Study On Curative Effects And Mechanism In Treating UC By A Prodrug Of 5-ASA And Approach The Role Of β2GPⅠ

Ulcerative colitis(UC) is an immunologic disease which is presumably resulted from the combined contribution of immunity,infection,environmental factors and genetic factors.But the exact etiology and pathogenesis is not clear at present.The role of immune factors is confirmed to be more important through many investigation,especially the disorder of cytokine(CK) network plays an important role in UC.It is more common in our country with the improvement of living standard and the change of diet.Because of the backwardness in investigation and slow progress in treatment,exploring the pathogenesis and new therapeutic tool,especially the safe and effective medicine,is of heated discussion.Part one:Current treatment strategies endeavor to control the underlying inflammation,and conventional anti-inflammatory medicines including 5-aminosalicylic acid(5-ASA),glucocorticoid,immuno-suppressive agent and immunomodulator,which are used widely.However,they have many adverse effects and recurrent attacks might occur after discontinuation.5-ASA is the main therapeutic medicine to treat UC in the active period.In order to strengthen its efficacy and degrade its toxicity,researchers have been trying to find a more effective prodrug.Balsalazide is a new prodrug of 5-ASA.Compared with the congener drug salfasalazine(SASP),which is a prodrug of 5-ASA,balsalazide possesses more favourable drug tolerance because of the non-existence of SP,decreasing the adverse effect such as digestive symptom or drug hypersensitivity.To investigate about its therapeutic effect and safety on Chinese patients,and the possible mechanism, we conducted animal experiments and clinical observation systematically,which is the first time in China.The role of some cytokines in UC was also investigated from a different point of view.Set up an oxazolone(OXZ)-induced colitis mice model and treat them with balsalazide,Compared with SASP.Detect their DAI score,macroscopic damage,histological score,MPO activity and the cytokines(IFN-γ,IL-4,IL-10)representing the Th1 or Th2 immunoreaction by enzyme-linked immunosorbent assay(ELISA) and RT-PCR to evaluate the colitis mice model's value in this study;investigate the efficacy and immunologic mechanism of balsalazied.The result shows that the DAI score,macroscopic damage score,histological score,MPO activity in OXZ induced colitis were significantly higher than control group(P<0.05),and the IL-4 level was obviously higher than the control group,but IFN-γwas lower,which indicated the seting up of an OXZ induced colitis mice model successful.In the intervention experiment with medicine,all kinds of detect index in the balsalazide group were ameliorated obviously compared with normal sodium(NS) group and OXZ group(P<0.05),at the same time surpassing the SASP group.The level of IFN-γfrom LPMC in SASP and balsalazide group were not significantly different from OXZ group on days 3 and 7 post-intervention(P>0.05).The level of IFN-γmRNA by RT-PCR in SASP and balsalazide group were significantly higher on days 3(P<0.05),but the level decreased on days 7.The level of IL-4 from LPMC and IL-4mRNA in OXZ group were significantly higher than normal control group(P<0.05).The levels of IL-4 and IL-4mRNA were significantly decreased in balsalazide group on days 3 and 7 post-intervention(P<0.05).The level of IL-10 from LMPC were significantly higher than normal control group(P<0.05),but the level of IL-10mRNA were significantly higher on days 3,which no significantly different from normal control group on days 7.The levels of IL-10 from LPMC in SASP and balsalazide group were significantly higher than OXZ group(P<0.05),especially in balsalazide group(P<0.05).The level of IL-10mRNA in balsalazide group was significantly higher than OXZ group on days 3.Furthermore,the up-regulation was more obvious on days 7.Still,the level in SASP were inferior to alsalazide group.The clinical trial portion:fouty-six patients with UC were randomly allocated into balsalazide group(n=24) or SASP group(n=22) in double-blind and double mimic trial and treated for eight weeks. The efficacy and adverse events of this drug were evaluated on the basis of the clinical improvements of symptoms and colonoscopic changes.And detecting the cytokines(IFN-γ,IL-4,IL-10)by ELISA and RT-PCR to investigate the efficacy and immunologic mechanism of balsalazied.Results:The rate of total efficiency rate in balsalazide group was higher than that in SASP group(96% vs 45%,P<0.01).The rate of drug adverse events in balsalazide group was lower than that in SASP group(4% vs 36%,P<0.05).The level of IFN-γmRNA in balsalazide and SASP group were not significantly different from normal control group prior therapy or after the weeks.The level of IL-4 from serum and IL-4mRNA from colon in all the groups were not significantly different(P>0.05).The levels of IL-10 from serum and IL-10mRNA from colon in balsalazide and SASP group were significantly higher than normal control group prior therapy and after 8 weeks,which were higher in balsalazide group than that in SASP group(P<0.05)as well.Conclusions:The setting-up of the OXZ induced colitis mice model was successful;OXZ induced colitis is T helper cell type 2(Th2) inflammation,which is similar to the human UC;balsalazide has good therapeutic efficacy to the OXZ induced colitis mice model;balsalazide is obviously effective and safe in the treatment of mildly to moderately active UC,which surpass SASP.The mechanism may be through inducing the proper doses of IL-10 to educe the dominance of anti-inflammatory reaction and recover the balance of Th1/Th2;the role of IFN-γwas not significant,maybe not necessary in UC;IL-4 participate in the morbility of UC at some stage. Part two: It has been about 40 years since Beta2-glycoproteinⅠ(β2GPⅠ) was discovered,and for about 10 years it has been a focus.But most researches involved were about blood clotting,regulation of fibrolysis system and the relationship with SLE and Antiphospholipid(APS). In the various complications,thromboembolic disease is very severe and become one of the three causes of death in UC.The internal hypercoagulable state is likely to generate microthrombus,which may be one of the most important pathogenesis,and the creation of abnormal immune antibody should not be neglected.The relationship betweenβ2GPⅠand the hypercoagulable state in UC has not been reported yet while the connection ofβ2GPⅠand the function of the blood clotting and the change of platelet in UC patients remain to be explored.By ELISA and flow cytometry(FCM),we detected the level and clinical significance of anti-beta2-glycoproteinⅠantibodies(anti-β2GPⅠ) and combination rate betweenβ2GPⅠand platelet in UC patients.The levels and the positive rates of anti-β2GPⅠof type IgM and IgG were detected in three groups:active UC,inactive UC and normal control groups by using ELISA.Results:1.There were both significant increase in the levels of IgM and IgG anti-β2GPⅠin active UC group and inactive UC group than in normal control group.There was also significant difference on the levels of anti-β2GPⅠof IgG between active UC group and inactive one(P<0. 05).2.On the positive rates of anti-β2GPⅠ,there was statistical significance between active UC group and inactive UC group;they had both remarkable difference with the normal control group,especially the active UC group(P<0.01).Combination rates ofβ2-GPⅠand platelet in the UC patients and in the normal control subjects were detected respectively.Results:There was significant difference in the combination rates ofβ2-GPⅠand platelet in the two UC groups and normal control group(P<0.01);meanwhile,there was also remarkable statistical significance between the active UC group and the remittent group(P<0.01).Conclusion:anti-β2GPⅠmaybe play a role in the pathogenesis of UC and is a possible reason for its hypercoagulabale state;the examination of combination rates betweenβ2GPⅠand platelet in the UC patients may predict their hypercoagulabale state to some degree.Our investigation established valuable theory evidence in both theory and practice for the screening of the efficacious drug,the study of hypercoagulable state and the pathogenesis of UC.