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 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 explore the safety of the elderly donors in living related donor kidney transplantation. Methods Forty-five elderly donors (51 - 78 years,study group) who underwent ne-phrectomy for living related donor kidney transplantation from April 1993 to December 2007 were retrospectively investigated. Clinical data including serum creatinine (SCr), glomerular filtration rate (GFR) in pre-and post-operation, operation complications and hospital stay time were analyzed and compared with the control group(62 cases, the donors age were younger than 50 years). Results The operations of all living donors were successful. The SCr and GFR in pre-operation were (82.16 ± 10.86)μmol/L, (85. 82±6.26)ml/min(study group)and (78. 66±10. 41)μmol/L, (88. 74±9. 44) ml/min (control group) respectively. There were no significant differences in SCr and GFR between the groups at different time points (P>0. 05). The average hospitalization time was 9 days in study group and 8 days in control group. There were no severe perioperative complications and no renal function failure was found in long-term following-up in study group. Conclusions Age is not the absolute contraindication of donor for living related donor kidney transplantation. The preoperative evaluation and careful operation can ensure the safety of elderly donors.

Objective To discuss the safety and efficacy of simultaneous bilateral percutaneous nephrolithotomy (SBPCNL) for bilateral renal or upper ureteral calculi. Methods Forty-eight cases (26 males, 22 females, 24-57 years )who underwent SBPCNL with pneumatic and ultrasonic power for bilateral renal or upper ureteral calculi were retrospectively reviewed. Clinical data including opera-tion time, blood loss, transfusion rates, length of hospital stay, stone free rate and complications were analyzed. Results The percutaneous renal access was successfully established under ultrasonic guid-ance in all patients. The average operation time was(105±18) rain(range 80-190 min). The average drop in hemoglobin was 21 g/L (range 5-54 g/L), with 5 patients requiring blood transfusion. In 43 patients, a single stage was performed on both sides, while 5 required the second stage PCNL on one side. A single tract was adopted on both sides in 44 patients, while 4 cases of the patients required two tracts on one side. No one required two tracts on both sides or more than one stage on both sides. The stone-clearance rate was 87.5 %. The average hospital stay was 6.5 d. There was no severe complica-tion occurred. Conclusion SBPCNL might be safe and effective for bilateral renal or upper ureteral calculi for selected patients.

Objective To report the experiertce of management of complicated renal stones by percu taneous nephrolithotripsy (PCNL) with pneumatic and ultrasonic power by ultrasound guidance. MethodsThree hundred and eighty two cases(218 males,164 females,4 74 years) who underwent PCNL by u sing the third generation Swiss LithoClast Master for kidney stones from 2004 to 2007 were retrospectivelyreviewed. Clinical data including operation time,stone free rate and complications were analyzed. ResultsPhaseⅠlithotripsy was performed in 397 sides and delayed phaseⅡlithotripsy in 8 sides. Twenty three casesunderwent simultaneous bilateral PCNL. The operation time ranged from 70 to 190 min,average time was(93±11)min. Nine cases needed blood transfusion. Severe complications did not occur during operations.Stone free rate was 91.8% (372/405). Residual stone fragment was found in 33 cases after delayed phase Ⅱlithotripsy and 14 cases received adjuvant extracorporeal shock wave lithotripsy. One hundred and forty sixcases were followed up for 3 to 24 months and showed no recurrence. Conclusion PCNL with pneumaticand ultrasonic power could be an efficient treatment for complicated kidney stones.

OBJECTIVETo investigate the expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) and GM-CSF/IL-3/IL-5 receptor common beta chain (beta c receptor) in an adult patient with idiopathic pulmonary alveolar proteinosis (PAP), so as to demonstrate the possible association of the GM-CSF and beta c receptor with the pathogenesis of human PAP.

METHODSThe GM-CSF levels were measured with a commercial ELISA kit (sensitivity 5 pg/ml) and the beta c receptor expression on the cell surface was detected by flow cytometry analysis. Reverse transcription polymerase chain reaction (RT-PCR) analysis was employed to detect the expression of the GM-CSF mRNA and the beta c receptor mRNA in peripheral blood mononuclear cells and alveolar macrophages. The entire coding regions of the GM-CSF cDNA and the beta c receptor cDNA were sequenced by the Sanger dideoxy-mediated chain termination method to detect possible mutations.

RESULTSThe patient with PAP failed to release the GM-CSF protein either from circulating mononuclear cells or from alveolar macrophages. The expression of the GM-CSF mRNA was normal after the stimulation of lipopolysaccharide, whereas a point mutation at position 382 of the GM-CSF cDNA from "T" to "C" was revealed by cDNA sequencing, which caused a change in amino acid 117 of the protein from isoleucine to threonine. The beta c receptor expression on the cell surface was normal, and the beta c receptor mRNA expression and the sequence of the entire coding region of the beta c receptor were also normal.

CONCLUSIONSThe decreased GM-CSF production is associated with the pathogenesis of human PAP. A point mutation of the GM-CSF cDNA may contribute to the decreased GM-CSF production in our adult PAP patient. The mutation of the beta c receptor in some of paediatric patients with PAP may not be a common problem in adult patients.

DNA, Complementary ; chemistry ; Granulocyte-Macrophage Colony-Stimulating Factor ; analysis ; biosynthesis ; Humans ; Male ; Middle Aged ; Pulmonary Alveolar Proteinosis ; etiology ; metabolism ; RNA, Messenger ; analysis ; Receptors, Cytokine ; biosynthesis ; genetics