| Background:IBS (irritable bowel syndrome) is a common disorder of gastrointestinal function,which is characterized by abdominal pain associated with bowel habit change. The etiology and pathogenesis is still unclear. There is an upward trend in incidence in recent years,and the patient is given priority to with the young and middle-aged people. Published studies have reported incidence of PI-IBS to range between 15% and 20% in western countries, and 5% and 10% in our country. Its high prevalence and the considerable effects on quality of life make PI-IBS a disease with high social cost. The study found that in recent years, the incidence of IBS increased significantly after intestinal infection. Patients were intestinal infection such as viruses, bacteria or parasites, and when the causative agent and mucosal inflammation receded, IBS symptoms appear,so this case was called PI-IBS (post- infectious irritable bowel syndrome). The majority of cases of PI-IBS meet the Rome II criteria for IBS with diarrhea (IBS-D), which proportion can reach more than 70 % .In recent years, clinical studies have shown that had intestinal mucosa barrier damage existed in IBS patients with different degrees. Intestinal inflammation reaction can affect intestinal mucosal epithelial barrier and intestinal mucosal immune system, which leaded to bowel function disorder, so as to induce state of IBS. The pathogenesis of PI-IBS is closely associated with sustained low intestinal inflammation, colon increased permeability, and abnormal immune mechanism.Persistent low-grade inflammation induced by intestinal inflammation,particularly acute gastroenteritis,can destroy the intestinal mucosa epithelial barrier function, and increase the permeability of intestinal mucosa epithelial barrier.Consequently consequently diarrhea was caused by malabsorption of water and sodium. Meanwhile, the excessive exposure of antigen, and the lack of brush border activate intestinal mucosal immune system, so as to the increase of various inflammatory and immune cells, including mast cells, lymphocytes, and endocrine cells, et al. These inflammatory and immune cells release a variety of cytokines, also increase inflammatory mediator expression. Gut flora disorder was also involved in low-grade inflammation, mainly reflected in the intestinal bacterial overgrowth. In addition, the improper application of antibiotics after intestinal infection, also can lead to intestinal flora imbalance.Prevention and cure of PI - IBS get more attention. However, to date, there are no successful treatments for PI-IBS, and management primarily aims at symptomatic relief. The advantages of Chinese medicine treatment include a multi-channel,multiple targets, and less side effect. Therefore, TCM has achieved remarkable curative effect in the treatment of IBS by treatment based on syndrome differentiation.The main clinical symptoms of PI-IBS are diarrhea-predominant IBS, characterized by abdominal pain or discomfort,stool frequency increased,loose stools,mucus stools, defecation urgency.These symptoms belongs to "pain", "diarrhea", under the category in TCM. Most scholars of TCM considered that spleen deficiency and dampness is the key of IBS pathogenesis, the dampness obstructing spleen-stomach is an important factor of IBS palindromia.Huoxiang Zhengqi powder originated from the ancient Chinese pharmacy book"Formularies of the Bureau of the People's Welfare Pharmacies". Its effect was good at removing dampness, so it was honoured as "effective medicine of removing dampness" by ancient doctors of TCM. Huoxiang Zhengqi oral liquid, a Chinese patent medicine,composed of 10 kinds of Chinese medical herbs,is modified based on Huoxiang Zhengqi powder and has been mainly used to treat gastrointestinal diseases in China. Clinical researches had proved that HXZQ could significantly relieve symptoms in Diarrehea-predominant pattern irritable bowel syndrome (IBS-D)patients and has definite clinical effect for treating IBS-D. Pharmacological research has shown that HXZQ has the activities of two-way regulating gastrointestinal motility, improving gastrointestinal dysfunction, lowering visceral sensitivity,anti-inflammation, and immune regulation. It suggested HXZQ may be an ideal drug for the prevention and cure of PI-IBS.At present, mechanism researchs about HXZQ used in the treatment of IBS focused on gastrointestinal hormone, gastrointestinal dynamics, visceral sensitivity,nerve -endocrine-immune network, and water metabolism. However, systematic study about its efficacy in PI-IBS has not been exposured and whether it could regulate or protect intestinal mucosal barrier function on PI-IBS is not yet unknown.Therefore, in this study, we used a rat model with acetic acid -induced PI-IBS and studied the relationship between pathogenesis of PI-IBS and abnormal function of intestinal mucosal barrier and the role of HXZQ oral liquid in regulating or/and protecting the intestinal mucosal barrier. Further aims were to investigate the potential protective mechanisms of HXZQ oral liquid on intestinal mucosa barrier and present experimental support for clinical applications.Objectives:1. Observe change of colonic mucosa barrier function in PI-IBS rats, so as to clarify pathogenesis of PI-IBS from the angle of the intestinal mucosal barrier function, and reveal the relationship between pathogenesis of PI-IBS and intestinal mucosal barrier dysfunction.2. Conduct pharmacodynamics study of HXZQ oral liquid in treatting PI-IBS,reveal the role of HXZQ oral liquid in regulating or/and protecting intestinal mucosal barrier, including mechanical barrier, immunologic barrier , and biological barrier.Investigate the potential effective mechanisms of HXZQ oral liquid on intestinal mucosa barrier and present experimental support for clinical applications.Methods:The rat model of intracolonic instillation of acetic acid (4%)-induced colitis is set up. According to general situation of animals, including defecate character,defecate changes in water content and abdominal withdrawal reflex (AWR) detection,and distal colon histopathological examination, whether model was successful was confirmed. After PI-IBS animal model establishment, the rats were randomly divided into six groups including normal control group, PI-IBS model group, trimebutine maleate tablet group(TMT group), Huoxiang Zhengqi low-dose group(low-dose of HXZQ), middle-dose group(middle-dose of HXZQ) and high dose group(high-dose of HXZQ) with 10 rats in each group,and treated with water(control group and PI-IBS model group), trimebutine maleate tablets ( 0.1g/kg/d, TMT group), HXZQ oral liquid (1.7, 3.3, and 6.6 ml/kg/d, low-dose, middle-dose and high dose of HXZQ).Administration dose of HXZQ oral liquid was established according to the usage and dosage criteria of Chinese pharmacopeia 2010. At the seventh day after acetic acid administration, agent was administered to each group through intragastric administration for 5 days. On the day next to the last treatment, the rats were sacrificed.Histological analysis of colonic mucosa were performed, intestinal epithelial ultrastructure was analyzed by transmission electron microscopy (TEM), the diamine oxidase (DAO) activity in serum was measured by ELISA assay, and zonula occluden-1 (ZO-1) and occludin expression levels in the intestinal mucosa were detected by immunohistochemistry and Western blotting, respectively. The above indexes were examined to explore the protective effect and mechanism of HXZQ oral liquid on intestinal mucosal mechanical barrier in acetic acid-induced PI-IBS rats.SIgA content and Thl and Th2 cytokines (IL,1??TNF-??IL-4?IL-10) content in serum was measured by ELISA assay,and NF-?B p65(Nuclear factor-icB p65),p38 MAPK (p38 mitogen-activated protein kinase), ICAM-1 were detected by immunohistochemistry. The above indexes were examined to explore the regulative effect and mechanism of HXZQ oral liquid on intestinal mucosal immune barrier in acetic acid-induced PI-IBS rats.Flora analysis of animal waste was analyzed and CR( colonization resistance)(the number of bifidobacteria and enterobacteria B/E value) was calculated aimed to explore the protective effect and mechanism of HXZQ oral liquid on intestinal mucosal biological barrier in acetic acid-induced PI-IBS rats.Results:1. On the first day,depression,loose stools,eat less,foul crissum began to appear in medication administration groups and model group. Since the fourth day,animal mental state and hair gloss were improved, but still loose stool. The seventh day, compared with normal group, poor activity, less luster fur, runny stool appeared in the rest groups and weight is lower than the normal control group (P < 0.05).During the treatment, remission is slow in HXZQ low-dose group. In HXZQ middle and high dose groups, symptoms improved obviously. The weight of model group is lower than that of normal group (P < 0.01), and defecate wet weight and water content were higher than that of normal group (P < 0.01). Some animals still have loose stools or half loose stools. Positive drug group, animal loose bowel disappeared,but the mental state,fur luster,and diet is not as good as HXZQ middle and high dose groups. According to HE staining microscopy results,colonic mucosa tissue of the normal group and model group rats were not seen obvious abnormity.2. Observation under transmission electron microscope (TEM): microvilli on the surface of the epithelial cells was sparse, uneven distribution and different length in model group. Even, microvilli appeared fractured. More mast cells were observed in epithelial cells, which was full of high electron density of particles and some cavity appeared after degranulation. The mast cells were in active degranulation state.Integrated epithelial cell membrane, the normal forms of cell, and microvilli arranged neatly were observed in HXZQ high, middle and low-dose groups. However, the effect of pairing damaged ultrastructure of colonic epithelial cells in high-dose group is most obvious.Serum DAO content in model group was higher than it of the normal group(P<0.05). Compared with PI-IBS model group,serum DAO content in high,middle low-dose groups were significantly decreased(P<0.05, P<0.01). The difference between the normal group and high, middle low-dose groups was not statistically significant. Immunohistochemistry and Western blotting analysis results show that ZO-1 and occludin dyeing was uneven distributed or fade in colonic mucosa tissues,protein expression significantly decreased (P<0.05, P<0.01) in PI-IBS model group.ZO-1 and occludin protein expression significantly enhanced in HXZQ high, middle,low-dose groups, and IOD value of ZO-1, occludin protein is significantly higher than model group (P<0.001). While, on ZO-1 and occludin protein expression, there was no statistically significant difference between TMT group and model group(P>0.05).3. Compared with normal group, serum SIgA levels significantly reduced in model group, TMT group and HXZQ low-dose group (P<0.01). The difference of serum SIgA levels among HXZQ middle-dose group, high-dose groups and normal group was no significant.The content of serum IL-1? in PI-IBS model group was obviously higher than that of normal group, IL-10 levels significantly lower than normal group (P <0.05,P<0.01); The difference between normal group with the rest of groups, IL-1? and IL-10 levels was not significant. Serum IL-10 levels of HXZQ middle, high-dose groups and model groups increased significantly, compared with model group(P<0.05, P<0.01). However, serum TNF-??IL-4content of each group had no significant difference.Immunohistochemical analysis showed that there are a large number of NF-?B p65 expression in colonic mucosa tissue in model group. IOD values of NF-?B p65 in HXZQ high, middle, low-dose groups were significantly lower than that of model group (P<0.05) , and compared with normal group, there was no statistically significant difference (P<0.05). Compared with model group, IOD values significantly decreased in TMT group,but still higher than that of normal group,the difference was statistically significant; p3 8 MAPK expression was weakly positive in normal group, and visible p38 MAPK expression in colonic mucosa tissue in model group. IOD value of HXZQ high, middle and low-dose groups and TMT group decreased significantly, compared wih model group (P<0.01); ICAM-1 protein expressed of intestinal mucosa lamina propria was in very small amounts in normal group. ICAM-1 protein expression of model group was significantly stronger than that of normal group. ICAM-1 protein expression IOD values of HXZQ middle,high-dose groups were significantly lower than model group, and there was no significant difference between HXZQ middle, high-dose groups with the normal group.4. In model group and medication administration groups, the number of bifidobacteria and lactobacilli changes over time (F=42.406, P = 0.000, F=9.78, P=0.000),the main effect exist on the time (F= 984,491,P= 0.000, F= 54.528,P=0.000),main effect exist between groups (F= 88.689, P= 0.000, F= 54.528, P = 0.000).On seventh day, the number of bifidobacterium and lactobacillus in model group and medication administration groups significantly reduced, compared with the normal group(P<0.001). On twelfth day, the number of bifidobacterium and lactobacillus in model group and medication administration groups were more than that on seventh day(F=51.517,P=0.000;F=26.881,P=0.000),but less than that of normal group(P<0.01 ). but the number in HXZQ low,middle,high dose groups still significantly higher than that in model group (P<0.001). However,the number of escherichia coli,enterococcus change is not obvious (F=0.599,P=0.701,F=0.910,P=0.484) , and there were no statistically significant difference between the groups. On seventh day,the B/E value of normal group rats>1, B/E value of rest of the groups<1, and less than normal group(P<0.001). On 12th day, B/E value in each treatment groups were significantly increased, compared with that of the seventh day(F=11.154,P=0.000).B/E value of medication administration groups is higher than that of model group, but still lower than that of normal group, the difference was statistically significant(P<0.01).Conclusion:1. In this study, the rat model of intracolonic instillation of acetic acid(4%)-induced colitis showed major features of PI-IBS after subsidence of the acute inflammation in colon. We found that PI-IBS model group animal appeared marked visceral hypersensitivity with no evidence of histological inflammation. The observations suggested that the features of PI-IBS could be identified in this model.Bowel dysfunction after the acute intestinal infection are similar with human PI-IBS,so it.can be used as a PI-IBS animal model for research.2. Intestinal mucosal mechanical barrier damage exists in PI - IBS model animal.It show changed epithelial cell ultrastructure of PI-IBS rat intestinal mucosa barrier,increased DAO content in serum and decreased tight junction protein ZO-1 and occludin expression levels. HXZQ liquid could repair damaged the ultrastructure of intestinal mucosal epithelial cells, repair the tight junction between epithelial cells,thereby lower the intestinal permeability.3. Immune barrier dysfunction exists in PI-IBS model animal. It showed declined intestinal mucosal humoral immune function, imbalance of proinflammatory and anti-inflammatory factor, activation of NF-?B p65, p38 MAPK pathway and the excessive expression of ICAM-1. HXZQ fluid could improve intestinal mucosal humoral immunity, regulate pro-inflammatory/anti-inflammatory factor levels, inhibit the expression of NF-?B p65?p38 MAPK?ICAM-1,thereby regulate intestinal mucosal immune system and play the role of treatment of PI-IBS.4. Intestinal flora imbalance exists in PI-IBS model animal. It showed less expression intestinal bifidobacterium and lactobacillus. HXZQ liquid could improve the number of intestinal bifidobacterium, lactobacillus in PI-IBS intestinal tract, have certain effect on adjusting intestinal flora balance. The adjustment effect may not be direct effects on intestinal flora, but rather indirectly effect on the intestinal flora through repairing intestinal mechanical barrier and regulating intestinal immune barrier. |
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