| Background and objective:Inflammatory bowel disease(IBD)is a kind of nonspecific chronic inflammatory disease in the intestinal tissue,including ulcerative colitis(UC)and Crohn’s disease(CD).To date,but its etiology is still unclear.Most scholars believe that IBD is mainly caused by environmental factors,genetic factors,infection factors,and immune dysfunction.Among them,the immune dysfunction plays a crucial role in the occurrence,development and sustainable development of intestinal tissue inflammation in IBD.It is clear that Toll like receptor 4(TLR4)is a important kind of pattern recognition receptors(PRR),which could recognize danger signals of pathogenic microorganism infection,inflammation or injury.TLR4 could mediate the innate immune response and adaptive immune response,so it is especially concerned with the role in the intestinal inflammation of IBD,but the mechanism and regulation mode remains to be further study.High mobility group box 1(HMGB1)is a kind of important and late inflammatory mediators involved in the occurrence,development and outcome of many chronic inflammatory diseases and autoimmune disease.It is also one of the endogenous ligands for TLR4.HMGB1 plays an important role in the inflammatory and immune response mediated by the abnormal TLR4 signaling pathway.It is reported that HMGB1 may play a role in the occurrence and development of IBD,but what is the role in IBD? Can regulation of HMGB1 improve the intestinal chronic inflammation of IBD? At present,there are few reports at home and abroad.To illustrate this problem,we constructed dextran sulfate sodium(DSS)-induced colitis model in mice to study the relationship of HMGB1/TLR4 signaling pathway and IBD first,then further study the role and its possible mechanism of HMGB1 in the intestinal inflammatory reaction of IBD using a HMGB1 inhibitor-ethyl pyruvate(EP).Our results provide an important theoretical and experimental basis for the further elucidation of the role of HMGB1/TLR4 signaling pathway in IBD and provide a novel therapeutic target for control and prevention of IBD.Method:1.Experimental groups:Thirty-six clean grade BALB/C mice,6~ 8 weeks old,half male and half female,were randomly divided into three groups(n=12 in each)as follows:(1)Normal group;(2)DSS group;(3)DSS+EP group.2.Establishment of IBD model:(1)Establishment of DSS model: BALB/C mice in DSS group and DSS+EP group were free drinking 5%DSS adlibium for 7 days.Acute colitis models were successfully established according to anyone of the symptoms:loose stools,positive occult blood and the bloody stools.Mice in normal group only were free drinking clean water for 7 days.After 7 days,all mice were sacrificed.(2)Administration of EP intervention: On the third day,the mice in DSS+EP group were received intra-peritoneal injection of EP(40mg/kg,dissolved in Ringer’s solution)every day for 5 days,0.4 ml each time.Mice in normal group and DSS group were only received intra-peritoneal injection of Ringer’s solution,every day for 5 days,0.4 ml each time.3.Observation and detection of every index:(1)To observe the body weight and disease activity index(DAI)of every group everyday;(2)To observe general morphology of colon in mice,and change of the pathohistology through HE stain;(3)To detect protein expression of Foxp3、HMGB1、TLR4、MyD88 and NF-κB p65 through the immunohistochemistry stain.Result:(1)Change of weight and DAI scores of mice(1)In the normal group,the weights of mice were increased continuously;The weights of mice in DSS group were gradually decreased and on the 7th day were to the lowest;The weights of mice in DSS+EP group were gradually decreased and on the 7th day were to the lowest,then rose in the following days.Weight percentages of the DSS group and DSS+EP group were higher than that of the normal group on the 5th days(P<0.01);In addition,weight percentages of the DSS+EP group was significantly higher than that of DSS group on the 5th day(P<0.01),but the weight percentages of the DSS+EP group was significantly lower than that of DSS group on the 7th day(P<0.01).(2)DAI scores of mice in each group had significant discrepancy on the 5th,the7th day of experiment.DAI score of mice in the normal group were significant lower than that of the DSS group and DSS+EP group(P<0.01);On the 5th or 7th day,DAI score of mice in the DSS+EP group was significant lower than that of the DSS group(P<0.01).(2)Pathological change of colon mucosa(1)Magnum shape of the colon mucosa: There were no hyperemia,bleeding,edema,erosion and ulcer in the colon mucosa of mice in the normal group;The colon mucosa of mice in the DSS group could be seen obvious hyperemia,edema,erosion,serious ulcers.Compared with the DSS group,the serious degree of mice in the DSS+EP group was slightly reduced.(2)Intestinal pathological changes were observed by HE stain in each group: The degrees of colon inflammation,the pathological depth and the crypt destruction of mice in DSS group and DSS+EP group were higher than those in the normal group(P<0.01);But the degrees of the colon inflammation,the pathological depth and the crypt destruction of mice in the DS+EP group were lower than those of the DSS group(P<0.01).(3)Foxp3 expression in colon mucosaThe expression of Foxp3 protein in the DSS group and DSS+EP group was lower than that in the normal group(P<0.01).The expression of NF-κB p65 protein in the colon mucosa of DSS+EP group was higher than that in the DSS group(P<0.01).(4)HMGB1expression in colon mucosaThe expression of HMGB1 protein in the DSS group and DSS+EP group was higher than that in the normal group(P<0.01).The expression of HMGB1 protein in the colon mucosa of DSS+EP group was lower than that in the DSS group(P<0.01).(5)TLR4 expression in colon mucosaThe expression of TLR4 protein in the DSS group and DSS+EP group was higher than that in the normal group(P<0.01).The expression of TLR4 protein in the colon mucosa of DSS+EP group was lower than that in the DSS group(P<0.01).(6)MyD88 expression in colon mucosaThe expression of MyD88 protein in the DSS group and DSS+EP group was higher than that in the normal group(P<0.01).The expression of MyD88 protein in the colon mucosa of DSS+EP group was lower than that in the DSS group(P<0.01).(7)NF-κB p65 expression in the colon mucosaThe expression of NF-κB p65 protein in the DSS group and DSS+EP group was higher than that in the normal group(P<0.01).The expression of NF-κB p65 protein in the colon mucosa of DSS+EP group was lower than that in the DSS group(P<0.01).Conclusion:1.In my experiment,we successfully established the DSS-induced experimental colitis model in mice.The intestinal inflammation was alleviated after the intervention of EP,suggesting that EP can alleviate the inflammatory response and has a certain therapeutic effect on IBD.2.After EP intervention,the protein expression of Foxp3 in colon mucosa of the experimental colitis mice was increased,suggesting that EP may participate in the regulation of Foxp3+ cell response.3.After EP intervention,the protein expression of HMGB1,TLR4,MyD88,and NF-κB p65 in colon mucosa of the experimental colitis mice were decreased,suggesting that EP alleviated the intestinal inflammation may through inhibiting HMGB1/TLR4/MyD88/NF-κB p65 signal pathway. |
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