Effect And Mechanism Of A Flagellum Protein Derivative CZLC331on Ulcerative Colitis

ObjectiveThe incidence of UC trends to raise year by year and it thrights the health ofhuman being. UC lacks effective clinical treatment and has been recognized asone of refractory diseases by WHO, as a result of its unclear pathogenesis andetiology. The further identifing of pathogenesis and developing of a newmedicament for curing of UC have been the emphasis and difficulty of UCresearch. CZLC331is a kind of bacteria flagellum protein derivatives and theactivator of TLR5. It can activate the signal pathway of TLR5-NF-κB. The studyfound that CZLC331can protect the mice gastrointestinal tract by inhibitingapoptosis, regulating the expression level of TLRs and other mechanisms. Atpresent, it’s believed that the cause of UC related with apoptosis imbalance andoxidative stress injury of T cells. Consequently, we proposed a hypothesis thatwhether CZLC331can prevent and cure UC by regulating the natural immunityof TLRs and inhibiting T cells apoptosis. Based on the establishment of miceulcerative colitis model. this research observed the effect of CZLC331on UC,investigated the possible mechanism of therapeutical effect and presentedevidences of pathogenesis and etiology of UC. All the effort of this research laya solid foundation for developing a new drug of ulcerative colitis.MethodsThe study established UC mice model by TNBS enema, replaced traditionalsilicon tube for the stomach needle, and refined the correlation technique and dosage of TNBS enema. Consequently, it can improve efficiency and stability ofestablishing rice model. Analysis of therapeutic effect of CZLC331: macroscopicobservation for general status, observation for the changes of mucosa andmuscularis from colon by photograph, observation for the pathological changesof colon tissue staining under microscope, observation for mice depressionindex by forced swimming test, tail suspension experiment and sugar waterconsumption experiment; Research for mechanisms of CZLC331: RNAextraction from mice colon, determination of TLRs expression in mice colon byRT-PCR, determination of IL-4、IL-6、IL-8、IL-10、TNF-α in mice plasma byELISA method, determination of NF-κB expression in mice colon byWestern-blot.ResultsAfter establishing UC model in mice successfully, UC mice showed someappearances such as: eated less, reduced physical activities and weight, furlacked luster, diarrhea, had blood in stool and so on. the mice colon hadhyperemia, edema, inflammation, ulceration. Edema, cell disorder, inflammatorycells infiltration and goblet cells deletion were detected by pathologicalexamination in UC mice colon. During the process of instablish mice model,traditional silicon tube and anesthetization were replaced for the stomachneedle and anesthesia machine respectively. Meanwhile, correlation techniquewere refined and the dosage of TNBS enema were identified. Consequently, itcan improve efficiency and stability of establishing rice model.Bacterial flagellum protein derivatives CZLC331had significant therapeuticeffect of mice UC. The general condition of mice improved obviously. Ulcer,edema and erosion reduced in mice colon. Pathological changes reduced: cellsin order, small amount of goblet cells and lymphocytes lack. With intraperitonealinjection of CZLC331, mice depression index decreased, which included forcedswimming experiment, the tail suspension experiment and sugar water preferences experiment, P<0.05: In treatment group the fixed time of swimmingwas64.7±10.35s, much lower than the model group (86.7±15.96s, P <0.01),Significantly higher than the control group(21±5.25s, P <0.01). In treatmentgroup mice the tail suspension experiment within the fixed time was62.5±13.92s, Significantly lower than the model group(111.3±41.11s, P <0.01),Significantly higher than the control group(16±8.34s, P <0.01). In treatmentgroup mice the sugar water percentage preference was66.5±7.29%,Significantly higher than the model group(34.5±4.64%, P <0.01), Significantlylower than those in the control group(77.2±7.68，P <0.01).RT-PCR showed that CZLC331can cut the TLRs expression in mice colon.model group and the treatment group was statistically significant differences inTLRS; CZLC331increased the expression of inflammation factors、TNF-α inplasma and suppression factors of inflammation were relief, but IL-4had noobvious changes; IL-4level in serum, treatment group(54.29±5.83ng/ml),control group(53.41±7.28ng/ml), model group (53.71±7.72ng/ml)had noobvious difference(P>0.05); IL-10level in serum, model group(59.36±14.46ng/ml) obviously decreased than control group (91.48±24.38ng/ml,P <0.05),treatment group(54.29±5.83ng/ml)had no obvious difference(P>0.05).IL-6level in serum, model group (47.10±15.72ng/ml) had a significantly higherthan treatment group(48.34±8.20ng/ml, P <0.05)and controlgroup(41.48±6.33ng/ml, P <0.05). IL-8level in serum,the group of12h, modelgroup(42.85±6.39ng/ml)had a significantly higher than control group(36.37±3.52ng/ml, P <0.05). TNF-α level in serum, treatment group(192.50±63.85ng/ml)had a significantly higher than control group(173.86±29.26ng/ml, P <0.05),model group(140.11±12.70ng/ml) had a significant reduction than controlgroup(P <0.05).Western-blot showed that CZLC331can also cut the NF-κBexpression in mice colon. ConclusionsThe study established UC mice model by TNBS enema; with the stomachneedle insteading of silicon tube. Meanwhile, it conducted a serial of techniquedevelopment. The refined method of establishing UC mice model. consequently,improved the efficiency and stability of establishing rice model. CZLC331had asignificant therapeutic effect on UC mice; There were obvious developments inthe general condition of mice,colonic mucosa damage,pathological changesand depression index. UC have relationship with TLRs, TLR5-NF-κB-TNFpathways, IL-4,10and other proinflammatory factor were all involved in theformation of UC. CZLC331therapeutic effect on UC mice might be closelyrelated to TLRs inflammation factors and TLR-NF-κB-TNF-α pathways.