Volume 93  ·   Issue 36  ·  2013

To explore the relationship between body fat distribution, insulin resistance,islet β cell function and metabolic disorders in adult population.

From February to November 2012, a total of 174 subjects aged 20-68 years were recruited.Their anthropometric parameters, blood biochemical indices and the results of oral glucose tolerance test (OGTT) and insulin releasing test (IRT) were collected.Body fat distribution was measured with dual energy X-ray absorptiometry (DEXA).

The values of trunk/total fat mass (T/B) and android/gynoid fat mass ratio (A/G) were positively correlated with blood pressure, blood lipid, plasma glucose, insulin resistance index (HOMA-IR) and high-sensitivity C-reactive protein.Compared with the group of normal metabolism, the group of metabolic disorders had higher T/B and A/G (P<0.05).After multiple stepwise regression analysis, the main influencing factors of ln HOMA-IR and ln HOMA-β were T/B and G respectively.Logistic regression showed that A (OR=3.01, 95% CI 1.86-8.17) was a risk factor for diabetes and A/G (OR=2.71, 95% CI 1.75-6.56) a risk factor for dyslipidemia.

Trunk and android fat deposition aggravates insulin resistance, metabolic disorders.And the main influencing factors of insulin resistance and islet β cell function are trunk and gynoid fat respectively.Android fat mass is a major risk factor for glycolipid metabolism.

To compare the morphological and functional differences of human primary preadipocytes from different fat depots and explore the effects of insulin glargine on their proliferation and differentiation.

Primary preadipocytes isolated from human subcutaneous and omental adipose tissue by collagenaseⅠ were passaged in vitro.Inverted phase contrast microscope was used to observe the morphological differences of two kinds of preadipocytes.Then two kinds of preadipocytes were cultured or induced to differentiation with different doses of insulin glargine.The methyl thiazolyl tetrazolium (MTT) assay was used to detect their proliferative differences.Reverse transcription-polymerase chain reaction (RT-PCR) was used to observe the effects of insulin on adipogenic gene expression.

(1) Both preadipocytes could be successfully cultured from adipose tissue and amplified in vitro.Subcutaneous preadipocytes were more slender and proliferated more quickly while omental preadipocytes were polygonal and aged easily. (2) MTT results showed that insulin glargine could inhibit the proliferation of omental preadipocytes in a dose-dependent fashion.After 72 h incubation, compared with negative control, the absorbance (A) value of 1000 nmol/L insulin glargine group decreased greatly (0.144±0.021 vs 0.267±0.040,P<0.01).But it had no effect on subcutaneous preadipocytes (0.305±0.045 vs 0.350±0.037,P>0.05). (3) Insulin at 500 nmol/L was a suitable concentration for inducing differentiation.RT-PCR analysis showed that, for subcutaneous adipocytes, adipogenic genes such as peroxisome proliferator-activated receptor gamma (PPARγ)(F=31.31,P<0.01) and CCAAT enhancer binding proteinα (C/EBPα)(F=9.86, P<0.05) had the highest mRNA expression while preadipocytic gene Pref-1 had the lowest expression at this concentration.But insulin dose had no obvious effect on PPARγor C/EBPαmRNA (P>0.05) for omental adipocytes.

Insulin glargine could inhibit the proliferation of omental preadipocytes, and enhance the differentiation of subcutaneous and omental preadipocytes.

To explore the role and mechanism of SIRT1 (Sirtuin1) in the differentiation of adipocyte.

SIRT1^-/- mice with C57BL/6J gene background were generated and litter-mate wild-type (WT) mice were used as controls.Body weight and fat content were detected and their epididymal fat pads were collected at 28 weeks old of age.The tip part of epididymal fat tissue was incubated in phosphate buffered saline (PBS) containing both BODIPY 558/568 (5μmol/L in PBS) for adipocytes and isolectin Alexa Fluor 488 (40μg/ml in PBS) for endothelial cells overnight.The stained cells were then visualized under confocal microscope.Reconstruction of 3D data sets was accomplished with image processing software Imaris.Immunohistochemistry was used to detect the protein level of endothelial cell marker CD31.Hematoxylin and eosin staining was performed for fixed epididymal fat tissue.Mouse embryonic fibroblast (MEF) cells were prepared from 13-14 days embryos of SIRT1^-/- or WT mice and differentiated into adipocytes.Then oil-red O staining was performed.

Compared with wild-type controls, both body weight (WT 42.1 g±1.6 g vs SIRT1^-/- 25.4 g±1.0 g,P<0.05) and epididymal fat mass (WT 13.4 g±1.0 g vs SIRT1^-/- 7.8 g±0.5 g, P<0.05) were much smaller in the SIRT1^-/- mice.HE staining of fat tissue exhibited a significant reduction in adipocyte size and extracellular matrix in SIRT1-/- mice.However, the adipogenesis ability of MEF cells was significantly enhanced in vitro in SIRT1^-/- MEF cells.Further study found that the density of vascular network decreased by 50% in the tip portion of epididymal fat pads of SIRT1^-/- mice (capillary density:WT 2.92%±0.03% vs SIRT1^-/- 1.34%±0.02%, P<0.05).CD31, a cellular marker of decreased angiogenesis, decreased significantly in epididymal fat pads of SIRT1^-/- mice.

SIRT1 knockout impairs adipocyte differentiation in SIRT1^-/- mice with C57BL/6J gene background through reduced angiogenesis but not adipogenesis.

To explore the prevalence and risk factors for dyslipidemia in diabetics with overweight or obesity.

Diabetics with overweight or obesity were recruited from 62 tertiary and secondary hospitals in Guangdong Province between August 2011 and March 2012.Dyslipidemia was diagnosed as total cholesterol (TC) ≥5.7 mmol/L or triglycerides (TG) ≥1.7 mmol/L or low-density-lipoprotein cholesterol (LDL-C) ≥3.6 mmol/L or high-densitylipoprotein cholesterol (HDL-C) <1.29 mmol/L in females or HDL-C<1.03 mmol/L in males.Binary Logistic regression was used to assess the associations between dyslipidemia and associated risk factors.

Dyslipidemia was detected in 3160/3593 (87.9%) diabetics with overweight or obesity.And the prevalence of hypertriglyceridemia, low blood HDL-C,hypercholesterolemia and high blood LDL-C was 52.5% (1888/3593), 54.1% (1945/3593), 33.1% (1188/3593) and 27.4% (985/3593) respectively.Among those with dyslipidemia, patients with simple and mixed dyslipidemia accounted for 34.1% and 53.9% respectively.In binary Logistic regression analysis, the presence of dyslipidemia were associated with female gender (OR=1.593, 95% CI 1.233-2.057), hemoglobin A1c (Hb A1c) (OR=1.120,95% CI 1.054-1.191), body mass index (OR=1.084,95% CI 1.022-1.150), hypertension (OR=1.331,95% CI 1.033-1.714), history of diabetes (OR=1.586,95% CI 1.186-2.120) and hyperuricacidemia (OR=2.270,95% CI 1.642-3.138).

The prevalence of dyslipidemia is quite high in diabetics with overweight or obesity.The controls of blood pressure, serum uric acid level, blood glucose and body weight may reduce the prevalence of dyslipidemia, prevent and delay the development of cardiovascular complications and reduce the mortality of diabetics with overweight or obesity.

To summarize the experiences of binding pancreaticogastrostomy in pancreatoduodenectomy.

Retrospective analysis was performed for the data of 36 patients undergoing pancreatoduodenectomy from January 2010 to January 2013.Binding pancreaticogastrostomy was used to reconstruct digestive tract.The postoperative complications were observed.And follow-ups were conducted on the patency of anastomotic stoma of pancreatogastrostomy and pancreatic duct.

All operations were successful.There were simple pancreatic fistula (n=7, 19.4%), upper gastrointestinal tract bleeding (n=2, 5.6%), delayed gastric emptying (n=1),intra-abdominal infection (n=1) and infection of incision (n=3).All were cured with conservative therapy.There was no instance of bile leakage, intra-abdominal bleeding, reoperation or in-hospital mortality.

Binding pancreaticogastrostomy causes no serious pancreatic fistula and complications.And it is a safe and reliable reconstructive approach with manageable follow-ups.

To compare the continuous positive airway pressure (CPAP) of automatic titration with that of manual titration.

A total of 58 patients with obstructive sleep apnea and hypopnea syndrome (OSAHS) diagnosed by overnight polysomnography at sleep center of First Affiliated Hospital, Guangzhou Medical University were studied between December 2010 and December 2012.Manual titration was performed under full polysmnography and auto-titration at home for 3-7 nights.

There were 52 males and 6 females with an age range of (48±11) years.CPAP pressure titrated by automatic device (10.0±2.2) cm H₂O (1 cm H₂O=0.098 k Pa) was significantly higher than that titrated manually (7.5±1.5) cm H₂O(P=0.000).Apnea-hyponea index decreased significantly from (54.0±21.0) events/h pre-treatment to (3.8±2.5) events/h posttreatment under manual titration (P<0.01).

CPAP pressure titrated by automatic device is usually higher than that titrated manually.Manual titration should be performed if a patient can not tolerate the CPAP pressure titrated by an automatic device.

To analyze Ki-67 expression and explore its significance in different molecular subtypes of breast invasive ductal carcinoma (IDC).

The expressions of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor-2 (HER-2) and Ki-67 were detected in 126 cases of IDC by immunohistochemical staining.Then the molecular subtype of each case of IDC was determined.Statistical analysis was performed to determine the relationship between Ki-67 expression and the molecular subtypes with clinicopathological features of IDC.

There was no statistically significant difference of Ki-67 expression in age and tumor size (P>0.05).However, significant difference existed in histological grading and lymph node metastasis (P<0.05).The expression level of Ki-67 was negatively correlated with ER expression (r=-0.273, P=0.002) and PR expression (r=-0.242, P=0.007) and positively with HER-2 expression (r=0.245, P=0.006).A low expression of Ki-67 was in Lumianl A subtype (17/17) and high expression in other molecular subtypes.Moreover, the rate of high expression (Ki-67 LI>50%) in each subtype progressively increased with the degree of molecular typing and Ki-67 expression in different molecular subtypes showed significant difference (P<0.05).

The expression level of Ki-67 is correlated with histological grading and molecular type of IDC.High expression of Ki-67 carries poor prognosis.Thus it is necessary to perform a variety of routine clinicopathological examinations, such as Ki-67, ER, PR and HER-2.

To explore the effects of hsa-miR-206 on the proliferation, migration and invasion of breast cancer.

The hsa-miR-206 mimics, inhibitors and their paired negative controls were transfected into human breast cancer cell line MDA-MB-231 by liposome.The proliferation of cell was evaluated by CCK-8 and the migration and invasion was detected by Transwell.Matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), breast cancer metastasis-suppressor 1 (BRMS-1) and connexin 43 (Cx43) were detected by both quantitative polymerase chain reaction (qPCR) and Western blot.The expression of miR-206 was detected by qPCR.Dual luciferase assay was detected to confirm the specific binding sites of miR-206 and Cx43.

(1) The proliferation activity of 206m-group cell (0.74±0.16) was significantly lower than that of control group (1.12±0.23)(t=-3.066,P=0.037) while that of 206i-group cell (1.43±0.26) was higher than that of control group (0.98±0.14)(t=3.635, P=0.022). (2) Transwell tests showed the migration and invasion of 206m-group cell decreased significantly (migration:0.56±0.01 vs 0.63±0.01,t=-23.00,P=0.002; invasion:0.79±0.01 vs 0.99±0.01,t=-21.200,P=0.002), but that of 206i-group cell increased significantly (migration:0.97±0.11 vs 0.61±0.09,t=32.787,P=0.001; invasion:1.10±0.01 vs 0.93±0.05, t=5.167, P=0.035). (3) The expressions of MMP-2,MMP-9 and Cx43 decreased and the expression of BRMS-1 increased in 206mgroup cell and vice versa in 206i group. (4) The expression of miR-206 in lymph node-negative group of clinical breast cancer sample was higher than that of lymph node-positive one.And there was statistical difference (Z=-2.098, P=0.003).And the expression of Cx43 was opposite. (5) Dual luciferase reporter assay confirmed the specific binding sites of hsa-miR-206 and Cx43.

Hsa-miR-206 has negative controls of proliferation, migration and invasion of breast cancer cell by targeting Cx43.

To explore the differences of colonic mucosal-associate bacterial diversity between the patients with Crohn′s disease (CD) and healthy controls.

Eight CD patients and 23 healthy controls were recruited from September 2010 to December 2011 at West China Hospital.One biopsy were taken from cecum of every patient with CD and healthy control by endoscopic examination.The diversity of colonic mucosa-associated microbiota was detected by terminalrestriction fragment length polymorphism (T-RFLP).Hierarchical cluster analysis were performed to compare the similarity of microbial communities between CD patients and healthy controls.Differences of bacterial diversity between two groups were also evaluated.The difference of predominant terminal-restrict fragments (T-RF) were analyzed and the bacterium predicted by predominant T-RFs were identified according to MiCA database.

Hierarchical cluster analysis showed that the mucosal microbial community of CD patients differed from healthy controls.And there were more similarities in the samples of same group than that of different groups.Compared with healthy control group, the richness of mocosal microbiota in CD patients was lower (HaeⅢ: 7±4 vs 10±8, P=0.048; MspⅠ+HaeⅢ: 20±10 vs 24±12, P=0.036).Shannon-Wiener index of CD patients was lower than healthy control (1.7±0.7 vs 2.0±0.5, P=0.220) with no significant difference.Species evenness and Simpson index of CD patients were significantly greater than healthy controls (0.84±0.14 vs 0.77±0.13, P=0.045; 0.25±0.16 vs 0.22±0.15, P=0.038).The T-RF of 37, 40 and 66 bp digested with MspⅠ enzyme predominated in CD patients.Relative quantitative analysis showed 35 bp T-RF digested with MspⅠ was significantly higher in CD patients than that in healthy controls (36.8% (23.0%, 55.4%) vs 14.3% (9.5%, 19.5%), P=0.001), and 74, 141, 486, 490 bp T-RFs were all significantly lower than healthy controls (3.2% (1.3%, 5.1%) vs 10.2% (5.4%,17.3%), P=0.001; 4.5% (1.7%, 7.1%) vs 10.8% (5.9%, 21.1%), P=0.007; 4.2% (1.6%, 5.3%) vs 7.6% (5.9%, 9.3%), P=0.022; 3.6% (2.4%, 6.1%) vs 18.3% (9.9%, 43.2%), P=0.008).The mucosal bacterial community composition in CD patients was predominated by Firmicutes, Proteobacterium and Actinobacterium.Compare with healthy control, Bacteroides were significantly reduced in CD patients while Firmicutes (e.g.Enterobacter sp.) and Actinomycetaceae significantly increased.

Dysbiosis of mucosal microbiota occurs in CD with decreases of richness and biodiversity.Increased Enterobacter sp., Actinobacterium and decreased Bacteroides may play an important role in the pathogenesis of CD.

To explore the effect of toxicity of sunitinib on the clinical outcome of patients with advanced renal cell carcinoma (RCC).

A total of 136 patients with advanced RCC were treated with sunitinib from 2008 to 2011.There were 91 males and 45 females with an average age of 56 years.Their 6-week therapy cycle was 4 weeks of sunitinib 50 mg daily followed by 2-week off-treatment (schedule 4/2).The median follow-up time was 15 months.Correlation between toxicities and overall survival (OS) was evaluated in a Cox model using log-transformed levels after adjusting for MSKCC model.Log-rank test and Cox proportional hazard model were used to assess the value of drug toxicity as the prognostic factors.

The increased hemoglobin on cycle 1 day 14 (HR:0.950, 95% CI:0.923-0.978) and the increased lymphocytes on cycle 1 days 28 and 42 (HR: 0.405,95% CI:0.203-0.809,HR:0.394,95% CI:0.179-0.867) were significantly associated with OS (P_adj=0.001, 0.014 and 0.022 respectively).Hypertension class Ⅲ/Ⅳ (HR:0.066,95% CI: 0.008-0.582), and the number of neutrophils screening and lymphocyte count ratio (HR:2.537, 95% CI:1.182-5.404) were the survival prognosis independent predictors.

Early hematopoietic toxicities may potentially predict the outcomes of advanced RCC after a therapy of sunitinib.

To explore the epidemiological profiles of invasive fungal disease (IFD) in hospitalized patients with hematological diseases during 2007- 2012.

A total of 419 IFD patients with hematological diseases from January 2007 to December 2012 were reviewed.All of them were analyzed with regards to diagnostic levels, infection sites and various related factors.

(1) A total of 233 cases (55.61%) were preliminarily identified as IFD,140 cases (33.41%) had a clinical diagnosis and 46 cases (10.98%) were confirmed cases of IFD. (2) Among 46 confirmed cases of IFD, there were agranulocytosis (n=43) and aspergillosis infection (n=36). (3) Respiratory tract was the most frequent infection site in all IFD patients (85.20%). (4) And chemotherapy-induced agranulocytosis was a major reason for IFD patients with hematological diseases.The number of IFD patients without chemotherapy had a rising trend. (5) The age group of IFD was during 41-60 years old. (6) All of them stayed on antibiotic therapy at the diagnosis of IFD.The numbers of antibiotics were two (205 cases,48.93%) and three (179 cases,42.72%). (7) The peak incidence of IFD was recorded in January, July and December.And June was another lower peak.

Agranulocytosis is the main reason for IFD patients with hematological disease.The data is important and valuable for the early diagnosis and therapy of IFD patients with hematological disease.

To explore the independent risk factors associated with one-year mortality in patients with atrial fibrillation (AF).

This study consecutively enrolled AF patients presenting to an emergency department at 20 Chinese hospitals from November 2008 to October 2011.Their baseline data and therapies were recorded.They were followed up for one year.Their major cardiovascular outcomes were recorded.And the predictors of one-year mortality were identified by uni- and multi-variate Cox regression analysis with baseline, therapy variables and follow-up therapy variables.

The one-year all-cause mortality was 13.8% among a total of 2016 AF patients.They were divided into mortality group (A,n=279) and survival group (B,n=1737).The baseline data of two groups were analyzed.The group A patients were older ( (76.1±11.6) vs (67.2±13.1) years, P<0.01) and had smaller body mass index compared with group B ((23.7±3.6) vs (22.3±3.4) kg/m²,P<0.01); the proportion of permanent AF and CHADS² score≥2 points was higher in the group A (71.8% vs 47.5%, P<0.01).History of heart failure, previous stroke, left ventricular systolic dysfunction, diabetes, dementia and chronic obstructive pulmonary disease (COPD) were in a higher proportion of group A (51.2% vs 35.1%, 26.3% vs 17.6%, 26.7% vs 17.9%, 21.0% vs 14.6%, 6.0% vs 1.6%,21.4% vs 10.1%,all P<0.01).With regards to drug treatment, usage of diuretics, digoxin and other anticoagulants (heparin, etc), the values were greater in group A (50.9% vs 42.2%, 41.3% vs 34.7%, 10.0% vs 5.9%, all P<0.01).The Kaplan-Meier survival curves showed that the mortality rate increased along with rising CHADS² score.Multi-variate Cox regression analysis showed that age (HR=1.053, 95% CI:1.040-1.066), permanent AF (HR=1.374, 95% CI:1.003-1.883), history of heart failure (HR=1.385, 95% CI:1.009-1.901), previous stroke (HR=1.345, 95% CI: 1.009-1.795), COPD (HR=1.379, 95% CI: 1.030-1.848), unused angiotensin Ⅱ receptor blocker (ARB) (HR=1.955,95% CI: 1.349-2.832), aspirin unused (HR=1.770,95% CI:1.375-2.278) and warfarin unused (HR=3.262, 95% CI: 1.824-5.834) were independent risk factors for one-year mortality of AF patients.

Age, history of heart failure, previous stroke, COPD history, ARB unused,aspirin and warfarin unused are independent risk factors for one-year all-cause mortality of AF patients.

To explore the protective role of osthole in intestinal ischemia-reperfusion (I/R) injury in mice and examine its underlying mechanism.

A murine model of intestinal I/R injury was established with clamping of superior mesenteric artery for 120 min and then clamping was relieved for 60 min.Forty-five SD male mice weighing 27- 31 g were randomly divided into 3 groups (n=15 each) :sham group (S), I/R injury group (I/R) and I/R plus osthole treatment group (Ost+).Intestinal wet/dry weight ratio, superoxide dismutase (SOD), malondialdehyde (MDA) in serum were examined by colourimetric assay and diamine oxidase (DAO) was examined by automatic biochemical analyzer, the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-2 were examined by enzyme-linked immunosorbent assay (ELISA).Intestinal barrier permeability was detected by Evans blue (EB) test.One-way ANOVA was used to analyze all experimental data variance.

Intestinal tissues wet/dry weight ratios, Evans blue content and Chiu′s score of I/R group mice were significantly higher than those of Sand Ost+ groups (0.80%±0.03% vs 0.77%±0.02% & 0.78%±0.02%, (0.11±0.04) vs (0.05±0.02) & (0.06±0.02) μg/mg, 3.42±0.73 vs 0.87±0.35 & 2.63±0.58,P<0.05 or P<0.01).Serum level of DAO, MDA,IL-1β & TNF-α of I/R group mice were significantly higher than those of S and Ost+ groups ((18.9±4.0) vs (14.5±2.3) & (16.0±2.6) U/L, (8.4±1.2) vs (6.9±1.7) & (6.1±2.4) μmol/L, (93±6) vs (51±4) & (67±5) ng/L, (467±31) vs (235±21) & (323±30) ng/L, P<0.01 or P<0.05).Serum SOD activity and IL-2 level were significantly lower than those of Sand Ost+ groups ((75±7) vs (93±16) & (89±5) U/ml, (95±16) vs (198±14) & (139±11) ng/L, all P<0.05).All parameters showed no significant difference between S and Ost+ groups (all P>0.05).

Treatment of osthle may protect murine intestinal tissue against intestinal I/R injury.And the mechanisms may be due to its actions of preventing oxygen stress and inflammatory responses.

To screen,identify and verify the serum specific protein of neuroblastoma in a tumor-bearing murine model and speculate about the source of cytochrome C.

NB cells (KP-N-NS) were inoculated subcutaneously into nude mice.And the sera samples of tumor-bearing mice (observation group,n=14) and controls (control group, n=25) were collected at 4 weeks to screen for and identify differentially expressed proteins.The method of surface-enhanced laser desorption-ionization time-of-flight mass spectrometry (SELDI-TOF-MS) was employed to screen the serum specific protein of neuroblastoma between two groups.Then serum protein targets were purified by high-performance liquid chromatography (HPLC) and identified by LC-MS/MS (LTQ).

By comparing protein peaks among two sera groups, we identified the peak (11 609) showing significant differential expression between two groups.The expression of peak (11 609) was 3338.4±1410.9 in observation group and 59.8±40.7 in control group.This peak was identified as murine cytochrome C.

Cytochrome C is a serum specific protein for neuroblastoma tumor and it comes from the apoptosis of non-tumor cells.

To explore the role of altered characteristics of intestinal dendritic cell (DC) in the induction of visceral hyperalgesia through the activation of mast cells in a rat model of irritable bowel syndrome (IBS).

A total of 20 Sprague-Dawley rats were divided into modeling and control groups (n=10 each).The IBS rat model was established by combining colorectal distention with restraint stress.Abdominal withdrawal reflex (AWR) was employed to evaluate visceral sensitivity.The surface marker of intestinal DC was analyzed by immunohistochemistry.Toluidine blue staining was used to determine the number of mast cells (MC).The expressions of interleukin (IL) -4 and IL-9 in colonic mucosa were measured by enzyme-linked immunosorbent assay (ELISA) and the level of proteinase-activated receptor-2 (PAR-2) was measured by Western blot.Mesenteric lymph node (MLN) DC and splenic CD4⁺/CD8⁺T cells were isolated and purified by magnetic label-based technique.Cytokine production of MLN DC co-culturing with CD4⁺or CD8⁺T cells was determined.

The number of colonic MC in modeling group was more than that in control group ((2.73±0.21) vs (1.13±0.10), P=0.000).The expressions of PAR-2, IL-4 and IL-9 in colonic mucosa were all higher than those in control group (2.13±0.81 vs 0.42±0.29, (7.2±1.2) vs (3.3±1.0) pg/ml, (7.3±1.3) vs (5.2±0.6) pg/ml, P=0.026, 0.000, 0.001).Co-cultured MLN DC with CD4⁺T cells showed a predominant IL-4 secretion in the modeling group ((1.22±0.33) vs (0.80±0.48) pg/ml, P=0.000).

Increased colonic DC may stimulate CD4⁺T cells to secrete a high level of IL-4 to cause the degranulation of mast cells and the generation of visceral hypersensitivity in IBS rats.