Electroacupuncture intervention of visceral hypersensitivity is involved in PAR-2-activation and CGRP-release in the spinal cord

Electroacupuncture (EA) relieves visceral hypersensitivity (VH) with underlying inflammatory bowel diseases. However, the mechanism by which EA treats ileitis-induced VH is not clearly known. To assess the effects of EA on ileitis-induced VH and confirm whether EA attenuates VH through spinal PAR-2 activation and CGRP release, goats received an injection of 2,4,6-trinitro-benzenesulfonic-acid (TNBS) solution into the ileal wall. TNBS-injected goats were allocated into VH, Sham acupuncture (Sham-A) and EA groups, while goats treated with saline instead of TNBS solution were used as the control. Goats in EA group received EA at bilateral Hou-San-Li acupoints for 0.5 h at 7 days and thereafter repeated every 3 days for 6 times. Goats in the Sham-A group were inserted with needles for 0.5 h at the aforementioned acupoints without any hand manipulation and electric stimulation. Visceromotor responses to colorectal distension, an indicator of VH, were recorded by electromyography. The terminal ileum and thoracic spinal cord (T11) were sampled for evaluating ileitis at days 7 and 22, and distribution and expression-levels of PAR-2, CGRP and c-Fos on day 22. TNBS-treated-goats exhibited apparent transmural-ileitis on day 7, microscopically low-grade ileitis on day 22 and VH at days 7–22. Goats of Sham-A, VH or EA group showed higher (P < 0.01) VH at days 7–22 than the Control-goats. EA-treated goats exhibited lower (P < 0.01) VH as compared with Sham-A or VH group. Immunoreactive-cells and expression-levels of spinal PAR-2, CGRP and c-Fos in the EA group were greater (P < 0.01) than those in the Control group, but less (P < 0.01) than those in Sham-A and VH groups on day 22. Downregulation of spinal PAR-2 and CGRP levels by EA attenuates the ileitis and resultant VH.


TNBS-induced ileal inflammation.
TNBS-administrated goats demonstrated mucoid or bloody diarrhea, sluggishness and gradual weight loss. As compared with Control group, the goats of VH, Sham-A and EA groups resulted in lower (P = 0.008, 0.045 and 0.049) body weight at 7 days [F (3, 32) = 5.380, P = 0.004]. The body weight did not differ (P > 0.05) among the VH, Sham-A and EA groups. The goats of VH group exhibited lower (P = 0.039) body weight than that of the Control at 22 days [F (3, 20) = 9.755, P = 0.0003]. The EA-treated goats showed greater (P = 0.0003 or 0.005) body weight than the goats of the VH or Sham-A group at 22 days (Fig. 1a). No obvious histopathological lesions were observed on saline-treated ilea at days 7 and 22. However, the TNBSinjected ilea showed hemorrhage, necrosis, edema, adhesion with intestinal loop or mesentery, ulcerations and thickened ileal wall on day 7 (Fig. 1b).The segments cranial (jejunum) and caudal (cecum and colon) to the TNBS-injected ileum did not reveal any pathological lesions. Microscopically, the TNBS-injected ilea exhibited disarranged intestinal villi, distorted epithelial crust, shorter crypt, fewer goblet cells, the increased neutrophils and lymphocytes, granulomatous lesions and discrete edema at days 7 and 22 (Fig. 2). However, the ilea of EAtreated goats revealed no histopathologically remarkable changes on day 22.
The ilea of VH group revealed remarkably higher (P < 0.01) macroscopic (U = 2.000, P = 0.007) and microscopic changes (U = 0.000, P = 0.003) scores than the control on day 7 (Figs. 1c, 2). As compared with control, the macroscopic (U = 7.500, P = 0.05 and U = 5.500, P = 0.026) and microscopic (U = 0.000, P = 0.003 and U = 4.000, P = 0.014) change scores in the ileum of VH and Sham-A groups were increased (P < 0.05, 0.01) on day 22. However, no difference (P > 0.05) in macroscopic and microscopic change scores was found between ileal samples of Control and EA groups. The ilea of Sham-A (U = 4.000, P = 0.014) and VH (U = 0.000, P = 0.003) groups exhibited an increased (P < 0.05, 0.01) change in microscopic scores as compared to the ilea of EA-treated goats. Likewise, the TNBS-injected ileal segments showed markedly higher (P < 0.01) MPO concentrations [F (3, 20) = 9.503, P = 0.000] than NaCl-injected ilea on day 7 (Fig. 1d). However, no changes (P > 0.05) in MPO concentrations among the four groups were found on day 22.     Sham-A, VH and EA groups showed higher mRNA levels of spinal PAR-2, CGRP and c-Fos compared with the Control. Spinal mRNA level of EA group was lower (P < 0.01) than those of VH or Sham-A group, but not different (P > 0.05) from that of the Control (Fig. 6).

Discussion
Visceral hypersensitivity (VH), an important symptom of IBDs and IBS, has received great interest in the recent years. However, VH originated from the distal part of the ileum still remains unexplored. Several researchers used the colitis model for studying the VH, which is not suitable for investigating the exact underlying pathogenesis of ileitis-induced VH because of its different anatomical location, the severity of inflammation, gut microbiome as well as spinal afferent pathways. Shah et al. 40 administered TNBS/ethanol solution into the rat's ileal lumen to induce transmural ileitis including granuloma and VH, which lasted over 20 days. Besides rodents, other animals have also been used for studying the ileitis and VH [41][42][43] . Goats are the appropriate animals for investigating the mechanism by which EA attenuates VH because of their profound sensitivity to EA stimuli and easier handling with minimal stress. Tahir et al. 42 injected TNBS/ethanol solution (1.2 ml; 30 mg TNBS in 40% ethanol) in goat's ileal wall through laparotomy and showed ileal inflammation for 21 days and VH for at least 28 days. Recently, MPO activity has been assayed as a biochemical indicator of inflammation. It is observed in the neutrophils and their oxidative products, particularly involved in the tissue damage process during the acute stage of  32 reported that the MPO concentration in TNBS-injected ileum of the goats was increased on day 7 rather than on day 22. Increasing sets of evidence show the importance of PAR-2 both in gastroenteritis 11,45,46 and inflammatory condition of the central nerve system 17,47,48 . PAR-2 has been expressed in two-thirds of sensory neurons, and 40% of PAR-2 expressing sensory neurons coexpress CGRP and substances-P 17 . Proteases such as trypsin, tryptase and cathepsin-s, activate PAR-2 and cause the release of neuropeptides from the primary spinal afferents to release their peripheral and central terminals. The peripherally released neuropeptides inflict neurogenic inflammation while their central releases result in hyperalgesia 5 . Interestingly, proteases that activate the neuronal PAR-2 have been derived from the blood and mast cells, or are recruited into the neurons and astrocytes [49][50][51][52] . Intracolonically infused PAR-2 agonist (SLIGRL-NH 2 ) results in the persistent VH, with an elevated spinal c-Fos expression as an indicator of the neuronal activation 53 . The majority of neurons showed increased CGRP expression after peripheral inflammation, and mice missing CGRP expression in the CNS failed to generate inflammation-induced hyperalgesia 54 . In this study, goats with TNBSinduced ileitis showed remarkably higher VMR to CRD and increased expressions of PAR-2 and CGRP in the SCDH. These studies indicate that intestinal VH development that may be caused by PAR-2 activated release of CGRP in CNS. However, the molecular involvement of spinal PAR-2 in ileitis-induced VH is unknown. It is well known that the PAR-2 is involved in signaling mechanism to regulate activity as well as expression of ion channels. The activated PAR-2 increases TRPV1 currents and regulates the TRPV1-induced Ca 2+ signal transduction in neuronal cells via a protein kinase-C (PKC) dependent manner. Furthermore, PAR-2 activation enhances the capsaicin-evoked CGRP release 5,18 , which has been proved to be remarkably involved in the nociception [55][56][57] . The c-Fos, a proto-oncogene protein, has been exclusively considered as a biological indicator of the activated central neurons or areas 34,35 . It can be rapidly expressed in the SCDH neurons after noxious stimulation. www.nature.com/scientificreports/ An earlier study 36 reported the increased expression level of c-Fos in laminae-I and -II or -V and -VI of the SCDH after nociceptive stimulation. It plays a crucial role in the development of a pain state as part of spinal cord adaptive response to both constant and subsequent nociceptive input. However, EA stimulation activates c-Fos-immunoreactive cells on the laminae-III and -IV of the SCDH 34,58 , which is somewhat different from those caused by noxious stimulation. It is important to note that both high and low frequencies of EA stimulations suppress nociception-induced spinal c-Fos expression 59 . Qi and Li 60 reported that EA significantly reduced chronic visceral hyperalgesia and provoked c-Fos expressions in SCDH superficial laminae. Therefore, c-Fos levels are commonly used to evaluate how EA modifies the neuronal regulatory mechanism underlying the neuropathic and inflammatory pain. In this study, c-Fos levels in the superficial laminae of the SCDH were increased during VH and ileitis, but decreased after EA treatment, indicating that intestinal inflammation-or VH-activated SCDH was the important site on which EA regulates. Accumulating sets of evidence supports the presence of VH due to cross-sensitization between the different internal organs in human [61][62][63] and experimental animals [64][65][66][67] . Furthermore, Pan et al. 68 described the sensitization of pelvic organs in rats with TNBS-induced colitis because colitis triggers the release of SP and CGRP in the urinary bladder. Likewise, CD pediatric patients coexisting functional GI disorders showed rectal hypersensitivity 2 . Our earlier studies 32,40,42 also reported VH in response to graded CRD in rats and goats with TNBS-induced ileitis. Researches demonstrate that VH across the organs is due to the central sensitization of spinal cord neurons receiving convergent input from different organs 69,70 or descending bulbospinal inhibition of sacral dorsal horn neurons in response to chronic intestinal tissue irritation 71 . Therefore, assessment of VMRs to CRD in TNBS-induced ileitis goats in our experiment is based on the rationale of central sensitization of spinal cord neurons receiving convergent input from both thoracolumbar and lumbosacral spinal afferents pathways.   76 demonstrated that EA stimulation at ST36 resulted in attenuation of the acute inflammation-induced visceral pain in rats. Furthermore, EA at ST36 relieved the visceral sensitivity and the gastrointestinal motility disorder in rats of IBS model 77 . Recently, Wan et al. 32 reported that EA at ST36 reduced pain behavioral manifestations and VH in goats. In this study, EA application at bilateral ST36 reduced the TNBSinduced ileitis and VMR to CRD of goats, which is similar to the findings of the earlier study 32 . These experiments clearly demonstrated that EA has the potential for attenuating VH. However, its underlying mechanisms need to be further explored. Hu et al. 78 reported EA at ST36 and Kulun (BL60) that attenuated inflammation-induced hyperalgesia and down-regulated the PAR-2 levels in dorsal root ganglion neurons. Sun et al. 33 described that EA attenuates VH by suppressing the spinal CGRP in diarrhea-predominant IBS rats. In this study, TNBS injection induced a remarkable VH, and higher expressions of PAR-2 and CGRP in the SCDH of goats, which were reversed by repeated EA. Our study suggests that EA intervention of VH is involved in its downregulation of spinal PAR-2 and CGRP.

Methods
Animal grouping. A total of 36 goats (20 males and 16 females with the dry period, i.e., not pregnant); 1-year-old and weighing 24.28 ± 5.01 kg were bought from Hubei Agricultural Academy of Science. Goats were fed on dried grass supplemented with a concentrated ration, and availed drinking water ad libitum. The clinically healthy goats were acclimatized to the surroundings for a period of 15 days. The experiments involving animals were performed according to the stipulated rules for experimental usage of laboratory animals (the regulation of the administration of affairs concerning experimental animals of P.R. China). All protocols were approved by the Laboratory Animal Research Center, Hubei and the ethical committee of Huazhong Agricultural University (Permit number: HZAUMO-2015-12). The goats were randomly allocated into the Control (n = 12), Sham-Acupuncture (Sham-A; n = 6), Visceral hypersensitivity (VH; n = 12), and Electroacupuncture (EA; n = 6). All experimental goats were injected with a solution of TNBS/ethanol and 0.9% NaCl into the ileal wall through laparotomy to induce VH on day 0. The 6 goats taken from each of the VH group (4 male and 2 female) and  for the resection of terminal ileum segments for H&E and MPO assay. Goats of all groups were restrained on sternal position. In goats of EA group, electroacupuncture (60 Hz, 3 mA) was used to stimulate the bilateral ST-36 acupoints for 0.5 h firstly at 7 days and thereafter repeated every 3 days for 6 times. Sham-acupuncture was conducted for 0.5 h by inserting needles at the acupoints. A balloon catheter was inserted colorectally, and its tubular end was attached with a Y-connector to a vacuum-pump and sphygmomanometer for measurement of distension pressure. Two nickel-steel needles were inserted 2-3 cm apart as electrodes into the left abdominal musculature. The electrodes were fastened on the skin individually and connected to an EMG-apparatus. VMRs to CRD pressures were recorded by EMG. The balloon was distended for 6 s with 20 mmHg and pressures were increased continuously in a ramp mode to 40, 60, 80 and 100 mmHg at an interval of 6 s. CRD and EMG were recorded three times at an interval of 3 min. Immediately, goats were euthanized to collect the terminal-ileum for evaluation of ileitis and spinal cord (T 11 ) for distribution and expression-levels of PAR-2, CGRP and c-Fos on day 22.
Scientific RepoRtS | (2020) 10:11188 | https://doi.org/10.1038/s41598-020-67702-2 www.nature.com/scientificreports/ minal ileum on moist sterile gauze. For the goats in Sham-A, VH and EA groups, 1.2 ml (30 mg TNBS; Sigma Aldrich, USA, mixed in 30% Ethanol) was injected at five points on the non-mesenteric side of ileum, just 15 cm anteriorly from the ileo-cecal junction, with a 30-G needle attached to a 2-CC syringe. The TNBS dose was ascertained based on our earlier experiment 79 . For goats in the Control group, an equal volume of 0.9% NaCl was injected into the ileal wall following the aforementioned method. The cranial and caudal margin of the injection area was marked with two loosely placed silk ligatures (3-0) in the mesentery for easy identification during sampling time. The abdominal incision was closed as usual. Once recovered from anaesthesia, the goats were fed on dried grass supplemented with concentrated ration and availed free access to drinking water. The goats were monitored daily, and their incised wounds were dressed with 1% povidone-iodine twice a day daily till healing. Goats were injected with Tramadol-HCl (4 mg/kg, IM; Hubei Qianjiang Pharmaceutical Co., Ltd, China) up to 3 days for the relief of post-surgical pain. For the prevention of infection, Ampicillin (10 mg/kg, IM; Wuhan Shu Ou Technology Co., Ltd, China) was administered twice a day for upto 5 days.
Assessment of ileal inflammation. The body weight, feed and water consumption and mortality rate of the experimental goats were recorded appropriately, while the fecal consistency and hemorrhage in feces were evaluated at days 0, 3 and 7. After overnight fasting, 6 goats from each of the VH and Control groups on day 7 underwent the same laparotomy procedure as described above for partial ileal samples for macroscopic and microscopic assessment and MPO assay. In brief, the mesenteric arteries and veins of the intestine to be resected were properly ligated with catgut, and the intestinal content was milked away. Two intestinal forceps were used to occlude the cranial and caudal ends of the intestinal segment. Approximately, a 5 cm ileum was resected from the previously marked area. After being washed in PBS solution, the intestinal segment was longitudinally incised and spread out on a sterilized drape to score the macroscopic lesions by two independent observers employing a 0-4 scale modifying the earlier criteria 79 . An anterior 2 × 2 cm intestinal segment was cut from the non-mesenteric side and fixed in a 4% buffered formaldehyde solution for the observation of microscopic lesions. A distal 2 × 2 cm non-mesenteric intestinal segment was weighed, frozen in liquid nitrogen (N 2 ) and stored at − 80 °C for measuring the MPO concentration. Parallelly, an experienced surgical team anastomosed the two intestinal segments together in an end-to-end pattern, and laparotomy incision was closed in a routine manner. Goats were kept in a quiet room and observed till completely recovered from anaesthesia. Postoperative care was performed as mentioned above. The goats were sent to farms after 2 weeks of recovery to resume their normal lives. The ileal tissue sections were performed following our previous report 40 . Briefly, the formaldehyde-fixed ileal specimens (2 × 2 cm) were embedded in paraffin, microtomed at 5 μm and mounted properly on polylysine coated slides. The three sequential slides were stained with H & E for observation (20 ×) with a microscope (Nikon Eclipse 80I, Nikon Corporation, Tokyo, Japan). The microscopic lesions were assessed and scored by two pathologists who were unaware of the experimental protocol using a 0-9 scale as described previously 80 . The distal ileal specimens were ground, homogenized at 4 °C in 1 ml PBS (pH 7.2) and centrifuged at 5,000 g at 4 °C for 10 min. The aliquots were separated to measure their protein concentrations using a Nanodrop Spectrophotometer (Thermo Fisher Scientific, Inc., USA). The MPO concentration was evaluated in triplicate utilizing its specific ELISA Kit (eBioscience, Inc., San Diego, CA 92,121, USA). The MPO values are expressed as pg/mg.

electroacupuncture.
A set of bilateral Hou-San-Li (i.e. equivalent to Zu-San-Li; ST-36 in humans) points described in veterinary medicine was chosen for EA 81 . Seven days after the surgery, a pair of sterile acupuncture needles (0.45 mm D × 7.5 cm L, Suzhou Medical Supplies, Co. Ltd., China) was inserted 2 cm deeper bilaterally at the Hou-San-Li points in hind legs of the goats of EA group. Here, we just inserted needles into the muscles but not implanted them surgically. This technique did not irritate the goats, and thereby VMR values were not interfered by them. The both bilaterally inserted needles were linked with the wires to the output terminal of an EA-machine (WQ-6F Electronic Acupunctoscope; Beijing Xindonghua Electronic Instrument Co., Ltd., Beijing, China). In this study, we used 60 Hz stimulation frequency because of its profound analgesic effect in goats 82 . After restraining the experimental goats in sternal recumbency, EA was applied at Hou-San-Li acupoints with a stimulation frequency of 60 Hz for 0.5 h. The intensity of stimulation was accustomed to a value that only induced mild muscular spasm at the acupoints and it was restricted below 3 mA so as to avoid the discomfort. In the Sham-A group, acupuncture needles were inserted at the same acupoints of the goats and kept for 0.5 h without any hand manipulation and electrical stimulation. The goats in Control and VH groups were restrained only for 0.5 h similarly as the EA-treated goats. The EA treatments were applied firstly on day 7 and thereafter repeated at an interval of 3 days, upto 6 times as reported earlier 32 .
Visceromotor response to colorectal distension. Electromyographies (EMGs) were performed for the quantitative measurement of visceromotor responses at different CRD pressures. It was recorded in all the goats immediately after EA treatment from 6 a.m. to 11 a.m. at days 7, 10, 13, 16, 19 and 22 following the previously described methods 40,83 . Initially, the goats were accustomed to the testing room for a period of 3 days. A balloon catheter (8 cm long), made from the finger of a latex surgical glove, was lubricated with paraffin jelly. It was introduced 10 cm colorectally via the anus and its tubular end was attached using the Y-tubing to a vacuumpump and sphygmomanometer for simultaneous distention of balloon and measurement of pressure. A pair of nickel-steel needles (0.45 mm D × 5 cm L) was introduced at a distance of 2-3 cm into the abdominal musculature of the left flank as electrodes. Thereafter, both electrodes were fastened to the skin and linked with a cable to an EMG-apparatus (Nanjing Medease Science and Technology Co. Ltd., China). The balloon was distended for 6 s with 20 mmHg and then to 40, 60, 80 and 100 mmHg in a continuous ramp mode at an interval of 6 s. Simultaneously, EMG was recorded for 6 s at each stage and then a 3 min break was done before another set Scientific RepoRtS | (2020) 10:11188 | https://doi.org/10.1038/s41598-020-67702-2 www.nature.com/scientificreports/ of distention for a total of 3 sets (Fig. 7). The EMG and mechanical irritation signals were quantified using the software (MedLab-U/4C501, Nanjing Medease Science and Technology Co., Ltd, China). The differences of total areas of EMG during distensions and resting periods were considered as VMRs to CRD, and are expressed as millivoltage per-second (mV/s).
Sample collection at the end of the experiment. Immediately after EMG, the xylidinothiazoline (3 mg/kg) was administered intravenously for euthanasia of the goats on day 22. Previously marked intestinal segment (5 cm) was excised and processed for assessing the inflammatory status with H & E and MPO concentration assay (as mentioned above in the assessment of ileal inflammation). The upper part of the eleventh thoracic spinal cord (T 11 ) was excised and fixed in 4% buffered formaldehyde for IHC. While the lower part of the same spinal cord was frozen in liquid N 2 and kept at − 80 °C for mRNA and protein extraction to be used for qRT-PCR and WB analysis, respectively.
Immunohistochemistry. The fixed spinal specimens were processed, embedded properly in paraffin, sectioned using a microtome (5 μm), and mounted on glass slides coated with polylysine. Four sequential slides were processed for the SAB-immunohistochemistry procedure 32 . Briefly, these slides were incubated with mouse anti-PAR-2 antibody (Santa Cruz Biotechnology, CA, USA; 1:100 diluted in PBS), rabbit anti-CGRP antibody (Abcam Inc., Cambridge, MA-02139-1517, USA; 1:100 diluted in PBS), rabbit anti-c-Fos antibody (Santa Cruz Biotechnology, CA, USA; 1:200 diluted in PBS) and PBS (the negative control) for 12 h at 4 °C, respectively. Thereafter, the slides were incubated with their specific secondary antibodies (SAP 102-anti-mouse IgG Kit and SA1022-anti-rabbit IgG Kit, Boster Biological Technology Ltd., Wuhan, China) for 1 h at normal laboratory temperature. Finally, the slides were observed under the microscope using DAB for 2-3 min at normal laboratory temperature.
Western blot analysis. The lower part of the T 11 spinal cord was weighed and triturated in liquid N 2, and its protein was extracted using the RIPA-lysis buffer (Beyotime Biotech, Nantong, China). Protein concentration was quantified using a Nanodrop Spectrophotometer (Thermo Fisher Scientific, Inc., USA). Twenty micrograms of protein were loaded in each lane and differentiated by 10% or 12% SDS-PAG gel based on their molecular weight. The protein was transferred to PVDF-membranes and blocked in 3% BSA solution for 2 h min at normal room temperature. The blocked membranes were incubated using their primary antibodies, mouse anti-PAR-2 (1:500 in PBS; Santa Cruz Biotechnology, CA, USA), rabbit anti-CGRP (1:1,000 in PBS, Abcam Inc., Cambridge, MA-02139-1517, USA), rabbit anti-c-Fos (1:500 in PBS, Santa Cruz Biotechnology, CA, USA) and rabbit anti-βactin (1:300, Santa Cruz Biotechnology, CA, USA), respectively, overnight at 4 °C. The membranes were washed and further treated with horseradish peroxidase conjugated secondary antibodies (goat anti-mouse-IgG/goat anti-rabbit IgG, 1:3,000 in PBS) for 1 h at normal room temperature. Visualization of the antigen-antibody complex was conducted with a horseradish peroxidase substrate (Millipore, Billerica, MA, USA) using the Image-Quant LAS 4,000 min CCD camera (GE Healthcare, Piscataway Township, NJ, USA). The bands were analysed using a Quantity One software (Bio-Rad), and the result is expressed as the immunoreactivity ratio of the target gene to β-actin. Statistical analysis. Data are represented as a mean ± SD. Data were analysed utilizing IBM-SPSS versus 23 (Armonk, NY: IBM Corp). Groups of comparisons were made using ANOVA, followed by Independent t test and Bonferroni post-hoc-test. The statistical differences in macroscopic and microscopic scores among groups were identified using Kruskal-Wallis analysis of variance followed by the rank-based Mann-Whitney U-test. A P < 0.05 was accepted as statistically significant.

Data availability
All the data generated during this study are statically analysed and illustrated as