Estrogen altered visceromotor reflex and P2X3 mRNA expression in a rat model of colitis
Introduction
About 4,000,000 people in the world suffer from inflammatory bowel disease (IBD) [1]. IBD is a group of inflammatory conditions of the colon and small intestine. The major types of IBD are Crohn's disease and ulcerative colitis. Though persons with IBD and/or irritable bowel syndrome (IBS) share similar signs and symptoms (abdominal pain, alterations in form, and frequency of stool) [2], visceral hypersensitivity plays an important role in IBS and may account for symptoms of urgency, bloating, and abdominal pain [3], [4], [5], [6]. In the current study, we used colonic trinitrobenzene sulfonic acid (TNBS) inflammation as a trigger for visceral hypersensitivity associated with ovariectomy and female hormones replacement.
It has been reported that a wide variety of signaling molecules are involved in initiating and maintaining the inflammatory response, including cations, amines, kinins, prostanoids, purines, cytokines and growth factors. Adenosine 5′-triphosphate (ATP) is an important candidate which plays a role in nociception and, in particular, inflammatory pain. Behavioral studies in rats [7], [8] and humans [9] have demonstrated that the pain-inducing effects of ATP are enhanced in states of inflammation. Nerve recordings also show exaggerated responses to ATP from inflammatory tissues [10]. ATP acts via both ionotropic P2X receptors and metabotropic P2Y receptors. P2X3 receptors are expressed in peripheral sensory nerve terminals and in small-diameter sensory neurons in dorsal root ganglia (DRG), which are known to supply, among other areas, the pelvic viscera [11], [12], [13]. P2X3 receptor-null mice show reduced formalin-induced pain behavior [14], [15] and P2X3 receptors are upregulated in colitis specimens obtained from patients with IBD [16] and in animal models of colitis [39] and IBS [17]. ATP released during distension from epithelial cells lining tubes (such as ureter or gut) and sacs (such as bladder) acts on P2X3 and/or P2X2/3 receptors on a subepithelial nerve plexus to initiate impulses that are relayed via the spinal cord to pain centers in the brain [13], [18].
It is also reported that the chronic pain conditions, such as IBS, are more prevalent in females than in males and the severity of pain fluctuates with the menstrual cycle [19], [20], suggesting that gonadal hormones may affect the perception of painful stimulations. However, clinical data on the association between IBD and female sex hormones are conflicting. On one hand, IBD is believed to flare during a first pregnancy and postpartum and, on the other hand, to be better controlled during subsequent pregnancies [21], [22], [23]. Another report suggests that female gender is a risk factor for relapse in ulcerative colitis [24]. Although study design and inclusion criteria may explain some of the discrepancies between studies, a causal relationship between sex hormones and IBD has not been clearly established.
Classical estrogen receptors, which consist of two subtypes (i.e., ERα and ERβ) have been described in vascular endothelium, fibroblasts, smooth muscle cells and gastrointestinal mucosal epithelial cells [25], [26], [27], [28]. Most of the effects of estrogen are mediated by binding of the hormone to these estrogen receptors (ERs) that act as nuclear transcriptor activators [26]. T cells, B cells and macrophages are known targets of estrogen [29], [30]. Besides, ERs are also widely distributed throughout the central and peripheral nervous systems [31], [32]. The peripheral sensory neurons express both estrogen receptor-α (ERα) and estrogen receptor-β (ERβ) [33], with ERα being selectively localized in small-diameter neurons. Thus it is reasonable to expect that estrogen modulates inflammation at the sensory ganglion level and also in the gastrointestinal system [34].
It has been reported that in ovariectomized rats P2X3 receptor mRNA levels are significantly increased in rat bladders [35], [36]. Our previous studies have also shown that estradiol selectively modulates homomeric P2X3 receptor-mediated transient currents without affecting homomeric P2X2 or heteromeric P2X2/3 receptor-mediated responses in rat cultured DRG neurons [37]. These results suggest estradiol may affect peripheral pain signal transduction at the sensory ganglion level via P2X3 receptors.
In this study, we examined changes to the visceromotor reflex (VMR) and P2X3 and P2X2 receptor mRNA levels in DRG from rats with or without ovariectomy after colitis, and in the female hormone-treated ovariectomized rats, to observe the possible modulatory effects by estrogen during colon inflammation.
Section snippets
Animals
In VMR and polymerase chain reaction (PCR) experiments, 24 female Sprague–Dawley rats (200–250 g) were randomly divided into four groups. Group 1 included six rats that had a sham operation, group 2 had six rats that had bilateral ovariectomy, group 3 had six rats that had bilateral ovariectomy following 17β-estradiol injection (daily, 30 μg/kg) 1 week after operation and groups 4 had six ovariectomized rats following daily complex (17β-estradiol, 30 μg/kg, progesterone, 50 μg/kg) injection 1 week
Differences of VMR among sham-op, ovariectomized, estrogen-treated and estrogen and progesterone-treated colitis rats
The magnitude of VMR was measured when applying colonic distension pressures of 20, 40, 60 and 80 mmHg. Compared with that in the sham-op colitis rats, the magnitude of the VMR in the ovariectomized colitis (OVX) rats was significantly decreased (p = 0.007, Fig. 1A). Estrogen produced a significant facilitation of the VMR (p = 0.002, Fig. 1A) compared with that in the ovariectomized colitis rats. There was no difference between ovariectomized and estrogen–progesterone-treated groups (p = 0.143, Fig. 1
Discussion
The VMR is a contraction of the abdominal muscles in response to colorectal distension, which provides a reliable noxious visceral stimulus in awake rats that is reproducible and produces measurable, reliable, physiological and behavioral responses [41]. In the present study, we first found that VMR was significantly decreased in OVX rats after colitis compared with that observed in sham-op rats after colitis, which was reversed after estrogen treatment. These data demonstrate that the reduced
Acknowledgement
Supported by the National Natural Scientific Foundation of China, No. 30570597
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Both authors contributed equally to this work.