Effect Of Jianpihuashi Granules On 5-HT, CRF And Their Receptors In The Colon Of Rats With Diarrhea-prominant Irritable Bowel Syndrome

Irritable bowel syndrome(IBS) is a common functional gastrointestinal disorder disease, and it is often accompanied by some symptoms such as abdominal pain, abdominal distension, bowel and stool abnormalities. According to the clinical manifestation, IBS can be divided into diarrhea, constipation, diarrhea constipation alternating, among which diarrhea Irritable bowel syndrome is a common type. The patients with diarrhea predominant irritable bowel syndrome mostly are accompanied by abdominal pain and diarrhea, and at the same time with irritability, anxiety, depression and other neurological disorders. So far, its etiology and pathogenesis are related to multiple factors. it can not be explained by the simplex physiological and pathological mechanism. In recent years, neural gastroenterology researchwith considered that D-IBS is related with brain gut axis dysfunction caused by mental stress, and cosoder that abnormality of the brain gut axis may be the main factor of leading to the enhancement of various stress responses and increasing the visceral sensitivity in patients with D-IBS, while the regulation of brain gut axis function depends on the expression of brain gut peptides and its receptors. The main excitatory neurotransmitters contain histamine, 5-HT, SP, CGRP, CRF, inhibitory neurotransmitters contain CCK, NO, NE, VIP and so on. This experiment is to study from the 5-HT and CRF. 5-HT is a monoamine neurotransmitter, which exist widely in CNS and the intestinal tract, and play an important role in the brain- gut conduction; CRF is a kind of important neuroendocrine peptides, an important factor involved in the stress response, which is mainly distributed in the organization of the CNS and peripheral, such as gastrointestinal tract, spleen, adrenal gland and so on.At present, western medicine based on symptomatic treatment, including gastrointestinal antispasmodic drugs, antidiarrheal, laxatives, intestinal motility feeling regulating drugs, antidepressants and so on. Although these drugs in relieving the symptoms have certain effect in the short term, the long-term effect is not satisfied, and it is easy to relapse after stopping the medicine, Or it may have strong side effects. for example, the tegaserod used to improving gastrointestinal symptoms was officially American FDA approved clinical in 2002, but it has been withdrawn from the market because of its cardiotoxicity. Chinese medicine treatment is more advantageous than western medicine in the aspects of alleviating the symptoms and improving quality of life in patients. JG derived from clinical prescription, and its curative effect is exact. This experiment is to establish the diarrheapredominant irritable bowel syndrome rat model by taking senna and applying the method of restraint stress. Preliminary experiments show that JG can significantly improve and mitigate D-IBS rats visceral hypersensitivity and gastrointestinal motility abnormalities,the experiments explain the mechanism according to the changes of 5-HT, CRF, CRF1 R in the central nervous system(brain, spinal cord). This experiment will explore its mechanism from the peripheral(colon) 5-HT, CRF and their receptors 5-HT3 R, 5-HT4 R, CRF1 R for research, combined with the previous experiment, and further provide experimental data for JG in the treatment of D-IBS and enrich the theory of Chinese medicine knowledge.Objective: This experiment is to investigate the peripheral mechanism of JG on colonic motility and visceral sensitivity of D-IBS model rats, through the influence of CRF, CRF1 R, 5-HT, 5-HT3 R, 5-HT4 R by JG on D-IBS rats colon.Methods: Totally 72 seven weeks old SD rats were randomly divided into six groups, 12 in each group, including the normal group(N), the model group(MO), the positive control group(PC), the low-dose group of Jianpihuashi Granules(L), the middle-dose group of Jianpihuashi Granules(M),the high-dose group of Jianpihuashi Granules(H). Fasting but allowing the water before the experiment, each group of the rats are given senna(0.3 g·m L-1 Crude drug) by gavage except the NC group in the first 14 days. The next 14 days, the upper limbs, chest and shoulders of rats are tied for two hours by scotch tape after filling senna decoction. After being tied for 1 hours, The model group are given saline by gavage. The low, middle, high-dose groups of Jianpihuashi Granules are given Jianpihuashi Granules(2.5 mg·m L-1, 5 mg·m L-1, 10 mg·m L-1) by gavage. The positive control group are given Dicetel(3mg·m L-1) by gavage. The normal group are only given saline by gavage in the whole process, and each solution intragastric volume is in accordance with 10 m L·kg-1. Rats are killed with Intraperitoneal injection of 10% chloral hydrate after 14 days pharmacological interventions, then take a segment of distal colon tissue quickly, with physiological saline cleaned, some of which are placed in-80 ℃ refrigerator, and some are fixed with paraformaldehyde for detecting index. The content of 5-HT and CRF in the rat colon are detected by enzyme linked immunosorbent assay(ELISA). The content of Positive cells of CRF in colonic mucosa, CRF1 R, 5-HT3 R, 5-HT4 R in colonic submucosa are detected by Immunohistochemical method. The expressions of CRF m RNA, 5-HT3 R m RNA, 5-HT4 R m RNA in the colon are detected by using reverse- transcription PCR.Results: 1 Effect of JG on 5-HT and its receptors in the colon of rats with D-IBS 1.1 Effect of JG on the content of 5-HT in the colon of rats with D-IBS Compared with the normal group, the content of 5-HT in colon of rats in model group is significantly increased(P< 0.05). Compared with the model group, three kinds of dose-group(L, M, H) of JG could reduce the content of 5-HT in colon of rats in different degrees(P< 0.05). 1.2 Effect of JG on positive cell expression of 5-HT3 R in colonic submucosa of rats with D-IBS Compared with the normal group, the content of positive cells expression of 5-HT3 R in colonic submucosa of rats in model group is significantlyincreased(P< 0.05). Compared with the model group, three kinds of dose-group(L, M, H) of JG could reduce the content of positive cells expression of 5-HT3 R in colonic submucosa of rats in different degrees(P< 0.05). 1.3 Effect of JG on positive cell expression of 5-HT4 R in colonic submucosa of rats with D-IBS Compared with the normal group, the content of positive cells expression of 5-HT4 R in colonic submucosa of rats in model group is significantly decreased(P< 0.05). Compared with the model group, three kinds of dose-group(L, M, H) of JG could increase the content of positive cells expression of 5-HT4 R in colonic submucosa of rats in different degrees(P< 0.05). 1.4 Effect of JG on the expression of 5-HT3 R m RNA in the colon of rats with D-IBS Compared with the normal group, the content of expression of 5-HT3 R m RNA in colon of rats in model group is significantly increased(P< 0.05). Compared with the model group, low-dose group of JG has the similar effect with it(P> 0.05), middle-dose group and high-dose group of JG could reduce the content of expression of 5-HT3 R m RNA in colon of rats in different degrees(P< 0.05). 1.5 Effect of JG on the expression of 5-HT4 R m RNA in the colon of rats with D-IBS Compared with the normal group, the content of expression of 5-HT4 R m RNA in colon of rats in model group is significantly decreased(P< 0.05). Compared with the model group, three kinds of dose-group(L, M, H) of JG could increase the content of expression of 5-HT4 R m RNA in colon of rats in different degrees(P< 0.05). 2 Effect of JG on CRF and its receptor in the colon of rats with D-IBS 2.1 Effect of JG on the content of CRF in the colon of rats with D-IBS Compared with the normal group, the content of CRF in colon of rats in model group is significantly increased(P< 0.05). Compared with the modelgroup, three kinds of dose-group(L, M, H) of JG could reduce the content of CRF in colon of rats in different degrees(P< 0.05). 2.2 Effect of JG on positive cell expression of CRF in colonic mucosa of rats with D-IBS Compared with the normal group, the content of positive cells expression of CRF in colonic mucosa of rats in model group is significantly increased(P< 0.05). Compared with the model group, three kinds of dose-group(L, M, H) of JG could reduce the content of positive cells expression of CRF in colonic mucosa of rats in different degrees(P< 0.05). 2.3 Effect of JG on positive cell expression of CRF1 R in colonic submucosa of rats with D-IBS Compared with the normal group, the content of positive cells expression of CRF1 R in colonic submucosa of rats in model group is significantly increased(P< 0.05). Compared with the model group, three kinds of dose-group(L, M, H) of JG could reduce the content of positive cells expression of CRF1 R in colonic submucosa of rats in different degrees(P< 0.05). 2.4 Effect of JG on the expression of CRF m RNA in the colon of rats with D-IBS Compared with the normal group, the content of expression of CRF m RNA in colon of rats in model group is significantly increased(P< 0.05). Compared with the model group, three kinds of dose-group(L, M, H) of JG could reduce the content of expression of CRF m RNA in colon of rats in different degrees(P< 0.05). 2.5 Effect of JG on the expression of CRF1 R m RNA in the colon of rats with D-IBS Compared with the normal group, the content of expression of CRF1 R m RNA in colon of rats in model group is significantly increased(P< 0.05). Compared with the model group, low-dose group of JG has the similar effect with it(P> 0.05), middle-dose group and high-dose group of JG could reduce the content of expression of CRF1 R m RNA in colon of rats in differentdegrees(P< 0.05). Conclusion: The mechanisms of JG in improving the abnormalities of colonic motility and visceral sensitivity of the rats with diarrhea-predominant irritable bowel syndrome have relations with increasing expressions of 5-HT4 R, 5-HT4 R m RNA and reducing expressions of 5-HT, CRF, CRF1 R, 5-HT3 R, CRF m RNA, CRF1 R m RNA, 5-HT3 R m RNA.