大黄多糖治疗溃疡性结肠炎的机制研究
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摘要
研究背景
溃疡性结肠炎(ulcerative colitis，UC)是一种病因不明的反复发作的慢性非特异性结肠炎症，为消化系统疾病中常见的难治性疾病。近年来，UC在我国的发病率呈快速增长趋势，越来越引起人们重视。目前，UC的治疗措施主要为抗炎抗免疫，例如糖皮质激素、免疫抑制剂、非甾体抗炎药的应用等，但临床治疗效果并不满意，且长期应用副作用严重。因此，急需寻找疗效良好、副作用较小的治疗方法。唐古特大黄为传统的胃肠道用药，临床上长期用于胃肠道疾病的治疗，疗效显著。本实验室前期分离纯化获得了唐古特大黄多糖(Rheum tanguticum polysaccharide， RTP)，发现RTP对大鼠溃疡性结肠炎疗效显著。为了深入研究RTP的作用机制，本课题从前期实验分离纯化得到的RTP中再分离得到了分子量较低的组分RTP-2，经过糖含量和仪器分析发现该组分为含有蒽醌的多糖复合物。为了进一步探讨RTP-2的生物活性，本课题采用酶解的方法，对RTP-2作进一步的酶降解分离纯化得到新的不含蒽醌的大黄多糖片段RTP-2A。然后，采用TNBS诱导的大鼠UC模型，将二者对溃疡性结肠炎治疗作用及其对肠道菌群失调的调节作用进行对照研究；并在细胞水平深入研究RTP-2、RTP-2A和大黄素(Emodin)对LPS致炎的巨噬细胞分泌的细胞因子的影响，以及RTP-2、RTP-2A与巨噬细胞甘露糖受体的结合特性。本课题旨在通过上述研究探讨含有蒽醌的RTP-2与其酶解后不含有蒽醌的多糖片段RTP-2A，在UC治疗、肠道菌群失调和免疫功能方面调节作用的差异，以期进一步探讨大黄多糖对结肠炎的治疗机制，为UC的治疗以及多糖药物研究提供新的线索。
实验方法
第一部分：RTP-2的酶解、分离和纯化：采用β-甘露聚糖酶酶解RTP-2，RTP-2为100~200KD且含有蒽醌的多糖复合物，本实验通过正交实验筛选最佳酶解工艺，以Sephadex G-100凝胶柱分离、纯化酶解得到的多糖片段，并通过HPLC检测纯化后多糖片段中蒽醌的含量。
第二部分：观察RTP-2及RTP-2A对TNBS诱导大鼠试验性结肠炎的治疗作用：采用40%乙醇和TNBS混悬后给大鼠灌肠复制模型，动物分组为：正常对照组(normal)和模型组(TNBS)给0.2ml/day生理盐水，RTP-2治疗组(RTP-2)200mg/kg/day，RTP-2A治疗组(RTP-2A)200mg/kg/day和DEX阳性对照组(DEX)0.2mg/kg/day；每组10只，造模后6h灌胃给药，连续给药5天，每天1次，治疗结束后，大鼠禁食不禁水24h，乙醚麻醉处死动物，解剖收集肛门以上约8cm结肠，用于疾病活动指数、溃疡指数及病理学检测及评分；比较RTP-2、RTP-2A与DEX对UC的疗效。
第三部分：临床UC患者粪便菌群及UC大鼠肠道菌群分析：采集临床确诊为UC且近四周未服用抗菌药物的患者的新鲜粪便（65例），参考《肠道菌群粪便涂片检查图谱》进行肠道菌群失调的分度诊断。结肠炎大鼠分组同第二部分为：正常对照组(normal)、模型组(TNBS) RTP-2治疗组，RTP-2A治疗组和Emodin阳性药对照组(Emodin)20mg/kg/day。TNBS复制模型，给药5天后，乙醚麻醉处死，在无菌操作台取盲肠段大鼠粪便，涂片检查肠道菌群失调的分度；通过实时定量PCR技术定量分析上述各UC大鼠盲肠粪便中双歧杆菌、乳酸杆菌、脆弱拟杆菌及肠球菌的变化情况，比较RTP-2、RTP-2A与Emodin对UC大鼠菌群失调的调节作用。
第四部分：观察RTP-2及RTP-2A对LPS诱导炎症因子分泌的拮抗作用：以巨噬细胞系RAW264.7作为研究对象，Man-FITC-BSA为配体，通过竞争性抑制试验，以荧光显微镜和多功能酶标仪定性、定量的观察巨噬细胞荧光染色情况，比较RTP-2、RTP-2A及MR单抗与RAW264.7中MR的结合能力的差异；然后，通过ELISA和Western blot的方法观察RTP-2及RTP-2A对LPS诱导RAW264.7分泌的细胞因子TNF-α及NO的影响，并对其可能的信号通路进行研究。
实验结果
1．通过β-甘露聚糖酶对RTP-2的酶解、分离和纯化，获得分子量分别为7000-11000、20000-35000、2000-3000的多糖片段RTP-2A，RTP-2B和RTP-2C，其中RTP-2A量最多，且未检测到蒽醌，其甘露糖含量为41.36%。
2．RTP-2、RTP-2A对TNBS诱导的大鼠试验性结肠炎疗效显著，RTP-2、RTP-2A和DEX对UC大鼠疾病活动指数没有差异(P＞0.05)，对于大体形态评分和组织学评分的作用：RTP-2＞RTP-2A＞DEX(P<0.05)，病理结果显示：RTP-2及RTP-2A可显著促进黏膜上皮的修复和再生，其病理评分为：RTP-2＞RTP-2A＞DEX(P<0.05)，含有蒽醌的RTP-2的抗炎作用优于不含蒽醌的RTP-2A。
3.65例UC患者中45例(69.3％)发生菌群失调，而TNBS诱导的UC大鼠均发生菌群失调，较UC患者的菌群失调分度更为严重。实时定量PCR结果提示，UC大鼠肠道益生菌双歧杆菌和乳酸杆菌明显减少，而条件致病菌脆弱拟杆菌和肠球菌过度增殖。RTP-2及RTP-2A均可逆转该菌群失调，使肠道菌群恢复平衡，且含有蒽醌的RTP-2效果更优，其对脆弱拟杆菌及肠球菌抑制作用优于RTP-2A组(P<0.05)。Emodin治疗组大鼠肠道双歧杆菌和乳酸杆菌未见明显变化，而对脆弱拟杆菌、肠球菌有抑制作用，与TNBS组大鼠相比有显著性差异(P<0.05)，但Emodin对双歧杆菌和乳酸杆菌促增殖的作用不及RTP-2与RTP-2A(P<0.05)。
4. RTP-2及RTP-2A与MR具有一定的亲和力，亲和力强弱为：MRmAb＞RTP-2A＞RTP-2。RTP-2、RTP-2A及Emodin均可拮抗LPS诱导巨噬细胞TNF-α及NO的分泌，拮抗LPS诱导的磷酸化p65蛋白表达的上调；该作用可被MR单抗阻断；但RTP-2的作用弱于RTP-2A(P<0.05)。
结论
1.在本实验室前期研究的基础上，将RTP进一步分离纯化得到分子量较低的组分RTP-2，经糖含量与仪器分析发现该组分为含有蒽醌的多糖复合物；为了探讨蒽醌对低分子量大黄多糖作用的影响，再将RTP-2进行酶解、分离和纯化，获得不含有蒽醌的RTP-2A；二者均为新的大黄多糖片段，未见文献报道。
2. RTP-2、RTP-2A及DEX对大鼠UC具有良好的治疗作用，RTP-2与RTP-2A主要通过对结肠粘膜异常免疫反应的调节和促进粘膜的修复、再生来治疗UC，而DEX对UC的治疗作用主要体现在炎症抑制上。RTP-2、RTP-2A抑制炎症反应的作用，可能与其抑制NF-κB的活化，降低TNF-α的分泌有关。
3. TNBS诱导的UC大鼠较临床UC患者肠道菌群紊乱更加严重。RTP-2及RTP-2A均可逆转TNBS诱导的UC大鼠的菌群失调，促进乳酸杆菌和双歧杆菌增殖，抑制脆弱拟杆菌及肠球菌增殖；而Emodin治疗组仅对脆弱拟杆菌和肠球菌有一定的抑制作用，对乳酸杆菌和双歧杆菌没有影响。提示RTP-2致病菌的抑制作用上优于RTP-2A可能与其含有蒽醌有关。
4. RTP-2和RTP-2A可通过MR的介导被巨噬细胞内吞入胞，通过降低P-p65的表达水平，抑制NF-κB p65的活化，从而拮抗LPS诱导的巨噬细胞产生的TNF-α和NO。
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.
溃疡性结肠炎(ulcerative colitis，UC)是一种病因不明的反复发作的慢性非特异性结肠炎症，为消化系统疾病中常见的难治性疾病。近年来，UC在我国的发病率呈快速增长趋势，越来越引起人们重视。目前，UC的治疗措施主要为抗炎抗免疫，例如糖皮质激素、免疫抑制剂、非甾体抗炎药的应用等，但临床治疗效果并不满意，且长期应用副作用严重。因此，急需寻找疗效良好、副作用较小的治疗方法。唐古特大黄为传统的胃肠道用药，临床上长期用于胃肠道疾病的治疗，疗效显著。本实验室前期分离纯化获得了唐古特大黄多糖(Rheum tanguticum polysaccharide， RTP)，发现RTP对大鼠溃疡性结肠炎疗效显著。为了深入研究RTP的作用机制，本课题从前期实验分离纯化得到的RTP中再分离得到了分子量较低的组分RTP-2，经过糖含量和仪器分析发现该组分为含有蒽醌的多糖复合物。为了进一步探讨RTP-2的生物活性，本课题采用酶解的方法，对RTP-2作进一步的酶降解分离纯化得到新的不含蒽醌的大黄多糖片段RTP-2A。然后，采用TNBS诱导的大鼠UC模型，将二者对溃疡性结肠炎治疗作用及其对肠道菌群失调的调节作用进行对照研究；并在细胞水平深入研究RTP-2、RTP-2A和大黄素(Emodin)对LPS致炎的巨噬细胞分泌的细胞因子的影响，以及RTP-2、RTP-2A与巨噬细胞甘露糖受体的结合特性。本课题旨在通过上述研究探讨含有蒽醌的RTP-2与其酶解后不含有蒽醌的多糖片段RTP-2A，在UC治疗、肠道菌群失调和免疫功能方面调节作用的差异，以期进一步探讨大黄多糖对结肠炎的治疗机制，为UC的治疗以及多糖药物研究提供新的线索。
实验方法
第一部分：RTP-2的酶解、分离和纯化：采用β-甘露聚糖酶酶解RTP-2，RTP-2为100~200KD且含有蒽醌的多糖复合物，本实验通过正交实验筛选最佳酶解工艺，以Sephadex G-100凝胶柱分离、纯化酶解得到的多糖片段，并通过HPLC检测纯化后多糖片段中蒽醌的含量。
第二部分：观察RTP-2及RTP-2A对TNBS诱导大鼠试验性结肠炎的治疗作用：采用40%乙醇和TNBS混悬后给大鼠灌肠复制模型，动物分组为：正常对照组(normal)和模型组(TNBS)给0.2ml/day生理盐水，RTP-2治疗组(RTP-2)200mg/kg/day，RTP-2A治疗组(RTP-2A)200mg/kg/day和DEX阳性对照组(DEX)0.2mg/kg/day；每组10只，造模后6h灌胃给药，连续给药5天，每天1次，治疗结束后，大鼠禁食不禁水24h，乙醚麻醉处死动物，解剖收集肛门以上约8cm结肠，用于疾病活动指数、溃疡指数及病理学检测及评分；比较RTP-2、RTP-2A与DEX对UC的疗效。
第三部分：临床UC患者粪便菌群及UC大鼠肠道菌群分析：采集临床确诊为UC且近四周未服用抗菌药物的患者的新鲜粪便（65例），参考《肠道菌群粪便涂片检查图谱》进行肠道菌群失调的分度诊断。结肠炎大鼠分组同第二部分为：正常对照组(normal)、模型组(TNBS) RTP-2治疗组，RTP-2A治疗组和Emodin阳性药对照组(Emodin)20mg/kg/day。TNBS复制模型，给药5天后，乙醚麻醉处死，在无菌操作台取盲肠段大鼠粪便，涂片检查肠道菌群失调的分度；通过实时定量PCR技术定量分析上述各UC大鼠盲肠粪便中双歧杆菌、乳酸杆菌、脆弱拟杆菌及肠球菌的变化情况，比较RTP-2、RTP-2A与Emodin对UC大鼠菌群失调的调节作用。
第四部分：观察RTP-2及RTP-2A对LPS诱导炎症因子分泌的拮抗作用：以巨噬细胞系RAW264.7作为研究对象，Man-FITC-BSA为配体，通过竞争性抑制试验，以荧光显微镜和多功能酶标仪定性、定量的观察巨噬细胞荧光染色情况，比较RTP-2、RTP-2A及MR单抗与RAW264.7中MR的结合能力的差异；然后，通过ELISA和Western blot的方法观察RTP-2及RTP-2A对LPS诱导RAW264.7分泌的细胞因子TNF-α及NO的影响，并对其可能的信号通路进行研究。
实验结果
1．通过β-甘露聚糖酶对RTP-2的酶解、分离和纯化，获得分子量分别为7000-11000、20000-35000、2000-3000的多糖片段RTP-2A，RTP-2B和RTP-2C，其中RTP-2A量最多，且未检测到蒽醌，其甘露糖含量为41.36%。
2．RTP-2、RTP-2A对TNBS诱导的大鼠试验性结肠炎疗效显著，RTP-2、RTP-2A和DEX对UC大鼠疾病活动指数没有差异(P＞0.05)，对于大体形态评分和组织学评分的作用：RTP-2＞RTP-2A＞DEX(P<0.05)，病理结果显示：RTP-2及RTP-2A可显著促进黏膜上皮的修复和再生，其病理评分为：RTP-2＞RTP-2A＞DEX(P<0.05)，含有蒽醌的RTP-2的抗炎作用优于不含蒽醌的RTP-2A。
3.65例UC患者中45例(69.3％)发生菌群失调，而TNBS诱导的UC大鼠均发生菌群失调，较UC患者的菌群失调分度更为严重。实时定量PCR结果提示，UC大鼠肠道益生菌双歧杆菌和乳酸杆菌明显减少，而条件致病菌脆弱拟杆菌和肠球菌过度增殖。RTP-2及RTP-2A均可逆转该菌群失调，使肠道菌群恢复平衡，且含有蒽醌的RTP-2效果更优，其对脆弱拟杆菌及肠球菌抑制作用优于RTP-2A组(P<0.05)。Emodin治疗组大鼠肠道双歧杆菌和乳酸杆菌未见明显变化，而对脆弱拟杆菌、肠球菌有抑制作用，与TNBS组大鼠相比有显著性差异(P<0.05)，但Emodin对双歧杆菌和乳酸杆菌促增殖的作用不及RTP-2与RTP-2A(P<0.05)。
4. RTP-2及RTP-2A与MR具有一定的亲和力，亲和力强弱为：MRmAb＞RTP-2A＞RTP-2。RTP-2、RTP-2A及Emodin均可拮抗LPS诱导巨噬细胞TNF-α及NO的分泌，拮抗LPS诱导的磷酸化p65蛋白表达的上调；该作用可被MR单抗阻断；但RTP-2的作用弱于RTP-2A(P<0.05)。
结论
1.在本实验室前期研究的基础上，将RTP进一步分离纯化得到分子量较低的组分RTP-2，经糖含量与仪器分析发现该组分为含有蒽醌的多糖复合物；为了探讨蒽醌对低分子量大黄多糖作用的影响，再将RTP-2进行酶解、分离和纯化，获得不含有蒽醌的RTP-2A；二者均为新的大黄多糖片段，未见文献报道。
2. RTP-2、RTP-2A及DEX对大鼠UC具有良好的治疗作用，RTP-2与RTP-2A主要通过对结肠粘膜异常免疫反应的调节和促进粘膜的修复、再生来治疗UC，而DEX对UC的治疗作用主要体现在炎症抑制上。RTP-2、RTP-2A抑制炎症反应的作用，可能与其抑制NF-κB的活化，降低TNF-α的分泌有关。
3. TNBS诱导的UC大鼠较临床UC患者肠道菌群紊乱更加严重。RTP-2及RTP-2A均可逆转TNBS诱导的UC大鼠的菌群失调，促进乳酸杆菌和双歧杆菌增殖，抑制脆弱拟杆菌及肠球菌增殖；而Emodin治疗组仅对脆弱拟杆菌和肠球菌有一定的抑制作用，对乳酸杆菌和双歧杆菌没有影响。提示RTP-2致病菌的抑制作用上优于RTP-2A可能与其含有蒽醌有关。
4. RTP-2和RTP-2A可通过MR的介导被巨噬细胞内吞入胞，通过降低P-p65的表达水平，抑制NF-κB p65的活化，从而拮抗LPS诱导的巨噬细胞产生的TNF-α和NO。
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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