Liting Zhu, Zhonghan Li, Bin Xu, Chen Xia, Jie Cheng, Xiaoren Xiang, Yi Zhu

1Department of Acupuncture and Massage, Second Clinical College, Nanjing University of Traditional Chinese Medicine, Nanjing 210046,Jiangsu Province, China

2National Key Laboratory of Acupuncture and Medicine of China , Nanjing 210029, Jiangsu Province, China

3Department of Acupuncture and Rehabilitation, Jiangsu Province Chinese Medicine Hospital, Nanjing 210029, Jiangsu Province, China

INTRODUCTION

Irritable bowel syndrome (IBS) is a functional bowel disorder related to disturbances in gastrointestinal motility and visceral sensitivity[1-3].At present, the treatment of IBS is symptomatic, and aimed at improving the quality of life of patients.Drugs used for IBS treatment include anticonvulsant drugs, antidiarrheal drugs,cathartic drugs, leavening agents and nerve regulating drugs, but their effects are limited[4].Previous studies of the pathological mechanisms underlying IBS subtypes have focused on hyperesthesia and motility abnormalities, but rarely on changes in various types of myenteric nerve plexus before and after IBS onset[5].In addition, many studies have used animal models to simulate IBS-diarrhea (IBS-D),but models of IBS-constipation (IBS-C) have rarely been used.

Acupuncture exhibits some effects in the treatment of functional gastrointestinal disorders, but the precise mechanisms remain poorly understood[6].Zusanli(ST 36)is a common acupoint for the clinical treatment of gastrointestinal disorders.Acupuncture at ST 36 can promote colon movement and this may be mediated by parasympathetic efferents[7-8].Another study has shown that electroacupuncture at ST 36 stimulates glutamatergic neurons in the brainstem, resulting in improvements in gastrointestinal motility and emptying[9].In this study, we tested the effects of electroacupuncture at ST 36on defecation and on neurons in the colonic myenteric plexus in rats with IBS-C, with the aim to reveal the underlying pathway.We used pinaverium bromide (trade name Dicetel) as a positive control drug, because pinaverium bromide is a highly selective L-type Ca2+channel inhibitor that acts on colon smooth muscle to normalize colonic motility in IBS.

RESULTS

Quantitative analysis of experimental animals

One week after arrival to the institution, 50 rats were randomly and evenly divided into five groups: blank control, IBS-C model,pinaverium bromide, electroacupuncture,and electroacupuncture + pinaverium bromide.The interventions in the latter three groups were given in the second week of the 14-day IBS-C period.Three rats from the pinaverium bromide group died for unknown reasons, so 47 rats were included in the final analysis.

Electroacupuncture at ST 36 improved defecation in IBS-C rats

The effects of electroacupuncture at ST 36 on defecation are shown in Table 1.

Table 1 Defecation of IBS-C rats in each group

Electroacupuncture at ST 36 or intragastric administration of pinaverium bromide significantly increased the number of stool drops in IBS-C rats (P＜0.05).The combination of the two methods produced a greater effect than acupuncture alone (P＜0.05).

Electroacupuncture at ST 36 increased the number of neurons and protein gene product 9.5 (PGP9.5)staining intensity in the colonic myenteric plexus(Tables 2, 3 and Figure 1)

Table 2 Effects of electroacupuncture at Zusanli(ST 36)on the number of neurons (neurons per 400 × visual field)in the colonic myenteric plexus

Immunohistochemical staining of PGP9.5, an enteric nervous system neuronal marker, showed that the number of neurons in the colonic myenteric plexus of IBS-C rats was significantly decreased (P＜0.05).Electroacupuncture at ST 36 alone, or in combination with pinaverium bromide, significantly increased the number of neurons in the colonic myenteric plexus (P＜0.05).Electroacupuncture at ST 36combined with pinaverium bromide produced a greater effect (P＜0.05)(Table 2 and Figure 1).

正常高水位加风浪和地震超高计算得防浪墙顶高位539.61 m，设计洪水位和校核洪水时计算的防浪墙顶高程分别为539.6 m和541.14 m。结果表明，校核洪水位工况为坝顶高程的控制工况。防浪墙顶高程为541.2 m，工程防洪能力满足规范要求。

Figure 1 Neurons in the rat colonic myenteric plexus(protein gene product 9.5 immunohistochemistry, ×400).(A-E) Model, electroacupuncture, pinaverium bromide,electroacupuncture + pinaverium bromide, and blank control groups.Dark blue cells in the dark brown region(arrows) are neurons of myenteric nerve plexus.The number of neurons was significantly increased in the electroacupuncture and pinaverium bromide groups.

PGP9.5 staining intensity in the colonic myenteric plexus was significantly decreased in IBS-C rats compared with control (P＜0.05).Electroacupuncture at ST36 alone or in combination with pinaverium bromide significantly increased the number of neurons in the colonic myenteric plexus (P＜0.05).Electroacupuncture at ST36 combined with pinaverium bromide produced a greater effect (P＜0.05).PGP9.5 expression was similar in the electroacupuncture + pinaverium bromide group and the blank control group (Table 3).

Table 3 Effects of electroacupuncture at Zusanli (ST 36) on the expression of protein gene product 9.5 in the colonic myenteric plexus

DISCUSSION

The purpose of this study was to determine whether there is a relationship between IBS-C symptoms and neuronal changes in the myenteric plexus in rats.We found that IBS-C caused a significant reduction in the number of neurons and PGP9.5 staining intensity in the colonic myenteric plexus.This indicates that a reduced number of neurons in the colonic myenteric plexus may contribute to the onset of IBS-C, consistent with previous research[5].Our results also showed that IBS-C can be induced in rats by intragastric administration of iced water, and faithfully simulates the clinical manifestations of IBS-C in patients.Thus, this animal model is suitable for the study of the pathological mechanisms of IBS-C.The colonic myenteric plexus is primarily composed by motor neurons and primary afferent neurons[10].A decreased activity of motor neurons is related to decreased secretion and defecation[11].Therefore, it is likely that the decrease in the number of PGP9.5-positive neurons observed in this study reflects a loss of motor neurons, leading to the decreased defecation seen in IBS-C rats.

It has been shown that electroacupuncture at ST 36can strengthen the motility of distal segments of the colon[12].Disperse-dense waves of electroacupuncture can decrease visceral hypersensitivity of IBS-C rats and exhibit additive effects that strengthen with increasing stimulation frequencies[13].PGP9.5 is the enteric nervous system neuronal marker.We found that electroacupuncture at ST 36using the disperse-dense wave setting of the apparatus can increase the number of neurons and PGP9.5 expression in the colonic myenteric plexus of IBS-C rats, indicating that this treatment can promote regeneration of the colonic myenteric plexus.

Pinaverium bromide can inhibit mast cell degranulation to decrease damage to intestinal tract neurons, thereby decreasing visceral hyperresponsiveness[14].We found that pinaverium bromide decreased constipation in IBS-C rats, indicating that symptomatic improvements can be related to a decrease in visceral hypersensitivity.

Taken together, our results show that electroacupuncture at ST 36 promotes the regeneration of neuron in the colonic myenteric plexus and improves defecation in IBS-C rats.In addition, the finding that pinaverium bromide increased stool drops of IBS-C rats indicates that visceral hypersensitivity is a primary cause of decreased defecation in this rat model.

MATERIALS AND METHODS

Design

A randomized controlled animal experiment.

Time and setting

This study was performed at Jiangsu Province Key Laboratory of Acupuncture, Nanjing University of Traditional Chinese Medicine between March and May 2011.

Materials

Fifty adult Sprague-Dawley rats (200±20 g) of specific pathogen-free grade and of either gender were provided by Shanghai SLAC Laboratory Animal Co., Ltd., China and raised at the Laboratory Animal Center at Nanjing University of Traditional Chinese Medicine.The experimental protocol was in accordance with theGuidance Suggestions for the Care and Use of Laboratory Animalsformulated by the Ministry of Science and Technology of China[15].

Methods

Establishment of the rat IBS-C model and eletroacupuncture treatment

Rats were treated daily with intragastrically administered ice-cold physiological saline (4 mL, 0-4°C) for a total of 14 days to establish the IBS-C model[7].An independent sample t test was performed to compare the number of neurons in the colonic myenteric plexus between the IBS-C model and control groups.A significant difference indicates success in IBS-C induction.

IBS-C was treated by electroacupuncture at ST 36 as follows:

Group Intervention Model Daily intragastric administration of 4 mL physiological saline within 8-14 days after IBS-C induction.Pinaverium bromide Daily intragastric administration of 4 mL 0.3% pinaverium bromide suspension (batch No.3008620359, Solvay Pharmaceuticals, Brussels, Belgium) within 8-14 days after IBS-C induction[10].Electroacupuncture Daily intragastric administration of 4 mL physiological saline and receiving electroacupunture treatment within 8-14 days after IBS-C induction[10].Electroacupuncture methods: rats were daily intragastrically administered pinaverium bromide or physiological saline, then according to Experimental Acupuncture Science[16],the acupoint ST 36 (located in the posterolateral knee joint, approximately 0.5 cm below capitulum fibulae).SDZ-II electroacupuncture apparatus(Huatuo brand, Suzhou Medical Apparatus Co., Ltd., Suzhou, Jiangsu Province, China) was used.The parameters of electroacupuncture apparatus included current intensity 3 mA, alternative dense wave 100 Hz/3 s and disperse wave 4 Hz/3s, with a needling depth of 7 mm, vertical to skin plane, once a day, 10 minutes once.Electroacupuncture +pinaverium bromide Daily receiving electroacupuncture at ST 36with the same method as above and intragastric administration of 4 mL 0.3% pinaverium bromide suspension[9] within 8-14 days after IBS-C induction.Blank control Daily intragastric administration of 4 mL physiological saline for a total of 14 days.

Rat defecation

At 7 and 14 days after IBS-C induction, a clean filter paper was placed on the bottom of the cage during daytime, and stool drops were collected for 3 and 6 hours[17].

PGP9.5 immunohistochemical staining of neurons in the colonic myenteric plexus

After 14 days of IBS-C, rats were fasted for 24 hours and then sacrificed by cervical dislocation.A median abdominal incision was made to expose the abdominal cavity and the whole segment of colon was harvested.The colon sample was washed with 0.01 mol/L phosphate buffered saline (PBS; pH 7.4) (Nanjing Jiancheng Bioengineering Institute, Nanjing, China)[6].The colon sample was engorged with 4%paraformaldehyde, the two ends were tightened, and the sample was immediately fixed in 4% paraformaldehyde(Nanjing Jiancheng Bioengineering Institute, Nanjing,China) for 6-8 hours at 4°C.Thereafter, the colon sample was washed three times in PBS, and the intestinal canal was cut into small segments with a size of approximately 1 cm.Ten segments from each group were selected, and two segments were embedded in paraffin wax and preserved at 4°C.The paraffin-embedded tissue samples were sliced into 5 μm sections with a cryostat microtome (Leica RM2145, Leica, Munich, Germany).Following dewaxing, sections were washed for 3×15 minutes in PBS.Hydrogen peroxide-methanol solution(0.3%) was added to eliminate the effects of endogenous peroxidases.Sections were then treated with primary antibody (rabbit anti-rat PGP9.5 monoclonal antibody,1: 400) for 30 minutes at room temperature, followed by secondary antibody (goat anti-rabbit IgG-horseradish peroxidase, 1: 1 000, pH 7.4) for 30 minutes at room temperature.Following addition of avidin-biotin-peroxidase complex (ABC) reagent,sections were incubated for 2 hours at room temperature,washed 3×5 minutes in PBS, and then rapidly washed three times with distilled water.Sections were treated with 0.01% H2O2-DAB (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) for 20 minutes at room temperature, and checked regularly under the light microscope (400 × magnification).After washes with running water, nuclei were counterstained with 0.5%methyl green.Ten visual fields were randomly selected under an optical microscope (400 × magnification,Olympus, Japan).The mean number of PGP9.5-positive neurons, the mean gray value, the mean absorbance and the integrated absorbance were calculated using the JD801D morphological microimage analysis system(Jiangsu JEDA Science-Technology Development Co.,Ltd., Nanjing, Jiangsu Province, China).

Statistical analysis

The data were statistically analyzed using SPSS 13.0 software (SPSS, Chicago, IL, USA) and were expressed as mean±SD.One-way analysis of variance was used for comparison between groups, and post-hoc pairwise comparisons were made using the Least Significance Difference test.P＜0.05 was considered statistically significant.

Author contributions:This study was designed by Liting Zhu,Bin Xu and Chen Xia, performed by Liting Zhu, Zhonghan Li,and evaluated by Jie Cheng, Xiaoren Xiang, Yi Zhu, who were blinded to the experimental design.

Conflicts of interest:None declared.

Funding:This study was supported by the State Key Development Program for Basic Research of China (No.2011CB505206) and Practice Innovation Development Program of College Students in Higher Education Institutions of Jiangsu Province (No.00485).

Ethical approval:This study received approval from the Committee of Laboratory Animal Management, Nanjing University of Traditional Chinese Medicine.

[1] Drossman DA.The functional gastrointestinal disorders and the Rome III process.Gastroenterology.2006;130(5):1377-1390.

[2] Kaptchuk TJ, Kelley JM, Conboy LA, et al.Components of placebo effect: randomised controlled trial in patients with irritable bowel syndrome.BMJ.2008;336(7651):999-1003.

[3] Lim B, Manheimer E, Lao L, et al.Acupuncture for treatment of irritable bowel syndrome.Cochrane Database Syst Rev.2006;(4):CD005111.

[4] Chang FY, Lu CL.Treatment of irritable bowel syndrome using complementary and alternative medicine.J Chin Med Assoc.2009;72(6):294-300.

[5] Xu JR, Luo JY, Shang L, et al.The plasticity of the enteric submucosal plexus in the irritable bowel syndrome rats.Zhonghua Xiaohua Zazhi.2007;27(2):107-110.

[6] Conboy LA, Wasserman RH, Jacobson EE, et al.Investigating placebo effects in irritable bowel syndrome: A novel research design.Contemp Clin Trials.2006;27:123-134.

[7] Chitkara DK, van Tilburg MA, Blois-Martin N, et al.Early life risk factors that contribute to irritable bowel syndrome in adults: a systematic review.Am J Gastroenterol.2008;103(3):765-774.

[8] Iwa M, Tateiwa M, Sakita M, et al.Anatomical evidence of regional specific effects of acupuncture on gastric motor function in rats.Auton Neurosci.2007;137(1-2):67-76.

[9] Iwa M, Nakade Y, Pappas TN, et al.Electroacupuncture improves restraint stress-induced delay of gastric emptying via central glutaminergic pathways in conscious rats.Neurosci Lett.2006;399(1-2):6-10.

[10]Wood JD.Enteric neuroimmunophysiology and pathophysiology.Gastroenterology.2004;127(2):635-657.

[11]Lomax AE, Fernández E, Sharkey KA.Plasticity of the enteric nervous system during intestinal inflammation.Neurogastroenterol Motil.2005;17(1):4-15.

[12]Iwa M, Matsushima M, Nakade Y, et al.Electroacupuncture at ST-36 accelerates colonic motility and transit in freely moving conscious rats.Am J Physiol Gastrointest Liver Physiol.2006;290(2):G285-292.

[13]Wang ZJ, Li WM.Effects of electroacupuncture on disorder of intestinal motility in a rat model of irritable bowel syndrome.Zhong Xi Yi Jie He Xue Bao.2010;8(9):883-887.

[14]Malysz J, Farraway LA, Christen MO, et al.Pinaverium acts as L-type calcium channel blocker on smooth muscle of colon.Can J Physiol Pharmacol.1997;75(8):969-975.

[15]The Ministry of Science and Technology of the People’s Republic of China.Guidance Suggestions for the Care and Use of Laboratory Animals.2006-09-30.

[16]Li ZR.Experimental Acupuncture Science.Beijing: China Press of Traditional Chinese Medicine.2007.

[17]Tang HM, Huang YH, Li DT, et al.Effect of Changji'an prescription on T lymphocytes and 5-hydroxytryptamine in rats with diarrhea predominant irritable bowel syndrome.Guangzhou Zhongyiyao Daxue Xuebao.2009;26(2):164-168.