol used in the HCl/ethanol-induced gastric ulcers in mice. present study was approved by the Animal Ethics Committee of Chongqing University of Education. Materials and methods Evaluation of antiulcer activity. The volume of gastric Preparation of raw tea and insect tea aqueous extracts. secretions from each mouse was measured with a 10-ml Fresh raw tea (Ilex kudingcha C. J. Tseng) and insect tea were measuring cylinder following sacrifice and the pH of gastric purchased from Guizhou Chishui Green Ecology Industry Co., juice was determined using a SevenEasy pH meter (Mettler Ltd. (Zunyi, China), freeze-dried and subsequently ground into Toledo GmbH, Greifensee, Switzerland). The stomachs were separate fine powders, the raw tea (Kuding tea) and insect tea harvested using hemostatic forceps, and the isolated stomachs was identified by Professor Huayi Suo (Southwest University, were inflated by injecting 10 ml 1% formalin solution for Chongqing, China). Powdered samples (1 kg) were added to 10 min to fix the tissues at 25˚C and opened along the greater 10 l ethanol (70%, vol/vol) and extracted at 37˚C for 2 h. Each curvature. Gastric tissue was observed using digital camera solution was filtered and condensed using a vacuum rotary (D550; Canon, Tokyo, Japan), and the gastric injury area was evaporator (1,490 x g for 10 min) (Eyela N-1100V-W; Tokyo measured using ImageJ 1.44 software (National Institutes of Rikakikai Co. Ltd, Tokyo, Japan) at 37˚C, freeze-dried and Health, Bethesda, MD, USA). The gastric injury inhibitory rate stored at -80˚C for further experiments. (%) = (1 - gastric injury area of sample treated mice / gastric injury area of control mice) x 100. Amino acid assay. A total of 800 mg of each type of tea was mixed with 3 ml sulfosalicylic acid solution (6%, w/v; Measurement of gastric neuropeptide levels. Serum levels of Shanghai Zhanyun Chemical Company Limited, Shanghai, MOT (H182; MOT assay kit), SP (H218; substance P assay kit), China), sonicated (26 kHz) for 60 min at 37˚C and cooled to VIP (H219; vasoactive intestinal peptide assay kit), SS (H092; 4˚C for 12 h. The extract was centrifuged at 13,000 x g for somatostatin assay kit) and ET-1 (H093; endothelin-1 assay kit) 2 min at 4˚C and filtered using a 0.45‑µm milliporefilter (EMD were measured with radioimmunoassay kits (all Beijing Puer Millipore, Billerica, MA, USA). The amino acids in raw tea Weiye Biotechnology Co., Ltd., Beijing, China) according to and insect tea were determined using an automatic amino the manufacturer's protocols. acid analyzer (L-8800; Hitachi, Ltd., Tokyo, Japan) equipped with a Hitachi Packed Column (2620MSC-PS; inner diameter, Measurement of superoxidase (SOD), nitric oxide (NO) and 4.6 mm; length, 80 mm) filled with a Na+-form sulfonic resin malondialdehyde (MDA) in gastric tissue. Gastric levels of (Langfang Miaoyang Chemical Company Limited, Langfang, SOD (A001-3), NO (A012) and MDA (A003-1) were respec- China). The amount of total amino acids were identified and tively measured using commercial ELISA kits (Nanjing quantified against a standard compound based on its reten- Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) tion time and peak areas. The amino acid standards were according to the manufacturer's protocols. purchased from National Institute for Food and Drug Control (Beijing, China). Protein extraction and western blot analysis. A total of 100 mg gastric tissue was washed with ice-cold PBS, homogenized Animal groups and acute gastric ulcer induction. A total with ice‑cold modified radioimmunoprecipitation assay buffer of 35 male ICR mice (6 weeks old, 25-30 g) were purchased (Sigma-Aldrich; Merck KGaA) and centrifuged at 13,000 x g from the Experimental Animal Center of Chongqing Medical for 30 min at 4˚C. Protein concentrations were determined University (Chongqing, China). Mice were housed with a stan- using a bicinchoninic acid protein assay kit (Bio-Rad dard 12-h light/dark cycle at 23±1˚C with relative humidity Laboratories, Inc., Hercules, CA, USA). For western blot anal- 50±5% and had ad libitum access to food and water.
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