Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by chronic abdominal pain with altered bowel habits [1
]. Visceral hypersensitivity is considered to be one of the most important underlying mechanisms and is a hallmark of IBS [2
]. Stress alters the visceral sensory function and has a significant impact on the development and exacerbation of IBS symptoms [3
At the same time, there have been several reports showing that compromised gut barrier function manifested by gut hyperpermeability is observed in some patients with IBS, and stress also enhances gut permeability [3
]. Impaired gut barrier leads to bacterial translocation causing increased lipopolysaccharide (LPS) and proinflammatory cytokines, which is also considered to be a pivotal feature of IBS [5
]. Actually, it was reported that circulating pro-inflammatory cytokines and LPS are increased in IBS patients [5
]. Moreover, we previously showed that LPS induced visceral hypersensitivity and gut barrier disruption via interleukin-6 (IL-6), IL-1 [7
], and toll-like receptor 4 (TLR4) signaling [4
Water avoidance stress (WAS) is a psychological stress protocol causing visceral hypersensitivity and gut hyperpermeability, and now WAS-loaded rats are generally accepted as the animal model of IBS [11
]. We have recently demonstrated that IL-6, IL-1β, and TLR4 pathways mediated these visceral changes induced by repeated WAS [4
], which is similar to the LPS-induced IBS model. These results suggest that TLR4-cytokine signaling is a significant contributor to IBS [13
Grape seed extract (GSE) is produced as a by-product of wine and grape juice. It is rich in polyphenolic compounds including proanthocyanidins, which have anti-oxidative and anti-inflammatory properties [14
]. These beneficial effects are considered to contribute to the prevention of cancer, cardiovascular diseases and diabetes [14
]. Recent studies showed that GSE supplementation inhibited intestinal hyperpermeability and the expression of proinflammatory cytokines in IL-10-deficient colitis [16
]. Furthermore, GSE was found to reduce plasma levels of LPS and proinflammatory cytokines with concomitant protection of gut barrier function mediated by tight junction (TJ) structure in animal models of obesity [18
]. Thus, we hypothesized that GSE improves visceral sensation and gut barrier function via suppressing the TLR4-cytokine signaling and maintaining TJ structure, which are beneficial factors in the treatment of IBS.
In the present study, we investigated the effects of GSE on visceral allodynia and colonic hyperpermeability induced by repeated WAS. Moreover, the actions of GSE in epithelial monolayers in vitro were also evaluated.
We previously demonstrated that repeated WAS induced visceral allodynia and colonic hyperpermeability, which were mediated via TLR4 and proinflammatory cytokine signaling [4
]. The current study showed that repeated WAS increased the colonic levels of IL-6, IL-1β and TLR4, and altered colonic TJ protein expression, which could impair gut barrier integrity. These results suggest that repeated WAS activates the TLR4-proinflammatory cytokine signaling to cause these visceral changes, which further supports our previous findings above.
The TLR4 pro-inflammatory cytokine system is thought to be involved in the pathophysiology of a certain proportion of IBS patients [5
]. TLR4 in the colonic tissue of IBS patients is elevated [27
], and TLR4 messenger RNA expression in the colonic mucosa is significantly correlated with the duration of symptoms in IBS patients [28
]. Although it has not yet been precisely determined how the elevated expression of TLR4 in the colon contributes to the pathophysiology, cytokine production triggered by TLR4 activation might evoke the visceral functional changes observed in IBS. We have also recently demonstrated that peripheral injection of IL-6 or IL-1β induced visceral allodynia [7
], possibly through the activation of cytokine receptors located in visceral afferent neurons [29
]. Additionally, proinflammatory cytokines also increase gut permeability via regulating TJ protein expression [22
]. In the present study, we showed that the colonic level of IL-6 or IL-1β was positively correlated with elevated colonic permeability, which is consistent with the evidence above.
TJ proteins are the main regulator of paracellular permeability. Knockdown of occludin which was reported to maintain TJ structure causes intestinal hyperpermeability with visceral hypersensitivity in mice, suggesting that an intestinal barrier mediated by TJ proteins plays a significant role in the development of visceral pain [31
]. In the present study, we showed that repeated WAS significantly increased claudin-2, which promotes leaky gut barriers [22
] and decreased claudin-3, which is involved in the maintenance of barrier function [23
]. These changes appear to be associated with increased colonic permeability in the present study. On the other hand, other tested TJ proteins, such as claudin-7 and ZO-1, were unchanged. Since only claudin-2 was significantly correlated with colonic permeability among these TJ proteins, claudin-2 is considered to be the most significant contributor in our stress model. Notably, it was shown that claudin-2 expression is higher in the ileum of IBS patients than healthy controls [32
Gut hyperpermeability leads to bacterial translocation and results in immune system activation and subsequent inflammation [33
]. In this process, LPS is released and proinflammatory cytokines are produced through LPS activation of TLR4 [5
], thereby further stimulating the TLR4-cytokine system and leading to a vicious cycle [4
]. In this scenario, visceral hypersensitivity appears to result from colonic inflammation induced by gut barrier impairment. We showed that the reduced threshold of VMR was significantly related to increased colonic permeability, which supports the validity of our hypothesis.
Incidentally, the corticotropin-releasing factor (CRF) modulates the gastrointestinal stress response and may significantly contribute to the pathophysiology of IBS [3
]. We previously demonstrated that repeated WAS-induced visceral changes were mediated via peripheral CRF receptors and TLR4, and peripheral administration of CRF mimicked these responses [4
]. Moreover, CRF increased TLR4 expression in macrophages [36
] and enhanced inflammatory cytokine production following LPS treatment [37
], which possibly stimulates visceral afferent nerves. In addition, other researchers showed that CRF increased claudin-2 by upregulation of TLR4 in intestinal epithelial cells in vitro
]. These findings suggest that activating peripheral CRF signaling triggered by stress modulates TLR4 signaling, which may explain the mechanism of visceral changes induced by repeated WAS.
It was demonstrated that GSE inhibited inflammatory responses via nuclear factor κB (NFκB) signaling in LPS-stimulated macrophages [39
]. Moreover, GSE supplementation improves colonic tissue damage, which is probably mediated by inhibiting inflammatory cytokine gene expression and NFκB signaling in IL-10 KO colitis mice [16
]. Therefore, we hypothesized that GSE attenuates the visceral changes triggered by repeated WAS through suppression of cytokine signaling, which was demonstrated to occur. GSE inhibited the increases in colonic IL-6 and TLR4 levels following stress. This is the first report showing the beneficial effects of GSE in a rat IBS model. Since NFκB is a key regulator of cytokine production and TLR4 expression in immune cells, GSE likely attenuates the TLR4-cytokine signaling to evoke the effects via inhibition of the NFκB pathway [41
We also found that GSE suppressed the elevated expression of claudin-2 in vivo
, which appears to be a direct mechanism of colonic barrier improvement in our IBS model, as previously described. Since IL-6 increases gut permeability by increasing expression of claudin-2 [22
], one of the mechanisms underlying GSE-mediated suppression of WAS-induced elevation of claudin-2 may be attributable to the inhibition of cytokine production.
Since GSE blocked the visceral changes in vivo
, we also explored the effects in vitro
using Caco-2 cells stimulated by IL-6 and IL-1β. These cytokines induced hyperpermeability and increased claudin-2 expression in Caco-2 cell monolayers. Unlike the results from the in vivo
animal model in the present study, the treatment reduced the level of claudin-7, which could also increase the permeability [25
]. Since factors other than inflammatory cytokines, such as CRF or mast cells, may influence the tightness of the intestinal TJ barrier [4
], the changes in TJ protein expression are thought to differ from the results in vivo
. Interestingly, GSE attenuated the increased paracellular permeability and also fully reversed the elevated claudin-2 expression. Suzuki et al. [22
] demonstrated that the IL-6-induced expression of claudin-2 in Caco-2 cells was mediated via intracellular signalings such as mitogen-activated protein kinase, kinase 1/extracellular signal-regulated kinase 1 and phosphoinositide 3-kinase. Although the cellular mechanisms of GSE remain to be elucidated, our results indicate that GSE inhibits the release of proinflammatory cytokines, as well as cytokine-induced changes in TJ protein levels, which may account for the strong effects of GSE on the visceral changes induced by repeated WAS.
The GSE used in this study contained proanthocyanidins at a level of >80%. It was reported that GSE composed of more than 85% proanthocyannidins inhibited LPS-induced NFκB signaling in macrophages [40
]. Additionally, it was shown that proanthocyanidins (>95% purity) extracted from grape seeds attenuated intestinal inflammation and NFκB activation in a drug-induced colitis model [45
]. Based on these lines of evidence, proanthocyanidins appear to be the main contributor to the effects of GSE in the present study, likely through the suppression of NFκB signaling.
The study has several limitations. The precise cellular or molecular mechanisms of GSE effects have not been determined. In addition, we did not show direct evidence that GSE inhibits NFκB signaling. Since the source of cytokines contributing to the visceral changes triggered by repeated WAS has not yet been determined, the cellular target of GSE also remains unknown. Further studies are needed to explore these issues.
Despite the above limitations, our results strongly suggest that GSE ameliorates the symptoms of IBS by improving visceral sensation and gut barrier integrity. Since GSE has been widely utilized as a supplement/medicine for blood vessel protection, its utilization for treating IBS is not expected to be challenging. Large-scale clinical trials that aim to evaluate the effectiveness of GSE in patients with IBS should be conducted in the future