Molecular and Cellular Biochemistry

, Volume 362, Issue 1, pp 43–53

Antinociceptive effect of VSL#3 on visceral hypersensitivity in a rat model of irritable bowel syndrome: a possible action through nitric oxide pathway and enhance barrier function

Authors

  • Cong Dai
    • Department of GastroenterologyFirst Affiliated Hospital, China Medical University
  • Stefano Guandalini
    • Section of GastroenterologyCeliac Disease Center, University of Chicago Comer Children’s Hospital
  • De-Hui Zhao
    • Department of GastroenterologyFirst Affiliated Hospital, China Medical University
    • Department of GastroenterologyFirst Affiliated Hospital, China Medical University
Article

DOI: 10.1007/s11010-011-1126-5

Cite this article as:
Dai, C., Guandalini, S., Zhao, D. et al. Mol Cell Biochem (2012) 362: 43. doi:10.1007/s11010-011-1126-5

Abstract

Irritable bowel syndrome (IBS) is a functional bowel disorder characterized by visceral hypersensitivity and altered bowel function. There are increasing evidences suggested that VSL#3 probiotics therapy has been recognized as an effective method to relieve IBS-induced symptoms. The aim of this study was to examine the effects of VSL#3 probiotics on visceral hypersensitivity (VH), nitric oxide (NO), fecal character, colonic epithelium permeability, and tight junction protein expression. IBS model was induced by intracolonic instillation of 4% acetic acid and restraint stress in rats. After subsidence of inflammation on the seventh experimental day, the rats were subjected to rectal distension, and then the abdominal withdrawal reflex and the number of fecal output were measured, respectively. Also, colonic permeability to Evans blue was measured in vivo, and tight junction protein expression was studied by immunohistochemistry and immunoblotting method. Rats had been pretreated with VSL#3 or aminoguanidine (NOS inhibitor) or VSL#3+ aminoguanidine before measurements. The rats at placebo group showed hypersensitive response to rectal distension (P < 0.05) and defecated more stools than control rats (P < 0.05), whereas VSL#3 treatment significantly attenuated VH and effectively reduced defecation. Aminoguanidine reduced the protective effects of VSL#3 on VH. A pronounced increase in epithelial permeability and decreased expression of tight junction proteins (occludin, ZO-1) in placebo group were prevented by VSL#3, but not aminoguanidine. VSL#3 treatment reduce the hypersensitivity, defecation, colonic permeability and increase the expression of tight junction proteins (occludin, ZO-1). As the part of this effect was lowered by NOS inhibitor, NO might play a role in the protective effect of VSL#3 to some extent.

Keywords

Irritable bowel syndromeProbioticVSL#3Intestinal barrierTight junctionVisceral hypersensitivityNitric oxide

Introduction

Probiotics have been defined as living organisms in food and dietary supplements which improve the health of the host beyond inherent basic nutrition [14]. Preventive and curative efficacy of probiotic treatments has been described in several gastrointestinal pathologies; and in the past decade, encouraging results have been obtained in inflammatory bowel disease using different strains. For example, treatment with several lactobacilli strains has been found to reduce the severity of experimental colitis in animals [57]. And some studies have shown that Lactobacillus farciminis can release spontaneously nitric oxide (NO) into the colonic lumen and reduce the severity of trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats [8]. Other studies have reported enhancement of intestinal barrier function by strengthening tight junctions between enterocytes and thereby preventing an increase in paracellular permeability and subsequent bacterial translocation [913]. And some studies showed that VSL#3 probiotics shares these properties as it prevents bacterial translocation and the increase in colonic paracellular permeability in dextran sodium sulfate (DSS)-induced colitis in rats [14, 15].

Clinically, probiotic therapy has also been recognized as an effective method to relieve IBS-induced symptoms [1618]. Irritable bowel syndrome (IBS) is a functional bowel disorder with unknown etiology and is characterized by abdominal pain or discomfort, diarrhea, constipation, gas, bloating, and nausea [19]. According to Rome III Criteria [20, 21], the most recently used criteria, IBS can be diagnosed based on at least 3 months, with onset at least 6 months previously of recurrent abdominal pain or discomfort associated with 2 or more of the following: (1) Improvement with defecation; (2) Onset associated with a change in frequency of stool; and (3) Onset associated with a change in form (appearance) of stool. Additionally, IBS patients are further subdivided through a classification based on the predominant symptoms and stool characteristics into diarrhea-predominant IBS (D-IBS), constipation-predominant IBS, and alternating symptoms [22].

Visceral hypersensitivity (VH) is a consistent finding in a considerable proportion of patients with IBS and may provide a physiological basis for the development of IBS symptoms. Gut hypersensitivity may lead to alterations in gut motility by disturbing regulatory reflex pathways and secretory functions. Some studies have demonstrated the important role of NO in pain transmission and an antinociceptive action of NO in visceral or peritoneal pain [2326].

Another character of D-IBS patients is an increase in intestinal permeability [27, 28]. The altered permeability of the epithelium is thought to increase the load of bacterial and dietary antigens in the lamina propria leading to the generation of diarrhea and visceral hypersensitivity [27]. Some studies showed that the redistribution and downregulation of several tight junction proteins were the molecular basis of increased epithelial permeability. The tight junction complex is constituted by transmembrane proteins, like occludin, and by linker proteins, like zonula occludens-1 (ZO-1), that are affiliated with the actin cytoskeleton [29, 30].

VSL#3 is a proprietary preparation consisting of a high concentration of eight different probiotic bacteria species. Several clinical studies have shown that VSL#3 improves symptoms such as abdominal pain or discomfort, diarrhea, and bloating in IBS patients [17, 18]. However, the exact mechanism of its action is still unknown. Recently, it has been reported that increasing the NO level in the extracellular space of the target tissue is one of the considerable involved mechanisms and this involvement has been clarified in recent molecular studies [31].

Based on this background, the aim of our study was to evaluate: (1) whether VSL#3 treatment prevents the visceral hypersensitivity to colorectal distension (CRD) and the increases of colonic paracellular permeability; (2) the involvement of NO in these effects; and (3) the effect of VSL#3 treatment on changes in tight junction proteins expression and distribution.

Materials and methods

Animals

Male Wistar rats, weighing 200–220 g, were obtained from the Experiment Animal Center, China Medical University. Rats were housed in plastic cages containing corn chip bedding and were maintained on a 12-h light–12-h dark cycle (07:00–19:00 h, light cycle; 19:00–07:00 h, dark cycle) with a room temperature of 22 ± 1°C and a humidity of 65–70%. Water and food were available ad libitum. All rats in the study were used strictly in accordance with the National Institutions of Health Guide for the Care and Use of Laboratory Animals. Our research has got the approval of China Medical University Animals Committee (Number: 2010-1423).

Induction of IBS model

IBS symptoms were produced as described previously [32]. Firstly, rats were lightly anesthetized with ether after an overnight fast, and colitis was induced by intracolonic instillation of 1 ml 4% acetic acid at 8 cm proximal to the anus for 30 s. Secondly, 1 ml phosphate-buffered saline was instilled to dilute the acetic acid and flush the colon. Rats were left to recover from colitis for 6 days and used for experiments 7 days post-induction of colitis [32]. Thirdly, the rats were placed in restraint cages (5 × 5 × 20 cm) for 3 h at room temperature [32].

Experimental protocol

Ten healthy rats without treatment served as controls.

In the placebo group, IBS model was induced as described above. And ten rats were treated once daily with 15 mg of placebo (corn starch alone) dissolved in 200 μl of PBS and administered via gastric tube after induction of IBS.

In the VSL#3 group, ten rats were treated once daily with VSL#3 after induction of IBS. Each sachet of VSL#3 (2.5 g) contained 450 billion freeze-dried bacteria. VSL#3 (15 mg, containing 2.7 billion bacteria) was dissolved in 200 μl of PBS and administered via gastric tube.

In the aminoguanidine group, ten rats were treated once daily with the aminoguanidine (100 mg/kg) via intraperitoneal injection after induction of IBS.

In the VSL#3+ aminoguanidine group, ten rats were treated once daily with VSL#3 via gastric tube after induction of IBS, and then were treated once daily with the aminoguanidine (100 mg/kg) via intraperitoneal injection.

These experimental operations were last 7 days. At 7 days, the feces excreted by each group were divided into three types—hard pellet, soft pellet, and formless stool—and counted separately.

Rectal distension procedure

At 7 day above experimental operations, ten rats from each group were used for studying visceral sensitivity to rectal distension. A disposable silicon balloon-urethral catheter for pediatric use was used for this purpose. The maximal inflation volume for the balloon was 1.0 ml and the length of the maximally inflated balloon was 1.2 cm. After an overnight fast, the rats were lightly anesthetized with ether, and the balloon was carefully inserted into the rectum until the pre-marked line on the catheter (2 cm distal from the end of the balloon) was positioned to the anus, and then the catheter was taped to the base of the tail to prevent displacement. After this procedure, the rats were placed in a transparent cubicle (20 × 8 × 8 cm) on a mirror-based elevated platform while still sedated and were allowed to recover and acclimate for a minimum of 30 min before testing. The catheter was connected to a pressure transducer via a 3-way connector. The signals from pressure transducer were processed and recorded on an IBM-compatible computer.

After the animals were fully awaken and acclimatized, ascending-limit phasic distension (0.1, 0.2, 0.3, 0.4, 0.6, 0.8, and 1 ml) was applied for 30 s every 4 min. The balloon was distended with pre-warmed (37°C) water. We chose this protocol as hypersensitivity was reported to be best elicited by rapid phasic distension [24]. In this experiment, the abdominal withdrawal reflex (AWR) was semiquantitatively scored as previously described [33] (Table 1).
Table 1

The AWR score standard

Score

The abdominal withdrawal reflex (AWR)

0

No behavioral response to distension

1

Brief head movements followed by immobility

2

Contraction of abdominal muscle without lifting of abdomen

3

Lifting of abdomen

4

Body arching and lifting of pelvic structure

After the experiments, the balloon was withdrawn and immersed in 37°C water. Since the compliance of balloon was not infinite, we measured intraballoon pressure at each distension volume in 37°C water, and digitally subtracted the value from that recorded during the rectal distension experiment to calculate the intrarectal pressure.

In vivo permeability to Evans blue

In rats of each group, colonic permeability to Evans blue was measured in vivo. This is a well-validated method for assessing epithelial permeability [34, 35]. After being lightly anesthetized with ether, spontaneously breathing rats were placed in a supine position on a heating pad and a laparotomy was performed. A small polyethylene tube was inserted into the proximal ascending colon and secured by a ligature. Via this tube the colon was gently flushed until all stools were rinsed out, and 1 ml of Evans blue 1.5% (Sigma-Aldrich) in PBS was instilled into the colon and left in place for 15 min. Then, the colon was rinsed with PBS, until the rectal washout was clear. Rats were euthanized, and the colon was rapidly taken out. It was rinsed again with several milliliters of PBS, followed by 1 ml of 6 mM N-acetylcysteine to eliminate dye sticking in the colonic mucus. The colon was opened and rinsed once more with PBS. The whole colon was placed in 2 ml N,N-dimethylformamide for 12 h to extract the Evans blue dye. The dye concentration in the supernatant was measured spectrophotometrically at 610 nm and given as extinction per gram colonic tissue.

Staining process of HE

A 0.5-cm sample was selected in the descending colon (5 cm above anus) and cleaned with normal saline, fixed with 10% in formalin, dehydrated, paraffin embedded, continuously slided, deparaffinized and rehydrated, hematoxylin and eosin staining, dehydrated in 70%, 90%, 95% ethanol, cleared in xylene, mounted in Permount or Histoclad, and checked under microscope to observe morphological change of rats’ colonic membrane.

Immunohistochemistry

Paraffin slides were deparaffinized and rehydrated, deparaffinized in xylene I, II, III for 10 min, dehydrated in 95%, 90%, 70% ethanol for 2 min, and then tissues sections were rinsed three times for 5 min each in 0.01 mol/l phosphate-buffered saline (PBS, pH 7.4), pre-incubated for 15 min with 0.3% H2O2, and then pre-incubated for 15 min with 5% normal goat serum and incubated overnight at 4°C with rabbit anti-occludin polyclonal antibody (rabbit anti-rat, 1:400). After rinsing with 0.01 mol/l PBS, sections were incubated for 15 min with secondary goat anti-rabbit immunoglobulin G at 37°C, rinsed three times for 5 min each in 0.01 mol/l PBS, incubated for 15 min with tertiary antibody at 37°C, and rinsed 3 × 5 min in 0.01 mol/l PBS. A peroxidase reaction was performed to visualize occludin immunolabeling by incubating with 0.05% 3,3-diaminobenzidine tetrahydrochloride for 3 min and stopping the reaction with 0.01 mol/L PBS. To assess antibody specificity, incubation with the primary antibody was omitted for some sections and no significant staining was observed in this case. Positive results showed brown and dark brown.

Similarly, tissues sections were incubated overnight at 4°C with rabbit anti-ZO-1 polyclonal antibody (rabbit anti-rat, 1:400), and then were stained by the same immunohistochemistry method. The positive expressing areas and density of occludin and ZO-1 were counted by Image-Pro Plus 6.0 software.

Immunoblotting

Using snap-frozen colon specimens with histologically intact epithelium, we stripped the mucosa from the underlying submucosal tissue, homogenized and sonicated it, and transferred it into ice-cold lysis buffer with a protease inhibitor cocktail for 60 min. Lysates were centrifuged and the protein content of the supernatant was determined by using the BCA protein assay kit. Depending on the antibody used, equivalent protein concentrations of 10–75 μg were loaded in each lane of SDS–polyacrylamide gels. Electrophoretically separated samples were transferred to an Immobilon transfer membrane. Membranes were incubated with the respective primary antibodies and a corresponding peroxidase-conjugated secondary antibody. Blots were visualized by chemiluminescence using Immobilon Western Chemiluminescent HRP substrate. After detection of specific tight junction, all membranes were stripped with Restore Western Blot Stripping Buffer, and an immunoblot for β-actin was performed to ensure equal protein loading in each lane. Densitometry was performed for each detected protein in each group.

Statistical analysis

Data were expressed as mean ± SD. Significant difference between the three groups in the values (AWR score) at each distension volume was statistically analyzed using ANOVA. The relationship between the intraballoon volume and intrarectal pressure was determined by linear regression analysis, and the estimated slope coefficients and intercepts were compared between groups using ANOVA. The number of fecal output was compared using ANOVA and further analyzed using Bonferroni or Tamhane’s T2 test. Immunohistochemistry and Western blot data were analyzed by one-way ANOVA. An LSD and S–N–K post hoc test was used after ANOVA analysis where appropriate. Differences with P < 0.05 were considered to be significant. Multiple comparisons between the groups were corrected by SPSS 16.0 statistics software.

Results

Defecation

Rats in the placebo group defecated 3.800 ± 0.632 hard pellets, 3.200 ± 0.632 soft pellets, 2.700 ± 0.483 formless stool, and a total of 9.700 ± 1.059 feces, while rats in the control group defecated 0.500 ± 0.527 hard pellets, 0.300 ± 0.483 soft pellets, 0.100 ± 0.316 formless stool, and a total of 0.900 ± 0.316 feces. Rats in the VSL#3 group defecated 2.400 ± 0.516 hard pellets, 1.800 ± 0.422 soft pellets, 1.200 ± 0.422 formless stool, and a total of 5.400 ± 0.843 feces. Rats in the aminoguanidine group defecated 3.600 ± 0.516 hard pellets, 2.800 ± 0.422 soft pellets, 2.600 ± 0.516 formless stool, and a total of 9.000 ± 0.943 feces. Rats in VSL#3+ aminoguanidine group defecated 3.500 ± 0.527 hard pellets, 2.600 ± 0.516 soft pellets, 2.400 ± 0.516 formless stool, and a total of 8.500 ± 0.527 feces.

The number of hard pellets, soft pellets, formless stool, and a total of feces output in placebo group and aminoguanidine group were significantly larger than the corresponding values in control group, VSL#3 group, and VSL#3+ aminoguanidine group (P < 0.05).

As shown in Fig. 1, rats in placebo group had significantly increased defecation (P < 0.05). VSL#3 and VSL#3+ aminoguanidine can effectively reduced defecation (P < 0.05), but aminoguanidine cannot effectively reduced defecation (P > 0.05).
https://static-content.springer.com/image/art%3A10.1007%2Fs11010-011-1126-5/MediaObjects/11010_2011_1126_Fig1_HTML.gif
Fig. 1

Number of hard pellets, soft pellets, formless stool, and the total number of defecations in each group

Visceral sensitivity

The nociceptive threshold (i.e., distension volume that produced the AWR of score 2) was 0.6 ml in control group and VSL#3 group, but it was lowered to around 0.3 ml in the placebo group, the aminoguanidine group, and VSL#3+ aminoguanidine group. The AWR score in rats at placebo group (2.000 ± 0.001, 2.800 ± 0.422, 3.200 ± 0.422, 3.300 ± 0.483) was significantly higher than that in control group (0.500 ± 0.527, 1.200 ± 0.422, 2.100 ± 0.316, 2.900 ± 0.316), VSL#3 group (0.600 ± 0.516, 1.400 ± 0.516, 2.300 ± 0.483, 3.000 ± 0.001), the aminoguanidine (2.100 ± 0.316, 2.900 ± 0.316, 3.300 ± 0.483, 3.400 ± 0.516) and VSL#3+ aminoguanidine group (1.900 ± 0.316, 2.700 ± 0.483, 3.100 ± 0.316, 3.200 ± 0.422), when distension volume was 0.3, 0.4, 0.6, and 0.8 ml (P < 0.05). And the AWR score in rats at VSL#3 group was significantly lower than that in the aminoguanidine and VSL#3+ aminoguanidine group (P < 0.05) (Fig. 2). In conclusion, rats in the placebo group showed hypersensitive response to the ascending-limit phasic rectal distension, and VSL#3 can effectively reduced visceral hypersensitivity. But the aminoguanidine and VSL#3+ aminoguanidine cannot effectively reduced visceral hypersensitivity.
https://static-content.springer.com/image/art%3A10.1007%2Fs11010-011-1126-5/MediaObjects/11010_2011_1126_Fig2_HTML.gif
Fig. 2

Summarized plots representing the rectal distension-induced AWR in each group

In order to examine whether the visceral hypersensitivity in rats was related to changes in rectal compliance, we compared the intraballoon volume-intrarectal pressure relationship of these five groups. The distension volume from 0.1 to 1.0 ml and the corresponding value of calculated intrarectal pressure were plotted for regression analysis. Intrarectal pressure was linearly increased as the balloon inflated (r = 0.999, P < 0.001 in control group; r = 0.997, P < 0.001 in placebo group; r = 0.993, P < 0.001 in VSL#3 group; r = 0.995, P < 0.001 in aminoguanidine group; r = 0.994, P < 0.001 in VSL#3+ aminoguanidine group). The fitted functions of the five groups were not significantly different from each other (slope coefficient: 72.059 ± 0.392 in control group; 73.374 ± 0.706 in placebo group; 74.110 ± 1.078 in VSL#3 group; 73.258 ± 0.848 in aminoguanidine group; 73.791 ± 1.005 in VSL#3+ aminoguanidine group; intercept: 14.557 ± 0.225 in control group; 12.847 ± 0.405 in placebo group; 14.661 ± 0.618 in VSL#3 group; 14.646 ± 0.486 in aminoguanidine group; 14.144 ± 0.576 in VSL#3+ aminoguanidine group) (Fig. 3).
https://static-content.springer.com/image/art%3A10.1007%2Fs11010-011-1126-5/MediaObjects/11010_2011_1126_Fig3_HTML.gif
Fig. 3

Summarized plots representing the relationship between intraballoon volume and intrarectal pressure in each group

Colonic epithelial permeability

Compared with rats in the control group (0.440 ± 0.108), a significant increase of Evans blue uptake into the colonic mucosa of placebo group rats (3.660 ± 0.178) was observed. And this significant increase of Evans blue uptake can be effectively prevented by VSL#3 and VSL#3+ aminoguanidine (0.770 ± 0.142, 0.800 ± 0.105). But this significant increase of Evans blue uptake cannot be effectively prevented by aminoguanidine (3.640 ± 0.126) (Fig. 4). This demonstrates that VSL#3 probiotics can ameliorates the leakiness of the colonic epithelium in IBS rats, and aminoguanidine cannot effectively prevent this effect.
https://static-content.springer.com/image/art%3A10.1007%2Fs11010-011-1126-5/MediaObjects/11010_2011_1126_Fig4_HTML.gif
Fig. 4

Colonic permeability to Evans blue in each group

Histology of colonic tissue

In each group, colonic membrane structure was intact, without epithelial sloughing, trimmed glands, and without red blood cells (RBC) and inflammatory cells appearing in the mucosa (Fig. 5).
https://static-content.springer.com/image/art%3A10.1007%2Fs11010-011-1126-5/MediaObjects/11010_2011_1126_Fig5_HTML.jpg
Fig. 5

a HE stain of control group rat colonic tissue (×10); b HE stain of placebo group rat colonic tissue (×10); c HE stain of VSL#3 group rat colonic tissue (×10); d HE stain of aminoguanidine group rat colonic tissue (×10); e HE stain of VSL#3+ aminoguanidine group rat colonic tissue (×10); f Immunohistochemistry for occludin of control group rat colonic membrane (×10); g Immunohistochemistry for occludin of placebo group rat colonic membrane (×10); h Immunohistochemistry for occludin of VSL#3 group rat colonic membrane (×10); i Immunohistochemistry for occludin of aminoguanidine group rat colonic membrane (×10); j Immunohistochemistry for occludin of VSL#3+ aminoguanidine group rat colonic membrane (×10); k Immunohistochemistry for ZO-1 of control group rat colonic membrane (×10); l Immunohistochemistry for ZO-1 of placebo group rat colonic membrane (×10); m Immunohistochemistry for ZO-1 of VSL#3 group rat colonic membrane (×10); n Immunohistochemistry for ZO-1 of aminoguanidine group rat colonic membrane (×10); o Immunohistochemistry for ZO-1 of VSL#3+ aminoguanidine group rat colonic membrane (×10)

The expression and distribution of tight junction proteins

In order to examine the effect of VSL#3, aminoguanidine and VSL#3+ aminoguanidine on protein expression and distribution of tight junction proteins, we first chose the method of immunohistochemistry. In the control group, the tight junction proteins occludin and ZO-1 were appropriately localized at the colonic epithelial apical cell–cell contacts both at the surface and in crypts, consistent with their distribution in tight junctions. But in placebo group and aminoguanidine group, there was a substantial loss of occludin, ZO-1 from tight junctions. This loss was manifested by discontinuities in membrane staining and a reduction in staining intensity, which in some areas led to a complete loss of staining. And VSL#3 could completely prevent the loss of those two tight junction proteins (occludin and ZO-1) (Fig. 5). The areas expressing occludin and ZO-1, as well as the density of occludin and ZO-1 in colonic tissue in placebo group, aminoguanidine group, are lower than in control group; while the areas expressing occludin and ZO-1, as well as the density of occludin and ZO-1 in VSL#3 group and VSL#3+ aminoguanidine group, are remarkably higher than in placebo group and aminoguanidine group (Table 2).
Table 2

Occludin and ZO-1 expression levels in rats’ colonic membrane (\( \bar{x} \) ± SD)

Group

n

Occludin

ZO-1

Area (μm2)

Density

Area (μm2)

Density

Control

10

9449.00 ± 173.05

42907.00 ± 2165.02

9372.40 ± 235.54

42696.20 ± 1626.39

Placebo

10

7940.80 ± 188.10*

37081.10 ± 2016.05*

6890.90 ± 197.85*

36480.70 ± 2301.59*

VSL#3

10

9145.10 ± 102.63*#

39931.20 ± 832.18*#

8816.30 ± 96.70*#

39376.50 ± 1210.46*#

Aminoguan

10

8243.80 ± 174.20*

37466.20 ± 1448.25*

7124.88 ± 165.47*

37290.70 ± 1845.24*

VSL#3+ Amin

10

9085.30 ± 143.27*#

39622.20 ± 794.32*#

8684.21 ± 143.20*#

38944.52 ± 1724.53*#

Compared with control group *P < 0.05, compared with placebo group #P < 0.05

To extend our observations of dramatic changes of tight junction proteins expression, those two proteins were analyzed by Western blotting in the colons of the five groups. Compared with rats in control group (0.904 ± 0.022, 0.509 ± 0.017), significant reductions in total protein for occludin and ZO-1 were observed in rats in placebo group (0.338 ± 0.023, 0.246 ± 0.017) and aminoguanidine group (0.328 ± 0.014, 0.236 ± 0.016). And the tight junction proteins expression in rats in VSL#3 group (0.837 ± 0.021, 0.538 ± 0.019) and VSL#3+ aminoguanidine group (0.727 ± 0.018, 0.474 ± 0.016) were significantly higher than those in placebo group and aminoguanidine group (Fig. 6, 7).
https://static-content.springer.com/image/art%3A10.1007%2Fs11010-011-1126-5/MediaObjects/11010_2011_1126_Fig6_HTML.gif
Fig. 6

Western blots of tight junction proteins (occludin and ZO-1); 1 control group; 2 placebo group; 3 VSL#3 group; 4 aminoguanidine group; 5 VSL#3+ aminoguanidine group

https://static-content.springer.com/image/art%3A10.1007%2Fs11010-011-1126-5/MediaObjects/11010_2011_1126_Fig7_HTML.gif
Fig. 7

Western blot densitometry of tight junction proteins (occludin and ZO-1) in each group. Immunoblots for β-actin were performed for each membrane to normalize epithelial protein loading between samples

Discussion

In the present study, we used a postinflammatory and restraint stress model to investigate the effects of VSL#3 administration on D-IBS. In the evaluation of IBS model, we utilized colorectal distention stimulator and the distention stress processes as the pace from lower to higher. And we also applied the AWR scoring system to objectively assess improvement of visceral hypersensitivity. In this model, 7 days after the instillation of acetic acid when there was no sign of inflammation in the colon, the rats still showed VH and defecated more stools. Their stool form was more soft and formless rather than hard compared with the control group. These findings are in accordance with the clinical observations in D-IBS patients.

Drug effects to be examined on all three IBS signs (VH, defecation, and epithelial barrier function) may be interpreted as the effect of drug on colitis rather than on IBS signs. To avoid this ambiguity, histopathological parameters of inflammation at 7 day after induction of IBS model were measured and the complete subsidence of inflammation was found in each group. From this study, we concluded that VSL#3 probiotics has neither a positive nor negative effect on the histopathological parameters of inflammation in the colon and does not impair the process of postinflammatory model establishment.

Considering all these precautions, we investigated the effects of VSL#3 and showed that VSL#3 significantly reduces VH and reduced defecation. We also investigated the role of NO in the protective effects of VSL#3 using an experimental model of IBS with NOS inhibitor aminoguanidine. Our results showed that NOS inhibitor significantly lower the effect of VSL#3 on VH, indicating that this effect of VSL#3 is at least partly mediated through NO synthesis.

NO is a key neurotransmitter in both short- and long-acting inhibitory motor neurons and plays a critical role in mediating gastrointestinal motility [31]. Several studies have shown that NOS neuronal activity considerably changes after inflammatory processes [36, 37]. Apart from its major role in the peripheral nervous system, as in enteric inhibitory nerves of the myenteric plexus, NO is thought to be an intracellular messenger or neurotransmitter in the central nervous system (CNS) [38]. It has been proposed that NO, due to its free diffusibility, acts as a retrograde transmitter in the CNS mediating some nervous paradigms, for example, long-term potentiation, and is the key neurotransmitter in descending inhibitory neurons modulating nociception at the spinal level [39]. Furthermore, evidence shows that NO is involved in the modulation of visceral perception, for example, an intraperitoneal injection of acetic acid in rats increases nitrergic neurons in specific regions of the brain, and NOS immune reactivity has been demonstrated in lumbosacral afferents and preganglionic neurons innervating the pelvic viscera [40, 41]. Human studies on IBS patients using functional MRI (fMRI) have shown some abnormal central processing of pain [42]. Therefore, several hypotheses can be made based on our results, which interpret the protective effects of VSL#3 through NO on VH in IBS at the myenteric plexus level, the CNS, and smooth muscles [43].

The previous studies suggest that the role of NO should be interpreted at the Enteric Nervous System (ENS) level rather than the CNS or muscle [38]. Our results would be more probably interpretable at the level of myenteric plexus.

Our results showed that NOS inhibitor per se cannot increase VH in VSL#3 untreated group. This finding is in accordance with a recent study which showed no significant difference in NO containing neurons of the colonic myenteric plexus between D-IBS rats and controls [41]. Thus, we hypothesize that basal NO synthesis is not significantly decreased in D-IBS, and it might be the positive effects of VSL#3 that decreases VH through increasing NO levels. Therefore, we speculated that the effects of VSL#3 on VH in the postinflammatory rat model of D-IBS are at least partly exerted through NO synthesis potentiation.

We also examined the effect of NOS inhibitor on defecation form (hard pellets, soft pellets, formless stool) in D-IBS rats. The effect of the NOS inhibitor on stool form was not significant, but NOS inhibitor diminished the protective effects of VSL#3 on stool form. NO plays a critical role in mediating gastrointestinal motility, and the gastrointestinal motility is very important for stooling. So the effects of VSL#3 on stooling can be inhibited by the aminoguanidine. However, the specific mechanism is not very clear. Further study will be conducted to explore the specific mechanism.

Moreover, our study shows that VSL#3 treatment can reduce colonic paracellular permeability and increase the expression of tight junction proteins (ZO-1 and occludin). We have established that this hypersensitivity of D-IBS model to distension also results from increased gut permeability. Increased colonic paracellular permeability resulting from the decreased expression of tight junction proteins. In this study, we showed that the expression of tight junction proteins (ZO-1 and occludin) was decreased, and VSL#3 treatment suppressed this effect. Therefore, our results suggest that probiotic-induced protection of epithelial barrier function is by prevention of changes in tight junction proteins expression and distribution. At the same time, we have found that the aminoguanidine cannot inhibited the effects of VSL#3 on permeability and tight junctions. So we speculated that there was no correlation between the effects of VSL#3 (permeability and tight junctions) and the aminoguanidine (the NOS inhibitor) from our study.

The protective effect of VSL#3 treatment on mucosal barrier integrity is in agreement with previous data showing that in vitro live probiotic strains interact with intestinal epithelial cells to protect them from the deleterious effects of enteroinvasive Escherichia coli by blocking the fall in transepithelial resistance and acting on tight junction proteins [12]. Therefore, we speculate that blockade of tight junction opening by VSL#3 may in turn prevent excessive uptake of luminal microbial antigens and bacterial products able to activate the submucosal immune system, which may sensitize terminals or sensory nerves through release of mediators.

According to the literature [44], the most attractive potential use of probiotics seems to be colonization of the gut and subsequent improvement of the intestinal microflora balance and fight against pathogens. Thus, we cannot exclude the fact that the ability to colonize and interact with the microflora could be involved in the beneficial effects of VSL#3 treatment observed in this study. Furthermore, a recent study by Verdu et al. [45] showed that perturbations in the gut flora and inflammatory cell activity enhanced visceral sensitivity and this effect was prevented by probiotic administration. Also, another study showed that disturbances of the commensal flora resulting from oral antibiotic administration in mice decreased the effects of acute stress-induced changes on colonic permeability, underlying the role of the flora in the responsiveness of epithelial cells [46].

In conclusion, in this study we showed that probiotic treatment prevented the hypersensitivity to distension and the increase in colonic paracellular permeability. It is possible that NO released by VSL#3 may affect colonic hypersensitivity. At the same time, VSL#3 exert a primary action on the colonic epithelial barrier through increased expression of tight junction proteins. All of these data suggest that VSL#3 probiotics may be of interest in the treatment of visceral hyperalgesia, particularly irritable bowel syndrome.

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© Springer Science+Business Media, LLC. 2011