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.

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.

Analgesic effects of electroacupuncture at ST25 and CV12 in a rat model of postinflammatory irritable bowel syndrome visceral pain.

BACKGROUND: Treatment with electroacupuncture (EA) at ST25 and CV12 has a significant analgesic effect on postinflammatory irritable bowel syndrome (PI-IBS) visceral pain. Enterochromaffin (EC) cells and serotonin (5-hydroxytryptamine (5-HT)) are important in the development of visceral hyperalgesia. OBJECTIVE: To investigate the analgesic effect and underlying mechanisms of EA at ST25 and CV12 on the treatment of trinitrobenzene sulfonic acid (TNBS)-induced PI-IBS visceral hyperalgesia in rats. METHODS: After EA at ST25 and CV12, changes in abdominal withdrawal reflex (AWR), electromyography (EMG) recordings, colonic EC cell numbers, and expression of tryptophan hydroxylase (TPH), 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) of TNBS-induced PI-IBS visceral hyperalgesia in rats were examined. RESULTS: The results of AWR tests and EMG recordings indicated a significant analgesic effect of EA stimulation at ST25 and CV12on PI-IBS visceral hyperalgesia (p<0.05). In addition, the increased EC cell numbers and colonic expression of TPH and 5-HT in rats with TNBS-induced PI-IBS visceral hyperalgesia were significantly reduced by EA (p<0.05). CONCLUSIONS: EA stimulation at ST25 and CV12 can attenuate visceral hyperalgesia. This analgesic effect may be mediated via reduction of both colonic EC cell number and 5-HT concentration.

Visceral and somatic pain modalities reveal NaV 1.7-independent visceral nociceptive pathways.

KEY POINTS: Voltage-gated sodium channels play a fundamental role in determining neuronal excitability. Specifically, voltage-gated sodium channel subtype NaV 1.7 is required for sensing acute and inflammatory somatic pain in mice and humans but its significance in pain originating from the viscera is unknown. Using comparative behavioural models evoking somatic and visceral pain pathways, we identify the requirement for NaV 1.7 in regulating somatic (noxious heat pain threshold) but not in visceral pain signalling. These results enable us to better understand the mechanisms underlying the transduction of noxious stimuli from the viscera, suggest that the investigation of pain pathways should be undertaken in a modality-specific manner and help to direct drug discovery efforts towards novel visceral analgesics. ABSTRACT: Voltage-gated sodium channel NaV 1.7 is required for acute and inflammatory pain in mice and humans but its significance for visceral pain is unknown. Here we examine the role of NaV 1.7 in visceral pain processing and the development of referred hyperalgesia using a conditional nociceptor-specific NaV 1.7 knockout mouse (NaV 1.7Nav1.8 ) and selective small-molecule NaV 1.7 antagonist PF-5198007. NaV 1.7Nav1.8 mice showed normal nociceptive behaviours in response to intracolonic application of either capsaicin or mustard oil, stimuli known to evoke sustained nociceptor activity and sensitization following tissue damage, respectively. Normal responses following induction of cystitis by cyclophosphamide were also observed in both NaV 1.7Nav1.8 and littermate controls. Loss, or blockade, of NaV 1.7 did not affect afferent responses to noxious mechanical and chemical stimuli in nerve-gut preparations in mouse, or following antagonism of NaV 1.7 in resected human appendix stimulated by noxious distending pressures. However, expression analysis of voltage-gated sodium channel α subunits revealed NaV 1.7 mRNA transcripts in nearly all retrogradely labelled colonic neurons, suggesting redundancy in function. By contrast, using comparative somatic behavioural models we identify that genetic deletion of NaV 1.7 (in NaV 1.8-expressing neurons) regulates noxious heat pain threshold and that this can be recapitulated by the selective NaV 1.7 antagonist PF-5198007. Our data demonstrate that NaV 1.7 (in NaV 1.8-expressing neurons) contributes to defined pain pathways in a modality-dependent manner, modulating somatic noxious heat pain, but is not required for visceral pain processing, and advocate that pharmacological block of NaV 1.7 alone in the viscera may be insufficient in targeting chronic visceral pain.

Downregulation of the spinal NMDA receptor NR2B subunit during electro-acupuncture relief of chronic visceral hyperalgesia.

The involvement of spinal NR2B, a N-methyl-D-aspartate (NMDA) receptor subunit, in the therapeutic effect of electro-acupuncture (EA) on chronic visceral hyperalgesia was investigated. Chronic visceral hyperalgesia was induced using an irritable bowel syndrome (IBS) model in rats. Graded colorectal distention (CRD) stimuli at strengths of 20, 40, 60 and 80 mmHg were applied, and behavioral tests were performed to measure the abdominal withdrawal reflex (AWR) in response to the CRD stimuli and assess the severity of the visceral hyperalgesia. Rats were randomly divided into four groups: normal intact (control) group, IBS model (model) group, EA-treated IBS rats (EA) group and sham EA-treated IBS rats (sham EA) group. For the EA treatment, electric stimuli were applied through needles inserted into two acupoints [Zu-san-li (ST-36) and Shang-ju-xu (ST-37)] in both hind limbs, while the sham EA treatment consisted of only the insertion of needles into these same acupoints without an application of electric stimuli. Our results showed that AWR scores of the model group responding to CRD stimuli of 20, 40, 60 and 80 mmHg were significantly increased. These increased scores subsequently decreased following EA treatment (P < 0.05) compared with those for the other groups. The expression of NR2B in the superficial laminae (SDH, laminae I and II), nucleus proprius (NP, laminae III and IV), neck of the dorsal horn (NECK, laminae V and VI) and central canal region (lamina X) at thoracolumbar (T13-L2) and lumbosacral (L6-S2) segmental level significantly increased in the model group versus the control group (P < 0.05) and significantly decreased after EA treatment (P < 0.05). There were no significant changes in neither AWR scores nor expression of the NR2B subunit in these spinal regions after the sham EA treatment. These results confirm that EA can relieve chronic visceral hyperalgesia in IBS model rats and suggest that such an effect is possibly mediated through the downregulation of the NR2B subunits of NMDA at the spinal level.

[Effect of Electroacupuncture and Moxibustion on Visceral Pain and Expression of VR 1 and HSP 70 of "Tianshu" (ST 25) Region in Colorectal Distension-induced Visceral Hypersensitivity Rats].

OBJECTIVE: To compare the effects of electroacupuncture (EA) and moxibustion (Moxi) on visceral pain and expression of vanilloid receptor subtype 1 (VR 1) and heat shock protein (HSP)70 in "Tianshu" (ST 25) region in colorectal distension (CRD)-induced visceral hypersensitivity (VHS) rats. METHODS: Fifty male SD rats were randomly divided into normal control, VHS model, 43℃-moxi, 46℃-moxi, 1 mA-EA and 3 mA-EA groups (n=10 in each group). The VSH model was established by CRD once daily for 14 days. EA or Moxi stimulation was applied to bilateral "Tianshu" (ST 25) for 10 min, once daily for consecutive 10 days. The abdominal withdrawal reflex (AWR) scores (0-4 points) were rated according to Al-Chaer's and coworkers' standards (2000) and the expression levels of VR 1 and HSP 70 in bilateral ST 25 area tissues detected by immunohistochemistry. RESULTS: The AWR scores for 20, 40, 60 and 80 mmHg CRD pressures were significantly increased compared to the normal control group (P<0.01) and notably decreased after 43℃- and 46℃-moxi, and 1 mA- and 3 mA-EA stimulation of bila-teral ST 25 in comparison with the model group (P<0.05, P<0.01), and the effect of 46℃-moxi was apparently superior to those of 1 mA-EA at 40 and 80 mmHg, and 3 mA-EA at 40 mmHg (P<0.05). After modeling, the expression of both VR 1 and HSP 70 (percentages of area of positive-cells) in ST 25 region had no significant changes (P>0.05). Compared to the model group, the expression levels of VR 1 in the 43℃-moxi and 46℃-moxi groups, and HSP 70 in the 43℃-moxi and 46℃-moxi, 1 mA-EA and 3 mA-EA groups were significantly up-regulated (P<0.01), but without obvious changes in the expression of VR 1 in the 1 mA-EA and 3 mA-EA groups (P>0.05). The effects of 46℃-moxi were considerably better than those of 43℃-moxi, 1 mA-EA and 3 mA-EA in up-regulating VR 1 and HSP 70 expression (P<0.05, P<0.01). No significant differences were found among the 43℃-moxi, 1 mA-EA and 3 mA-EA groups in the expression of VR 1 and HSP 70 (P>0.05). CONCLUSIONS: Moxibustion at 43℃ and 46℃ and EA at 1 mA and 3 mA, especially the 46℃-moxi, can relieve visceral pain in visceral hypersensitivity rats, which may be related to their effects in up-regulating expression of VR 1 and HSP 70 in "Tianshu" (ST 25) area.

Effects of electroacupuncture on corticotropin-releasing hormone in rats with chronic visceral hypersensitivity.

AIM: To investigate the effect of electroacupuncture on corticotropin-releasing hormone (CRH) in the colon, spinal cord, and hypothalamus of rats with chronic visceral hypersensitivity. METHODS: A rat model of chronic visceral hypersensitivity was generated according to the internationally accepted method of colorectal balloon dilatation. In the 7(th) week after the procedure, rats were randomly divided into a model group (MG), electroacupuncture group (EA), and sham electroacupuncture group (S-EA). After treatment, the abdominal withdrawal reflex (AWR) score was used to assess the behavioral response of visceral hyperalgesia. Immunohistochemistry (EnVision method), ELISA, and fluorescence quantitative PCR methods were applied to detect the expression of CRH protein and mRNA in the colon, spinal cord, and hypothalamus. RESULTS: The sensitivity of the rats to the colorectal distension stimulus applied at different strengths (20-80 mmHg) increased with increasing stimulus strength, resulting in increasing AWR scores in each group. Compared with NG, the AWR score of MG was significantly increased (P < 0.01). After conducting EA, the AWR scores of the rats were decreased compared with MG rats. The relative expression of CRH mRNA in the colon, spinal cord, and hypothalamus of MG rats was significantly increased compared with NG rats (P < 0.01). CRH mRNA in the colon and spinal cord of EA and S-EA rats was decreased to varying degrees (P > 0.05) compared with normal rats (NG). However, the decrease in EA compared with MG rats was statistically significant (P < 0.01). The average optical density of CRH expression in the colon of the MG rats was significantly enhanced compared with NG (P < 0.05), while the average optical density of CRH expression in the EA and S-EA rats was significantly decreased compared with MG rats (P < 0.01, P < 0.05, respectively). Compared with MG rats, the CRH concentration in the spinal cord of EA rats was significantly reduced (P < 0.01), but there was no significant change in S-EA rats (P > 0.05). CONCLUSION: Electroacupuncture at the Shangjuxu acupoint was able to significantly reduce the visceral hypersensitivity in rats, and regulated the expression of CRH protein and mRNA in the colon, spinal cord and hypothalamus at different levels, playing a therapeutic role in this model of irritable bowel syndrome.

Evidence of a role for GTP cyclohydrolase-1 in visceral pain.

BACKGROUND: The enzyme guanosine triphosphate-cyclohydrolase-1 (GCH-1) is a rate limiting step in the de novo synthesis of tetrahydrobiopterin (BH4) a co-factor in monoamine synthesis and nitric oxide production. GCH-1 is strongly implicated in chronic pain based on data generated using the selective GCH-1 inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP), and studies which have identified a pain protective GCH-1 haplotype associated with lower BH4 production and reduced pain. METHODS: To investigate the role for GCH-1 in visceral pain we examined the effects of DAHP on pain behaviors elicited by colorectal injection of mustard oil in rats, and the pain protective GCH-1 haplotype in healthy volunteers characterized by esophageal pain sensitivity before and after acid injury, and assessed using depression and anxiety questionnaires. KEY RESULTS: In rodents pretreatment with DAHP produced a substantial dose related inhibition of pain behaviors from 10 to 180 mg/kg i.p. (p < 0.01 to 0.001). In healthy volunteers, no association was seen between the pain protective GCH-1 haplotype and the development of hypersensitivity following injury. However, a substantial increase in baseline pain thresholds was seen between first and second visits (26.6 ± 6.2 mA) in subjects who sensitized to esophageal injury and possessed the pain protective GCH-1 haplotype compared with all other groups (p < 0.05). Furthermore the same subjects who sensitized to acid and possessed the haplotype, also had significantly lower depression scores (p < 0.05). CONCLUSIONS & INFERENCES: The data generated indicate that GCH-1 plays a role in visceral pain processing that requires more detailed investigation.