26 - World Journal of Gastroenterology

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Clostridium; PY Agar for Clostridium perfringens; LBS Agar for Lactobacillus and Lactobacillus crispatus (L. crispatus); and BL Agar for Bifidobacterium. All aerobic plates were incubated at 37℃ for 24 h, followed by incubation for 12 h at room temperature. The LBS plates were incubated in a candle-jar atmosphere of 10% CO2. Anaerobes were grown in an anaerobic jar at 37℃ for 72 h. The number of colonies from each plate was recorded. Amplification of Lactobacillus 16S rDNA for denaturing gradient gel electrophoresis DNA from Lactobacillus cultures was extracted according to the protocol of BioTeke Corporation (Beijing, China) and stored at -20℃. Universal bacterial primers (Invitrogen Biotechnology, Shanghai, China) HAD1-GC (CGCCCGGGGCGCGCCCCGGGCGGGGCGGGG- GCACGGGGGGACTCCTACGGGAGGCAGCAGT- 3′) and HAD2(5′-GTATTACCGCGGCTGCTGGCAC- 3′) [23] were used to amplify V2 to V3 regions of the bacterial 16S rDNA. PCR reactions were run at 95℃ for 5 min, followed by 38 cycles of amplification at 94℃ for 40 s, 52℃ for 40 s, and 72℃ for 50 s, and a 7-min extension at 72℃. Denaturing gradient gel electrophoresis Denaturing gradient gel electrophoresis (DGGE) was performed with the DCode Universal Detection System (Bio-Rad) that utilized 16 cm × 16 cm × 1 cm gels. Eight percent polyacrylamide gels were prepared and run with 1 × TAE buffer (2 mol/mL Tris base, 1 mol/mL glacial acetic acid, and 50 mmol/mL EDTA) diluted from 50 × TAE buffer (Sigma, Beijing, China). The denaturing gradient was formed with two 8% acrylamide (acrylamide/ bis, 37.5:1) stock solutions (Bio-Rad). The gels contained a 30%-70% gradient of urea and formamide that increased in the direction of electrophoresis. A 100% denaturing solution contained 40% (v/v) formamide and 7.0 mol/mL urea. The electrophoresis was conducted with a constant voltage of 110 V at 60℃ for 12 h. Gels were stained with ethidium bromide solution (5 μg/mL) for 30 min, washed with deionized water, and viewed by UV transillumination (Bio-Rad). Amplification and sequencing of the 16S V2-V3 region Single predominant bands of the DGGE gel were selected by cutting with a sterile scalpel, and added to 50 μL deionized sterile water (Fermentas Life Sciences, Shenzhen, China). The gel pieces were placed at 4℃ for 24 h, centrifuged at 12 000 × g for 10 min, and the supernatants were used as templates for PCR amplification. Universal bacterial primers (Invitrogen Biotechnology) HAD1(5′- TCCTACGGGAGGCAGCAGT-3′) and HAD2(5′- GTATTACCGCGGCTGCTGGCAC-3′) [23] were used for amplification. For each PCR amplification, 5 μL template was added to 45 μL PCR reaction mixture (Fermentas Life Sciences) that contained 5 μL 10 × PCR buffer, 5 μL 25 mmol/L MgCl2, 1.5 μL 10 mmol/L dNTPs, 1.5 μL 100 ng/μL each primer, and 2.5 U Taq DNA polymerase. WJG|www.wjgnet.com Zhao X et al . Lactobacillus shift in distal esophagus Reactions were run at 95℃ for 5 min, followed by 36 cycles of amplification at 94℃ for 30 s, 56℃ for 40 s, and 72℃ for 40 s, and a 10-min extension at 72℃. Wizard PCR Preps DNA Purification System (Promega, Beijing, China) were used to purify the PCR products. The purified products were sent to Beijing Genomics Institute of Technology for sequencing. Blast sequences were obtained through GenBank BLAST searches (http://www. ncbi.nlm.nih.gov/blast). Statistical analysis Statistical analysis was performed using SPSS for Windows version 15.0. Results were expressed as measurement and enumeration data. Data are presented as means and SDs. For statistical comparisons, the t test and χ 2 test were performed and P < 0.05 was considered statistically significant. RESULTS High-fat diet resulted in elevated body weight and serum lipid with no significant histological change in vivo There was no significant change in body weight in the high-fat diet and normal control groups at the end of the first week. Since the second week, the body weight and Lee’s index increased weekly (Figure 1A and B). Accordingly, serum triglyceride and total cholesterol in the highfat diet group were significantly increased compared with the normal control group (Figure 1C). HE staining of esophageal mucosa showed no musculature damage or mucosal injury in the distal esophagus in the high-fat diet or normal control group (Figure 1D). High-fat diet led to microfloral shift in distal esophagus based on bacterial culture Bacteria in the distal esophagus were cultured on selective media. In the high-fat diet group, S. aureus was absent, and the numbers of total anaerobes and lactobacilli were reduced significantly in the distal esophagus compared with the normal control group. There was no obvious difference between the common and adaptive feeding groups for composition of cultivable bacteria in the distal esophagus of Sprague-Dawley rats (Table 2). Determination of Lactobacillus species shift in distal esophagus of high-fat diet-fed rats based on DGGE and sequencing 16S rDNA of cultivable Lactobacillus from the high-fat and common diet groups was amplified by PCR using universal bacteria primers HAD1-GC and HAD2 (Figure 2A). The amplified products of 16S rDNA were separated by DGGE. The two groups shared dramatically different bands, with bands A, C, D and E in the high-fat diet group, and bands B, C, D and E in the common diet group (Figure 2B). The purified bands were sequenced and BLASTed online with the V2-V3 region. The composition of Lactobacillus species in the distal esophagus in the common diet group was Lactobacillus gasseri (L. gasseri), Lac- 3153 July 14, 2011|Volume 17|Issue 26|
A 400 Body weight (g) B Lee’s index C Mean serum lipid (mmol/L) Zhao X et al . Lactobacillus shift in distal esophagus 350 300 250 200 150 100 50 0 330 320 310 300 290 280 270 260 6 5 4 3 2 1 0 Figure 1 No significant change in distal esophageal mucosa in Sprague-Dawley rats fed a high-fat diet. A: Body weight changes of age- and sex-matched rats in the high-fat diet and normal control groups (n = 10 each); B: Lees index changes for the rats in (A) (n = 10); C: Mean serum lipid for the rats in (A); D: Histological analysis of representative distal esophagus from the rats in (A) (Original magnification, hematoxylin-eosin staining, 100 × or 400 ×). tobacillus jensenii (L. jensenii) and Lactobacillus reuteri (L. reuteri), whereas the composition shifted to L. crispatus, L. gasseri and L. reuteri in the high-fat diet group (Figure 2C and D). DISCUSSION 2 3 4 5 6 7 t /wk 2 3 4 5 6 7 t /wk TG TCH Accumulating evidence has shown that obesity is a risk factor in the development of GERD [1-8,24,25] . However, is not well established how obesity affects the incidence of GERD. According to the published experimental data, no musculature damage has been reported in the distal esophagus of obese rats or mice. However, different components of the microbiota have been identified between obese and lean animals/humans. Based on this evidence, the purpose of this study was to analyze the microbiota composition and focus on how the shift in WJG|www.wjgnet.com Normal High-fat Normal High-fat Normal High-fat D High-fat 100 × High-fat 400 × Normal 100 × Normal 400 × microflora occurred in the distal esophagus of rats fed a high-fat diet. To date, the role of microbiota in the distal esophagus under conditions of weight gain remains obscure. Some studies have shown that high-fat/high-sugar chow dietfed, germ-free mice gain significantly less weight than their control littermates. The absence of microbiota has a protective role against weight gain in mice that consume a Western-style diet. Thus, body weight gain could be associated with a shift in the microbiota in mice fed a westernstyle diet. On the other hand, current research has demonstrated that there is a complex residential microbiota shift in the distal esophagus of patients with esophagealreflux-related disorders, such as GERD, reflux esophagitis and Barrett’s esophagus [14,15,26] . The possible explanations are that the reflux content might contain gastric bac- 3154 July 14, 2011|Volume 17|Issue 26|
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World Journal of Gastroenterology W
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Robert JL Fraser, Daw Park Jacob Ge
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Italy Donato F Altomare, Bari Piero
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Fernando Azpiroz, Barcelona Ramon B
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S Contents EDITORIAL REVIEW ORIGINA
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Contents APPENDIX FLYLEAF EDITORS F
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of HCC. Multimodal approaches with
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15S): A4577 63 Gruenwald V, Wilkens
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cause of liver disease in pediatric
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Table 1 Published studies on the as
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genesis through the presence of abn
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Manini R, Natale S, Vanni E, Villan
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Murray KF, Abrams SH, Rosenthal P,
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localization, the risk was 4.5 (95%
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The role of chemokines in CAC is fu
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MM, Rotter JI, Nicolae DL, Cho JH.
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Online Submissions: http://www.wjgn
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