| Part One:The Effect and Mechanism of Inflammatory Factors on Ulcerative Colitis CarcinogenesisObjective:To compare different methods for establishing the mice models induced by azoxymethane(AOM)and dextran sulfate sodium(DSS),and further explore the effect and mechanism of inflammatory factors-NF-κB and TNF-a on ulcerative colitis carcinogenesis on the established mice model.Method and Groups:Firstly,to compare the different establishment methods of mice models,C57BL/6 mice were divided into 4 groups.Group 1 served as AOM+ 3 cycles DSS,which was given AOM 12.5mg/kg by intraperitoneal injection,and one week later 2.5%DSS in drinking water was fed for 5 days.The DSS feeding was repeated every 3 weeks,for 3 cycles in total.Group 2 served as AOM+ 1 cycle DSS,which was given AOM in the same way and received only 1 cycle DSS in total.Group 3 served as normal saline(NS)+ 3 cycles DSS,which was given NS with the same dose by intraperitoneal injection and received 3 cycles DSS.Group 4 served as normal control.Secondly,to explore the effect and mechanism of NF-κB and TNF-α on AOM/DSS induced mice model,C57BL/6 mice were divided into 6 groups.Group 1-5 served as intervention groups,which were given AOM and DSS by the established methods above and received different interventions.Group 1 was given NF-κB antisense oligonucleotide by enema every 2 weeks.Group 2 was given TNF-a monocolonal antibody by intraperitoneal injection every 2 weeks.Group 3 was given NS by enema with the same dose.Group 4 was given NS by intraperitoneal injection with the same dose.Group 5 served as model control,which was given AOM and DSS in the same way without intervention.Group 6 served as normal control.Mice were sacrificed in the end of the 12th week.Macroscopic evaluation of tumor load(TL)as well as pathologic evaluation was carried out to assess carcinogenesis severity.TNF-a and IL-6 levels in colon tissue were determined by ELISA.The transcription activity of NF-κB were assessed by EMSA.The distribution and protein expression level of β-catenin in cell nucleus were determined by immunohistochemistry.The transcription activity of TCF-4 were assessed by EMSA.Results:In comparison of different modeling methods,the tumor formation rate of AOM+3 cycles DSS group(n=11)and AOM+1 cycle DSS group(n=14)was both 100%,but the mortality of mice was lower in AOM+1 cycle DSS group.Therefore,we adopted the method of AOM+1 cycle DSS to establish the UC carcinogenesis mice model.The tumor load(0.92 ± 0.17cm)of NF-κB antisense oligonucleotide group(n=8)was significantly lower than model control group(n=12,TL=2.07 士 0.33cm)(p<0.05).EMSA showed the transcription activity of NF-κB was inhibited by NF-κB antisense oligonucleotide enema.The level of TNF-a and IL-6 measured by ELISA was significantly lower than model control group(p<0.05).The tumor load(1.45 ± 0.27cm)of TNF-a monocolonal antibody group(n=9)was lower than model control group(n=12,TL=2.20 ± 0.34cm),but the difference of two groups didn’t show the statistic significance(p>0.05).The level of TNF-a in TNF-a monocolonal antibody group was verified to be significantly lower than model control group by ELISA(p<0.05),and the same with the level of IL-6(p<0.05).The transcription activity of NF-κB declined in TNF-a monocolonal antibody group.The β-catenin expression in nucleus of colon mucosa cell in NF-κB antisense oligonucleotide group and TNF-α monocolonal antibody group was both significantly lower than model control group(p<0.05),what’s more,the transcription activity of TCF-4 both declined in two groups,which showed the down-regulation of Wnt pathway.Conclusion:AOM intraperitoneal injection followed by 1 cycle DSS was a better method to build the UC carcinogenesis mice model.Inflammatory factors-NF-κB and TNF-a played important roles in ulcerative colitis carcinogenesis mice model induced by AOM/DSS.NF-κB antisense oligonucleotide and TNF-a monocolonal antibody down-regulated Wnt/β-catenin pathway by inhibition of NF-κB activity,and therefore inhibit the occurrence of UC carcinogenesis.Part Two:The Inhibition effect and Mechanism of Probiotics on Ulcerative Colitis CarcinogenesisObjective:To explore the intervention effect and mechanism of probiotics on ulcerative colitis carcinogenesis on AOM/DSS induced UC carcinogenesis mice model.Method and Groups:C57BL/6 mice were divided into 7 groups.Group 1-6 were given AOM 12.5mg/kg by intraperitoneal injection,and one week later 2.5%DSS in drinking water was fed for 5 days to establish UC carcinogenesis model.Group 1 was 5-ASA group,which was given 5-ASA 75mg/kg/d by lavage every day;Group 2 was Saccnaromyces boulardii group,which was given Saccharomyces boulardii 5×107 CFU/d by lavage every day;Group 3 was VSL#3 group,which was given VSL#3 1.5x109 CFU/d by lavage every day;Group 4 was 5-ASA+ Saccharomyces boulardii group,which was given 5-ASA 75mg/kg/d and Saccharomyces boulardii 5×107 CFU/d by lavage every day;Group 5 was 5-ASA+ VSL#3 group,which was given 5-ASA 75mg/kg/d and VSL#3 1.5 x109 CFU/d by lavage every day;Group 6 served as model control group;Group 7 served as normal control.Mice were sacrificed in the end of the 12th week.Macroscopic evaluation of tumor load(TL)as well as pathologic evaluation was carried out to assess carcinogenesis severity.TNF-α,IL-6 and IL-10 levels in colon tissue were determined by ELISA.The transcription activity of NF-κB were assessed by EMSA.The distribution and protein expression level of β-catenin in cell nucleus were determined by immunohistochemistry.The transcription activity of TCF-4 were assessed by EMSA.Results:(1)The tumor load of 5-ASA group(n=20,TL=0.43±0.14cm),Saccharomyces boulardii group(n=19,TL=0.20±0.07cm),VSL#3 group(n=20,TL=0.25±0.07cm),5-ASA+ Saccharomyces boulardii group(n=20,TL=0.53±0.11cm),5-ASA+ VSL#3 group(n=19,TL=0.46±0.11cm)were significantly lower than model control group(n=19,TL=0.97±0.19cm)(p<0.05).(2)The level of TNF-a and IL-6 measured by ELISA in 5-ASA group,Saccharomyces boulardii group,VSL#3 group,5-ASA+Saccharomyces boulardii group,5-ASA+VSL#3 group were significantly lower than model control group(p<0.05),and on the other hand,the level of IL-10 significantly elevated than model control group(p<0.05).(3)EMSA showed that the transcription activity of NF-κB declined in 5-ASA group,Saccharomyces boulardii group,VSL#3 group,5-ASA+Saccharomyces boulardii group,5-ASA+VSL#3 group than model control group.(4)The β-catenin expression in the nucleus of colon mucosa cell in 5-ASA group,Saccharomyces boulardii group,VSL#3 group,5-ASA+Saccharomyces boulardii group,5-ASA+VSL#3 group were significantly lower than model control group(p<0.05).(5)The transcription activity of TCF-4 declined in 5-ASA group,Saccharomyces boulardii group,VSL#3 group,5-ASA Saccharomyces boulardii group,5-ASA+VSL#3 group than model control group,which showed the down-regulation of Wnt pathway.Conclusion:Probiotics and 5-ASA can inhibit the tumor formation of UC carcinogenesis induced by AOM/DSS in mice.To analyze the mechanism,probiotics may down-regulate the pro-inflammatory factor-TNF-α,IL-6 and up-regulate the level of anti-inflammatory factor-IL-10,further inhibit the transcription activity of NF-κB,and finally down-regulate the Wnt/β-catenin pathway by inhibiting the nuclear import ofβ-catenin and suppressing the transcription activity of TCF-4,consequently prevent the process from inflammation to carcinogenesis.Aim:To assess the validity of Asia-Pacific Colorectal Screening(APCS)score score in asymptomatic Chinese population,and to identify other risk factors associated with advanced colorectal neoplasm(ACN).Methods:Asymptomatic subjects who underwent colonoscopy screening between 2012 and 2014 in Beijing were enrolled.APCS scores based on questionnaires were used to stratify subjects into high-,moderate-,and average-risk tiers.Cochran-Armitage test for trend was used to assess the association between ACN and risk tiers.Univariate and multivariate logistic regression was performed with ACN as the outcome,adjusting for APCS score,body mass index,alcohol consumption,self-reported diabetes,and use of non-steroidal anti-inflammatory drugs as independent variables.Results:The average age of enrolled subjects(N=1,010)was 53.5(Standard Deviation 8.4)years.The prevalence of ACN was 4.1%overall;and in the high-,moderate-,and average-risk tiers the prevalence was 8.8%,2.83%and 1.55%,respectively(p<0.001).High-risk tier had 3.3-and 6.1-fold increased risk of ACN as compared to those in the moderate-and average-risk tiers,respectively.In univariate analysis,high-risk tier,obesity,diabetes,and alcohol consumption were associated with ACN.In multivariate analysis,only high-risk tier was an independent predictor of ACN.Conclusion:APCS score can effectively identify a subset of asymptomatic Chinese population at high risk for ACN.Further studies are required to identify other risk factors,and the acceptability of the score to the general population will need to be further examined. |
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