O-GlcNAc And IBD And The Intervention Mechanism Of Pulsatilla Decoction On IBD

Background:Inflammatory bowel disease (IBD) including ulcerative colitis (UC) and Crohn’s disease (CD),which is an idiopathic chronic infammatory bowel disease.Its main manifestations are diarrhea, mucus bloody stool,abdominal pain and tenesmus. As the pathogenesis of IBD is not completely clear, there is not one kind of treatment medicine of remarkable curative effect and small side effect at present. Therefore, finding new and effective drugs for the treatment is a hot topic in this research field.Pulsatilla Decoction treatment of IBD lead to a lower incidence of adverse reactions, the security is higher.This experiment can provide theoretical evidence for pulsatillae decoction treat-ing IBD,and providing the theoretical direction for the clinical medication. Addition of O-linked N-acetylglucosamine (O-GlcNAc) to the hydroxyl group of serine/threonine residues (O- GlcN acylation) is a post-translational modification common to multicellular eukaryotes. Its physiolo--gical significance as a major stress sensor that inhibites the inflammatory response, inhibites cell apoptosis, reduces the amount of protein degradation, adjusts the body’s immunity, etc. we mainly discusses the relationship between protein O- GlcNAc modification and inflammatory bowel disease.Part Ⅰ Establishment of Inflammatory bowel disease model induced by TNBS in ratObjective:To establish a TNBS induced inflammatory bowel disease model in rat and to ana-lyze its dosage effect, finding a reasonable dose of building model.Methods:Totally 50 rat were divided into 5 groups by random,10 rat in each group.then the con--trol group was instilled the ethylalcohol.The others groups were instilled by 25mg/kg,50mg/kg, 100 mg/kg,150 mg/kg TNBS dissolved in 500g/L ethylalcohol. They disease activity index were recorded.All rat were killed at days 7 after establishing models to observe the colon macroscopic damage index、tissue damage index, and detection the TNF-α level in serum, myeloperoxidase (MPO) activity of intestines was evaluated.Results:A total of 3 rat were died in 150 mg/kg TNBS group during establishment, other group did not see death.1.Once mechanical injury were found in contral group and 25mg/Kg TNBS group,the symptom began to relieve 3 days after induced.50 mg/kg-150 mg/kg TNBS groups had continue inflammatory injury, including poor general condition, DAI reduce, diarrhea, hemafecia, etc.2. The score of DAI、CMDI、TDI in 50 mg/kg-150 mg/kg TNBS group were significantly higher than in contral group and 25mg/Kg TNBS group(P<0.01), and with the increase of dosage of TNBS,the DAI、CMDI and TDI score is higher, the effect reached the peak in 150 mg/kg TNBS group. However, there is no difference between contral group and 25mg/Kg TNBS group(P>0.01).3. The TNF-α level in 50-150 mg/kg TNBS groups were significantly higher than contral group and 25mg/Kg TNBS group(P<0.01), the effect reached the peak in 150 mg/kg TNBS group.4. MPO activity in intestines:The activity was significantly increased in the 50-150mg/kg TNBS groups as compared with the control group and 25mg/kg TNBS group.Conclusion:500 g/L alcohol containing 100 mg/kg TNBS induced rat inflammatory bowel disease is an ideal method.Part Ⅱ To study the relativity between 0-GlcNAc Modification and inflammatory bowel disease in rats and the intervention mechanism of Pulsatilla Decoction on Inflammatory bowel disease in ratsObjectivet:To explore the effect of Pulsatilla Decoction and O-GlcNAc Modification in the treatment of TNBS induced inflammatory bowel disease in rat.Methods:50 rats were randomy divided into the model group, Pulsatillae Decoction(PD) group, Glucosamine (GlcN) group, Glutamine (Gln) group and Alloxan (ALX) group of 10 in each.Be used 500 g/L alcohol containing 100 mg/kg TNBS method copied IBD rat model. After gavage 7d, The severity of colitis was evaluated using the DAI, CMDI, TDI score, TNF-α、IFN-γ、IL-6、 IL-27、IL-4、IL-10 levels in serum were determined by ELISA.The expression of Calprotectin in intestinal was determined by Western blot. O-GlcNAc modification and NF-κB p65 in PBMC was determined by Flow cytometry(FCM).Results:1.The score of DAI,CMDI,TDI and the serum TNF-α、IFN-γ、IL-6、IL-27 level and PBMC NF-κB p65 positive expression and the expression of calprotectin in intestinal in PD group were significantly lower than that of the model group (P<0.01). Meanwhile,IL-4 and IL-10 level were significantly higher (P<0.01)2.Compared with model group, treatment with Gln and GlcN significantly improved symptoms, reduced DAI,CMDI and TDI scores, and decreased the levels of serum TNF-α,IFN-γ,IL-6,IL-27. Gln and GlcN decreased the expression of NF-κB p65 and the expression of calprotectin in intestinal more significantly than model (P<0.01). Meanwhile, increase the content of IL-4 and IL-10 and up-regulation the O-GlcNAc modification in PBMC (P<0.01)3. Compared with the model group, the ALX can increase the DAI,CMDI,TDI scores and the levels of serum TNF-α,IFN-β,IL-6,Il-27,reduce the level of IL-4 and IL-10, decrease the content of O-GlcNAc modification in PBMC, increase the content of NF-κB p65 in PBMC and calpro--tectin in intestinal.Conclusion:1.Protective effects of PD on intestinal mucosal immunity of IBD may be related to reduc--ing the expression of NF-κBp65, decreasing the content of proinflammatory cytokine,increasing the content of anti-inflammatory agents, to make the cytokine to reach the equilibrium state, regulating the organism’s immune response, improving the intestinal mucosal damage.2.GlcN an Gln attenuates intestinal mucosal damage through 0-GlcNAc protein modifica--tion in IBD rats. The mechanism may be to inhibit the relative activity of NF-κB p65, and modulate the release of cytokines, reducing inflammatory reaction, promoting mucosal healing. However, O-GlcNAcase(Alloxan) can completely reverse this effect.3、PD with increased 0-GlcNAc protein modification can inhibit the activity of NF-κB p65, modulate the release of cytokines, reducing mucosal damage, promoting mucosal healing.