Moxibustion ameliorates chronic inflammatory visceral pain via spinal circRNA-miRNA-mRNA networks: a central mechanism study.

This study aimed to unveil the central mechanism of moxibustion treating chronic inflammatory visceral pain (CIVP) from the angle of circRNA-miRNA-mRNA networks in the spinal cord. The rat CIVP model was established using a mixture of 5% (w/v) 2,4,6-trinitrobenzene sulfonic acid and 50% ethanol at a volume ratio of 2:1 via enema. Rats in the moxibustion group received herb-partitioned moxibustion at Tianshu (ST25, bilateral) and Qihai (CV6) points. The abdominal withdrawal reflex (AWR), mechanical withdrawal threshold (MWT), and thermal withdrawal latency (TWL) were adopted for pain behavior observation and pain sensitivity assessment. The circRNA, miRNA, and mRNA expression profiles were detected using the high-throughput sequencing technique. Relevant databases and bioinformatics analysis methods were used to screen for differentially expressed (DE) RNAs and build a circRNA-miRNA-mRNA (competing endogenous RNA) ceRNA regulatory network. The real-time quantitative PCR was employed to verify the sequencing result. CIVP rat models had a significantly higher AWR and lower TWL and MWT than normal rats. Between normal and model rats, there were 103 DE-circRNAs, 16 DE-miRNAs, and 397 DE-mRNAs in the spinal cord. Compared with the model group, the moxibustion group had a lower AWR and higher TWL and MWT; between these two groups, there were 118 DE-circRNAs, 15 DE-miRNAs, and 804 DE-mRNAs in the spinal cord. Two ceRNA networks were chosen to be verified. As a result, moxibustion's analgesic effect on visceral pain in CIVP rats may be associated with regulating the circRNA_02767/rno-miR-483-3p/Gfap network in the spinal cord and improving central sensitization.

Peripheral neural mechanism of visceral pain and acupoint sensitization in 2, 4, 6-trinitrobenzene sulfonic acid-induced colitis model mice. / ç»“è‚ ç‚Žæ¨¡åž‹å°é¼ å† è„ç—›åŠç©´ä½æ•åŒ–çš„å¤–å‘¨ç¥žç»æœºåˆ¶æŽ¢è®¨.

OBJECTIVES: To explore the neural mechanism of visceral pain and related somatic (acupoints) sensitization by using in vivo calcium imaging of dorsal root ganglia (DRG) neurons. METHODS: Eight BALB/c mice were randomly divided into control and model groups, with 4 mice in each group. The colitis model was induced by colorectal perfusion of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) once daily for 7 days. Mice of the control group received colorectal perfusion of normal saline once daily for 7 days. The location and area of the somatic neurogenic inflammation (cutaneous exudation of Evans blue ï¼»EBï¼½) of the 2 groups of mice were observed after intravenous injection of EB. For pain behavioral tests, sixteen C57BL/6J mice were randomly divided into control and model groups, with 8 mice in each group, and a Von Frey filament was used to stimulate the referred somatic reactive regions in colitis mice, and the number of avoidance and paw withdraw reaction within 10 tests was recorded. For in vivo DRG calcium imaging tests, 24 Pirt-GCaMP6s transgenic mice were randomly and equally divided into control group and colitis model group. The responses of the neurons in L6 or L4 DRG to colorectal distension (CRD), lower back brushing, or mechanical stimulation at the hindpaw were observed using confocal fluorescence microscope. RESULTS: Compared with the control group, the area of EB exudation spot in the hindpaw and lower back regions was increased in the colitis model group (P<0.05), and the avoidance or paw withdraw numbers induced by Von Frey stimulation at the lower back and hindpaw were increased (P<0.01, P<0.05), indicating that colitis induced regional skin (acupoints) sensitization in the lower back and hindpaw regions. Compared with the control group, the percentage of L6 DRG neurons activated by 60 mm Hg CRD in the colitis model mice were apparently increased (P<0.01), the activated neurons mainly involved the medium-sized DRG neurons (P<0.01). In Pirt-GCaMP6s transgenic mice, following brushing the skin of the receptive field (lower back) of L6 DRG neurons, the fluorescence intensity of the brushing-activated DRG neurons and small, medium and large-sized neurons were significantly higher in the colitis model group than those in the control group (P<0.001, P<0.01, P<0.05). After brushing and clamping the skin of the right hindpaw (receptive field of L4 DRG neurons), the percentages of the activated L4 DRG neurons were obviously higher in the colitis model group than those in the control group (P<0.01, P<0.05), while there were no significant changes in the proportion of small, medium and large-sized neurons between the control and colitis model groups. CONCLUSIONS: Colitis may lead to body surface sensitization at the same and adjacent neuro-segments as well as to an increase of the number and activity of the responsive lumbar DRG neurons, among which the L6 DRG neurons at the same neuro-segment as the rectum colon showed an increase in the number of responders and intensity of calcium fluorescence signal while L4 DRG neurons at the level adjacent to the rectum colon showed an increase in the number of responders, suggesting that there may be different mechanisms of peripheral neural sensitization.

NGF/PI3K/TRPV1 pathway mediates the regulation of visceral pain by acupuncture at "Shangjuxu" (ST37) in irritable bowel syndrome rats with chronic visceral hyperalgesia. / NGF/PI3K/TRPV1通路介导针刺"上巨虚"è°ƒèŠ‚è‚ æ˜“æ¿€ç»¼åˆå¾æ ¢æ€§å† è„ç—›æ•æ¨¡åž‹å¤§é¼ å† è„ç—›.

OBJECTIVES: To investigate the effect of manual acupuncture at "Shangjuxu"(ST37) on nerve growth factor(NGF)/phosphatidylinositol 3-kinase(PI3K)/transient receptor potential vanilloid 1(TRPV1) signaling pathway in rats with chronic visceral hyperalgesia of irritable bowel syndrome (IBS), so as to explore its underlying mechanism in treating IBS chronic visceral hyperalgesia. METHODS: IBS chronic visceral hyperalgesia model was established by colorectal dilation stimulation for 2 weeks for SD pups at 8 d after birth, which were fed until 8-week age after the stimulation. Then the verified successfully modeled adult rats were randomly divided into model, Shangjuxu, and non-acupoint groups, with 6 rats in each group, and 6 unmodeled rats were selected as normal group. On the next day of model evaluation, rats in the Shangjuxu group received acupuncture at right ST37 while rats in the non-acupoint group received acupuncture at the non-meridian and non-acupoint point in the right hypochondrium, both for 15 min, with manual twisting of mild reinforcing and reducing performed for 30 s at an interval of 5 min, once a day, for a total of 7 d. Abdominal withdrawal reflex(AWR) score was used to evaluate the degree of chronic visceral pain in rats. Western blot and real-time fluorescence quantitative PCR were used to detect the colonic protein and mRNA expressions of NGF, tropomyosin receptor kinase A (TrkA), PI3K and TRPV1. The positive expressions of PI3K and TRPV1 proteins in the colon of rats were detected by immunohistochemistry method. RESULTS: Compared with the normal group, AWR scores corresponding to 4 pressure levels of 20, 40, 60 and 80 mm Hg, mRNA and protein expressions of NGF, TrkA, PI3K and TRPV1 in colon tissue, and positive expressions of PI3K and TRPV1 in colon tissue were significantly increased(P<0.05) in the model group. After intervention, compared with the model group, rats in the Shangjuxu group had reduced AWR scores corresponding to 4 pressure levels of 20, 40, 60 and 80 mm Hg, lower colonic mRNA and protein expressions of NGF, TrkA, PI3K and TRPV1, and decreased positive expressions of PI3K and TRPV1 in colon tissue(P<0.05), while there were no significant differences in the above indexes of the non-acupoint group. CONCLUSIONS: Manual acupuncture at ST37 can alleviate IBS chronic visceral hyperalgesia in rat and its analgesic effect may be related to regulating NGF/PI3K/TRPV1 signaling pathway.

[Study on the mechanism of electroacupuncture repairing intestinal barrier via regulating mast cell in rats with diarrhea-predominant irritable bowel syndrome].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on mast cell activation-related substances and intestinal barrier function in diarrhea-predominant irritable bowel syndrome (IBS-D) model rats, so as to explore its underlying mechanisms. METHODS: Thirty female SD rats were randomly divided into control group, model group and EA group, with 10 rats in each group. IBS-D model was established by chronic unpredictable mild stress combined with senna solution gavage. Rats in the EA group received EA treatment (2 Hz/15 Hz，0.1-1.0 mA) at "Zusanli" (ST36), "Taichong"(LR3) and "Tianshu"(ST25), 20 min per day, for a total of 14 days, with sides alternated daily. Visceral pain threshold was used to evaluate visceral hypersensitivity, diarrhea index was used to evaluate diarrhea degree. After all treatments, the pathological scores of colon were recorded after HE staining, the contents of cholecystokinin (CCK), substance P (SP), tryptase (TPS) and adenosine triphosphate (ATP) in colon were detected by ELISA, and the expressions of colonic tight junction protein ZO-1 and occludin were detected by Western blot. RESULTS: Compared with the control group, the visceral pain threshold, the expression levels of colonic ZO-1 and occludin proteins decreased (P<0.01), while the diarrhea index, the contents of colonic CCK, SP, TPS and ATP were significantly increased (P<0.01) in the model group. After intervention, in comparison with the model group, the visceral pain thre-shold, the protein expression levels of colonic ZO-1 and occludin protein increased (P<0.01), while the diarrhea index, the contents of colonic CCK, SP, TPS and ATP were significantly decreased (P<0.01) in the EA group. CONCLUSION: EA can significantly alleviate the symptoms of visceral hypersensitivity and diarrhea in IBS-D rats. Its mechanism may be related to down-regulating colonic CCK, SP, TPS and ATP, inhibiting mast cell activation and degranulation, and up-regulating colonic barrier tight junction proteins.

Epigenetic mechanisms underlying stress-induced visceral pain: Resilience versus vulnerability in a two-hit model of early life stress and chronic adult stress.

BACKGROUND: Women with a history of early life stress (ELS) have a higher risk of developing irritable bowel syndrome (IBS). In addition, chronic stress in adulthood can exacerbate IBS symptoms such as abdominal pain due to visceral hypersensitivity. We previously showed that sex and the predictability of ELS determine whether rats develop visceral hypersensitivity in adulthood. In female rats, unpredictable ELS confers vulnerability and results in visceral hypersensitivity, whereas predictable ELS induces resilience and does not induce visceral hypersensitivity in adulthood. However, this resilience is lost after exposure to chronic stress in adulthood leading to an exacerbation of visceral hypersensitivity. Evidence suggests that changes in histone acetylation at the promoter regions of glucocorticoid receptor (GR) and corticotrophin-releasing factor (CRF) in the central nucleus of the amygdala (CeA) underlie stress-induced visceral hypersensitivity. Here, we aimed to investigate the role of histone acetylation in the CeA on visceral hypersensitivity in a two-hit model of ELS followed by chronic stress in adulthood. METHODS: Male and female neonatal rats were exposed to unpredictable, predictable ELS, or odor only (no stress control) from postnatal days 8 to 12. In adulthood, rats underwent stereotaxic implantation of indwelling cannulas. Rats were exposed to chronic water avoidance stress (WAS, 1 h/day for 7 days) or SHAM stress and received infusions of vehicle, the histone deacetylase inhibitor trichostatin A (TSA) or the histone acetyltransferase inhibitor garcinol (GAR) after each WAS session. 24 h after the final infusion, visceral sensitivity was assessed and the CeA was removed for molecular experiments. RESULTS: In the two-hit model (ELS + WAS), female rats previously exposed to predictable ELS, showed a significant reduction in histone 3 lysine 9 (H3K9) acetylation at the GR promoter and a significant increase in H3K9 acetylation at the CRF promoter. These epigenetic changes were associated with changes in GR and CRF mRNA expression in the CeA and an exacerbation of stress-induced visceral hypersensitivity in female animals. TSA infusions in the CeA attenuated the exacerbated stress-induced visceral hypersensitivity, whereas GAR infusions only partially ameliorated ELS+WAS induced visceral hypersensitivity. CONCLUSION: The two-hit model of ELS followed by WAS in adulthood revealed that epigenetic dysregulation occurs after exposure to stress in two important periods of life and contributes to the development of visceral hypersensitivity. These aberrant underlying epigenetic changes may explain the exacerbation of stress-induced abdominal pain in IBS patients.

Guanylyl cyclase C ameliorates visceral pain: an unsuspected link.

Visceral pain associated with irritable bowel syndrome afflicts 15% of the US population. Although treatments are limited, guanylyl cyclase C (GUCY2C) agonists alleviate pain and constipation. Until now, it was assumed that the activation of GUCY2C and production of cGMP in enterocytes stimulated fluid secretion and reduced visceral sensation. The recent discovery that a subtype of enteroendocrine cells (EECs) known as neuropod cells synapse with submucosal neurons unveiled a pathway for communicating gut signals to the nervous system. In this issue of the JCI, Barton et al. report that GUCY2C is enriched in neuropod cells and is involved with sensory nerve firing. Selective deletion of GUCY2C in mouse models suggests that defective GUCY2C neuropod-cell signaling underlies visceral pain. These studies introduce possibilities for dissociating the secretory and analgesic effects of GUCY2C agonism. Although further work remains, unveiling the role of neuropod cells is a major step in understanding visceral pain.

Intestinal neuropod cell GUCY2C regulates visceral pain.

Visceral pain (VP) is a global problem with complex etiologies and limited therapeutic options. Guanylyl cyclase C (GUCY2C), an intestinal receptor producing cyclic GMP(cGMP), which regulates luminal fluid secretion, has emerged as a therapeutic target for VP. Indeed, FDA-approved GUCY2C agonists ameliorate VP in patients with chronic constipation syndromes, although analgesic mechanisms remain obscure. Here, we revealed that intestinal GUCY2C was selectively enriched in neuropod cells, a type of enteroendocrine cell that synapses with submucosal neurons in mice and humans. GUCY2Chi neuropod cells associated with cocultured dorsal root ganglia neurons and induced hyperexcitability, reducing the rheobase and increasing the resulting number of evoked action potentials. Conversely, the GUCY2C agonist linaclotide eliminated neuronal hyperexcitability produced by GUCY2C-sufficient - but not GUCY2C-deficient - neuropod cells, an effect independent of bulk epithelial cells or extracellular cGMP. Genetic elimination of intestinal GUCY2C amplified nociceptive signaling in VP that was comparable with chemically induced VP but refractory to linaclotide. Importantly, eliminating GUCY2C selectively in neuropod cells also increased nociceptive signaling and VP that was refractory to linaclotide. In the context of loss of GUCY2C hormones in patients with VP, these observations suggest a specific role for neuropod GUCY2C signaling in the pathophysiology and treatment of these pain syndromes.

Spinal VGLUT3 lineage neurons drive visceral mechanical allodynia but not sensitized visceromotor reflexes.

Visceral pain is among the most prevalent and bothersome forms of chronic pain, but their transmission in the spinal cord is still poorly understood. Here, we conducted focal colorectal distention (fCRD) to drive both visceromotor responses (VMRs) and aversion. We first found that spinal CCK neurons were necessary for noxious fCRD to drive both VMRs and aversion under naive conditions. We next showed that spinal VGLUT3 neurons mediate visceral allodynia, whose ablation caused loss of aversion evoked by low-intensity fCRD in mice with gastrointestinal (GI) inflammation or spinal circuit disinhibition. Importantly, these neurons were dispensable for driving sensitized VMRs under both inflammatory and central disinhibition conditions. Anatomically, a subset of VGLUT3 neurons projected to parabrachial nuclei, whose photoactivation sufficiently generated aversion in mice with GI inflammation, without influencing VMRs. Our studies suggest the presence of different spinal substrates that transmit nociceptive versus affective dimensions of visceral sensory information.

Piezo2 channels expressed by colon-innervating TRPV1-lineage neurons mediate visceral mechanical hypersensitivity.

Inflammatory and functional gastrointestinal disorders such as irritable bowel syndrome (IBS) and obstructive bowel disorder (OBD) underlie the most prevalent forms of visceral pain. Although visceral pain can be generally provoked by mechanical distension/stretch, the mechanisms that underlie visceral mechanosensitivity in colon-innervating visceral afferents remain elusive. Here, we show that virally mediated ablation of colon-innervating TRPV1-expressing nociceptors markedly reduces colorectal distention (CRD)-evoked visceromotor response (VMR) in mice. Selective ablation of the stretch-activated Piezo2 channels from TRPV1 lineage neurons substantially reduces mechanically evoked visceral afferent action potential firing and CRD-induced VMR under physiological conditions, as well as in mouse models of zymosan-induced IBS and partial colon obstruction (PCO). Collectively, our results demonstrate that mechanosensitive Piezo2 channels expressed by TRPV1-lineage nociceptors powerfully contribute to visceral mechanosensitivity and nociception under physiological conditions and visceral hypersensitivity under pathological conditions in mice, uncovering potential therapeutic targets for the treatment of visceral pain.

Unraveling the role of Epac1-SOCS3 signaling in the development of neonatal-CRD-induced visceral hypersensitivity in rats.

AIMS: Visceral hypersensitivity in irritable bowel syndrome (IBS) is widespread, but effective therapies for it remain elusive. As a canonical anti-inflammatory protein, suppressor of cytokine signaling 3 (SOCS3) reportedly relays exchange protein 1 directly activated by cAMP (Epac1) signaling and inhibits the intracellular response to inflammatory cytokines. Despite the inhibitory effect of SOCS3 on the pro-inflammatory response and neuroinflammation in PVN, the systematic investigation of Epac1-SOCS3 signaling involved in visceral hypersensitivity remains unknown. This study aimed to explore Epac1-SOCS3 signaling in the activity of hypothalamic paraventricular nucleus (PVN) corticotropin-releasing factor (CRF) neurons and visceral hypersensitivity in adult rats experiencing neonatal colorectal distension (CRD). METHODS: Rats were subjected to neonatal CRD to simulate visceral hypersensitivity to investigate the effect of Epac1-SOCS3 signaling on PVN CRF neurons. The expression and activity of Epac1 and SOCS3 in nociceptive hypersensitivity were determined by western blot, RT-PCR, immunofluorescence, radioimmunoassay, electrophysiology, and pharmacology. RESULTS: In neonatal-CRD-induced visceral hypersensitivity model, Epac1 and SOCS3 expressions were downregulated and IL-6 levels elevated in PVN. However, infusion of Epac agonist 8-pCPT in PVN reduced CRF neuronal firing rates, and overexpression of SOCS3 in PVN by AAV-SOCS3 inhibited the activation of PVN neurons, reduced visceral hypersensitivity, and precluded pain precipitation. Intervention with IL-6 neutralizing antibody also alleviated the visceral hypersensitivity. In naïve rats, Epac antagonist ESI-09 in PVN increased CRF neuronal firing. Consistently, genetic knockdown of Epac1 or SOCS3 in PVN potentiated the firing rate of CRF neurons, functionality of HPA axis, and sensitivity of visceral nociception. Moreover, pharmacological intervention with exogenous IL-6 into PVN simulated the visceral hypersensitivity. CONCLUSIONS: Inactivation of Epac1-SOCS3 pathway contributed to the neuroinflammation accompanied by the sensitization of CRF neurons in PVN, precipitating visceral hypersensitivity and pain in rats experiencing neonatal CRD.

The pathological involvement of spinal cord EphB2 in visceral sensitization in male rats.

Patients with post-traumatic stress disorder (PTSD) are usually at an increased risk for chronic disorders, such as irritable bowel syndrome (IBS), characterized by hyperalgesia and allodynia, but its subsequent effect on visceral hyperalgesia and the mechanism remain unclear. The present study employed single prolonged stress (SPS), a model of PTSD-pain comorbidity, behavioral evaluation, intrathecal drug delivery, immunohistochemistry, Western blotting, and RT-PCR techniques. When detecting visceral sensitivity, the score of the abdominal withdrawal reflex (AWR) induced by graded colorectal distention (CRD) was used. The AWR score was reduced in the SPS day 1 group but increased in the SPS day 7 and SPS day 14 groups at 40 mmHg and 60 mmHg, and the score was increased significantly with EphrinB1-Fc administration. The EphB2+ cell density and EphB2 protein and mRNA levels were downregulated in the SPS day 1 group and then upregulated significantly in the SPS day 7 group; these changes were more noticeable with EphrinB1-Fc administration compared with the SPS-only group. The C-Fos-positive reaction induced by SPS was mainly localized in neurons of the spinal dorsal horn, in which the C-Fos-positive cell density and its protein and mRNA levels were upregulated on SPS days 7 and 14; these changes were statistically significant in the SPS + EphrinB1-Fc group compared with the SPS alone group. The present study confirmed the time window for the AWR value, EphB2 and C-Fos changes, and the effect of EphrinB1-Fc on these changes, which suggests that spinal cord EphB2 activation exacerbates visceral pain after SPS.

Delta opioid receptors on nociceptive sensory neurons mediate peripheral endogenous analgesia in colitis.

BACKGROUND: Inflammatory visceral pain is endogenously controlled by enkephalins locally released by mucosal CD4+ T lymphocytes in mice. The present study aimed at identifying opioid receptor(s) expressed on nociceptive sensory nerves involved in this peripheral opioid-mediated analgesia. METHODS: The peripheral analgesia associated with the accumulation of CD4+ T lymphocytes within the inflamed colonic mucosa was assessed in conditional knockout mice specifically deleted for either of the two opioid receptors for enkephalins (i.e., µ (MOR) and δ (DOR) receptors) in Nav1.8-expressing sensory neurons in the dextran sulfate sodium (DSS)-induced colitis model. RESULTS: Endogenous analgesia is lost in conditional knockout mice for DOR, but not MOR at the later phase of the DSS-induced colitis. The absence of either of the opioid receptors on sensory nerves had no impact on both the colitis severity and the rate of T lymphocytes infiltrating the inflamed colonic mucosa. CONCLUSION: The key role of DOR on primary afferents in relieving intestinal inflammatory pain opens new therapeutic opportunities for peripherally restricted DOR analgesics to avoid most of the side effects associated with MOR-targeting drugs used in intestinal disorders.

Environmental enrichment prevents stress-induced epigenetic changes in the expression of glucocorticoid receptor and corticotrophin releasing hormone in the central nucleus of the amygdala to inhibit visceral hypersensitivity.

INTRODUCTION: Stress is a known trigger for the symptoms of irritable bowel syndrome (IBS), a gastrointestinal (GI) disorder that presents with abnormal bowel habits and abdominal pain due to visceral hypersensitivity. While behavioral therapies have been used to attenuate IBS symptoms, the underlying mechanisms by which these therapies interact with stress-induced pathology remains to be delineated. Here we use a rat model to test the hypothesis that exposure to environmental enrichment (EE) inhibits stress-induced changes within the brain-gut axis to prevent visceral and somatic hypersensitivity and colonic hyperpermeability. METHODS: Female rats (n = 8/group) were housed in EE one week before and one week during exposure to water avoidance stress (WAS) while controls were housed in standard cages (SH). One day after the final WAS exposure, colonic and somatic sensitivity were assessed by the visceromotor response (VMR) to colorectal distension (CRD) and withdrawal threshold elicited by an electronic von Frey on the hind paw of the rats respectively. All rats were returned to SH for 3 weeks before colonic and somatic sensitivity were reassessed on day 28. The rats were then immediately euthanized and the spinal cord was collected to assess changes in neuronal activation (assessed via ERK phosphorylation) in response to noxious CRD. A separate cohort of animals (n = 8/group) that did not undergo behavioral assessments was euthanized the day after the final WAS exposure and the central nucleus of the amygdala (CeA) was collected to investigate WAS and EE induced epigenetic changes at the glucocorticoid receptor (GR) and corticotrophin releasing hormone (CRH) promoter. The colon from these rats was also collected to assess colonic permeability via changes in transepithelial electrical resistance (TEER) in vitro. RESULTS: Exposure to stress persistently increased VMR to CRD (P < 0.01) and decreased the hind paw withdrawal threshold (P < 0.001) in female rats. WAS also decreased TEER in the colon tissue of female rats (p = 0.05). In the CeA, WAS induced a decrease in histone acetylation at the GR promoter but increased histone acetylation at the CRH promoter and reduced GR-CRH interactions in the CeA. Analysis of the spinal cord showed that WAS increased CRD-evoked ERK phosphorylation in the dorsal horn. Exposure to EE prevented WAS-induced changes in the CeA, dorsal horn and colon respectively to prevent visceral and somatic hypersensitivity. CONCLUSION: Our data reveals that behavioral therapies can produce long lasting molecular and epigenetic changes that can prevent stress-induced pathologies even after completion of the therapy. These results highlight the potential mechanisms by which behavioral therapies may ameliorate visceral pain associated stress-related pathologies such as the irritable bowel syndrome.

Essential role of Cav3.2 T-type calcium channels in butyrate-induced colonic pain and nociceptor hypersensitivity in mice.

Given the role of Cav3.2 isoform among T-type Ca2+ channels (T-channels) in somatic and visceral nociceptive processing, we analyzed the contribution of Cav3.2 to butyrate-induced colonic pain and nociceptor hypersensitivity in mice, to evaluate whether Cav3.2 could serve as a target for treatment of visceral pain in irritable bowel syndrome (IBS) patients. Mice of ddY strain, and wild-type and Cav3.2-knockout mice of a C57BL/6J background received intracolonic administration of butyrate twice a day for 3 days. Referred hyperalgesia in the lower abdomen was assessed by von Frey test, and colonic hypersensitivity to distension by a volume load or chemicals was evaluated by counting nociceptive behaviors. Spinal phosphorylated ERK was detected by immunohistochemistry. Cav3.2 knockdown was accomplished by intrathecal injection of antisense oligodeoxynucleotides. Butyrate treatment caused referred hyperalgesia and colonic hypersensitivity to distension in ddY mice, which was abolished by T-channel blockers and/or Cav3.2 knockdown. Butyrate also increased the number of spinal phosphorylated ERK-positive neurons following colonic distension in the anesthetized ddY mice. The butyrate-treated ddY mice also exhibited T-channel-dependent colonic hypersensitivity to intracolonic Na2S, known to enhance Cav3.2 activity, and TRPV1, TRPA1 or proteinase-activated receptor 2 (PAR2) agonists. Wild-type, but not Cav3.2-knockout, mice of a C57BL/6J background, after treated with butyrate, mimicked the T-channel-dependent referred hyperalgesia and colonic hypersensitivity in butyrate-treated ddY mice. Our study provides definitive evidence for an essential role of Cav3.2 in the butyrate-induced colonic pain and nociceptor hypersensitivity, which might serve as a target for treatment of visceral pain in IBS patients.

Receptors involved in dexketoprofen analgesia in murine visceral pain.

Various animal models, especially rodents, are used to study pain, due to the difficulty of studying it in humans. Many drugs that produce analgesia have been studied and there is evidence among which NSAIDs deserve to be highlighted. Dexketoprofen (DEX) provides a broad antinociceptive profile in different types of pain; therefore, this study was designed to evaluate the profile of antinociceptive potency in mice. Analgesic activity was evaluated using the acetic acid abdominal constriction test (writhing test), a chemical model of visceral pain. Dose-response curves for i.p. DEX administration (1, 3, 10, 30 and 100 mg/kg), using at least six mice in each of at least five doses, was obtained before and 30 min after pre-treatment with different pharmacological agents. Pretreatment of the mice with opioid receptor antagonists was not effective; however, the serotonin receptor antagonist and nitric oxide synthase inhibitor produce a significant increase in DEX-induced antinociception. The data from the present study shows that DEX produces antinociception in the chemical twisting test of mice, which is explained with difficulty by the simple inhibition of COX. This effect appears to be mediated by other mechanisms in which the contribution of the NO and 5-HT pathways has an important effect on DEXinduced antinociception.

IRE1α-XBP1 signaling in leukocytes controls prostaglandin biosynthesis and pain.

Inositol-requiring enzyme 1[α] (IRE1[α])-X-box binding protein spliced (XBP1) signaling maintains endoplasmic reticulum (ER) homeostasis while controlling immunometabolic processes. Yet, the physiological consequences of IRE1α-XBP1 activation in leukocytes remain unexplored. We found that induction of prostaglandin-endoperoxide synthase 2 (Ptgs2/Cox-2) and prostaglandin E synthase (Ptges/mPGES-1) was compromised in IRE1α-deficient myeloid cells undergoing ER stress or stimulated through pattern recognition receptors. Inducible biosynthesis of prostaglandins, including the pro-algesic mediator prostaglandin E2 (PGE2), was decreased in myeloid cells that lack IRE1α or XBP1 but not other ER stress sensors. Functional XBP1 transactivated the human PTGS2 and PTGES genes to enable optimal PGE2 production. Mice that lack IRE1α-XBP1 in leukocytes, or that were treated with IRE1α inhibitors, demonstrated reduced pain behaviors in PGE2-dependent models of pain. Thus, IRE1α-XBP1 is a mediator of prostaglandin biosynthesis and a potential target to control pain.

Decreased miR-325-5p Contributes to Visceral Hypersensitivity Through Post-transcriptional Upregulation of CCL2 in Rat Dorsal Root Ganglia.

Chronic visceral hypersensitivity is an important type of chronic pain with unknown etiology and pathophysiology. Recent studies have shown that epigenetic regulation plays an important role in the development of chronic pain conditions. However, the role of miRNA-325-5p in chronic visceral pain remains unknown. The present study was designed to determine the roles and mechanism of miRNA-325-5p in a rat model of chronic visceral pain. This model was induced by neonatal colonic inflammation (NCI). In adulthood, NCI led to a significant reduction in the expression of miRNA-325-5p in colon-related dorsal root ganglia (DRGs), starting to decrease at the age of 4 weeks and being maintained to 8 weeks. Intrathecal administration of miRNA-325-5p agomir significantly enhanced the colorectal distention (CRD) threshold in a time-dependent manner. NCI also markedly increased the expression of CCL2 (C-C motif chemokine ligand 2) in colon-related DRGs at the mRNA and protein levels relative to age-matched control rats. The expression of CXCL12, IL33, SFRS7, and LGI1 was not significantly altered in NCI rats. CCL2 was co-expressed in NeuN-positive DRG neurons but not in glutamine synthetase-positive glial cells. Furthermore, CCL2 was mainly expressed in isolectin B4-binding- and calcitonin gene-related peptide-positive DRG neurons but in few NF-200-positive cells. More importantly, CCL2 was expressed in miR-325-5p-positive DRG neurons. Intrathecal injection of miRNA-325-5p agomir remarkably reduced the upregulation of CCL2 in NCI rats. Administration of Bindarit, an inhibitor of CCL2, markedly raised the CRD threshold in NCI rats in a dose- and time-dependent manner. These data suggest that NCI suppresses miRNA-325-5p expression and enhances CCL2 expression, thus contributing to visceral hypersensitivity in adult rats.

Quercetin Attenuates Visceral Hypersensitivity and 5-Hydroxytryptamine Availability in Postinflammatory Irritable Bowel Syndrome Rats: Role of Enterochromaffin Cells in the Colon.

Intestinal enterochromaffin (EC) cell hyperplasia and increased 5-hydroxytryptamine (5-HT) availability play key roles in the pathogenesis of abdominal hypersensitivity of irritable bowel syndrome (IBS). This study aims to study the effect of quercetin on visceral pain and 5-HT availability in postinflammatory IBS (PI-IBS) rats. PI-IBS model rats were administered quercetin by gavage at doses of 5, 10, and 20 mg/kg for 14 days. Compared with normal rats, the visceral pain threshold of PI-IBS rats was markedly decreased and the abdominal motor response to colon distension was markedly increased. The EC cell count and 5-HT level, as well as tryptophan hydroxylase (TPH) protein, were all significantly elevated in PI-IBS rats, while the 5-HT reuptake transporter (serotonin transporter) was reduced. Genes that are responsible for enteroendocrine cell differentiation, that is, Ngn3 and pdx1, were significantly increased in the PI-IBS group. Quercetin treatment markedly elevated the pain threshold pressure and decreased the visceral motor response of PI-IBS animals; and EC cell density and 5-HT level, as well as TPH expression, in the PI-IBS group were all reduced by quercetin. Quercetin treatment also significantly reduced colonic expression of Ngn3 and pdx1 of PI-IBS. Findings from the present study indicated that the analgesic effect of quercetin on PI-IBS may result from reduction of 5-HT availability in the colon, and the regulatory role of quercetin in endocrine progenitors may contribute to reduced EC cells.

Dietary ascorbic acid restriction in GNL/SMP30-knockout mice unveils the role of ascorbic acid in regulation of somatic and visceral pain sensitivity.

Cav3.2 T-type Ca2+ channels are expressed in the primary afferents and play a pronociceptive role. The activity of Cav3.2 is enhanced by H2S, a gasotransmitter, and suppressed by ascorbic acid (vitamin C) through metal-catalyzed oxidation of the Zn2+-binding His191 in Cav3.2. Since rodents, but not humans, are capable of synthesizing ascorbic acid, the present study examined the role of ascorbic acid in nociceptive processing, using the mice lacking GNL/SMP30, an enzyme essential for ascorbic acid biosynthesis. Intraplantar and intracolonic administration of NaHS, an H2S donor, caused somatic allodynia and referred hyperalgesia, respectively, and repeated treatment with paclitaxel produced neuropathic allodynia in wild-type mice, all of which were suppressed by ascorbic acid or T-type Ca2+ channel blockers. Dietary ascorbic acid restriction caused dramatic decreases in plasma and tissue ascorbic acid levels in GNL/SMP30-knockout, but not wild-type, mice. The ascorbic acid restriction enhanced the somatic and visceral hypersensitivity following intraplantar and intracolonic NaHS, respectively, and paclitaxel-induced neuropathy in GNL/SMP30-knockout mice, while it had no such effect in wild-type mice. Together, our data unveil the critical role of ascorbic acid in regulating somatic and visceral pain sensitivity and support accumulating clinical evidence for the usefulness of ascorbic acid in pain management.

MiRNA-29a modulates visceral hyperalgesia in irritable bowel syndrome by targeting HTR7.

Some patients with irritable bowel syndrome (IBS) have visceral hypersensitivity, which contributes to their abdominal pain. miRNA-29 was detected to be significantly upregulated in colonic tissues of patients with IBS. However, it is unknown whether miRNA-29a is involved in the visceral hypersensitivity pathogenesis of IBS. This study aimed to investigate whether miRNA-29a participates in visceral hypersensitivity in IBS. We investigated miRNA-29a in intestinal biopsies collected during endoscopy of patients with IBS (n = 10) and healthy volunteers (control) (n = 10). In addition, a water avoidance stress (WAS)-induced visceral hypersensitivity IBS mouse model was established. The abdominal withdrawal reflex (AWR) scores of mice in response to colorectal distention were used to assess visceral sensitivity. Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) was used to measure miRNA-29a levels. Immunofluorescence, RT-qPCR and western blot were used to measure 5-HT7 receptor (HTR7) levels. Bioinformatic analysis and luciferase reporter assays were used to detect the direct relationship between miRNA-29a and HTR7. Finally, alterations in the levels of HTR7 and miRNA-29a were measured in the human intestinal epithelial cell line NCM460 after transfection with miRNA-29a inhibitor or mimic. Intestinal tissues from patients with IBS and WAS-induced IBS mice had increased levels of miRNA-29a, but reduced levels of HTR7. MiRNA-29a knockout resulted in overexpression of HTR7 and attenuated visceral hyperalgesia in WAS-induced IBS mice. HTR7 was a direct target of miRNA-29a. Based on analyses of intestinal tissue samples from patients with IBS and WAS-induced miRNA-29a-/- mice, miRNA-29a plays a role in the visceral hyperalgesia pathogenesis of IBS, probably through regulating HTR7 expression.