Electroacupunture Relieves Pelvic Pain by Antagonizing Mecp2-Dependent Epigenic Modification of Bdnf Mediates Estrogen-Fasciliated Uterus-Colon Crosstalk in Rats

As an important content of alternative and complementary medicine, acupuncture therapy has been proved to be effective in relieving pain. In clinical scenario, it is interesting that patients of irritable bowel syndrome (IBS) have a high morbidity of lower urinary tract dysfunction with chronic pelvic pain (CPP). To mimic this observation, our laboratory has established an animal model, in which acute colon irritation reflexively sensitizes the visceral motor response (VMR) of the urethra in across-organ manner. Using our animal model we have demonstrated exogenous estradiol (E2) facilitated the crosstalk between the colon and urethra though its impact on the spinal neural plasticity, a mechanism possibly underlying the exaggerated incidence of IBS/CPP with pelvic pain in patients suffering from endometriosis. Nevertheless, whether E2 exhibits this effect through epigenic modulation via DNA methylation and histone acetylation has yet been established. We therefore, examined and compared the colon-urethra crosstalk between the ovariectomized (OVX) and sham-operated (SHAM) rats. Moreover, the levels of histone acetylation, and histone acetyl transferase (HAT), histone deacetylase (HDAC) and DNA methyltransferase (DNMT) expression in the dorsal horn of both groups in response to intra-colonic mustard oil (MO) instillation were assayed. We also tested whether E2 supplement therapy could reverse the ovarietomy-associated deficits we observed. Moreover, we administered with ICI-182,780 (a non-selective estrogen receptor antagonist), tichostatin A (TSA; a non-selective HAT inhibitor) and 5-Aza-2′-deoxycytidine (5-AZA; a non-selective DNMT inhibitor) to pharmacologically blocked the E2 receptor, HAT, and DNMT, respectively to confirm their contributions to the E2-faciliated colon-urethra crosstalk. Although the mechanism is no clear, a very recent study has linked the therapeutic effect of acupuncture to modifications of epigenetic control. We according tested the possibility that acupuncture could relief the estrogen-facilitated pelvic pain by antagonism of estrogen-dependent epigenic modification. In this experiment, acupoints that have been demonstrated to effectively relief urogenital pain, including Sanyinjiao (SP 6), Xuehai (SP 10), and Xuanzhong (GB 39) were adapted to animals from human acupuncture chart, and were stimulated daily for 3 weeks using a stimulator. The colon-urethra crosstalk was assayed and compared in estrogen supplement animals received electroacupunctue stimulation in specific versus non-specific acupoints. Moreover, the levels of histone acetylation, and histone acetyl transferase (HAT), histone deacetylase (HDAC) and DNA methyltransferase (DNMT) expression in the dorsal horn in response to intra-colonic mustard oil (MO) instillation in all groups were also assayed. Next, because methyl-CpG binding protein 2 (MeCP2)-dependent modification of brain-derived neurotrophic factor (BDNF) crucially participates in the impact of estrogen on plasticity in the CNS, we hypothesized the E2-dependent epgenic modification on the colon-urethra crosstalk is mediated by the MeCP2-associated BDNF pathway in the spinal cord. Accordingly, we examined the phosphorylation state of MeCP2 and the expression of BDNF in the dorsal horn after intra-colonic MO instillation in OVX compared to the SHAM group. On the other hand, in view of the interaction between MeCP2, sin3A, and HDACs has demonstrated to mediate MeCP2-assoicated epigenic modification, ChIP analysis was used to assay the association of these proteins in response to intra-colonic MO instillation. Also, in the OVX group, we daily administers with high-dose exogenous E2 to reverse the impairments in MeCP2 phosphorylation and BDNF expression. Moreover, ICI-182,780, TSA or 5-AZA were intrathecally injected to the SHAM group to confirm role of MeCP2-associated BDNF in E2-dependent epigenic modification. For there is so far no commercial MeCP2 antagonist available, and the BDNF exhibits it neural effect mainly through TrkB receptor, we pharmacologically inhibit TrkB receptor in the SHAM animals using ANA-12 to further confirm the role of BDNF. It is interesting that a recent study has demonstrated, in animal preparations, electoacupuncute successfully attenuated pain by decreasing spinal level of BDNF. We accordingly test the hypothesis that whether the electroacuture could ameliorate estrogen-facilitated colon-urethra crosstalk by diminishing spinal MeCP2-dependent BDNF cascade in rats. For this purpose, the colon-urethra crosstalk and spinal MeCP2 phosphorylation and BDNF expression were compared in estrogen supplement animals received electroacupunctue stimulation in specific versus non-specific acupoints. Finally, we tested whether the colon-urethra crosstalk varies across the estrus cycle, and whether such a cyclic fluctuation is mediated by an estrogen-dependent epigenic modification of BDNF via MeCP2 in the spinal cord. Hence, the colon-urethra crosstalk was evaluated in rats were in either the proestrus (high estradiol and low progesterone levels) or metestrus (low estradiol and high progesterone levels) stage. Also, the spinal levels of histone acetylation, MeCP2 phosphorylation, and HAT, HDAC, and BDNF expression as well as the association between MeCP2, sin3A, and HDACs in response to intra-colonic MO instillation was examined and compared between the proestrus and metestrus stage. In addition, the colon-urethra crosstalk and expressions of candidate proteins was assayed and compared in proestrus animals received electroacupunctue stimulation in specific versus non-specific acupoints. We hope our study could provide evidence supporting for the hypothesis that the menstrual cycle-associated fluctuation of colon-urethra crosstalk involves the estrogen/MeCP2/bdnf-dependent epigenic modification. Moreover, our electroacupuncture model could provide strategies for developing possible treatments for pelvic pain caused by crosstalk between pelvic organs.