Journal of Ethnopharmacology 160 (2015) 211–218

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Research Paper

Comparison of the anti-ulcer activity between the crude and bran-processed Atractylodes lancea in the rat model of gastric ulcer induced by acetic acid Yan Yu a,b, Tian-Zhu Jia b,n, Qian Cai b,n, Ning Jiang c, Ming-yue Ma b, Dong-yu Min a, Yuan Yuan d a

Experimental Center of Traditional Chinese Medicine, The Affiliated Hospital, Liaoning University of Traditional Chinese Medicine, Shenyang 110847, China School of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian 116600, China Laboratory of Molecular Biology, School of Basic Medical Science, Liaoning University of Traditional Chinese Medicine, Shenyang 110847, China d Institute of Traditional Chinese Medicine of Liaoning Province, Shenyang 110034, China b c

art ic l e i nf o

a b s t r a c t

Article history: Received 13 June 2014 Received in revised form 20 September 2014 Accepted 13 October 2014 Available online 4 December 2014

Ethnopharmacological relevance: The rhizome of Atractylodes lancea (AL, Compositae, Chinese name: Cangzhu; Japanese name: Sou-ju-tsu) has been used traditionally for the treatment of various diseases such as digestive disorders, rheumatic diseases, and influenza in China, Korea and Japan. The crude AL and AL bran-processed are both listed in the Chinese Pharmacopoeia. However, the differences between the effects of the crude and AL bran-processed on gastric ulcer were poorly understood, and the mechanisms for the treatment of gastric ulcer were not clear. This study aimed at comparing the antiulcer effects between the crude AL and AL processed in acetic acid induced model in rats and evaluating the mechanisms of action involved in the anti-ulcer properties of AL. Materials and methods: The model of gastric ulcer was imitated by acetic acid in rats, and AL was gavaged. The serum and gastric tissues were collected. The levels of epidermal growth factor (EGF), trefoil factor2 (TFF2), tumor necrosis factor-α (TNF-α), interleukin 6, 8 (IL-6, 8) and prostaglandin E2 (PGE2) in serum and gastric tissues were determined by the double-antibody sandwich enzyme-linked immunosorbent assay (ELISA), and the mRNA expressions of EGF, TFF2, TNF-α, and IL-8 in stomach were analyzed by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Meanwhile, histopathological changes were evaluated by hematoxylin and eosin (HE) stain. The protein expressions of EGF, TFF2, TNF-α, and IL-8 were examined by immunohistochemistry in stomach. Results: The results demonstrated that the damage of gastric tissue was obviously alleviated and the productions of TNF-α, IL-8, IL-6, and PGE2 and the mRNA expressions of TNF-α, and IL-8 were notably inhibited. Furthermore, the productions of EGF and TFF2 and the mRNA expressions of EGF and TFF2 were significantly stimulated by both crude AL and AL processed in a dose-dependent manner. Compared with the crude AL, the processed AL was more effective. Conclusion: The AL processed had more satisfactory effects in treatment of gastric-ulcer than the crude AL. The anti-ulcer effects of AL could be attributed to the anti-inflammatory properties via downregulating TNF-α, IL-8, IL-6 and PGE2 and to the gastroprotective effects via up-regulating EGF and TFF2. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Atractylodes lancea (AL) Processing Compositae Traditional Chinese medicine (TCM) Gastric ulcer Mechanism

1. Introduction

Abbreviations: AL, the Atractylodes lancea; EGF, epidermal growth factor; TFF2, trefoil factor2; TNF-α, tumor necrosis factor-α; IL, interleukin; PGE2, prostaglandin E2; ELISA, the enzyme-linked immunosorbent assay; RT-PCR, reverse transcription polymerase chain reaction; HE, hematoxylin and eosin; mRNA, messenger ribonucleic acid; TCM, traditional Chinese medicine n Corresponding authors. Tel.: þ 86 18909816763; fax: þ86 24 31961931. E-mail addresses: [email protected] (T.-Z. Jia), [email protected] (Q. Cai). http://dx.doi.org/10.1016/j.jep.2014.10.066 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

Gastric ulcer is one of the major gastrointestinal disorders. Its incidence increases due to rapid development and civilizational constraints. Although the exact etiology of the disease is unknown, an imbalance between defensive and aggressive factors results in peptic ulcer (Bose et al., 2003; Laine et al., 2008; Klein et al., 2010). The Atractylodes lancea rhizome (Compositae) is known to be widely distributed in China. The dried rhizome, which is listed in the Chinese, Korean, and Japanese pharmacopoeias (Kitajima et al., 2003), has been used for stomach disorders as well as for diuretic

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and analgesic purposes in traditional Japanese-Sino medicine, and used as a traditional remedy against digestive disorders, rheumatic diseases, night blindness, and influenza in China (Resch et al., 2001; Nakai et al., 2003). According to the traditional Chinese classics, like Compendium of Materia Medica (Ben Cao Gang Mu) or Treatise on Cold-induced and Miscellaneous Diseases (Shang Han Za Bing Lun), processing (pao zhi) played an important role in preparing Chinese medicines because the herbal properties were changed by different processing methods. Stir-frying with wheat bran was one of the common processing methods. In the Chinese Pharmacopoeia, the crude AL and AL bran-processed are both listed. As we knew, there was little information available in the literature about comparing anti-ulcer activities of the crude AL and AL processed and evaluating the mechanisms involved. In this study, we focused on determining variations in the anti-ulcer effects of the crude AL and AL processed by stir-frying with bran in acetic acid induced model of gastric ulcer in rats and evaluating the mechanisms of action involved in the anti-ulcer properties of AL.

2. Material and methods 2.1. Plant materials The rhizomes of the crude AL, cultivated in Zhenjiang City, Jiangsu Province in China, were purchased in the traditional Chinese medicine market, Bozhou City, Anhui Province, China, in April, 2013, which were identified as the rhizome of Atractylodes lancea (Thunb.) DC. by Prof. Yanjun Zhai, working in School of Pharmacy, Liaoning University of Traditional Chinese Medicine (TCM). The AL processed was prepared by stir-frying with bran according to the procedure recorded in the Pharmacopoeia of Peoples Republic of China (2010). Briefly, 30 g wheat bran was put into a heated pot which was hot enough to make the bran smoke immediately. 200 g rhizomes of Atractylodes lancea were thrown into the pot as soon as possible, and then stir-fried quickly for about 1 min until the surface is dark yellow. The rhizomes were removed and cooled down while the bran was sifted out. Voucher specimens were deposited at the experimental center of TCM, the Affiliated Hospital of Liaoning University of TCM, Shenyang, People's Republic of China. The rhizomes of AL were crushed into fine powder. The powder was suspended in normal saline in three different concentrations for use.

2.2. Chemicals Omeprazole was purchased from Jilin Province Dongbeiya Pharmaceutical Co., Ltd., (Jilin, China) (Drug approval number: H20057570). Sanjiu Weitai granule (a Chinese patent drug for the treatment of gastric ulcer in clinical practice) was purchased from China Resources Sanjiu Medical & Pharmaceutical Co., Ltd. (Shenzhen, China) (Drug approval number: Z44020705). They were used as positive control drugs, ground into powder and suspended in normal saline before use. Rat ELISA Kits were obtained from R&D Techno Co. (MN, USA). PrimeScripts RT reagent Kits with gDNA Eraser for real-time PCR were provided by TaKaRa Biotech Co. (Dalian, China). Brilliant III Ultra-Fast SYBRs Green QPCR Master Mix was provided by Agilent Technologies (USA). Primers for target genes were synthesized by Bejing Genomics Ins. (Beijing, China). Trizol Reagent was purchased from Invitrogen Corporation (CA, USA). Immunohistochemical kits were purchased from Zymed (CA, USA).

2.3. Animals Eight-week-old Sprague-Dawley rats of both sexes (SPF grade), weighing 220–240 g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (SCXK 2010-0001, Liaoning, China), housed for 1 week under normal laboratory conditions at 2471 1C and 5575% relative humidity and maintained on a standard laboratory diet and tapwater ad libitum. The rats were deprived of food with access to water ad libitum for 24 h and kept in cages with raised floors of wide mesh to prevent coprophagy before the experiments. Rats were treated according to the European Community Guidelines for Animal Experimentation. All animal studies were approved by the Ethic Committee for Animal Research of the Affiliated Hospital of Liaoning University of TCM. 2.4. Acetic acid-induced gastric ulcer and drug administration After one week's habituation, all rats were randomly divided into the following 10 groups each consisting of 8 animals (4 female and 4 male): (1) sham-operated group (SO), (2) model group (MD), (3) omeprazole group (OM, omeprazole at the dose of 13 mg/kg), (4) Sanjiu Weitai granule (SJ, Sanjiu Weitai granule at the dose of 2.08 g/kg), (5) low dose group of the crude AL (LC, the crude AL at the dose of 0.625 g/kg), (6) middle dose group of the crude AL (MC, the crude AL at the dose of 1.25 g /kg), (7) high dose group of the crude AL (HC, the crude AL at the dose of 2.5 g/kg), (8) low dose group of the processed AL (LP, the processed AL at the dose of 0.625 g/kg), (9) middle dose group of the processed AL (MP, the processed AL at the dose of 1.25 g/kg) and (10) high dose group of the processed AL (HP, the processed AL at the dose of 2.5 g/kg). Gastric ulcer was induced by acetic acid treatment in rats according to the method of Okabe et al. (1971) and Okabe and Pfeiffer (1972) with some modifications. After 24 h of food deprivation and being prevented coprophagy, the rats were anaesthetized with 10% chloral hydrate (4 ml/kg body wt., i.m.). A laparotomy was performed through a left subcostal incision. The stomach was gently exteriorized and clamped 3 mm away from pylorus with gastric ulcer preparation tweezers (a pair of tweezers with two rings 9 mm in diameter, patent number: 2009200151947, invented by Laboratory of Molecular Biology, School of Basic Medical Science, Liaoning University of TCM, Shenyang, China). The 0.18 ml mineral oil (to protect the other side of the stomach wall and avoid adherence to the ulcerated region) and 0.02 ml 60% acetic acid (v/v) in the same syringe were injected into the subserosal layer in the glandular part of the anterior wall in the clamping region and allowed to remain for 45 s. The solution was aspirated off and washed with normal saline to avoid adherence to the external surface of the ulcerated region. The opened abdomen was then closed and the rats were fed normally. A sham control group underwent the surgical procedure of ulcer induction with the application of saline instead of acetic acid. Rats, except in SO and MD groups, were administered positive control drugs or AL above-mentioned via intragastric (ig) two times each day, 10 ml/kg per time respectively for 10 consecutive days, beginning on the 2nd day after the operation, while rats in SO and MD groups were only administered the same amount of normal saline. On the 12th day after the operation, the animals were anaesthetized with 10% chloral hydrate (4 ml/kg body wt., i.p.) and the abdomens were opened. Blood was collected from the abdominal aorta of rats and allowed to settle for 2 h at room temperature, and then centrifuged at 3000 rpm for 15 min. The supernatant clear serum used for ELISA assay was then stored at  70 1C until the measurement. The stomachs were removed and cut open along the greater curvature and washed in ice-cold saline. The ulcer tissue was cut into three parts. One part was immediately fixed

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with a 4% paraformaldehyde solution for pathological analysis and immunohistochemical examination. One part was kept in trizol for mRNA level assays, while another part was used for ELISA assays and kept in a  70 1C freezer until analysis.

2.5. Assay for serum and stomachs levels of EGF, TFF2, PGE2, TNF-α, IL-6 and IL-8 Serum and the gastric tissues were thawed at 4 1C before use. The gastric tissues, weighing 100 mg, were homogenized in 900 μl PBS and centrifuged for 20 min at 3000 rpm. Supernatants were transferred to other tubes. The serum and stomachs levels of EGF, TFF2, PGE2, TNF-α, IL-6 and IL-8 were measured by commercially available double-antibody sandwich ELISA kits according to the manufacturer's instructions in an automated ELISA reader (Infinite M200, TECAN, Austria) at 450 nm.

2.6. The mRNA expressions of EGF, TFF2, TNF-α and IL-8 in the rat gastric tissues determined by quantitative real time reverse transcriptase-polymerase chain reaction (RT-PCR) 2.6.1. Total RNA isolation The mRNA expressions of EGF, TFF2, TNF-α and IL-8 were determined by real-time RT-PCR in the gastric tissues. Samples of gastric tissues, weighing about 100 mg, were homogenized in 1 ml trizol. After precipitation at 12,000g for 10 min at 4 1C, total RNA was isolated utilizing a Trizol total extraction kit based on phenol/chloroform extraction and isopropanol precipitation according to the manufacturer's instructions. The total RNA concentrations were estimated by absorbance at 260 nm, and the qualities of the isolated RNA were assessed by using the 260/280 nm absorbance ratio (1.8–2.0 indicates a highly pure sample) (Pan et al., 2002).

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2.7. Pathological observe in gastric tissue Gastric tissues were fixed in 4% paraformaldehyde solution. After fixation, the tissue samples were dehydrated with alcohol and xylene, and embedded in paraffin. Sections of 5 mm thickness were affixed to slides, deparaffinized, stained with hematoxylin and eosin (HE) for general histopathology examination under a light microscope (BS-51,Olympus, Tokyo, Japan), and assessed by an experienced histologist, who was unaware of the experimental groups.

2.8. Immunohistochemical staining for EGF, TFF2, TNF-α and IL-8 The gastric tissue paraffin sections (5 μm) were dewaxed in xylene and hydrated through standard graded ethanol solutions. The procedure was conducted according to the kit instructions. Sections were rinsed 2 times for 5 min each in PBS, incubated in 3% H2O2 solution for 10 min to inactivate endogenous peroxides and then heated in citric acid sodium solution in a microwave oven at 100 1C to retrieve antigen for 10 min. The sections were incubated with 10% normal goat serum for 20 min and then with rabbit anti-EGF, -TFF2, -TNF-α or -IL-8 (1:100) at 4 1C overnight. Subsequently, slides were rinsed in PBS and incubated in the biotin-labeled corresponding secondary antibody at 37 1C for 15 min. After washing, the sections were then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with diaminobenzidine (DAB) as substrate. In addition, a semi-quantitative analysis was conducted for the immunohistochemistry images. For each image, 10 visual fields were randomly selected, and the Olympus image pick up system and JD801 mode microscopic image analysis system were used to determine the optical density value.

2.9. Statistical analysis 2.6.2. Synthesis of cDNA and polymerase chain reaction 0.5 μg RNA was reversely transcribed to cDNA using the PrimeScripts RT reagent Kit with gDNA Eraser according to the supplier's instructions (37 1C 15 min, 85 1C 5 s). DNA amplification was carried out by using a Brilliant Ultra-Fast SYBR Green QPCR Master Mix kit under the following conditions: After initial denaturation for 3 min at 95 1C, 40 amplification cycles (denaturation for 5 s at 95 1C, annealing for 1 min at 60 1C) were performed in a Stratagene Mx3000p PCR System (Agilent, German). The primers for the sequences of the genes are shown in Table 1. Melting curves were used to verify the specificity of the PCR products. Data were analyzed by using the comparative cycle threshold (CT) method as means of relative quantitation with normalized to an endogenous reference (β-actin) (Livak and Schmittgen, 2001).

All statistical calculations were performed using SPSS 10.0 software (SPSS, Chicago, IL, USA). Results were expressed as mean 7standard deviation (S.D.). Comparisons between groups were made by one-way analysis of variance (ANOVA) followed by Fisher's protected LSD multiple comparison test or Tamhane's T2 test. Differences with P o0.05 were considered as statistically significant.

Table 1 Informations of PCR primers.

After treatment with the crude AL and AL processed, EGF, TFF2, PGE2, TNF-α, IL-6 and IL-8 in serum and stomachs were detected by ELISA. As shown in Tables 2 and 3, compared with SO group, higher levels of TNF-α, IL-6, IL-8, and PGE2 and lower levels of EGF, and TFF2 in serum and stomachs of MD rats were found (Po0.05). The crude AL and AL processed significantly decreased the levels of TNF-α, IL-6, IL-8 and PGE2 (P o0.05 in MC, HC, MP and HP groups) and increased the levels of EGF (P o0.05 in MC, HC, MP and HP groups), TFF2 (Po0.05 in MC, HC, MP and HP groups) in a dosedependent manner. Again, the difference of MC versus MP or HC versus HP was significant (P o0.05). The AL processed showed more remarkable EGF-, TFF2-, PGE2-, TNF-α-, IL-6- and IL-8regulation effects than the crude AL.

Gene symbol GenBank accession Sequence TFF-2

NM_053844

EGF

NM_012842

IL-8

NM_011339

TNF-α

NM_012675

β-actin

NM_031144

Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

50 -CACTTCCAAACCAAGCGTCG-30 50 -CAGCAGTGCCCTTCAGTAGT-30 50 -ATCAAGCACGGCACGATTTG-30 50 -GCATGTAGGTGCACGACTCT-30 50 -CTAGGCATCTTCGTCCGTCC-30 50 -TTGGGCCAACAGTAGCCTTC-30 50 –ACGTCGTAGCAAACCACCAA-30 50 -AAATGGCAAATCGGCTGACG-30 50 -CGCGAGTACAACCTTCTTGC-30 50 -CGTCATCCATGGCGAACTGG-30

3. Results 3.1. Effects of AL on the levels of EGF, TFF2, PGE2, TNF-α, IL-6 and IL8 in serum and stomachs

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Table 2 Levels of TFF2, EGF, PGE2, IL-6, IL-8 and TNF-α in serum. groups

TFF2 (pmol/L)

EGF (ng/mL)

PGE2 (ng/L)

IL-6 (ng/L)

IL-8 (ng/L)

TNF-α (ng/L)

SO MD OM SJ LC MC HC LP MP HP

1103.75 7 65.46n 160.177 26.86 934.81 7 78.85n 848.557 85.63n 184.127 9.67 255.747 29.11n 615.177 54.57n 248.007 34.64n 630.747 39.47n 772.98 7 77.53n

14.197 1.85n 2.447 0.24 14.60 7 0.37n 7.90 7 0.55n 3.05 7 0.70 4.217 0.88n 5.577 0.74n 4.63 7 0.50n 8.127 1.03n 9.337 1.09n

47.30 7 5.37n 244.637 26.00 128.81 7 5.01n 111.62 7 15.39n 218.707 15.27 156.83 7 3.85n 153.977 4.26n 220.45 7 11.16 90.50 7 3.09n 89.707 6.94n

15.81 7 1.27n 77.36 7 7.30 23.29 7 1.93n 24.517 1.56n 76.197 3.61 48. 727 4.45 n 46.43 7 3.91n 67.497 5.81 34.737 1.90n 32.86 7 2.07n

67.197 5.07n 308.25 7 9.18 87.197 8.03n 103.09 7 6.09n 273.60 7 19.18n 194.75 7 12.09n 185.647 6.85n 271.98 7 9.92n 164.597 13.91n 149.687 10.62n

25.7074.32n 110.92 74.75 43.19 75.13n 47.69 76.74n 109.22 76.68 77.58 77.67n 73.6777.56n 99.08 77.33n 49.95 74.93n 44.11 76.35n

Levels of TFF2, EGF, PGE2, IL-6, IL-8 and TNF-α in serum of rats in SO (sham-operated group), MD (model group), OM (omeprazole group, 13 mg/kg), SJ (Sanjiu Weitai granule, 2.08 g/kg), LC (low dose group of the crude AL, 0.625 g/kg), MC (middle dose group of the crude AL, 1.25 g/kg), HC (high dose group of the crude AL, 2.5 g/kg), LP (low dose group of the processed AL, 0.625 g/kg), MP (middle dose group of the processed AL, 1.25 g/kg) and HP (high dose group of the processed AL, 2.5 g/kg) were assayed by ELISA kits as described in the text. Values were expressed as mean 7 SD (n ¼8). n

Indicates a significant difference compared with the model group (P o 0.05).

Table 3 Levels of TFF2, EGF, PGE2, IL-6, IL-8 and TNF-α in stomachs. groups SO MD OM SJ LC MC HC LP MP HP

TFF2 (pmol/L)

EGF (ng/mL) n

1153.157 186.00 217.53 7 27.84 911.487 114.14n 830.28 7 137.03n 241.007 36.85 425.58 7 67.33n 650.147 89.11n 364.117 71.13n 652.417 67.06n 844.737 79.11n

n

14.51 70.84 2.34 70.36 14.21 70.64n 7.44 70.75n 2.43 70.58 3.62 70.87n 5.03 71.17n 4.37 70.93n 7.59 71.01n 8.83 71.15n

PGE2 (ng/L)

IL-6 (ng/L) n

59.86 74.99 255.30 732.72 141.58 715.30n 123.88 716.44n 227.49 713.34 168.47 710.47n 160.09 711.65n 224.86720.19 92.26 711.66n 85.76 76.19n

IL-8 (ng/L) n

10.95 7 0.79 58.187 5.19 16.79 7 3.96n 20.117 5.01n 64.227 12.83 44.127 3.70n 39.87 7 5.37n 48.787 7.50 27.047 4.41n 21.28 7 1.43n

TNF-α (ng/L) n

106.17 73.81 419.93 745.33 143.58 79.25n 148.76 77.81n 444.79 757.05 255.69 710.48n 245.92 77.38n 363.21 726.51 199.53 76.29n 188.7776.17n

33.677 4.09n 116.337 10.72 54.007 7.74n 59.84 7 9.00n 112.85 7 18.68 82.247 13.24n 71.977 9.73n 102.747 13.55 51.167 10.42n 40.84 7 16.00n

Levels of TFF2, EGF, PGE2, IL-6, IL-8 and TNF-α in stomachs of rats in SO (sham-operated group), MD (model group), OM (omeprazole group, 13 mg/kg), SJ (Sanjiu Weitai granule, 2.08 g/kg), LC (low dose group of the crude AL, 0.625 g/kg), MC (middle dose group of the crude AL, 1.25 g/kg), HC (high dose group of the crude AL, 2.5 g/kg), LP (low dose group of processed AL, 0.625 g/kg), MP (middle dose group of processed AL, 1.25 g/kg) and HP (high dose group of processed AL, 2.5 g/kg) were assayed by ELISA kits as described in the text. Values were expressed as mean 7 SD (n¼ 8). n

Indicates a significant difference compared with model group (Po 0.05).

3.2. Effects of AL on the mRNA expression of EGF, TFF2, TNF-α and IL-8 in the gastric tissues

3.4. Effects of AL on the expression of EGF, TFF2, TNF-α and IL-8 in gastric tissues by immunohistochemical staining

A dissociation curve analysis of EGF, TFF2, TNF-α, IL-8 or β-actin showed a single peak. The 260/280 nm absorbance ratios were all between 1.8 and 2.0. The mRNA expressions of EGF and TFF2 after 10 days of AL treatment were significantly higher than those in MD group in a dose-dependent manner. However, the mRNA expressions of TNF-α and IL-8 were significantly lower than those in MD group in a dose-dependent manner. These differences were statistically significant (Po0.05) (Fig. 1). Again, the difference of MC versus MP or HC versus HP was significant (P o0.05). The processed AL showed better TNF-α-, IL-8-decreacing and EGF-, TFF2- increasing effects compared with the crude AL.

After treatment with the crude AL and AL processed, the protein expressions of EGF, TFF2, TNF-α, and IL-8 in gastric tissues were examined by immunohistochemistry. The optical density values were determined by the Olympus image pick up system and a JD801 mode microscopic image analysis system. As shown in Fig. 3, the protein expressions of EGF and TFF2 in AL groups were higher than those in MD group in a dose-dependent manner. However, the protein expressions of TNF-α and IL-8 in AL groups were lower than those in MD group dose-dependently. Again, the difference of HC versus HP was significant (P o0.05). Compared with the crude AL, the AL processed showed better TNF-α-, IL-8-decreasing and EGF-, TFF2- increasing effects.

3.3. Effects of AL on gastric injury by histological analysis

4. Discussion

Histological analysis of gastric tissues is shown in Fig. 2. In model group, gastric tissues histopathological changes exhibited inflammatory cell infiltration, congestion, edema in mucosa, and extensive deep damage (Fig. 2B). Treatment with AL inhibited acetic acid induced ulcer, congestion, edema, and hemorrhage in gastric mucosa.

Gastric ulcer is a multifactorial etiological disease. Several factors play significant roles in gastric ulcerogenesis including stress, trauma, sepsis, hemorrhagic shock, burns, Helicobacter pylori, steroidal and non-steroidal drugs etc. (Saxena and Singh, 2011). Nowadays gastric ulcer is largely distributed among populations with a relatively higher incidence in elderly people. Its

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Fig. 1. The mRNA expressions of EGF (A), TFF2 (B), TNF-α (C) and IL-8 (D) in the gastric tissues of rats in SO (sham-operated group), MD (model group), OM (omeprazole group, 13 mg/kg), SJ (Sanjiu Weitai granule, 2.08 g/kg), LC (low dose group of the crude AL, 0.625 g/kg), MC (middle dose group of the crude AL, 1.25 g/kg), HC (high dose group of the crude AL, 2.5 g/kg), LP (low dose group of the processed AL, 0.625 g/kg), MP (middle dose group of the processed AL, 1.25 g/kg) and HP (high dose group of the processed AL, 2.5 g/kg). Fold changes of mRNA levels of target genes related to endogenous reference β-actin were calculated. Data were expressed as mean7 SD (n¼ 8). The (n) indicates a significant difference compared with model group (Po 0.05).

incidence increases mainly because of the growing use of tobacco, non-steroidal anti-inflammatory drugs (NSADs), complemented by alcohol and stress (Taylor and Blaser, 1991; Ishihara and Ito, 2002; Sheeba and Asha, 2006). The processing (pao zhi) plays a very important role in traditional Chinese medicine (TCM). Science of processing Chinese materia medica is a discipline researching about the processing methods and mechanisms of Chinese materia medica. LeiGong's Treatise on preparation and broiling of materia medica (Leigong PaozhiLun), written by Lei Xiao in the southern and northern dynasties, was the first book about processing Chinese materia medica in China. It recorded 17 kinds of processing methods, which have been considered as the classical criterions in processing Chinese materia medica till now. Today there have been a large number of classic books about processing written in different periods. It has been considered that the herbal properties are changed with more efficiency or less toxicity or even both after processing. Adjuvant medicines are frequently used in processing. In Leigong PaozhiLun, among all medicines, more than two thirds were processed with adjuvant medicines (Zhang and Zhang, 2010). Wheat bran is one of the most commonly used adjuvant medicines. In antiquity, there were almost 20 kinds of methods about processing of the Atractylodes lancea rhizome, of which stir-frying with wheat bran was commonly used. Currently, except for branprocessed product, other processed ones have been rarely used. In the Chinese Pharmacopoeia (2010 edition. Vol I), only two products (the crude AL and bran-processed AL) are listed. According to the reports of Nogami et al. (1985A, 1985B, 1986), Atractylodes lancea was effective in gastric ulcer. But until now, there has been little information available about the comparison of the anti-ulcer activity between the crude AL and bran-processed

AL. The present study was carried out to compare the anti-ulcer effect of the crude AL and AL processed with bran in rats where gastric lesions were induced by acetic acid. The so-called acetic acid ulcer model, which most closely approximates human ulcers in terms of both pathological features and healing mechanisms, is frequently utilized to investigate the pathophysiology of gastric ulcers, the efficacy of anti-ulcer drugs and the mechanisms of ulcer healing (Okabe and Amagase, 2005; Rodrigues Orsi et al., 2012). It was previously demonstrated that the damage caused by acetic acid penetrated in gastric mucosa not only in the mucous membrane and submucous layer but also in the muscular layer. These ulcers became chronic within 2–3 days and healed completely within 2–3 weeks without perforation or penetration to the surrounding organs (Okabe et al., 1971; Günal et al., 2003). The ulcer induction procedure is simple, readily resulting in ulcers of consistent size and severity with a 100% success rate. Furthermore, the ulcers respond well to various anti-ulcer drugs (Kang, et al., 2010). In this study, we utilized this type of experimental ulcer model to compare the anti-ulcer effects of the crude AL and AL processed and evaluate the mechanisms of anti-ulcer action. PGE2, which is generated by cyclooxygenase-2 (COX-2) catalyzing the metabolite of arachidonic acid, is an important cell growth and regulatory factor, whereas in excess, it becomes one of the strongest inflammatory mediators in inflammatory reactions because high concentration of PGE2 increases vascular permeability. Inhibition of PGE2 will reduce the degree of the inflammatory reaction (Form and Auerbach, 1983; Gao et al., 2013). TNF-α, an earliest and primary endogenous pro-inflammatory cytokine, is regarded as a parameter of local and systemic inflammation reaction. IL-6 and IL-8 are known to be pro-inflammatory cytokines that possess a multitude of biological activities linked to the

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Fig. 2. Microscopic observation of the stomachs of rats treated with saline, positive drugs and AL for 10 consecutive days. Microscopy magnification 10  (A) sham-operated group, (B) model group, (C) omeprazole group (13 mg/kg), (D) Sanjiu Weitai granule (2.08 g/kg), (E) low dose group of the crude AL (0.625 g/kg), (F) middle dose group of the crude AL (1.25 g/kg), (G) high dose group of the crude AL (2.5 g/kg), (H) low dose group of the processed AL (0.625 g/kg), (I) middle dose group of the processed AL (1.25 g/kg), and (J) high dose group of the processed AL (2.5 g/kg).

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Fig. 3. The protein expression of EGF (A), TFF2 (B), TNF-α (C) and IL-8 (D) in the gastric tissues of rats in SO (sham-operated group), MD (model group), OM (omeprazole group, 13 mg/kg), SJ (Sanjiu Weitai granule, 2.08 g/kg), LC (low dose group of the crude AL, 0.625 g/kg), MC (middle dose group of the crude AL, 1.25 g/kg), HC (high dose group of the crude AL, 2.5 g/kg), LP (low dose group of the processed AL, 0.625 g/kg), MP (middle dose group of the processed AL, 1.25 g/kg) and HP (high dose group of the processed AL, 2.5 g/kg). The optical density values were determined by the Olympus image pick up system and JD801 mode microscopic image analysis system. Data were expressed as mean 7 SD. The (n) indicates a significant difference compared with model group (Po 0.05).

immune-pathology of acute or chronic inflammatory diseases (Strieter et al., 1993). EGF plays an important role in the maintenance of the integrity of the gastric mucosa and in the acceleration of gastric ulcer healing. It significantly reduces the area of gastric ulcer and increases the gastric blood flow (Machowska et al., 2008). TFF2, mainly in the stomach (Rasmussen et al., 1992; Poulsen et al., 1999), is one of the members of the trefoil factor family, known for its role in the process of restitution after mucosal injury and protecting the integrity of the epithelial barrier (Mashimo et al., 1996; Farrell et al., 2002; Xue et al., 2010) and in the immune system by protecting the organism from consequences of infection. In addition, TFF2 deficiency has been shown to exacerbate inflammatory response upon gastric injury (Shah et al., 2012). In our study, the average levels of PGE2, TNF-α, IL-6 and IL-8 increased remarkably and the levels of EGF and TFF2 decreased markedly in serum and stomachs of the model group. A decrease in the level of PGE2, TNF-α, IL-6 and IL-8 and an increase in the levels of EGF and TFF2 were observed after treatment with the crude and branprocessed AL in a dose-dependent manner. The AL processed showed more remarkable effectiveness than the crude AL with statistical difference at the dose of 1.25 g/kg and the dose of 2.5 g/kg (Po0.05). In addition, our further study found that the mRNA expressions of TNF-α and IL-8 were decreased and the mRNA expressions of EGF and TFF2 were increased in gastric tissues in all the treatment groups, among which the processed AL groups exhibited obvious advantage over the crude AL groups with statistical difference at the dose of 1.25 g/kg and the dose of 2.5 g/kg (Po0.05). There was also a trend of better effects of the processed AL than crude

AL at the dose of 0.625 g/kg which did not reach statistical significance. The results of protein expressions of EGF, TFF2, TNF-α, and IL-8 in stomach examined by immunohistochemistry were in coincidence with the above-mentioned results. In this study, histological findings indicated that both the crude and processed AL produced marked reduction in the intensity of gastric tissue damage produced by acetic acid as compared with model group and their effects were comparable to those of Sanjiu Weitai granule even omeprazole at the dose of 2.5 g/kg. Being that, the AL processed was more effective.

5. Conclusion The current study first reported the potential differences between the crude AL and bran-processed AL in the effects on gastric ulcer induced by acetic acid in rats. The results provided experimental evidence to support the traditional use of this plant in the treatment of gastrointestinal disease and provided a scientific foundation for the rational clinical application of AL. Based on the above data, both the crude and processed AL significantly alleviated the damage of the gastric tissue and the effects of processed AL were more potent than those of the crude AL. The anti-ulcer action of AL could be attributed partly to the potent anti-inflammatory properties via down-regulating the productions of TNF-α, IL-8, IL-6 and PGE2 and to the gastroprotective effects via up-regulating the productions of EGF and TFF2. However, further studies are needed to investigate the active compounds and the mechanisms involved in the anti-ulcer effect produced by AL.

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Comparison of the anti-ulcer activity between the crude and bran-processed Atractylodes lancea in the rat model of gastric ulcer induced by acetic acid.

The rhizome of Atractylodes lancea (AL, Compositae, Chinese name: Cangzhu; Japanese name: Sou-ju-tsu) has been used traditionally for the treatment of...
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