| Background:Ulcerative colitis (UC) is a recurrent, chronic and nonspecific intestinal inflammation.It is a refractory and common disease of digestive system. In recent years, the incidence ofUC is increasing rapidly in our country. It is believed that the UC involves several reasonssuch as hereditary, immune abnormalities, the protective layer of intestinal out of orderand so on. At present, therapies are directed at the inflammatory disease, which isdefective on some extent.Traditional chinese medicine polysaccharide(TCMP) has important pharmacologicaleffects. As a traditional medicine treatment of gastrointestinal diseases, rheum hasobviously efficacy by years of clinical verification. Purified by water boiled and ethanol precipitated method, RTP is an important component of effectiveness, which is verificatedby our study through medicinal chemistry and pharmacology experiments. We also foundthat rhubarb polysaccharide (RTP) has obviously therapeutical effect on ulcerative colitisinduced by TNBS in rats. By separation and purification of RTP, we get lower molecularcomponent RTP-2(containing anthraquinone). There are studies show that polysaccharidecomplex have good biological activity. And there is listed drug, such as Niferex.Therefore, we plan to enzyme RTP-2to get smaller molecular weight fragments(RTP-2A) and remove the anthraquinone in RTP-2. Then on the animal level, we studythe therapeutic effect of RTP-2and RTP-2A on the ulcerative colitis rats induced byTNBS and the improvement of the intestinal flora imbalance. On the cellular level westudy immunoregulation on macrophage cytokine of RTP-2and RTP-2A and the bindingactivity with mannose receptor. The study is the basis of the mechanism for the TCMPstudy and can promote the development of new polysaccharides drug.Methods:Part I: By the enzymolysis effect of β-mannanase we enzyme RTP-2(containsanthraquinone), and orthogonal test to get the best enzymolysis process. Then use theSephadex G-100gel column to separate and purify the enzymatic polysaccharidefragments, the anthraquinone in the polysaccharide fragments are detected by HPLC.Part II: Copy the classic model, the TNBS induced ulcerative colitis rats model: TNBSand40%alcohol were mixed to enema. The experimental group: Control group (normal),model group (TNBS)0.2ml/day saline, RTP-2treatment group (RTP-2, n=10)200mg/kg/day, RTP-2A treatment group (RTP-2A, n=10)200mg/kg/day anddexamethasone positive drug treatment group (DEX, n=10).6h after modeling, dosing forthe first time. The medicine was given for5days.5days later, each group were abrosia butunforbidden water for24hours. Anesthesia executed and collected the colon8cm awayfrom the anus for pathology testing, then we compared the therapeutic effect of UCbetween RTP-2, RTP-2A and the clinical commonly used medicine dexamethasone.Part III: Copy the classic model, the TNBS induced ulcerative colitis rat model. Controlgroup (normal), model group (TNBS)0.2ml/day saline, RTP-2treatment group (RTP-2, n =10)200mg/kg/day, RTP-2A treatment group (RTP-2A, n=10)200mg/kg/day and emodinpositive drug treatment group (Emodin, n=10)20mg/kg/day. After modeling6h, dose forthe first time. The medicine was given for5days.5days later, each group were abrosiabut unforbidden water for24hours. Anesthesia executed and cecal segment rat feces,smears taken in sterile console. We collected the UC patients, who had not takenantibiotics in late4weeks. Gathering and smearing the fresh fece follow the reference"intestinal feces smear test atlas" and diagnose the dysbacteriosis index. Meanwhile, weused the Realtime RT-PCR methods to quantitatively observe the situation of thedysbacteriosis of the feces in TNBS induced ulcerative colitis rat model with or withoutthe treament of RTP-2or RTP-2A.Part IV: Based on the RAW264.7cells, we use fluorescent microscope and multi-functionmicroplate to study the binding ability between mannose receptor (MR) of RAW264.7with RTP-2and RTP-2A in vitro through the receptor-ligands competitive combinedexperiment. By the method of ELISA and Western blot we investigated the effects ofRTP-2and RTP-2A on the LPS induced inflammation cell factors on RAW264.7, such asTNF-α and NO. Then we investigated the possible downstream signaling pathways.Results:1The RTP-2is hydrolysed by β-mannanase and through seperation and purification. Weobtain the polysaccharide fragments RTP-2A, RTP-2B and RTP-2C with the molecularweight of7000-11000,20000-35000,2000-3000respectively, wherein the RTP-2A is thelargest part, with mannose content of41.36%, without anthraquinone detected by HPLC.2Based on successfully copied the ulcerative colitis model induced byTNBS in rats, wecompared the therapeutic effect between RTP-2, RTP-2A and DEX on UC. The resultsshow that RTP-2, RTP-2A and DEX have the same effect on the DAI index(P＞0.05), thescore of Gross morphology and pathology is RTP-2＞RTP-2A＞DEX (P<0.05). Theeffect of inhibiting inflammation is DEX＞RTP-2＞RTP-2A.3The results of the65cases of UC patients show: most patients (69.2%) have differentextent of dysbacteriosis, while the dysbacteriosis of the TNBS induced rat models is moreserious. The probiotics of the TNBS induced rat colitis models, including lactobacillus and bifidobacterium significantly reduce, while the pathogenic or conditional pathogenbacteria proliferate greatly. While RTP-2and RTP-2A can reverse the dysbacteriosis ofTNBS induced rat ulcerative colitis, and RTP-2is better than RTP-2A (P<0.05).4According to the fluorescent microscope and multi-function microplate, we studied thebinding ability between mannose receptor (MR) of RAW264.7and RTP-2and RTP-2A,which is MRmAb＞RTP-2A＞RTP-2(P<0.05). The ELISA and Western blot results showthat RTP-2and RTP-2A can antagonist the LPS induced inflammation through theproduction of TNF-α and NO produced by macrophages. Western blot results show thatRTP-2and RTP-2A can antagonism the rising level of NF-κB p65induced by LPS onmacrophage. This antagonism action can be blocked by MR antibody. The effect of RTP-2is weaker than that of RTP-2A and Emodin (P<0.05).Conclusion:1Through HPLC we detect RTP-2containing anthraquinone, which can be separated andpurified by enzyme and get RTP-2A, whose molecular is7000-11000. The two fragmentsare brand new RTP, which are not reported.2The effect on mucosal repair and regeneration of RTP-2and RTP-2A is better than DEX,but DEX is better in inhibiting inflammation development. The effect of RTP-2in thethree treatment groups is the best and may be related to the anthraquinone.3The Bifidobacterium and Lactobacillus in the TNBS induced UC model rats colondecreased significantly, while the Bacteroides fragilis and Enterococcus have greatproliferation. RTP-2and RTP-2A both can reverse the dysbacteriosis of TNBS inducedulcerative colitis in rats. The anthraquinone in RTP-2may have played a role in thisprocess.4RTP-2and RTP-2A’s immune modulating effect may be mediated by MR to antagonizethe proinflammatory of LPS induced secretion of cell factors, such as TNF-α and nitricoxide. NF-κB p65protein plays an important role in the signal transduction process. Butthe effect of RTP-2is weaker than that of RTP-2A, which is inconsistent with the resultsthat RTP-2is better than RTP-2A. There may be relationship with the molecular of RTP-2and intestinal flora. |
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