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 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 inhibitory effect of IFN-γ on acute allergic airway inflammation induced by IL-33 in mice.Methods Twenty-four female C57BL/6 mice (6-8 weeks) were randomly divided into four groups: IL-33 model group, IFN-γ treatment group, IL-33+IFN-γ treatment group and PBS control group.A mouse model of acute allergic airway inflammation was induced by IL-33.Samples of bronchial alveolar lavage fluid (BALF) and lung tissues were collected.Group 2 innate lymphoid cells (ILC2s) and eosinophils were analyzed by flow cytometry.Levels of IL-5 and IL-13 in the supernatants of lung homogenate and BALF were measured by ELISA.Expression of IL-5, IL-13 and ST2 at mRNA level was detected by real-time PCR.Pathological changes in lung tissues were observed following hematoxylin and eosin (HE) and periodic acid-Schiff (PAS) staining.Results Compared with the PBS control group, no infiltration with inflammatory cells, goblet cell hyperplasia or mucus secretion was observed in the IFN-γ group;the numbers of ILC2s and eosinophils were not affected by IFN-γ;the levels of IL-5 and IL-13 in the supernatants of BALF and lung homogenate, and the expression of IL-5, IL-13 and ST2 at mRNA level in lung tissues were not significantly changed by IFN-γ (P>0.05).Compared with the PBS control group, massive infiltration with inflammatory cells, excessive mucus secretion, increased numbers of ILC2s and eosinophils, up-regulated levels of IL-5 and IL-13 in the supernatants of BALF and lung homogenate, and enhanced expression of IL-5, IL-13 and ST2 at mRNA level in lung tissues were detected in the IL-33 model group (P<0.05).Compared with the IL-33 model group, the combined treatment with IL-33 and IFN-γ significantly alleviated inflammatory cell infiltration, inhibited mucus secretion, reduced the numbers of ILC2s and eosinophils, down-regulated the levels of IL-5 and IL-13 in the supernatants of BALF and lung homogenate, and suppressed the expression of IL-5, IL-13 and ST2 at mRNA in lung tissues (P<0.05).Conclusion IFN-γ can inhibit the proliferation of eosinophils and ILC2s induced by IL-33, and reduce the secretion of IL-5 and IL-13, which indicates that IFN-γ has an inhibitory effect on acute allergic airway inflammation induced by IL-33 in mice.

Objective To investigate the clinical value of double contrast-enhanced ultrasonography (DCUS)in diagnosing Lauren classification of advanced gastric carcinoma.Methods Fifty-eight patients with gastric cancer proved by endoscopic biopsy underwent preoperative DCUS examination,and the findings were compared with postoperative pathologic results via hematoxylin-eosin and alcian blue-periodic acid schiff(AB-PAS)staining.Results In 58 patients,34 cases were diagnosed as intestinal type and 24 cases as diffuse type by pathology after operation.In intestinal type,30 cases were enhanced as global homogeneous intensity by using DCUS.another 2 cases as"lamellar"model and the other 2 cases as heterogeneous intensity.However,in diffuse type,22 cases were enhanced heterogeneously with"barrier"signs in 5 cases,"map"signs in 9 cases and"lamellar"signs in 8 cases.the remaining 2 cases of diffuse type enhanced as homogeneous model.The sensitivity and specificity of heterogeneous enhancement in diagnosing diffuse type of advanced gastric cancer was 91.7%and 88.2%respectively,and the Youden's index was 0.8.Conclusions DCUS could be considered as a new valuable method to distinguish the Lauren classification and evaluate the invasiveness,metastasis and prognosis in patients with gastric carcinoma.

Objective To investigate the clinical value of double contrast-enhanced ultrasonography (DCUS)in Borrmann classification of advanced gastric carcinoma preoperatively. Methods A total of 69 patients with advanced gastric carcinoma were examined by ultrasound after taking oral contrast agent and bolus injection of SonoVue preoperatively.A comparison with final pathologic results was made after surgery. Results The accuracy of oral contrast-enhanced ultrasonography and DCUS in Borrmann classification of advanced gastric cancer were 78.3%and 91.3%,respectively.There was a significant difference between two methods(X2=4.087,P＜0.05).In addition,Kappa value of two methods were 0.73 and 0.90,respectively.Conclusions DCUS is a new valuable method for Borrmann classification with its high accuracy preoperatively.

Objective To investigate the value of oral and intravenous contrast enhanced ultrasonography-double contrast ultrasonography (DCUS) in the evaluation of differentiation degree of gastric cancer. Methods Fifty-three patients who had been diagnosed as gastric cancer by gastroscope and confirmed by pathology after operation were examined by DCUS, and they were divided into moderately to well differentiated group and poorly differentiated group. The correlation of size and enhancement features of the lesions was compared with differentiation degree of gastric cancer. Results The size and enhancement distribution of lesions were markedly correlated with the degree of differentiation. The baseline intensity(BI)in poorly differentiated group was lower than that in well and moderately differentiated group[(0.52±1.12)dB vs (1.53±1.87) dB, P<0.05], but enhanced intensity(EI) was higher than that in the second group [(26.22±3.69)dB vs (23.69±5.02)dB, P<0.05]. Conclusions DCUS could initial evaluate the degree of differentiation of gastric cancer before operation.

Objective This study examined the effects of repaglinide on the proliferation, differentiation, and apoptosis of mouse pre-osteoblasts MC3T3-E1 subclone 14 cell under high glucose condition.Methods MC3T3-E1 cells cultured in vitro for 48h were divided into the following groups: control group and repaglinide with different concentrations (0.01, 0.1, 1, and 10 μmol/L).Cell proliferation was measured by CCK-8 assay.mRNA levels of type I collagen(COL-Ⅰ), osteopontin(OPN) and alkaline phosphatase(ALP) were assayed using quantitative real-time PCR.Expression of apoptosis related proteins (Bcl-2, Bax) was measured by western blot analysis.Results (1)Compared with control group, the proliferation rate of repaglinide with different concentrations increased, while the proliferation rate of the 1 μmol/L repaglinide group significantly higher than that of the control group(P<0.05).(2) There was no significant difference on the COL-Ⅰ mRNA expression among the repaglinide groups and the control group(P>0.05).Compared with control group, the OPN and ALP mRNA expression increased significantly in the 1 μmol/L repaglinide group(P<0.05), while the OPN expression was significantly declined in the 10 μmol/L repaglinide group(P<0.05).(3)The protein expression of Bcl-2 was positively related with repaglinide concentration(P<0.05), while the protein expression of Bax went down in the 1 and 10 μmol/L repaglinide groups(P<0.05).Conclusion When exposed to high glucose concentration, repaglinide in a certain concentration range may promote the proliferation and differentiation of MC3T3-E1 subclone 14, while restrain its apoptosis.

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 assess the value of approximal side angle of carotid atherosclerotic plaques in patients with cerebral infarction by ultrasonography.Methods One hundred and seventy six patients with carotid atherosclerotic plaques were examined with uItrasonography and divided into two groups:cerebral infarction group (n=67)and none cerebral infarction group(n=109).Vessel area and residual lumen area of carotid artery were measured in axial view using linear probe.Area stenosis rate was calculated.The approximal side angle and thickness of plaque were measured respectively.The results of two groups were compared.Results The approximal side angle of plaque of cerebral group was significantly higher than that of none cerebral infarction groups(P<0.05).The sensitivity and specificity for approximal side angle of plaque(cutoff value:13.0°)were 79.1%,71.6%,respectively.There were no significant difference of area stenosis rate and plaque thickness between two groups(P>0.05).Conclusions The approximal side angle of plaque can be regarded as a novel index to assess the stability of plaque.