basic study alterations in serotonin, transient receptor ...€¦ · irritable bowel syndrome (ibs)...

Hai-Lian Shi, Chu-Hsuan Liu, Li-Li Ding, Yu Zheng, Xiao-Yan Fei, Lu Lu, Xue-Ming Zhou, Jian-Ye Yuan, Jian-Qun Xie

Hai-Lian Shi, Chu-Hsuan Liu, Yu Zheng, Xiao-Yan Fei, Lu Lu, Jian-Ye Yuan, Jian-Qun Xie, Institute of Digestive Diseases, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China Hai-Lian Shi, Li-Li Ding, Institute of Chinese Materia Medica, Shanghai Key Laboratory of Complex Prescription, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, ChinaXue-Ming Zhou, Experimental Animal Center, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, ChinaAuthor contributions: Shi HL and Liu CH carried out all the experiments, analyzed the data and wrote the paper, and contributed equally to this study; Ding LL performed the HPLC analysis; Yuan JY and Xie JQ designed the experiments, analyzed the data, revised the paper, and contributed equally to this study; Zheng Y, Fei XY, Lu L and Zhou XM performed parts of the experiments and provided valuable suggestions for this study; all authors have read and approved the final manuscript.Supported by Innovation Program of the Shanghai Municipal Education Commission, No. 12YZ065; National Natural Science Foundation of China, No. 81072786, No. 81473630 and No. 81202665; Longhua Medical Project, No. D-09; High level Project of the University of Educational Commission of Shanghai, China, No. 2008GSP19; and Shanghai Leading Academic Discipline Project, No. J50305.Institutional Review Board statement: This study has been reviewed and approved by the Institutional Review Board of Shanghai University of Traditional Chinese Medicine.Institutional Animal Care and Use Committee statement: All animal experiments were performed according to the protocols approved by the University Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine (IACUC protocol number: 2014022).Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and

the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/Correspondence to: Jian-Ye Yuan, Associate Professor, Institute of Digestive Diseases, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, 530 Lingling Road, Xuhui District, Shanghai 200032, China. [email protected] Telephone: +86-21-64385700Fax: +86-21-64398310Received: September 22, 2014 Peer-review started: September 24, 2014First decision: October 29, 2014Revised: December 7, 2014Accepted: January 30, 2015Article in press: January 30, 2015Published online: April 28, 2015

AbstractAIM: To determine the molecular mechanisms of Shugan decoction (SGD) in the regulation of colonic motility and visceral hyperalgesia (VHL) in irritable bowel syndrome (IBS).

METHODS: The chemical compounds contained in SGD were measured by high-performance liquid chromatography. A rat model of IBS was induced by chronic water avoidance stress (WAS). The number of fecal pellets was counted after WAS and the pain pressure threshold was measured by colorectal distension. Morphological changes in colonic mucosa were detected by hematoxylin-eosin staining. The contents of tumor necrosis factor (TNF)-α in colonic tissue and calcitonin-gene-related peptide (CGRP) in serum were measured by ELISA. The protein expression of serotonin [5-hydroxytryptamide (5-HT)], serotonin transporter (SERT), chromogranin A (CgA) and CGRP in

ORIGINAL ARTICLE

4852 April 28, 2015|Volume 21|Issue 16|WJG|www.wjgnet.com

Basic Study

Alterations in serotonin, transient receptor potential channels and protease-activated receptors in rats with irritable bowel syndrome attenuated by Shugan decoction

Submit a Manuscript: http://www.wjgnet.com/esps/Help Desk: http://www.wjgnet.com/esps/helpdesk.aspxDOI: 10.3748/wjg.v21.i16.4852

World J Gastroenterol 2015 April 28; 21(16): 4852-4863 ISSN 1007-9327 (print) ISSN 2219-2840 (online)

© 2015 Baishideng Publishing Group Inc. All rights reserved.

In recent years, laxatives or antidiarrheal agents, with or without spasmolytics, as well as serotonergic agents [e.g., tegaserod, a serotonin 5-hydroxytryptamine (5-HT4) agonist, and alosetron, a 5-HT3 antagonist], have achieved some therapeutic effects, but have not obtained satisfactory results due to their adverse effects[3]. Thus, to date, there are no standard therapeutic agents to treat IBS. Acupuncture and traditional Chinese medicine (TCM) have attracted increased attention due to their potential in the treatment of IBS. The efficacy of acupuncture on visceral hypersensitivity and related motility dysfunction in IBS has been demonstrated[4]. In China, there are many clinical formulas of TCM used in the treatment of IBS[2,5], for example, TongXieYaoFang (TXYF), is a classic TCM prescription[2] consisting of white attractylodes rhizome (Baizhu), white peony root (Baishao), dried old orange peel (Chenpi) and Ledebouriella root (Fangfeng). Shugan decoction (SGD) is another formula used to treat patients with diarrhea-predominant IBS, which is composed of TXYF and Radix bupleuri (Chaihu), and can alleviate visceral pain and improve bowel habits[6,7]. SGD shows an excellent therapeutic effect on patients who lack coordination between the liver and the spleen (TCM syndrome type)[6]. However, the functional mechanism of SGD is poorly understood.

Visceral hypersensitivity and gastrointestinal dysmotility are two primary characteristics of IBS. Transient receptor potential vanilloid type 1 (TRPV1) and TRP ankyrin-1 (TRPA1) have been shown to play an important role in visceral hypersensitivity, and are correlated with severity of abdominal pain in patients with IBS and inflammatory bowel disease (IBD), as well as IBS-like symptoms in rats with water avoidance stress (WAS)[8-12]. Alterations in protease-activated receptor (PAR)2 and PAR4 have also been indicated in visceral pain in IBS patients and stressed mice[13-16]. Serotonin (5-HT) in both the central and peripheral nervous systems, is involved in visceral hypersensitivity and gut motility disorders which can be attenuated by acupuncture and TCM[2,4,5,17]. Although their mechanisms have not been fully elucidated, regulation of the 5-HT signaling pathway has been shown to play an important role in relieving the symptoms of IBS. In recent studies, it was demonstrated that many types of endocrine cells are affected in IBS patients[18-20]. Lower densities of cells positive for 5-HT and peptide YY are found in the colon of patients with IBS, compared with healthy individuals. In addition, the density of chromogranin A (CgA) is reduced in the colon of patients with IBS[18-20].

A suitable animal model is important for elucidating the pathophysiological process of IBS. Previous studies have shown that repeated exposure to WAS induces sustained visceral hypersensitivity and gastrointestinal motility disorders in rats[21]. Therefore, WAS-exposed rats could reflect the typical characteristics of IBS. In the present study, the efficacy of SGD and its underlying mechanisms of action on visceral hypersensitivity and

Shi HL et al . Shugan decoction in rats with IBS


colon tissue was measured by immunohistochemistry.

RESULTS: SGD inhibited colonic motility dysfunction and VHL in rats with IBS. Blockers of transient receptor potential (TRP) vanilloid 1 (TRPV1) (Ruthenium Red) and TRP ankyrin-1 (TRPA1) (HC-030031) and activator of protease-activated receptor (PAR)4 increased the pain pressure threshold, whereas activators of PAR2 and TRPV4 decreased the pain pressure threshold in rats with IBS. The effect of SGD on pain pressure threshold in these rats was abolished by activators of TRPV1 (capsaicin), TRPV4 (RN1747), TRPA1 (Polygodial) and PAR2 (AC55541). In addition, CGRP levels in serum and colonic tissue were both increased in these rats. TNF-α level in colonic tissue was also significantly upregulated. However, the levels of 5-HT, SERT and CgA in colonic tissue were decreased. All these pathological changes in rats with IBS were attenuated by SGD.

CONCLUSION: SGD alleviated VHL and attenuated colon motility in IBS, partly by regulating TRPV1, TRPV4, TRPA1, PAR2, 5-HT, CgA and SERT, and reducing CGRP and TNF-α level.

Key words: Shugan decoction; Visceral hyperalgesia; Serotonin; Transient receptor potential; Protease-activated receptor; Serotonin transporter; Calcitonin-gene-related peptide; Tumor necrosis factor-α

© The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.

Core tip: The present study demonstrated that Shugan decoction alleviated visceral hyperalgesia and attenuated colon motility in a rat model of irritable bowel syndrome, partly by regulating the expression of transient receptor potential (TRP) vanilloid 1, TRP vanilloid 4, TRP ankyrin-1 and protease-activated receptor 2, serotonin, chromogranin A and serotonin transporter, and reducing the levels of calcitonin-gene-related peptide and tumor necrosis factor-α.

Shi HL, Liu CH, Ding LL, Zheng Y, Fei XY, Lu L, Zhou XM, Yuan JY, Xie JQ. Alterations in serotonin, transient receptor potential channels and protease-activated receptors in rats with irritable bowel syndrome attenuated by Shugan decoction. World J Gastroenterol 2015; 21(16): 4852-4863 Available from: URL: http://www.wjgnet.com/1007-9327/full/v21/i16/4852.htm DOI: http://dx.doi.org/10.3748/wjg.v21.i16.4852

INTRODUCTIONIrritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by altered bowel evacuation, bloating and visceral pain, without anatomical or biochemical abnormalities[1,2]. The pathological mechanism of IBS is not well understood.

colonic dysmotility were evaluated using WAS in a rat model of IBS.

MATERIALS AND METHODSDrug and materialsWhite attractylodes rhizome (Baizhu), white peony root (Baishao), dried old orange peel (Chenpi), ledebouriella root (Fangfeng), and Radix bupleuri (Chaihu) were purchased as crude herbs from the Yanghetang Pharmacy (Shanghai, China). Rabbit anti-CgA polyclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States); the SABC kit was purchased from Boston Biochem (Wuhan, China). Methanol was obtained from Merck (Darmstadt, Germany). Triple deionized water was from Millipore (Bedford, MA, United States) and was prepared for all aqueous solutions. Ruthenium Red, capsaicin, HC-030031, polygodial, AY-NH2, RN1747 and AC55541 were obtained from Tocris Bioscience (MI, United Kingdom). Saikosaponin d, penoniflorin, poncirin, synephrine, atractylenolide Ⅲ, isofraxidin, hesperidin and neohesperidin were purchased from Shanghai Research and Development Centre for Standardization of Chinese Medicines (Shanghai, China). Calcitonin-gene-related peptide (CGRP) ELISA kit was purchased from Cusabio Biotech (Wuhan, China). Tumor necrosis factor (TNF)-α ELISA kit was obtained from ExCell (Shanghai, China). Rabbit anti-5-HT antibody was obtained from ShangHai Xiangsheng Biotechnology Co. Ltd. (Shanghai, China).

Preparation of SGD and TXYF extractsSGD is comprised of five types of herbs with different mass ratios, which are listed in Table 1, and the total mass used was 31.5 g (common dose for adult humans). TXYF contained four types of herbs with a total mass of 25.5 g dry herbs, which are also listed in Table 1. The dry powder of an aqueous extract of SGD or TXYF was prepared by the Herbal Chemistry Laboratory at Shanghai University of Traditional Chinese Medicine. SGD and TXYF extracts were prepared as follows. Briefly, 315 g crude SGD and 255 g crude TXYF were powdered and soaked in 8 volumes of water for 12 h, boiled for 2 h, filtered, and the liquid collected. The procedure was then repeated. The liquid from the two procedures was mixed together and evaporated under vacuum until the aqueous phase was extracted completely. After drying in a vacuum

drying oven, 125 g SGD powder and 100 g TXYF powder were obtained separately.

Chromatography analysis The samples were determined using the Agilent 1100 chromatographic system, consisting of a solvent degasser, a quaternary gradient pump, a diode array detector, and a data station with analytical software (Chemstation 8.03; Agilent, SF, CA, United States). The separation was performed on a Kromasil C18 analytical column (250 mm × 4.6 mm, 5 µm) and maintained at 25 ℃. The mobile phase was a mixture of methanol/0.1% phosphoric acid aqueous solution (85:15, v:v) with a flow rate of 1 mL/min. The injection volume was 10 µL and the detection wavelength was set at 210 nm. The standard solutions of saikosaponin d, penoniflorin, poncirin and atractylenolide Ⅲ were prepared with methanol.

Animals and treatmentsMale Sprague-Dawley rats (150-200 g body weight) were purchased from SLAC Laboratory Animal Co. Ltd. (Shanghai, China), and fed in the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine. All animals were maintained at 22 ℃ with a 12-h light/dark cycle and had free access to rodent chow and water. All animal experiments were performed according to the protocols approved by the Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine. The rats were randomly divided into the control and WAS groups. After 3 d, the WAS rats were randomly divided into five subgroups: (1) IBS model group; (2) SGD-H group (1.97 g/kg); (3) SGD-M group (0.98 g/kg); (4) SGD-L group (0.49 g/kg); and (5) TXYF group (0.68 g/kg). SGD or TXYF extract was dissolved in distilled water and rats were treated by intragastric administration 1 h before WAS on day 4 and maintained for 7 d. The rats in the control and model groups were treated with distilled water.

WAS WAS was induced as described previously[21], with minor modifications. The rats were placed on an island-like plastic platform (10 cm × 8 cm × 8 cm), which was fixed in the center of a plexiglass pool (45 cm × 25 cm × 25 cm) filled with fresh tap water (25 ℃) up to 7 cm (1 cm below the top of the platform), for 1 h once daily and repeated for 10 consecutive days.


Table 1 Formulation of Shugan decoction and TongXieYaoFang

Chinese name Latin name Common name Drug (dry, g) Formula names

Baizhu Rhizoma Atractylodis Macrocephalae White attractylodes rhizome 9.0 TongXieYaoFang Shugan decoctionBaishao Radix Paeoniae Alba white peony root 6.0Chenpi Pericarpium Citri Reticulatae dried old orange peel 6.0Fangfeng Radix Saposhnikoviae Ledebouriella root 4.5Chaihu Radix bupleuri Radix bupleuri 6.0


collected. The level of calcitonin-gene-related peptide (CGRP) in serum was measured by ELISA (Cusabio Biotech), and the level of TNF-α in colonic tissue was determined by ELISA (ExCell), according to the manufacturer’s instructions.

ImmunohistochemistryThe sections were deparaffinized in xylene and an ethanol gradient. The endogenous peroxidase was quenched in methanol: 30% H2O2 (9:1) for 10 min, and microwaved for 10 min in citrate buffer (10 mmol/L, pH 6.0) for antigen retrieval. Sections were blocked in 5% bovine serum albumin at 37 ℃ for 1 h, followed by incubation with goat anti-rabbit polyclonal CgA antibody (Santa Cruz Biotechnology) at 1:30 dilution, or rabbit anti-5-HT polyclonal antibody (Xiangsheng, Shanghai, China) at 1:100 dilution overnight at 4 ℃, then followed by incubation with secondary antibody-peroxidase conjugate at 37 ℃ for 1 h. PBS was used for washing after each step. Subsequent visualization was performed using diaminobenzidine as a chromogen (Dako, Denmark).

Positive expression was displayed by brown color, and the cell nuclei were stained blue by hematoxylin. Positive expression of CgA and 5-HT in the colon was observed and photographed under a light microscope at × 200 magnification. Five randomly selected × 200 magnification fields were evaluated using a BH2 microscope (Olympus, Tokyo, Japan) equipped with a Nikon 4500 digital camera. The area and optical density (OD) of CgA- and 5-HT-positive cells in each field were counted and processed by the computer-aided automatic image analysis system (Qiu Wei, Shanghai, China). The immunohistochemical index was calculated as the average integral OD: [(positive area × OD)/ total area].

Statistical analysisEach value represents mean ± SD. Differences between two groups were analyzed using the Student’s t test, and statistical comparisons among more than two groups were subjected to One-way analysis of variance (ANOVA) followed by Dunnett’s test using GraphPad Prism 5.0. P < 0.05 was considered statistically significant.

RESULTSChemical compounds in water extract of SGD The ingredients in the water extract of SGD were determined by high-performance liquid chromatography (HPLC) analysis. Although many chemical compounds were detected by HPLC-MS/MS analysis (data not shown), we could not obtain all the standard chemical compounds shown in the HPLC-MS/MS analysis, thus, we were only able to confirm the following chemical ingredients in the water extract of SGD: saikosaponin d, penoniflorin, poncirin, and atractylenolide Ⅲ (Figure 1). The other chemical ingredients should be analyzed further.

Stress procedures were performed between 09:00 and 11:30 am to minimize diurnal variations in response.

Measurement of fecal pellet outputFecal pellet output was used to estimate distal colonic motility as a validated index[22]. Fecal pellets found in the pool were counted at the end of 1 h WAS. Control animals were left in the home cage for 1 h to count the fecal pellets.

Colorectal distension and pain pressure threshold Colorectal distension (CRD) induces contraction of the abdominal and hind limb muscles, and is termed abdominal withdrawal reflex (AWR), which has been validated as a quantitative method to determine visceral sensitivity in rats[10,23]. Behavioral responses to CRD were assessed by measuring the pressure threshold of CRD, that is, the minimum pressure that induced the first AWR. Immediately after the end of 1 h WAS on the 10th day, the rats were lightly anesthetized with ether while a deflated latex balloon (5 mm diameter at full inflation) was inserted intra-anally into the descending colon and rectum with its end 1 cm proximal to the anal sphincter. The balloon catheter (2 mm diameter) was fixed at the root of the tail. The rats were then placed into a small Lucite cubicle (20 cm × 8 cm × 8 cm) and allowed to recover from anesthesia for 30 min before testing. To measure the pressure threshold of CRD, the colorectal balloon was progressively inflated with an increment of 5 mmHg until pain behavior was observed, namely, at the first sign of obvious contraction in the abdominal wall. The measurement was repeated three times with at least 5-min intervals for recovery. During the measurement, the investigators were blinded to the treatment groups.

Hematoxylin-eosin stainingRats were deeply anesthetized with sodium pento-barbital, and the colonic tissues were excised and fixed in 10% formaldehyde solution. After being paraffin embedded and sectioned, the paraffin slides were deparaffinized in xylene Ⅰ, Ⅱ and Ⅲ for 15, 10 and 10 min, respectively, and dehydrated in 100%, 95%, 85% and 75% ethanol for 5 min, respectively. The sections were stained with hematoxylin-eosin (HE), dehydrated in 95%, 85% and 75% ethanol, cleared in xylene, and finally mounted with Permount Mounting Medium (Shanghai, China). Morphological changes in the colonic mucosa were observed and photographed under a light microscope (200 × objective) equipped with a Nikon color digital camera system.

ELISA Blood samples were centrifuged at 3000 rpm, at 4 ℃ for 10 min. Serum was collected and immediately frozen in liquid nitrogen and stored at -80 ℃ for further study. The frozen colonic tissues were homogenized and lysed in tissue lysis buffer, and centrifuged at 12000 g, at 4 ℃ for 10 min. The supernatant was



Effects of water extract of SGD on WAS-induced visceral hyperalgesia and colon hypermotilityAt the end of 10 d of WAS stimulation, the AWR pressure threshold of CRD was measured. The AWR

pressure threshold of rats in the IBS model group was significantly lower than that of rats in the control group (P < 0.01), which demonstrated that visceral hyperalgesia (VHL) was successfully induced by WAS (Figure 2). The AWR pressure threshold of SGD-treated rats was significantly higher than that of the IBS model rats (P < 0.01). TXYF was able to reverse the AWR pressure threshold in model rats (P < 0.01). However, SGD-H and SGD-M had a greater effect on AWR pressure threshold than TXYF (P < 0.01, P < 0.01, respectively).

As shown in Figure 3, compared with control rats, the number of fecal pellets produced by model rats was significantly increased (P < 0.01), then attenuated by the water extract of SGD (P < 0.01). TXYF also decreased the number of fecal pellets produced by model rats (P < 0.01). However, SGD-H had a better effect on fecal pellet output than TXYF (P < 0.05).

TRP channels and PARs involved in visceral hypersensitivity induced by WASBlockers of TRPV1 (Ruthenium Red) and TRPA1 (HC-030031), or the PAR4 agonist (AY-NH2) increased the AWR pressure threshold in IBS model rats (P <


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Figure 1 Analysis of the chemical constituents of Shugan decoction by high-performance liquid chromatography. HPLC chromatogram of SGD and standard reference monitored at 210 nm; Peak 1: saikosaponin d; Peak 2: penoniflorin; Peak 3: poncirin; Peak 4: atractylenolide Ⅲ. A: HPLC chromatogram of SGD; B: HPLC chromatogram of saikosaponin d; C: HPLC chromatogram of penoniflorin; D: HPLC chromatogram of poncirin; E: HPLC chromatogram of atractylenolide Ⅲ. SGD: Shugan decoction; HPLC: High-performance liquid chromatography.

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0.01, P < 0.01, P < 0.05, respectively) (Figure 4A). In contrast, agonists of PAR2 (AC55541), TRPV1 (capsaicin), TRPV4 (RN1747) and TRPA1 (polygodial) decreased AWR pressure threshold in model rats (P < 0.01, P < 0.01, P < 0.01, P < 0.01, respectively) (Figure 4A and B). The effect of SGD on AWR pressure threshold was weakened or abolished by agonists of PAR2 (AC55541), TRPV1 (capsaicin), TRPV4 (RN1747) and TRPA1 (polygodial) (P < 0.01, P < 0.01, P < 0.01, P < 0.01, respectively).

Effects of water extract of SGD on CGRP level in serum and colonic mucosa of WAS rats ELISA and immunohistochemistry indicated that CGRP in serum (P < 0.01) or in colonic tissue of WAS rats was elevated, compared with control rats. The water extract of SGD and TXYF reduced CGRP expression in

serum (P < 0.05, P < 0.01, respectively) (Figure 5A) and colonic tissue (Figure 5B) of WAS rats.

5-HT and CgA expression in colonic mucosa of WAS ratsTo determine whether the expression of 5-HT and CgA in the colonic tissue of rats was affected by WAS, the expression of 5-HT and CgA was detected by immunohistochemistry. As shown in Figure 6, after repeated exposure to WAS, the expression of 5-HT or CgA in colonic mucosa of IBS model rats was lower than that in the colonic mucosa of control rats (P < 0.05, P < 0.05, respectively). Treatment with the water extract of SGD increased expression of 5-HT and CgA in the colonic mucosa of IBS model rats (P < 0.01, P < 0.01, respectively). TXYF increased the expression of CgA (P < 0.01), but not 5-HT (P > 0.05) in colonic mucosa.


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Figure 3 Colon transit measured by fecal pellet output in 1 h. A: Colon transit measured by fecal pellet output in 1 h on day 3; B: Colon transit measured by fecal pellet output in 1 h on day 10. Data are presented as mean ± SD, bP < 0.01 vs control; dP < 0.01 vs irritable bowel syndrome; eP < 0.05 vs TongXieYaoFang. Statistical comparisons were performed using One-way ANOVA followed by Dunnett’s test using GraphPad Prism 5.0. (n = 10). IBS: Irritable bowel syndrome; SGD: Shugan decoction; TXYF: TongXieYaoFang.

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Figure 4 Transient receptor potential channels and protease-activated receptors involved in water avoidance stress-induced visceral hyperalgesia. A: TRP channels and PARs involved in WAS-induced VHL; B: TRP channels and PARs involved in the effect of SGD on VHL. The blockers or activators of TRP channels and PARs were given by intraperitoneal injection after WAS on day 10. One hour later, rats were subjected to colorectal distension and AWR pressure threshold measurement. Ruthenium Red: blocker of TRPV1, 5 mg/kg; HC-030031: blocker of TRPA1, 5 mg/kg; AY-NH2: activator of PAR4, 1 mg/kg; RN1747: activator of TRPV4, 5 mg/kg; AC55541: activator of PAR2, 0.78 mg/kg; capsaicin: activator of TRPV1, 5 mg/kg; Polygodial: activator of TRPA1, 5 mg/kg. Ruthenium Red was dissolved in distilled water. Other blockers or activators were dissolved in 10% dimethylsulfoxide/5% Tween 80/ 85% saline. Data are presented as mean ± SD, bP < 0.01 vs control; cP < 0.05, dP < 0.01 vs IBS. Statistical comparisons were performed using One-way ANOVA followed by Dunnett’s test using GraphPad Prism 5.0. (n = 5-7). TRP: Transient receptor potential; PAR: protease-activated receptor; WAS: Water avoidance stress; VHL: Visceral hyperalgesia; IBS: Irritable bowel syndrome; SGD: Shugan decoction.



Figure 5 Calcitonin-gene-related peptide level in serum and colonic tissues. A: CGRP level in serum measured by ELISA; B: CGRP expression in colonic tissues measured by immunochemistry. Data are presented as mean ± SD, bP < 0.01 vs control; cP < 0.05, dP < 0.01 vs IBS. Statistical comparisons were performed using One-way ANOVA followed by Dunnett’s test using GraphPad Prism 5.0. (n = 3 or 4). CGRP: Calcitonin-gene-related peptide; IBS: Irritable bowel syndrome; SGD: Shugan decoction; TXYF: TongXieYaoFang.

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Protein expression of serotonin transporter in colonic mucosa of WAS ratsFigure 7 shows that the protein expression of serotonin transporter (SERT) was significantly decreased in colonic tissues of IBS model rats, compared with control rats. After treatment with the water extract of SGD or TXYF, SERT expression in colonic tissues was increased.

TNF-α level in colonic tissue or serum of WAS ratsAlthough HE staining showed no inflammation in the colonic tissues of rats with chronic WAS (data not shown), Figure 8 shows that the level of TNF-α in colonic tissue of IBS model rats was higher than that in control rats (P < 0.05). The water extract of SGD, but not TXYF significantly reduced the TNF-α level in colonic tissue of WAS rats (P < 0.05, P > 0.05, respectively).

DISCUSSIONIn this study, we found that the water extract of SGD attenuated VHL by inhibiting the activation of TRPV1, TRPV4, TRPA1 and PAR2 and regulating 5-HT, SERT and CgA expression, as well as TNF-α and CGRP levels, which may contribute to the improvement in VHL and abnormal colonic motility in WAS rats.

Currently, the detailed pathological mechanism of IBS is not clear. Due to the difficulty in removing

colonic tissue samples from IBS patients, animal models of IBS are pivotal in clarifying the pathogenesis of IBS. To date, repeated WAS and neonatal maternal separation have been used to create animal models of IBS to study the pathological process or to evaluate the efficacy of western medicines or TCM[21,23]. In previous studies, rats with repeated exposure to WAS developed persistent VHL, exhibited anxiety-like behaviors, and increased fecal pellet output[21,23]. In our study, the AWR pressure threshold in response to CRD in model rats was lower than that in control rats, which indicated that the WAS rats suffered from VHL. The number of fecal pellets excreted after 1 h exposure to WAS in model rats was increased compared with control rats, which showed that abnormal colonic motility was present in the model rats. The water extract of SGD significantly improved this dysfunction (visceral hypersensitivity and abnormal colonic motility).

Abnormal pain perception or visceral hypersensitivity is considered to be an important pathological basis underlying the symptoms of some IBS patients[11]. The mechanism of action of SGD on VHL was also investigated in the present study. TRPV1 is widely expressed in the colonic afferent dorsal root ganglia (DRG) neurons and throughout the gastrointestinal tract. TRPV1 is a ligand-gated ion channel that modulates the sensation of pain and thermal hype-ralgesia as well as colonic hypersensitivity[24,25]. TRPA1 is


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Figure 6 Endocrine cells in colonic tissues. A, C: 5-HT levels in colonic tissues measured by IHC; B, D: CgA expression in colon tissues measured by immunohistochemistry. Data are presented as mean ± SD, aP < 0.05 vs control; dP < 0.01 vs IBS, Statistical comparisons were performed using One-way ANOVA followed by Dunnett’s test using GraphPad Prism 5.0.; fP < 0.01 vs TXYF, difference between SGD-M and TXYF was analyzed by Student’s t test using GraphPad Prism 5.0. (n = 3-5). 5-HT: 5-hydroxytryptamide; IHC: Immunohistochemistry; IBS: Irritable bowel syndrome; SGD: Shugan decoction; TXYF: TongXieYaoFang.


coexpressed with TRPV1 in primary sensory neurons[26], and has attracted the attention of researchers investigating gut sensation. TRPA1 deletion markedly reduces colitis-induced mechanical hyperalgesia in mice[27]. Moreover, activation of TRPA1 is proposed to contribute to the somatic hyperalgesia triggered by tissue damage and inflammation[28]. In WAS rats, protein expression of TRPV1 and TRPA1 in the colonic afferent DRG is significantly upregulated[10]. In our previous studies, increased expression of TRPV1 was also found in the DRG and colonic tissue of WAS rats

with IBS-like symptoms[23,29]. Some studies have shown that increased TRPV1 expression is also present in the colonic tissue of IBS patients[11,30]. However, evidence of the involvement of TRPV1 in visceral perception in IBS and in WAS rats with IBS-like symptoms is still lacking. In our previous studies, SGD decreased the elevated expression of TRPV1, substance P and CGRP in the DRG and colonic tissue[23,29]. However, whether TRPV1 is involved in the therapeutic effect of SGD on VHL of WAS-induced IBS-like symptoms has not been clarified. In a neonatal maternal separation IBS murine model, expression of the TRPV4 gene was significantly increased[31]. However, the role of TRPV4 in visceral perception in IBS is not well understood. In the present study, blockers of TRPV1 and TRPA1 and an activator of TRPV4 showed that TRPV1, TRPV4 and TRPA1 are involved in WAS-induced VHL. PARs play an important role in the modulation of visceral pain[32]. PAR2 is highly expressed in the gastrointestinal tract and implicated in the genesis of hyperalgesia in a number of models of colitis[32]. Activation of PAR2 in the lumen affects visceral pain[33]. In rats, the intracolonic infusion of PAR2 agonists (SLIGRL and trypsin) induces delayed hypersensitivity to colonic distension[34]. In contrast, PAR4 may mediate antinociceptive activity. PAR4 activation significantly increases the nociceptive threshold in response to noxious stimuli[35]. Thus, blockade of PAR2 at the periphery and/or inhibition of colonic luminal protease activity may be new targets for treating gut hypersensitivity and IBS. In the present study, activators of PAR2 and PAR4 showed that


C

SGD-M

IBS

TXYF

Figure 7 Serotonin transporter expression in colon tissue measured by immunochemistry. IBS: Irritable bowel syndrome; SGD: Shugan decoction; TXYF: TongXieYaoFang.

a

c,e

b200

150

100

50

0

TNF-α

(pg

/mg

prot

ein)


Figure 8 Tumor necrosis factor-α level in colonic tissue. Data are presented as mean ± SD, aP < 0.05, bP < 0.01 vs control, Statistical com-parisons were performed using One-way ANOVA followed by Dunnett’s test using GraphPad Prism 5.0.; cP < 0.05 vs IBS; eP < 0.05 vs TXYF, difference between SGD-M and TXYF was analyzed by the Student’s t test using GraphPad Prism 5.0. (n = 4). TNF: Tumor necrosis factor; SGD: Shugan decoction; IBS: Irritable bowel syndrome; TXYF: TongXieYaoFang.


activation of PAR2, but not PAR4 was involved in WAS-induced VHL. However, PAR4 activation increased AWR pressure threshold. Moreover, activators of TRPV1, TRPV4, TRPA1 and PAR2 also demonstrated that SGD attenuated VHL in WAS rats by inhibiting TRPV1, TRPV4, TRPA1 and PAR2. Whether there is crosstalk between TRPV1, TRPV4, TRPA1 and PAR2 warrants further study.

5-HT is an important neurotransmitter which is involved in mediating psychological stress, VHL and gastrointestinal dysmotility by influencing the sympathetic, parasympathetic, and enteric nervous systems[18,36]. In healthy individuals, 5-HT is released mainly from enterochromaffin (EC) cells in the gastrointestinal tract and is inactivated via reuptake by SERT from the mucosa into nerve fibers[37]. Altered SERT expression and function in IBS could result in abdominal hypersensitivity and abnormal colonic motility[5,18]. Previous studies indicated that mucosal 5-HT and SERT were both decreased in ulcerative colitis, diarrhea-predominant IBS (IBS-D), and constipation-predominant IBS (IBS-C)[18]. However, Kerckhoffs et al[38] reported that IBS-C patients have increased mucosal 5-HT concentration and EC cell numbers, whereas IBS-D patients have reduced mucosal 5-HT concentration. Other studies have also shown that SERT expression was increased in the colon together with an increased concentration of 5-HT in neonatal maternally separated rats[5]. Thus, altered SERT expression in different states may be associated with different processes of IBS development, which should be investigated in further studies. In the present study, compared with control rats, expression of SERT and 5-HT was significantly decreased in the colonic mucosa of WAS rats. The water extract of SGD upregulated the expression of SERT and 5-HT in the colon. The above results indicated that the water extract of SGD regulated the expression of 5-HT and SERT to attenuate IBS-like symptoms. These results also suggested that the decreased level of 5-HT in the colon possibly resulted from reduced biosynthesis of 5-HT, but not a change in 5-HT reuptake by SERT.

Endocrine cells are also important in the pathological process of IBS[20]. Recent research demonstrated that colonic endocrine cells are altered in patients with IBS[18-20]. Increased numbers of mast cells in the terminal ileum are observed in patients with IBS[39]. It is reported that there are low densities of 5-HT-immunoreactive (EC) cells, peptide-YY-immunoreactive cells and CgA-immunoreactive (enteroendocrine) cells in the colon of patients with IBS[20]. In the present study, low expression of CgA and 5-HT was seen in the colonic mucosa of WAS rats, which indicated low densities of CgA-positive and 5-HT-positive endocrine cells. These changes were improved by the water extract of SGD.

Although HE staining results did not show inflam-mation in colonic tissue in model rats, the level of TNF-α in colonic tissue was significantly increased, but not serum TNF-α levels, and was decreased by the water extract of SGD. Foley et al[40] proved that

TNF-α decreased SERT function and expression in Caco 2 cells. In the present study, the change in SERT and TNF-α was similar. However, whether there was a correlation between the expression of SERT and the level of TNF-α in this model requires clarification. In other research, TNF-α stimulated the expression and secretion of CGRP from rat trigeminal ganglion neurons[41]. In the present study, serum and colonic tissue CGRP levels were both increased in model rats after repeated exposure to WAS, accompanied by an increase in the level of TNF-α in colonic tissue. Following treatment with the water extract of SGD, the alterations in TNF-α and CGRP levels in model rats was attenuated.

In conclusion, the water extract of SGD enhanced the decreased AWR pressure threshold in response to CRD and attenuated abnormal colonic transit mainly by inhibiting TRPV1, TRPV4, TRPA1 and PAR2, but also by regulating 5-HT, SERT and CgA, as well as possibly reversing the imbalance in colonic 5-HT-positive and CgA-positive endocrine cells, thus decreasing the level of CGRP and TNF-α.

ACKNOWLEDGMENTSWe thank Dr. Ying Dong for help in statistical analysis.

COMMENTSBackgroundIrritable bowel syndrome (IBS) is a functional gastrointestinal disorder and not an organic disease. It is characterized by altered bowel evacuation, bloating and visceral pain, without anatomical or biochemical abnormalities. Currently, there are no standard drugs to treat IBS. Traditional Chinese medicine (TCM) has shown promising effects on IBS, however, the mechanism of action requires clarification. In this investigation, the efficacy of Shugan decoction (SGD) and its underlying mechanisms in IBS were evaluated in IBS-like rats with visceral hypersensitivity and colonic dysmotility. Research frontiersThe pathological mechanism of IBS is not well understood. Visceral hypersensitivity and gastrointestinal dysmotility are two pivotal characteristics of IBS. Transient receptor potential vanilloid type 1 (TRPV1), TRP ankyrin-1 (TRPA1), protease-activated receptor (PAR)2 and PAR4, as well as serotonin [5-hydroxytryptamine (5-HT)] in the central and peripheral nervous systems play important roles in abdominal pain severity and gut motility disorders in IBS and inflammatory bowel disease. In the present study, SGD alleviated visceral hyperalgesia (VHL) and attenuated colon motility in model rats, partly by regulating TRPV1, TRPV4, TRPA1, PAR2, 5-HT, chromogranin A (CgA), serotonin transporter (SERT) and reducing calcitonin-gene-related peptide (CGRP) and tumor necrosis factor (TNF)-α level.Innovations and breakthroughsTRPV1, TRPA1, PAR2, PAR4, 5-HT and 5-HT-positive endocrine cells are involved in abdominal pain severity and gut motility disorders in IBS patients. The effect of TCM on the above molecules, as well as its functional mechanism, is poorly understood. In the present investigation, the water extract of SGD attenuated the decrease in pain pressure threshold in response to colorectal distension and attenuated abnormal colonic transit, mainly by inhibiting TRPV1, TRPV4, TRPA1 and PAR2, and regulating 5-HT, SERT and CgA, as well as possibly reversing the imbalance in colonic 5-HT-positive and CgA-positive endocrine cells, decreasing the level of CGRP and TNF-α.ApplicationsIncreased stress in daily life has led to an increase in IBS, and the development of therapeutic agents is urgently needed. TCM has some beneficial effects on IBS, however, its use in IBS patients is hindered by our lack of understanding


COMMENTS


of its mechanism of action. In the present investigation, SGD relieved VHL and attenuated abnormal colonic transit, mainly by inhibiting TRPV1, TRPV4, TRPA1 and PAR2, regulating 5-HT, SERT and CgA, as well as possibly reversing the imbalance of colonic 5-HT-positive and CgA-positive endocrine cells, decreasing the level of CGRP and TNF-α. Thus, the present study shows the beneficial therapeutic effect and functional mechanism of TCM such as SGD in IBS patients.TerminologyIBS is a functional gastrointestinal disorder, and is characterized by altered bowel evacuation, bloating and visceral pain, without anatomical or biochemical abnormalities. TongXieYaoFang (TXYF) is a classic TCM prescription and consists of white attractylodes rhizome (Baizhu), white peony root (Baishao), dried old orange peel (Chenpi) and Ledebouriella root (Fangfeng). SGD is another formula used to treat patients with diarrhea-predominant IBS, which is composed of TXYF and Radix bupleuri (Chaihu), and can alleviate visceral pain and improve the bowel habits of patients with a lack of coordination between the liver and the spleen (TCM syndrome type). TRPV1 is widely expressed in the colonic afferent dorsal root ganglia neurons and throughout the gastrointestinal tract. TRPV1 is a ligand-gated ion channel that modulates pain sensation and thermal hyperalgesia as well as colonic hypersensitivity. TRPA1 is coexpressed with TRPV1 in primary sensory neurons. PAR2 is highly expressed in the gastrointestinal tract and implicated in the genesis of hyperalgesia in a number of models of colitis. 5-HT is an important neurotransmitter, which alters visceral perception and motor dysfunction in IBS by influencing the sympathetic, parasympathetic, and enteric nervous systems.Peer-reviewThe authors described Shugan-decoction attenuates the alteration of 5-HT, TRP channels and PAR receptors of rats exposed to water avoidance stress. The article is informative and well-presented.

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P- Reviewer: Chiba T, Pehl C S- Editor: Ma YJ L- Editor: Webster JR E- Editor: Ma S



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