Objective To investigate the possible influence of Lewis B expression in Helicobacter pylori (H.pylori) on bacterial adhesion property, and determine the relationship between babA 2 gene in H.pylori and peptic ulcer. Methods The bacterial adhesion property in a total of 78 H.pylori strains was performed using adherence assay in vitro, and the frequency of babA 2 gene was determined by polymerase chain reaction (PCR). Results Among 47 H.pylori strains isolated from peptic ulcer patients, 18 (38.3%) were positive for babA 2 gene, while babA 2 was positive in 14 (45.2%) of 31 strains isolated from non ulcer dyspepsia patients ( P =0.427). Among 31 H.pylori strains expressing Lewis B, 24 (77.4%) were positive for adherence assay, compared with 38 (80.9%) of 47 H.pylori strains without expression of Lewis B ( P =0.463). Conclusions (1) There is no association between babA 2 status in H.pylori strain and peptic ulcer in our population; (2) The expression of Lewis B in H.pylori does not interfere with bacterial adhesion property, and thus supports that the gastric epithelium is the receptor for Lewis B of H.pylori.

Objective To detect the expression of CRF and CRF receptors in colonic mucosa of DSS induced colitis in mice model and to study the effect of CRF and CRF receptors on the development.Methods Six to eight weeks healthy female BALB/c mice were divided into control group and DSS group.Setting up DSS colitis model and colitis was evaluated by the disease activity index(DAI) and histological score.The immunofluorescence technique was used to assay the CRF1 and CRF2 receptors expression in colonic mucosa.The expression of CRF and CRF receptors protein were analyzed by western blotting.Results DSS colitis was set up successfully with significant inflammation in colonic mucosa by the disease activity index (DAI) and histological score.Immunofluorescenee staining evidenced that expression of CRF1 receptor in DSS colitis group has no significant deviation compared to control group(P ＞ 0.05),while the expression of CRF2 receptor was elevated in DSS colitis group compared to control group (P ＜ 0.05).CRF2 receptor was localized in epithelial cells and mononuclear cells in the lamina propria.The levels of CRF and CRF2 receptor protein by western blotting were higher in in DSS colitis group compared to control group (P ＜ 0.05).The level of CRF1 receptor protein in DSS colitis group had no significant deviation compared to control group(P ＞ 0.05).Conclusion The higher expression of CRF and CRF2 in colonic mucosa of DSS colitis may participate in the development of colitis.

Objective To investigate the effect of corticotrophin-releasing factor (CRF) on the regulation of Toll-like receptor 4/NF-κB signaling pathway expression in human intestinal epithelial cell line HT-29. Methods HT-29 cells were divided into four groups, normal control group, LPS group (LPS 20 μg/ml stimulated for 24 h), CRF group (CRF 20 ng/ml stimulated 24 h) and CRF+ LPS group (CRF incubated for 12 h then changed to LPS for another 12 h). After stimulation, the expression of TLR4 mRNA of each group was examined by reverse transcriptase polymerase chain reaction (RT-PCR). Total cell protein were extracted and the expression of TLR4 and NFκB p65 at protein level were detected by western blotting.Cell culture supernatant was collected and the secretion of interleukin-8 was detected by enzyme-linked immunoasorbent assay (ELISA). Results The expression of TLR4 in LPS group at mRNA and protein level were 0.31±0.04 and 0.48±0.17,there was no significant difference compared with normal control group (0.28±0.02 and 0.45±0.12,t＝0.216 and 0.712 , P＞0.05 ). In CRF group which were 1.05±0.06 and1. 08±0.21, significantly higher than normal control group (t＝3.721 and 3.802, P＜0.05). In CRF+LPS group which were 1.68±0.05 and 1.81±0. 18,significantly higher than CRF group (t＝4. 816 and 3. 918, P＜0.05).The results of NF-κB p65 expression at protein level and interleukin-8 expression of cell culture supernatant were consistent with the results of TLR4 expression at mRNA and protein level.Conclusion CRF not only activate TLR4/NF-κB signaling pathway in human intestinal epithelial cell,but enhance the reaction of intestinal epithelial cell to LPS as well, which resulting in increased interleukin-8 secretion.

Objective To detect the dynamic Th17 cells in a murine model of irritable bowel syn-drome ( IBS) and to study the effect of aryl hydrocarbon receptor ( Ahr) on Th17 cells activation .Methods Thirty BALB/c male mice were randomly divided into three groups including experiment group ,control group and Ahr antagonist group .A murine model of IBS was established by perfusing three nitrobenzene sulfonic acid (TNBS) into the colon of mice.Equal volume of saline was used to set up the control .The mice in Ahr antagonist group were intraperitoneally injected with 10 μg Ahr antagonist for four consecutive days .All mice were evaluated for visceral hypersensitivity and colonic mucosal inflammation .Mesenteric lymph nodes (MLNs) and peripheral blood mononuclear cells (PBMCs) were detected by flow cytometry through staining Th17 cells.The distribution of Ahr and IL-17A in colon and the number of Th17 cells activated by Ahr (Ahr and IL-17A double positive ) were detected by double immunofluorescence staining .Results ( 1 ) The percentage of Th17 cells in MLNs was significantly increased in experiment group followed by those in Ahr antagonist group and control group (P<0.05).(2)Compared with control group,the number of Th17 cells in peripheral blood samples was significantly increased in experiment group and Ahr antagonist group ( both P<0.05 ) ,but there was no difference between Ahr antagonist group and experiment group ( P=0.642 ) .( 3 ) The number of Ahr-activated Th17 cells ( Ahr+IL-17A+) was significantly increased in experiment group (10.00±1.58) as in comparison with that in control group (3.80±0.83,P<0.05),but the number was de-creased with Ahr antagonist intervention ( 5.80 ±0.83 , P<0.05 ) .Conclusion The number of activated Th17 cells was increased in MLNs and peripheral blood samples from mice with IBS .Ahr played an important role in the activation of Th17 cells in intestines.However,the number of Ahr-activated Th17 cells in intestinal mucosa and the proportion of Th 17 cells in MLNs could be down-regulated through blocking Ahr .

Objective To investigate the role of intestinal immune dysfunction in the pathogenesis of irritable bowel syndrome(IBS) and to study the effects of Clostridium butylicum on the regulation of intestinal immune disorders.Methods A total of 50 male 6-week-old C57BL/6 mice were randomly divided into three groups,including the experimental group (n =20),the control group (n =20) and the Clostridium butylicum group(n =10).A mouse model of constipation-predominant IBS (C-IBS) was established by perfusing sodium butyrate solution(200 μl,concentration of 500 mmol/L) into the mouse colon twice a day for three consecutive days.The mice in control group were intrarectally perfused with normal saline enema (200 μl).Two hours before the perfusion of sodium butyrate into colon,the mice in Clostridium butylicum group were given Clostridium butylicum 500 μl(viable cell concentration of 1×109 CFU/ml) by oral gavage once a day for six days.The colorectal distention test(CRD) was carried out for evaluation of clinical parameters.HE staining of intestinal tissue section was performed for histopathological assessment of colonic mucosal inflammation.Intestinal intraepithelial lymphocytes (IELs) and lamina propria mononuclear cells (LPMCs) were isolated and analyzed by flow cytometry to evaluate the correlation between IBS and intestinal immune dysfunction/abnormal activation of intestinal immune cells in mouse model of C-IBS,and to assess the regulatory effects of Clostridium butylicum on the intestinal immune disorder.Results (1) Compared with the control group,the mice in experimental group showed a significant change in physiological parameters,histological structure of colon,inflammatory cells infiltration and low-grade inflammatory state.There was a significant increase in scores of CRD and a decrease in lowest sensory threshold (t=8.926 and t=6.103,both P＜0.001) ; (2) There was a decrease in the numbers of DC in IELs (t =2.878 and t =3.086,both P＜0.05),but an increase in the numbers of macrophage (t=3.191,P＜0.05) and the memory T cells in mice with IBS (t=3.071,P＜0.05) as compared with that in control group; (3)DCs were decreased (t=2.880 and t=2.664,both P＜0.05),but memory T cells were increased (t =3.732 and 2.682,P＜0.01 and P＜0.05) in the LPMCs of mice in experimental group; (4)There was no significant difference in the physiological index between the mice in control group and the Clostridium butylicum group.Levels of memory T cells,macrophages and DCs in the IELs were close to the normal level (6 d,t =1.103,0.0213,0.418,all P＞0.05),and levels of macrophages and DCs in the LPMCs of mice in the Clostridium butylicum group were also similar to that in the control group (6 d,t =0.782,0.347,both P＞0.05) ; (5) Compared with the mice in experimental group,the level of memory T cells in LPMCs of mice treated with Clostridium butylicum was dramatically declined (6 d,t=2.346,P=0.0470,P＜0.05),however,which was still higher than that of mice in control group (6 d,t =2.233,P =0.0476,P＜0.05).The intestinal immune function was restored to normal level with Clostridium butylicum intervention.Conclusion The pathophysiologic mechanism of IBS might be closely related to the abnormal activation of intestinal immune cellsand disordered functional state in the intestinal mucosa.Clostridium butylicum could regulate the intestinal immune homeostasis and restore the physiological function of gastrointestinal tract.

Objective To investigate the mechanism of the permeability of intestinal mucosa in the pathogenesis of irritable bowel syndrome (IBS) and the interventional effects of Clostridium butyricum combined with glutamine.Methods According to random number method,fifty BALB/c mice were divided into control group,experimental control group,glutamine group,Clostridium butyricum group and combination group.IBS mice model was established by water-avoidance stress (WAS) experiment.The defecating time of mice and fecal water content were detected by dyed stool after mice gavaged with methylcellulose (1.5％).The pathological injury of intestine was assessed by hematoxylin and eosin staining.The visceral sensitivity was evaluated by colorectal distention test (CRD).The changes of the permeability of intestine was evaluated by detecting the changes of serum D-lactic acid (D-LA),level of diamine oxidase (DAO),expressions of intestinal epithelial cells (IEC) cell tight junction protein (TJ) (occludin-1,claudin-1,zonula occludens-1 (ZOL-1)) at protein level.The interventional effects of Clostridium butyricum combined with glutamine were evaluated.t test was performed for comparison between groups,and analysis of variance was used for comparison among multi-groups.Results Compared with the control group,the defecating time of experimental control group was significantly shorten ((100.40±14.80) min vs (75.88±12.20) min and water content of fecal significantly increased ((54.76±9.98)％ vs (74.95±7.15)％,t =3.692 and 4.023; P=0.002 and 0.002).The lowest threshold of visceral sensitivity significantly decreased ((40.87 ± 4.82) mmHg (1 mmHg=0.133 kPa) vs (27.80±3.18) mmHg; t=8.761,P＜0.01),while the mucosal pathological injury score significantly increased (0.50±0.15 vs2.60±0.97; t＝6.034,P＜0.01).The level of D-LA ((1 476±246.8) ng/L vs (913.6±90.1) ng/L)) and DAO ((3 391.0±256.9) vs (5 096.0±725.2) ng/L) significantly increased (t=40.920 and 29.810; both P＜0.05),and the expression of tight junction protein ZOL-1 (0.165±0.005 vs0.119±0.003),occludin-1 (0.104±0.016 vs 0.022±0.006) significantly decreased (t=19.830 and 19.830; both P＜0.01).Compared with the experimental control group,after intragastric intervention the defecating time of glutamine group,Clostridium butyricum group and combination group increased ((90.50±3.78),(97.56±8.79) and (99.89±11.90) min and water content of fecal decreased ((69.33±6.71)％,(58.07±8.97)％ and (56.74±8.12)％) and the differences were statistically significant (F=10.020 and 8.740; both P＜0.01).The results of Clostridium butyricum group and combination group were good (F=2.481 and 4.874; both P＜0.05).And the lowest threshold of visceral sensitivity significantly increased ((31.80±2.69),(36.04±5.06) and (38.93±3.30) mmHg; F=2.420,P＜0.05),the result of combination group was the best (F=3.550,P＜0.01).Jejunal mucosal injury was significantly reduced (2.00 ± 0.94,1.30 ± 0.68 and 1.30±0.48; F＝11.350,P＜0.01).After intragastric intervention,serum levels of D-LA ((1 370.0± 78.9),(1 066.0±155.5) and (1 039.0±129.0) ng/L) and DAO ((4 808.0±477.4),(3 713.0± 595.0) and (3 725.0±615.9) ng/L) of glutamine group,Clostridium butyricum group and combination group significantly decreased (F＝37.480 and 27.670; both P＜0.01).The level of ZOL-1(0.126± 0.014,0.125±0.006,0.138±0.004) and occludin 1 (0.037±0.013,0.073±0.028,0.078±0.027) of glutamine group,Clostridium butyricum group and combination group significantly increased,and the differences were statistically significant (F=5.867 and 10.630; both P＜0.05).The change of ZOL-1 of combination group was more than that of Clostridium butyricum group (t =5.457,P ＜ 0.05).Conclusions WAS experiment can induce visceral hypersensitivity,increase the permeability of intestine and reduce the function of intestinal epithelial barrier.Clostridium butyricum and glutamine are effective in the recovery of visceral hypersensitivity and the permeability of mucosal epithelia cells.

Objective To investigate the effects of Bifidobacterium infatn ison the expression of in -testinal corticotropin releasing factor ( CRF) receptors and how the peripheral CRF receptors activate mast cells in a murine model of irritable bowel syndrome (IBS).Methods Thirty BALB/c male mice were ran-domly divided into three groups including control group , model group and Bifidobacterium infantis group. The mouse model of IBS was established by using chronic restraint stress .Mice in Bifidobacterium infantis group received daily intragastrical administration of Bifidobacterium infantis for 14 days.Mice in control and model groups were treated with equal volume of saline .Then all mice were killed after the assessment of weight and abdominal withdrawal reflex ( AWR) .The levels of histamine , tryptase and tumor necrosis fac-tor-α( TNF-α) in serum samples were detected by ELISA .The expression of CRF in colonic mucosa was analyzed by immunohistochemistry .The expression of CRF-R1 and CRF-R2 in mast cells and the number of mast cells in colonic mucosa were detected by double immunofluorescence staining assay .The expression of CRF-R1 and CRF-R2 at mRNA level in colon were detected by reverse transcription polymerase chain reac-tion ( RT-PCR) .Results Compared with control group , the levels of histamine , tryptase and TNF-αin pe-ripheral blood samples , the expression of CRF-R1 and CRF-R2 at mRNA level , and the number of mast cells, CRF-R1+mast cells and CRF-R2+mast cells in colonic mucosa were increased significantly in model group (P<0.05), but were remarkably down-regulated with the treatment ofB ifidobacterium infantis (P<0.05).Conclusion Bifidobacterium infantis could reduce the activation of mast cells in a murine model of IBS by inhibiting the expression of CRF-R1 and CRF-R2 in intestinal mast cells .

Objective To investigate the effects of two cholesterol-lowering probiotics, DM9054 (Lac-tobacillus Rhamnosus GG, LGG) in combination with 86066 (Lactobacillus plantarum WCFS1, LP), on the metabolism of bile acid via a rat model of non-alcoholic fatty liver disease (NAFLD) and the possible mecha-nism. Methods Twenty-one SD male rats were randomly divided into three groups including control group, NAFLD model group and probiotics intervention group. Rats in the control group received normal diet. The rat model of NAFLD was established by feeding rats with chronic high fat diet (45% of calories derived from fat di-et) for 20 weeks. Rats in the probiotics intervention group were given high fat diet together with cholesterol-low-ering probiotics through oral gavage. General indexes of each group including body weight and the levels of tri-glyceride, cholesterol and CK18-M30 in serums samples were detected. The expression of cholesterol 7-alpha hydroxy-lase (CYP7A1), fibroblast growth factor receptor 4 (FGFR4), farnesoid X receptor (FXR), fibroblast grwoth factor 15 (FGF15) and apical sodium-dependent bile acid transparter(ASBT) at mRNA level were de-tected by using real-time polymerase chain reaction (real-time PCR). Western blot assay was used to detect the protein expression of CYP7A1, FXR in liver tissues and ASBT in ileum tissues. The expression of FXR in liver and ileum tissues were analyzed by immunohistochemistry. Results Rats with NAFLD showed loss of body weight and decreased levels of the serological markers after treating with the probiotics (P<0. 05). Compared with the rats in model group, enhanced expression of CYP7A1 and inhibited expression of FXR in liver tissues, activated FXR-FGF15 pathway in ileum tissues as well as down-regulated expression of ASBT in ileum tissues were detected in rats receiving probiotics intervention (P<0. 05). No significant difference in the expression of FGFR4 at mRNA level was observed between NAFLD rats with or without probiotics intervention (P>0. 05). Conclusion Probiotics intervention might up-regulate the expression of CYP7A1 by suppressing the FXR path-way in liver tissues and inhibiting the expression of ASBT in ileum tissues. Treating NAFLD rats with cholester-ol-lowering probiotics could activate the FXR-FGF15 pathway in ileum tissues and enhance the metabolism of bile acid, which contributed to the alleviation of NAFLD.

Objective To analyze the roles and mechanisms of lactitol and Bifidobacterium infantis in the treatment of rat constipation and to investigate their effects on aquaporin3 (AQP3) and interstitial cells of Cajal (ICC) in colon tissues.Methods Thirty SD male rats were recruited in this study,6 of which were randomly selected as the control and the rest were given 4 mg/kg.d of loperamide for 5 consecutive days to construct the rat model of constipation.The rats with constipation were randomly divided into four groups including model group,lactitol treatment group,Bifidobacterium infantis treatment group and lactitol in combination with Bifidobacterium infantis treatment group.General indexes including food intake,water intake,body weight,fecal water content and intestinal transit rate of each rat were measured after receiving corresponding treatments for 7 consecutive days.The levels of substance P (SP) and vasoactive intestinal peptide (VIP) in serums samples were detected by ELISA.The expression of protein kinase A (PKA) and neurokinin-1 receptor (NK-1) at mRNA level in colon tissues were detected by real-time polymerase chain reaction (real-time PCR).Western blot assay and real-time PCR analysis were used to detect the expression of AQP3 and c-kit at protein and mRNA levels,respectively.Results Compared with the rats in model group,the levels of fecal water content and intestinal transit rate,the concentrations of SP and VIP in serums samples,the expression of PKA and NK-1 at mRNA level and the expression of AQP3 and c-kit at mRNA and protein levels were significantly increased in rats from the three treatment groups (P＜0.05).The most effective treatment was lactitol in combination with Bifidobacterium infantis,followed by the lactitol treatment and then the Bifidobacterium infantis treatment.Conclusion The combination therapy with lactitol and Bifidobacterium infantis increased the serum levels of SP and VIP in rats with constipation.SP could enhance the contraction of gastrointestinal smooth muscles and improve the intestinal motility by binding to the NK-1 receptor on the membrane of ICC.VIP could promote the absorption of water in intestinal tracts,soften stools and alleviate constipation by upregulating the expression of AQP3 at both protein and mRNA levels via the cyclic adenosine monophosphate-PKA (cAMP-PKA) signaling pathway.

Objective To investigate the effectiveness of Lactobacillus paracasei N1115 combined with fructooligosaccharides (FOS) in the treatment of nonalcoholic fatty liver disease(NAFLD) in a mouse model and to analyze the possible mechanism.Methods Fifty male C57BL/6 mice were randomly divided into five groups and respectively given normal diet (ND group), high-fat diet (HFD group), HFD containing Lactobacillus paracasei N1115 (HFD+L) (2.2×109 CFU/ml), HFD containing FOS (HFD+FOS) (4 g/kg per day) and HFD containing Lactobacillus paracasei N1115 and FOS for 16 consecutive weeks.Levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), lipopolysaccharide (LPS) and diamine oxidase (DAO) in serum samples from each group were measured.Expression of tight-junction proteins (Claudin-1 and Occludin), p38 and phosphorylated p38 (p-p38) in intestinal tissues were analyzed.Results Compared with the HFD group, the HFD+FOS+L group showed decreased levels of TC, TG, LDL, LPS and DAO in serum samples, but increased serum HDL level (P<0.05).Moreover, combined treatment with Lactobacillus paracasei N1115 and FOS alleviated liver lipid deposition, significantly increased the expression of Claudin-1 and Occludin in intestine and inhibited the phosphorylation of p38 (P<0.05).Conclusion Lactobacillus paracasei N1115 combined with FOS may increase the expression of Claudin-1 and Occludin through inhibiting the phosphorylation of intestinal p38, which is conducive to maintaining the intestinal mucosal barrier integrity and alleviating NAFLD.