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Effect of Shugan Jianpi Granule

Effect of Shugan Jianpi Granule on Gut Mucosal Serotonin-positive Cells in Patients with Irritable Bowe Syndrome of Stagnated Gan-qi Attacking Pi Syndrome Type
                     WANG Zai-jian l, LI Hui-xia 2, WANG Jing-hua 1, and ZHANG Fan 1
   
ABSTRACT  Objective: To evaluate the effect of Shugan Jianpi Granule , SJG) on the number of gut mucosal serotonin-positive cells (5-HT+C) in patients with irritable bowel syndrome (IBS) of stagnated Gan-qi attacking Pi (SGAP) syndrome type. Methods: Twenty-four patients were randomized equally into three groups. All were treated with the basic conventional treatment by cognition-behavior therapy with assistance of lactein 3 tablets thrice a day. Additionally, 24 g of SJG was given three times a day to group A, and the same dosage of SJG and Smecta 15 g thrice a day was given to group B, while no additional treatment was given to the control group. The number of 5-HT+C was measured respectively before and two weeks after treatment by immunohistochemical method. Results: The number of 5-HT+C decreased after treatment in all the three groups (P<0.05), but the decrement was more significant in the two test groups than in the control group (P<0.05 and P<0.01, respectively), while comparison of 5-HT+C between the two test groups showed insignificant difference (P>0.05). Conclusion: SJG can reduce the number of 5-HT+C in IBS patients of SGAP syndrome type, and its effect is enhanced when used in combination with Smecta.
KEY WORDS  Shugan Jianpi Granule, irritable bowel syndrome, serotonin
   
Irritable bowel syndrome (IBS) is a clinical syndrome involving digestive functional changes without intestinal constitutive and biochemical abnormalities; that is, no pathologically based organic change could be found. Researches showed that the occurrence of IBS of the diarrhea type (IBS-D) is closely related with the abnormal secretion of serotonin, an intestinal neural transmitter related with mental psychic factor(1).
    
The Chinese drug Shugan Jianpi Granule SJG) possesses the actions of stretching Gan ,strengthening Pi regulating qi and activating blood circulation, and is commonly used for the treatment of diarrhea, abdominal pain, etc., caused by Gan stagnation, Pi deficiency and qi-blood stasis, and IBS of stagnated Gan-qi attacking Pi (SGAP) syndrome type is one of the indications of SJG.
    
This study was a controlled trial designed for evaluating the effect of SJG on the number of gut mucosal serotonin-positive cells (5-HT+C) in IBS patients of SGAP syndrome type by way of observing the change in the number of 5-HT+C before and after SJG treatment.

METHODS

Diagnosis Standard and Clinical Materials
Patients with diagnosis of IBS-D up to the Rome ? standard(2), and with TCM syndrome of SGAP matching the standard in the integrative medical diagnosis and treatment program (draft)(3) were selected.
     
All the 24 patients enrolled were outpatients visiting the clinics of digestive medicine, the Third Hospital Affiliated to Beijing University of Chinese Medicine from March 2007 to August 2007. They were distributed into 3 groups equally according to the sequence of their visit. The 8 patients in group A were 4 males and 4 females, aged between 21-45 years, 31.8 ± 15.6 years on average, with the
disease course of 3-19 months, 13.1 ±8.7 months on average; the 8 patients in group B were 3 males and 5 females, aged between 28-39 years, 35.2±16.4 years on average, with the disease course of 4-28 months, 15.3± 12.7 months on average; the 8 patients in the control group were 4 males and 4 females, 18-46 years old, with the mean of 32.2±14.8 years. The three groups were insignificantly different in general conditions like age, sex, and course of disease (P>0.05).

Treatment
Treatment was implemented by an appointed specialist trained by the neurological department. The basic therapy given to all patients in the three groups included mainly recognition-behavior treatment, with the auxiliary measures of diet-guidance and oral administration of lactein tablet, three times a day, 3 tablets each time.
    
Additionally, for patients in group A, SJG was given three times a day, 24 g each time, which is a Chinese medicine consisting of nutgrass galingale rhizome, white atractylodes rhizome, tangerine peel, notoginseng, etc., made by the pharmacy department of Beijing University of Chinese Medicine; for patients in group B, besides the SJG given to group A, 15 g of Smecta (a product of Bofu-Yipusheng Pharmaceutical Co., Ltd., batch No. H20000690) thrice a day was also given; no additional treatment was given to the patients in the control group. The observation period lasted over 2 weeks for all the three groups.

Indexes and Methods of Observation

Sample Collection
Colonoscopic examination on fasting condition was performed in all patients before and after treatment to take 2-3 pieces of biopsy from the sigmoid-rectal conjuncture, and then the sample tissues were fixed by 4% formaldehyde, gradient alcohol dehydrated, dimethylbenzene hyalinized and paraffin embedded.

Immunohistochemical Stain
The paraffin embedded tissue was sectioned into 5 µ m successive slides, dewaxed in routine, managed with 3% H202 to block the endogenic peroxidase, rinsed with PBS, and incubated with 10% goat serum under 37? for 15 min. With the serum discarded, the sample was co-incubated with rabbit anti-human 5-HT multi-colony antibody (primary antibody, product of Zhongshan Company, China) under 37? for 1 h and under 4? overnight, then incubated in turn with 25-50 µg/mL biotin labeled secondary antibody (SP-9000, product of Zhongshan Company, China) under 30? for 40 min; followed by incubation with 25-50 µg/mL horse radish peroxidase labeled streptavidin, and after PBS rinsing, colored by 0.05% diaminobenzidine, terminated with water in time, hematoxylin stained, then hyalinized to make a neutral resin slide.
     
The blank negative control was made in the same procedure but PBS was used instead of primary antibody; the tissue of the appendix was used as the positive control.
5-HT+C Count
     
The stained 5-HT+C presented principally in cell plasma, showed itself to be brown-yellow, yellow or jasmine, which indicated the different degrees of positive responses such as strong positive, positive and weak positive respectively. Cells failing to be colored were regarded as serotonin negative cells.
     
The number of 5-HT+C in 5 high power visual fields (upper, lower, left, right, and middle) in each slide was counted with an Olympus laser cofocusing scanning microscope type FV500, and their sum was brought into analysis.

Statistical Analysis
Paired t-test was used for comparison of data before and after treatment in the same group, variance analysis for difference between groups, and X2 test for classified variable like age and sex. All were managed by SAS 6.12 software.

RESULTS
Changes in Immunohistochemical Figures
The positive colored site was mainly located at the plasma of glandular cells. Before treatment, 0-3 5-HT+C in each glandule with a few infiltration of lymphocytes and neutrophils around it could be seen in all samples of the three groups. After
treatment, the 5-HT+C and infiltration were reduced in all groups, but the reduction in group B was more significant, and the lymphocytes and neutrophils around the glandular organ were reduced significantly (Figure 1).

Comparison of the Number of 5-HT+C
The number of 5-HT+C in the three groups were different insignificantly before treatment (P>0.05), but it reduced in all groups after treatment, showing significant difference as compared with those before treatment (P<0.05). Inter-group comparison showed that significant difference was only shown in the
comparison between the control group and the two test groups (P<0.05, P<0.01), while insignificance was shown between the two test groups (P>0.05). However, the decrease in group B seemed rather more evident (Table 1).

DISCUSSION
IBS is one of the functional intestinal diseases often encountered. As a neuro-transmitter closely related with mental psychic factor, the action of 5-HT in IBS has been studied deeply. On the account that it is the main inflammatory mediator for intestinal secretion and abnormal feeling, many drugs for IBS treatment targeted on various subtypes of 5-HT receptors have been synthesized and effectively applied in clinical practice, among which, the 5-HT3 receptor antagonist displays a more prominent effect for IBS-D(1).
    
Through literature review, Gershon, et al(4) held that various degrees of 5-HT signal transmission abnormality exist in all IBS patients and other intestinal dysfunction patients, which might be the pathogenetic basis for IBS. Our previous study showed that gut mucosal concentration of 5-HT and its related neuro-endocrinal cells, as intestinal pheochromocyte, play an important role in the pathogenesis of IBS(5,6).
     
Traditional Chinese medicine (TCM) holds that IBS-D belongs to the categories of "loosing bowel", "abdominal pain" and "melancholia". For its pathogenesis, ancient doctors suggested the theory of "Gan-disease passing to Pi" in such ancient medical books as "Nan-jing  and "Jingui Yaolue "; while recent reports indicated that the often seen TCM syndrome of IBS-D is stagnated Gan-qi attacking Pi, and other syndromes would always develop from it(7); Gan and Pi are acting and impacting mutually in the pathological process. The transporting and transforming function of Pi depends on the catharsis of Gan, its hyperfunction or hypofunction could induce the dysfunction of Pi to form the pathological basis of IBS-D.
    
SJG is a pure Chinese herbal compound medicine made on the basis of experiences accumulated for many years, which consists of ten odd kinds of Chinese drugs, such as nutgrass galingale rhizome, white atractylodes rhizome, tangerine peel and notoginseng, ect. SJG has the effects of smoothening Gan, strengthening Pi, regulating qi and activating blood circulation, and is applied in clinical practice for the treatment of diarrhea, abdominal pain and distention, poor appetite, etc. Experimental studies showed that SJG can regulate the concentration of mediators of inflammation of tissues in the animal model of ulcerative colitis to inhibit the inflammation of the intestinal tract(8); and compared with the Western medicine, the pure Chinese preparation has a better efficacy in treating patients with IBS-D, with fewer adverse reactions found(9).
     
On the basis of the syndrome differentiation criteria suggested by Chen ZS, et al(3), IBS patients of SGAP type matching the Rome ? diagnostic standard of diarrhea type were enrolled in this study. The results showed that before treatment, the numbers of 5-HT+C in the three groups were not different significantly. After treatment, although the number decreased in all the three groups, the decrement in the two test groups was more statistically significant than that in the control group, while the difference between the two test groups was insignificant. Accordingly, it was deduced that SJG possibly impacts on the genesis of IBS-D, but it is not the only influencing factor. SJG possibly plays its action through 5-HT+C, the vital endocrinal cell in 5-HT signal transmitting path, and the combining of SJG with Smecta might be cooperative and could enhance the effect on the path.
     
Besides, the number of 5-HT+C also decreased significantly in the control group, in which patients were treated with simple cognition-behavior therapy and diet-control, which further illustrated that the influencing factors on the gut mucosal 5-HT signal transmitting path might be multiple. This result coincides with the outcome of the authors' previous study(6). Since it has been reported that the intestinal content of 5-HT in the fasting condition is different from that of postprandial(10), to avoid the influence of errors on the results, all the sampled biopsy for the test in this study were obtained in fasting condition.
     
All problems concerning changes of other cytokines and chemical mediators of gut mucosal 5-HT signal transmitting path in IBS patients, and molecular mechanisms that could affect the number of 5-HT+C remain to be further studied.
     
In sum, SJG, as a Chinese composite preparation for treatment of IBS patients of SGAP type or diarrhea type, might have an effect on the intestinal neuro-endocrinal network partly by way of influencing the number of gut mucosal 5-HT+C, thus realizing its clinical effect on patients. Besides, when SJG is used in combination with conventional recognition-behavior therapy and symptomatic treatment of Western medicine, it shows an apparent synergistic action on 5-HT+C.

REFERENCES
1.  Costedio MM, Hyman N, Mawe GM. Serotonin and its role in colonic function and in gastrointestinal disorders. Dis Colon Rectum 2007; 50:376-388.
2.  Drossman DA. The functional gastrointestinal disorders and the Rome ? process. Gastroenterology.2006; 130:1377-13 90.
3.  Chen ZS,  Zhang WD, Wei BH, et al. Schedule for diagnosis and treatment for irritable bowel syndrome with integrative Chinese and Western medicine (Draft). Chin J Integr Tradit West Med (Chin) 2005;25(3):282-284.
4.  Gershon MD, Tack J. The serotonin signaling system: from basic understanding to drug development for functional GI disorders. Gastroenterology 2007; 132:397-414.
5.  Wang ZJ, Ouyang Q, Zou YG, et al. Comparison of mucosal serotonin content in patients with ulcerative colitis and irritable bowel syndrome. Chin J Intl Med (Chin) 2005;44(11 ):851-852.
6.  Wang Z J, Ouyang Q, Wei B, et al. Alteration of intestinal mast cells in cases with overlap syndrome inside ulcerative colitis and irritable bowel syndrome. J Clin Intl Med (Chin) 2005;22(10):674-677.
7.  Han MH, Chen TQ. Exploration on evolvement rule and syndrome differentiation of  diarrea-predominant irritable bowel syndrome. Guangming J Tradit Chin Med (Chin) 2007;22(3):35-36.
8.  Qin ZS, Wang Y, Zhao Y, et al. Effect of Shugan Jianpi Granule on VIP and IL-10 in rats with ulcerative colitis. Jilin J Tradit Chin Med (Chin) 2007;27(3):48-50.
9. Tao Y, Chen DM, Dong MX, et al. The clinical study on Shugan Decoction in treating diarrhea-predominant irritable bowel syndrome. Chin Arch Tradit Chin Med (Chin) 2007;25(10):2190-2193.
10. Atkinson W, Lockhart S, Whorwell P J, et al. Altered 5-hydroxytryptamine signaling in patients with constipation and diarrhea-predominant irritable bowel syndrome. Gastroenterology 2006; 130:34-43.

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There are various listservs which can be useful. I only know one for confocal microscopy:
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