In this study, RT-PCR and immunohistochemistry were used to clarify that PAR2 and PAR4 were present in the colonic mucosa of patients with PI-IBS. Subsequently, PAR2 and PAR4 status of mast cells in PI-IBS was also examined. The results described here provide the first evidence that immunoreactivity for PAR4, but not for PAR2, significantly changes in mast cells of PI-IBS compared with the normal controls.
As intestinal inflammation is associated with the generation and release of proteases that are potential activators of PARs, there have been considerable interests in the role of PARs in inflammatory diseases of the intestine, where proteases from the circulation, inflammatory cells, and the intestinal lumen could alter neuronal excitability and induce alterations in motility, secretion, and pain perception.27,28 These studies about PARs have important implications for the pathophysiology of PI-IBS, especially in the maintenance mechanisms of visceral hypersensitivity after gastrointestinal infection. However, the information on the localization of PARs in intestinal tissue is lacking in PI-IBS.29 We found a clear expression of both PAR2 and PAR4 in normal human intestine and PI-IBS patients. Furthermore, location studies in the intestines showed the highest concentrations of PARs were also expressed in the human intestinal submucosa apart from their existences in the human intestinal mucosa. However, it is yet unknown what is the function of PARs in this intestinal compartment. It could be assumed that PARs plays a role in a wide range of processes including endothelial cell functions, platelet activation and homeostasis, somatic and visceral nociceptive response, and gastrointestinal functions, such as intestinal motility and inflammatory response.30–32 It is therefore important to obtain the information about PARs expression and distribution in human intestines. In our study, PAR2 mRNA expression and immunoreactivity did not show significant differences between PI-IBS and the control group, while PAR4 expression significantly decreased in PI-IBS. No major changes in the PARs expression pattern were seen. There was also no relation with the grade of symptom. These data suggested PAR4 might play a new role in the modulation of visceral nociception in PI-IBS. Some recent studies have reported that the PAR4 agonist had antinociceptive effects.8,33,34 Augéet al. provided the evidence for a role of PAR4 agonists modulate colonic nociceptive response, intracolonic administration of the PAR4 agonist, was able to significantly inhibit PAR2 agonist- and transient receptor potential vanilloid-4 (TRPV4) agonist-induced allodynia and hyperalgesia in response to colorectal distension. The role of endogenous PAR4 in visceral pain processing was also confirmed using PAR4 deficient mice, which exhibited increased nociceptive behaviors in response to intracolonic administration of mustard oil compared to controls.8,35 Although the underlying mechanism has not been established, PAR4 activation by cathepsin G can exert the antinociceptive effect by participating in the regulation of intestinal permeability, visceral sensitivity, and possible micro-inflammation in the mucosa of IBS patients.36 Moreover, Asfaha et al. showed that PAR4 was expressed in sensory neurons isolated from the DRG that expressed the sensory neuropeptides CGRP and SP, and the PAR4 agonist reduced the calcium signal of DRG neurons in response to KCl, suggesting that PAR4 activation could inhibit the nociceptive signal in DRG neurons.33 In our studies, PAR2 was not significantly different in PI-IBS compared with the control group. The result is not in accordance with the concept that PAR2 represents an important receptor mediating the symptoms of IBS.12,15,37,38 The difference between the conflicting results may be attributed to the different counting methods or the use of animal species.
Trypsin, tryptase, and PARs AP evoke transient depolarization of submucosal neurons followed by a prolonged hyperexcitability that can last for several hours.39 This remarkable long-term hyperexcitability also can be observed after degranulation of mast cells.40,41 So, we did a further experiment to obtain the information about PARs expression and distribution in mast cells. In our study, we did not find an increase in the number of mast cells present in the biopsies from controls or PI-IBS patients, but we detected a significant increase in the activity of mast cells in PI-IBS compared with control biopsies. It suggests that more mast cells are actively degranulating in tissues of IBS patients than the normal control group, which is consistent with the study done by Cenac et al.37 Tryptase could be in direct contact with enteric or primary afferent nerves, and induce hyperexcitability.19,22,42 Thus, activated mast cells induce long-term excitability of submucosal neurons. As the expression of PARs on mast cells has not been investigated, possible physiological/pathophysiological roles for PAR2 and PAR4 in mast cells degranulation are poorly understood. Using double immunofluorescence technique, we confirmed a clear expression of both PAR2 and PAR4 on mast cells. Our study showed that immunoreactivity for PAR4 significantly decreased in mast cells of PI-IBS, it is also found that the PAR4 immunoreactivity decreases followed by the increase in the activity of mast cells in PI-IBS rather than normal controls. Whereas, PAR2 was not significantly different in PI-IBS compared with the control group. The present results highlight PAR4 may be another potential endogenous suppressor to visceral hypersensitivity in PI-IBS. By and large, a hypothesis is proposed: gastrointestinal infection may induce mast cell activation, while mast cell induced visceral hypersensitivity is mediated by a mechanism involving the activation of the PARs pathway; PARs might further change the release of a range of mediators by mast cells contributing to the persistent sensitization of nociceptors and increased intestinal permeability. Although there is no direct evidence supporting this hypothesis, the demonstration of such a relationship may have important implications for understanding the maintenance mechanisms of peripheral sensitization in PI-IBS.
Reverse transcriptase polymerase chain reaction and immunohistochemical techniques are used to demonstrate the distribution of PAR2 and PAR4 in human colons. The newly discovered role of PAR4 in modulating visceral pain develops our growing understanding of the colonic hypersensitivity and their potential role as therapeutic targets for PI-IBS. Further studies have to be done to obtain more detailed information about the precise role of the PARs on the gastrointestinal epithelia function.