Pharmacodynamic Mechanism Of Semen Crotonis Pulveratum And Rhubarb In Rats With Ulcerative Colitis And Their Exploresion Of Effective Material Base

Object: Ulcerative colitis is a chronic non-specific colitis disease, which belongs to “diarrhea" areas in traditional Chinese medicine. The Pathogenesis remains unclear. The incidence rate shows a continuous elevation tendency worldwidely. Study on etiology about this disease is particular importance. Semen Crotonis Pulveratum and Rhubarb as antidiarrheal drugs are widely used in acute and chronic diarrhea treatment.Previous studies have shown that a small dose of a single product Semen Crotonis Pulveratum, Semen Crotonis Pulveratum and rhubarb at a certain percentage of compatibility have a clear antidiarrheal efficacy. It has been reported that proprietary Chinese medicines including rhubarb, the Croton cream compatibility are effective clinical treatment of UC, but their mechanism and the material basis are still reported rarely in a bottleneck with multi-component, multi-target and diversity characteristics. Therefore, the subject of TNBS/ethanol-induced ulcerative colitis in rats and H2O2-induced apoptosis in IEC-6 cells model, choose Semen Crotonis Pulveratum ethanol crude extracts, the main ingredients of rhubarb, Emodin, Gallic Acid as the object of study to explore the traditional drugs on the pharmacological mechanism and role in the treatment of ulcerative colitis, to clear rhubarb,croton cream effective material basis.Methods: 1.Rats model of ulcerative colitis is induced by coloclysis of mix TNBS and alcohol solution 2.From the immunological point, to explore the pathogenesis of ulcerative colitiss, study Rhubarb and Semen Crotonis Pulveratum therapeutic effect and further clarify the effective material basis of rhubarb.The Wister male rats were randomly divided into model group, Rhubarb + Semen Crotonis Pulveratum compatibility group, Semen Crotonis Pulveratum with Rhubarb ingredients compatibility group(Semen Crotonis Pulveratum + Emodin group, Semen Crotonis Pulveratum and Gallic Acid group) and normal control group. Rats of treatment groups were respectively administered orally for 7 days。Rat clinical situation was observed. Rats were sacrificed in the first 11. collected the new blood、 serum, colon tissue samples. Evaluated CMDI score and HE staining. Then the peripheral blood concentration of CD+CD25+Tregs and CD4+CD25+Foxp3 were tested by Flow cytometry and theConcentration of IL-6 and IL-10 in serum by enzyme-linked immunosorbent assay and the expression of ICAM-1, COX-2 in intestinal mucosa by immunohistochemical test. 3.To investigate Roles of the different concentrations of Semen Crotonis Pulveratum ethanol crude extract, Gallic acid, emoting on IEC-6 cell apoptosis induced by H2O2.The serum-starved IEC-6 cells were stimulated by H2O2 with or without compatibility of different concentrations of Semen Crotonis Pulveratum alcohol extracts and Emotin, Gallic Acid, respectively.Caspase-3 expression was detected by Western blot.Cell proliferation and apoptotic ratio was examined with MTT assay and flow cytometric analysis and the Tunnel method,respectively. DNA fragments were detected with agarose gel electrophoresis.Results: 1. Clean-level environmental conditions, a model of ulcerative colitis was established s UCcessfully.Rats presented diarrhea swiftly after establishment of ulcerative colitis models, diarrhea、bloody stools, mucous stools the next day and lasted for a comparatively long time. Some intestinal m UCosa kept congestion, edema, and seen occasionally an amount of inflammatory cell infiltration under light microscope conditions. 2. Compared with normal control group, these figures in model group of peripheral blood CD4+CD25 +Tregs and CD4+CD25+Foxp3 were lower,higher of IL-6 concentration while lower of IL-10 concentration; higher of mucosa ICMA-1、COX-2 expression. The difference between the two groups was significant(P <0.05). After treatment for 7 days, The figures were higher in the rat peripheral CD4+CD25+Tregs and CD4+CD25+Foxp3; lower in IL-6 concentration 、higher in IL-10 concentration, lower in ICAM-1, COX-2 expression.The difference was significant(P <0.05) between Rhubarb + Semen Crotonis Pulveratum compatibility group&Semen Crotonis ulveratum + Gallic Acid group and the model group. These figures in Semen Crotonis Pulveratum + Emotin compatibility group appeared insignificant difference with model group(P> 0.05). 3. After 4h stimulation with 200μmol/L H2O2, IEC-6 cells were activated significantly and activated caspase-3 expression was enhanced. In MTT assay, Semen Crotonis Pulveratum alcohol extracts and Gallic Acid, the Emotin respectively compatibility promoted cell proliferation obviously in a concentrationdependent manner of croton cream. DNA fragments by agarose gel electrophoresis, the Tunnel and flow cytometry results showed that H2O2 induced IEC-6 cell apoptosis. This effect was antagonized by Semen Crotonis Pulveratum alcohol crude extracts of emoting and Gallic Acid in certain concentrations. Inhibited apoptosis related Caspase-3 activation in Semen Crotonis Pulveratum alcohol extracts and Gallic Acid and had no relation in high Semen Crotonis Pulveratum alcohol crude extract and low concentrations of Emoting compatibility.Conclusion: 1. Clean-level environmental conditions, the TNBS+ethanol of low concentration were the best concentration for experimental model. The SPF-class environment condition, was not suitable of the experimental study of this subject since intestinal mucosal injury in rats recovered quickly for the absence of pathogenic microorganisms 2. Abnormalities of the intestinal mucosal immune and cytokine imbalance might be involved in pathogenesis of UC. Rhubarb and Semen Crotonis Pulveratum might have a therapeutic effect by promoting Tregs,reconstructing the balance between pro-inflammatory and anti-inflammatory cytokines,down-regulating the expression of ICAM-1, COX-2 and Semen Crotonis Pulveratum + Gallic acid achieve the similar therapeutic effect. Tip: Gallic Acid maybe the effective material basis for of e antidiarrheal of Rhubarb and Semen Crotonis Pulveratum compatibility t. Semen Crotonis Pulveratum +Emodin may improve the pathological damage, but the effect of immune regulation is not obvious. 3. The Semen Crotonis Pulveratum ethanol crude extracts and Gallic Acid,Emotin, respectively compatibility canpromote cellproliferation; inhibit apoptosis. To sum up, rhubarb, Semen Crotonis Pulveratum through the inhibition of local intestinal inflammation, the balance of the peripheral cytokine network, inhibition of apoptosis, reach antidiarrheal and protect the intestinal mucosa, and treat ulcerative colitis.Gallic Acid, Emotin, Semen Crotonis Pulveratum alcohol extracts may be the effective material basis of compatibility in the treatment of UC.