Chang’ Anerhao Decoction For The Treatment Of Trinitrobenzene Sulfonic Acid(TNBS) Induced Post-inflammation Irritable Bowel Syndrome (PI-IBS)Involving Intestinal Mucosal Immune Barrier In A Rat Model

[Background]IBS (irritable bowel syndrome, IBS) is functional gastrointestinal disorders, and its main symptoms are abdominal pain, abdominal discomfort associated with altered bowel habits, stool abnormalities. More and more research with PI-IBS(post-infectious/post-inflammatory irritablebowel syndrome). Now that, after gastrointestinal infection, the presence of low-grade inflammation in the gut or no inflammation, in a continuous state of immune activation, resulting in symptoms of IBS.After nearly10years of clinical observation literature analysis, western medicine mainly by regulating gastrointestinal motility, improve intestinal flora mainly symptomatic treatment, Chinese medicine believes that Ganyupixu is the most important syndromes, Tongxieyaofang is the main treatment, and there is a significant clinical effect. An Chang’anerhao Decoction used in this study is based the Tongxieyaofang and strengthen its role of Yiqianchang and Wenyangguse. An early study of Chang’anerhao Decoction found on immune function in rats with IBS has a significant improvement. The main features of PI-IBS disease is the presence of a low degree of intestinal inflammation, persistent immune activation status. Therefore, the Chang’anerhao Decoction treatment of PI-IBS, and elucidate its mechanism of action of PI-IBS has important significance.[Objective]After discussing the progress of modern western medicine treatment of IBS, and analyzing the clinical research in western medicine treatment of IBS literature, we discuss the current problems and advantages, to provide reference for clinical and scientific research. Conducted Chang’anerhao Decoction pharmacodynamic experiments clearly its pharmacological effects; mother-infant separation, colon trinitrobenzene sulfonic acid infusion, and chronic restraint to create a rat model of PI-IBS, and give Chang’anerhao Decoction different doses of intervention to observe its effect, and on the mucosal immune barrier mechanism. [Research]Experiment I:Chang’anerhao Decoction pharmacodynamic study in miceObjective:An analgesic anti-inflammatory effects observed Chang’anerhao Decoction and its impact on gastrointestinal motility and immune function.Materials and Methods:ICR mice Intestinal propulsive movement experimentsand neostigmine cause intestinal motility experiments to observe the effects on gastrointestinal motility Chang’anerhao Decoction; using hot plate analgesia test and xylene-induced ear edema in mice experiments to observe the anti-inflammatory and analgesic effects of Chang’anerhao Decoction;usingDelayed type hypersensitivity (DTH) and carbon clearance index determination experiments to observe an impact on immune function.Results:In the intestinal propulsive movement experiments and neostigmine cause intestinal motility experiments, Compared with the normal group,there was a significant difference in the rate of Chang’anerhao Decoction low-dose group, advance-dose group, and high dose group(P<0.01). Compared with the normal group, Chang’anerhao Decoction, middle dose group and high dose group, ear swelling was significantly reduced (P<0.01), swelling of the low-dose group decreased but not statistically significant (P=0.058>0.05); In the hot plate analgesia test, compared with the normal group, after the administration60s, Chang’anerhao Decoction medium and high dose groups with higher threshold (P<0.05), after administration120s, the threshold value of Chang’anerhao Decoction high-dose group is higher than the normal group (P<0.05). In the delayed type hypersensitivity test, compared with the model group, Chang’anerhao Decoction low, ear swelling, the high-dose group were significantly lower (P<0.001). Chang’anerhao Decoction low, medium and high dose groups compared with the control group, there is a trend significantly enhanced carbon clearance index in normal mice, but did not reach statistical significance (P>0.05).Experiment Ⅱ:Chang’anerhao Decoction for the treatment of trinitrobenzene sulfonic acid (TNBS) induced post-inflammation irritable bowel syndrome (PI-IBS) involvingintestinal mucosal immune barrier in a rat model. Objective:In this study, from a mechanical barrier, immune barrier, mucosal permeability three areasto observe Chang’anerhao Decoction effect on the PI-IBS rat intestinal mucosal barrier.Mainly from the regulation of cellular and humoral immunity to observe Chang’anerhao Decoction on immune barrier mechanism of action,and CD8, IL-4/IL-1β, SIgA plasma cell activation, SIgA levels of secreted in the intestine was observed indicators, to clear Chang’anerhao Decoction of the immune barrier mechanism.Materials and Methods:We combined mother-infant separation, colon trinitrobenzene sulfonic acid infusion, and chronic restraint to create a rat model of PI-IBS, Experimental animals were divided into model group, Chang’anerhao Decoction, the high dose group, middle dose group, low dose group, Tongxieyaofang group, atractylenolide I group, pinaverium bromide group and normal control group.Daily09:00am, the normal control group and model group were given saline gavage, the treatment group were given atractylenolide I, Tongxieyaofang and Chang’anerhao Decoction, according to body weight of rats fed0.8ml/100g stomach administration, continuous14d.Results:(1)The basic situation and intestinal tissue morphology:①After modeling, the degree of activity in rats, significantly reduced gloss coat. After treatment, bright white fur, increased activity.②Effect on food intake in rats:before treatment (after modeling), a significant reduction in model group and the treatment group than the control group eating (P<0.001, p<0.01); significantly reduced after treatment model group than the control group eating (p<0.01), the amount of each drug group and the normal group consumed a considerable intake (P>0.05).③Effect on weight gain in rats, before treatment (after modeling), body weight of rats model group and the treatment group less than the normal group(P<0.01, p<0.05); After treatment, the model group and each treatment group lighter weight than the normal group(p>0.05),After treatment, the amount of weight growth model rats, less than the normal group (P<0.01), the amount of weight gain in rats with normal growth of the amount of each drug group considerably (P>0.05).④Effect on stool water content,before treatment (after the model) model group and the treatment group stool water content is higher than normal, but not reach statistical significance(P>0.05).After treatment, Chang’anerhao Decoction low-dose, dose, atractylenolide I, Tongxieyaofang stool water content was significantly lower than that in the model group (P<0.01, p<0.05).⑤HE staining of colon tissue, the normal group, the treatment group and the untreated group were not found3points and3points or more lesions;there are two points in the model group lesions in different vision,Chang’anerhao Decoction low doses, Tongxieyaofang, atractylenolide I, field observation pinaverium bromide group, has rated the performance of mild inflammation of1minute.HE rated by statistical analysis, we can see that in the model group was significantly higher score than the normal group (P<0.001), Chang’anerhao Decoction dose group and low dose group have no significant difference between the normal group (P>0.05), HE score the rest of the treatment group were lower than the model, but did not reach statistical significance (P>0.05), HE score is still higher than the normal group for each treatment group after treatment (P<0.01).⑥Activation of mast cells (MC) count, No significant difference (P>0.05) Chang’anerhao Decoction dose group and the normal group, model group and the treatment group than the normal group MC count was significantly increased (P<0.001, P<0.01).⑦Pheochromocytoma (EC) count,Chang’anerhao Decoction dose group and the normal group no significant difference (P>0.05). A significant increase in the model group and the treatment group than in the normal group(P<0.001).(2)The efficacy of PI-IBS cases disease characteristics and features of the syndrome:①Effects on visceral sensitivity,before treatment model group and the treatment group AWR injection significantly lower than the normal group (P<0.001), model group after treatment AWR injection significantly lower than the normal group (P<0.001), model group lower after treatment than before treatment (P<0.05), Each treatment group improved significantly than before AWR water after treatment (P<0.001, P<0.01).②Effect on serum myeloperoxidase (MPO) activity level, before treatment (after successful model) model higher serum MPO activity, MPO activity levels after treatment with no significant difference (p>0.05). Model group, the serum levels of MPO activity compared each treatment group and the control group was significantly higher, but did not reach statistical significance (p>0.05).﹕ucrose preference test, sucrose preference model group was significantly lower than the normal group (P<0.001), Chang’anerhao Decoctionmedium and high dose groups, atractylenolide I,Pinaverium bromide groupwas significantly higher sucrose preference value and no difference with the normal group (p>0.05).④By open-field test, observe the effect on the degree of activity in rats, the number of cells before treatment to wear (after the model is completed) significantly reduced (P<0.01, P<0.05) in the model group and each treatment group than the normal group, model group after treatment than to wear the number of cells normal significantly reduced (P<0.01), Stand number in each group before treatment than normal group was significantly reduced. Chang’anerhao Decoction dose group and the normal group no difference after treatment (P>0.05),Increase the number of each treatment group to stand, but still with the normal group there were significant differences (P<0.001-0.05).Modification of the number of doses of medication before Chang’anerhao Decoction group Tongxieyaofang group, a significant reduction pinaverium bromide group than in the control group (P<0.05), less than in the other groups the normal group, but did not reach statistical significance (P>0.05). After treatment Chang’anerhao Decoction dose group, high dose group increased the number of modifications.⑤Rats D-xylose metabolism, before treatment, each group of urinary D-xylose levels significantly lower than the normal group (P <0.001). Model group before treatment, after treatment did not change. After treatment, each treatment group and urinary D-xylose content was no difference between normal (P>0.05).(3)Mechanism of action for PI-IBS rat intestinal mucosal barrier:①From the perspective of a mechanical barrier, observed the colonic epithelial mucosa of rats observed closely connected, visible group colonic epithelial cell model partial agenesis, cytoplasmic swelling, swelling and vacuolization of mitochondria, closely connected to open at1.2times TEM; Chang’anerhao Decoctionhigh-dose, medium-dose group colonic epithelial tight junction integrity, closely connected; Chang’anerhao Decoction, the low-dose group colonic epithelial cells are closely connected, the connection gap widened slightly; Tongxieyaofang group colonic epithelial cell tight junctions closely connected to the slit normal; atractylenolide I set visible intestinal epithelial tight junctions connecting the gap wider; pinaverium bromide group of intestinal epithelial tight junction complexes complete visibility, connecting the gap looser; complete normal colonic epithelial cells, cell membrane integrity tight junction complexes (tight junctions, gap junctions and desmosomes) complete, closely connected.②ffects on mucosal permeability, serum plasma endotoxin (LPS) level of model group was significantly higher than the normal group (P<0.001); each treatment group after treatment werelower thanmodelgroup significantly (P<0.05, P<0.01),and is slightly higher than the normal group (P <0.05, P<0.01).(3)Effects on intestinal mucosal immune barrier, medication former model group and the treated groups CD4count is lower than the normal group (P <0.001); among the treatment groups, Chang’anerhao Decoction high dose=dose> low dose=Tongxieyaofang=atractylenolide1=pinaverium,CD8counts were significantly lower for each treatment group and the normal group than in the untreated group(P<0.001);Chang’anerhao Decoction high dose group, middle dose group and low dose group and the normal group no significant difference (P>0.05),CD4/CD8ratio, each treatment group and the normal group was significantly higher than that in the model group (P<0.001);There was no significant difference between groups Tongxieyaofang and the normal group (P>0.05), Chang’ anerhao Decoction, and atractylenolide I and Pinaverium bromide group was slightly lower than the normal group, and had a statistically significant difference (P<0.05).Normal group and the treatment group IL-4levels were significantly higher than the model group (P<0.001), after treatment intervention can increase the level of IL-4, Chang’anerhao Decoction higher level than the other treatment groups, but did not reach statistical Learn the difference (P>0.05), the normal group and the treatment group IL-1β levels were significantly lower than that in the model group (P<0.001, P<0.05), after treatment intervention can reduce the levels of IL-1β, Chang’anerhao Decoction lower than other treatment groups, but did not reach statistical significance (P>0.05).Treatment group and the normal group, the number of plasma cells activated SIgA than the model group was significantly increased (P<0.001), more than two-fold;Each treatment group SIgA activated plasma cell count is slightly lower than the normal group (P<0.001); SIgA activated plasma cell count, Chang’anerhao Decoction dose, low dose, Tongxieyaofang, atractylenolide group I than Chang’anerhao Decoction high dose group, slightly higher pinaverium bromide group (P<0.01).Chang’anerhao Decoction group SIgA dose and low-dose levels of mucus in the inner wall of the intestine was significantly higher than that in the model group (P<0.05), each of the other treatment groups SIgA levels than the model group, but did not reach statistical significance (P>0.05).PGE2levels, the normal group, Chang’anerhao Decoction high dose group, middle dose group and low dose group was significantly higher than that in the model group (P<0.001, P <0.05), Chang’anerhao Decoction dose> normal> Chang’anerhao Decoction high dose=Chang’anerhao Decoction low dose=Tongxieyaofang> atractylenolide I=pinaverium bromide group (P<0.05).[Conclusion]Chang’anerhao Decoction has pharmacodynamic effects, its high, medium and low dose group, the role of regulating gastrointestinal motility; can significantly inhibit the delayed type hypersensitivity; dose and high-dose anti-inflammatory and analgesic may prolong analgesia time; high, medium and low doses in normal mice significantly enhanced carbon clearance index trend.Neonatal maternal separation, and colon trinitrobenzene sulfonic acid infusion, and chronic stimulus bound to create a rat model of PI-IBS, this model compliance with PI-IBS disease characteristics and spleen and liver stagnation syndrome characteristics. Mainly in visceral hypersensitivity, depression, increased stool water content, no inflammation or mild inflammatory diseases such as colon tissue pathological features, and there is reduction in food intake, D-xylose metabolism decreases, reducing activity, depression, etc. After Chang’anerhao Decoction treatment,disease and syndrome characteristics significantly improved, and the epithelial barrier of the colon mucosa has a good effect, make tight junctions between cells of the intestinal epithelial tight fitting, reducing the permeability of the colonic mucosa, prevent outside macromolecules (bacteria, viruses, etc.) invasion,and raise the level of protective factors and reduce the level of inflammatory damage factor, activates immune SIgA plasma cells, activating the secretion of SIgA to promote its intestine, thereby activating cellular immunity to humoral immune self-protection mechanism.Ultimately reduce intestinal inflammation, reduce visceral hypersensitivity, stool water content, etc. play a protective role in intestinal barrier, so as to achieve the purpose of treatment of PI-IBS.