| BACKGROUND The role of Aspirin in tumor prevention and adjuvant therapy has been paid more and more attention.Many reports have shown that it can be used to reduce the progression and recurrence of colorectal cancer.However,the mechanism of anti-tumor is still controversial.A large number of studies have confirmed that intestinal microecological imbalance is an important risk factor for colorectal cancer,intestinal flora can affect the body's immune cells and cytokines.Based on the above study,we investigate whether aspirin prevents the occurrence and development of colorectal cancer by interfering with changes in intestinal flora and immune cells and cytokines in mice.OBJECTIVE To investigate the immunological mechanism of aspirin in the prevention of colorectal cancer in mice modelMETHOD 1 Aspirin intervening colorectal adenoma-cancer mouse model(normal C57 BL/6N mouse model,APCMin/+ mice,C57 BL/6N colorectal cancer mouse model);2 Pyrophosphate sequencing was used to detect the changes of intestinal flora in mice with colorectal adenoma-cancerous mouse model?different doses of aspirin intervention,monitoring at different time points?.3 Multicolored flow cytometry was used to detect the changes of various immune cells in the mouse model of colorectal adenoma –cancer.4 Cytokine liquid chromatography was used to detect the changes of various cytokines in the model of mice with colorectal adenoma-carcinoma sequence.5 Statistical methods of i biological information and Spearman,Benjamini-Hochberg was used for intestinal flora analysisPart One Study on the Changes and Immune Mechanism of Aspirin Intervention in Intestinal Flora of C57 BL /6N MiceRESULT 1 Intestinal flora changes The effect of ASA on the intestinal microflora structure was observed by ASA intervention.First,ASA was able to affect the intestinal microflora structure.This change was dose-dependent including low?200 mg/kg?,medium?400 mg/kg?,high?600 mg/kg?.The intestinal flora structure of the group was different from that of the normal mice.Secondly,the intestinal flora structure under the influence of ASA was changed dynamically.With the prolongation of ASA feeding time,in treatment group the structure of the flora was significantly different from that of the control group;there was a significant time inflexion;thirdly,through the comparison of the flora structure between different groups and different time points,at the level of phylum,composition of Deferribacteres and Actinobacteria was obviously increasing in high dose of ASA group,at the level of geuns,different flora among four groups after ASA treatment was Mucispirillum,Bifidobacterium,Lachnospiraceae.At the time point the main different flora were Bacteroides,Lactococcus,Faecalibacterium,A llobaculum,Alistipes,Mucispirillum and Ruminococcus2 which had higher abundance in high-dose ASA group.It meaned that ASA did change the structure of intestinal flora of normal C57 mice.2 Immune cell changes The proportion of CD11c + MHC?+ DC cells in low dose of aspirin group was significantly lower than that in control group,but there was no significant difference in B cells among treatment groups.The percentage of CD4 + T-bet + Th1 cells in the low dose of aspirin group was significantly higher than that in control group?P <0.05?.The percentage of CD4 + IL4 + Th2 cells in the medium dose of aspirin group was significantly higher than that in the high dose aspirin group.The percentage of CD4 + IL17 + Th17 cells in the low dose of aspirin group was significantly higher than that in high dose of aspirin group,and the percentage of CD4 + IL17 + T cells in high dose of aspirin group was significantly lower.The proportion of CD4 + Foxp3 + Treg cells in the low dose of aspirin group was significantly higher than that in medium and high-dose of aspirin group,and the ratio of CD4 + Foxp3 + T cells in the medium dose of aspirin group and high-dose aspirin group was significantly lower.There was no significant difference in Tfh cells among treatment groups.It indicated that aspirin could influence immune cells such as CD11c+MHC?+DC?CD4+T-bet+Th1?CD4+IL-4+Th2?CD4+IL-17+Th17?CD4+Foxp3+Treg in model of normal C57 mice.3 Cytokine changes At the 6th week,the expression level of GM-CSF in high dose of aspirin group was significantly higher than that in low dose ones.Also,at the 6th weeks,the expression level of IL-6 in low dose of aspirin group was significantly higher than that at 0th week.At the 19 th week the expression level of IL-17 A in medium dose of aspirin group was significantly higher than that in high dose of aspirin group.At the 12 th week,the expression level of IL-1?in medium dose of ASA group was significantly higher than that at 0th week;at the same time the level of TNF-?,IL-6,IL-10,IL-15 were significantly higher than that in control group low and high dose of aspirin group,however,the expression level of IL-23 in control group was significantly higher than that in low and medium dose of aspirin group at the 12 th week.At the 27 th week the expression level of IL-2 in control group was higher than that in medium dose of aspirin group and IL-17 A in middle dose ones was significantly higher than that in other three groups,at the same time IL-23 in medium dose ones was higher than that at the 0th week.It indicated that different time and dose could influence the expression level of different cytokines and also meaned that different dose of ASA could intervene the expression of GM-CSF?IL-2?IL-6?IL-15?IL-17 A and IL-23 in different time.Part Two Study on the Changes and Immune Mechanism of Aspirin Intervention in Intestinal Flora of APCMin/+MiceRESULT 1 Intestinal flora changes Changes of intestinal flora in APCMin/+mice treated with ASA were these.Firstly,the rate of progression of whole disease in the experimental group was significantly slowed down,and the rate of tumor formation and size of adenoma were significantly decreased compared with the control group.So aspirin had obvious antitumor effect.Secondly,the effect of ASA on intestinal flora showed that structure and diversity of the flora in experimental group were significantly higher than those of the control group.At the key time point of the pathological changes at 8th and 16 th week,the diversity of intestinal flora structure between two groups were different from each other.Changes in flora were not only consistent with the progression of the disease itself,but also significantly correlated with the dynamic changes of interventional drug and time.Thirdly,analysis of changes in composition of the flora showed that Bacteroidetes were significantly different from the control group at the level of the phylum,which gradually increased with the development of the disease.,at the level of the genus,the harmful bacteria were significantly increased in the control group such as Allobaculum,Porphyromonadaceae,while abundance of Lactobacillus and Alistipes?butyrate producing bacteria?in the experimental group after ASA intervention were significantly increased.It indicate that ASA could influence the intestinal flora of APCMin/+mice.2 Immune cell changes In the APCMin/+mouse model of aspirin intervention,CD11c + MHC? + DC cells decreased slightly in the aspirin intervention group,while the CD80 + CD86 + DC cells were significantly increased.The CD19 + GL-7 + B cells in the ASH group were significantly CD4 + IL4 + Threga,CD4 + Foxp3 + Treg and CD4 + CXCR5 + Tfh cells in the aspirin intervention group were significantly different from the control group?P <0.05?.CD4 + + T cells were significantly increased;suggesting that aspirin affects the number and function of Th17 cells in the APCMin/+mouse model 3 Cytokine changes Aspirin intervened in the APCMin/+mouse model,the expression level of TNF-? was significantly lower at 16 th week in the aspirin intervention group than 0th week.Part Three Study on the Changes and Immune Mechanism of Aspirin Intervention in Intestinal Flora of C57 BL/6N Colorectal Cancer MiceRESULT 1 Intestinal flora changes Through ASA intervention in CAC mice model we found that: First,ASA especially at high dose?400mg?has a significant anti-tumor effect to colitis-associated cancer induced by AOM and DSS : the numbers of tumor and inflammatory symptoms were Significantly reduced;Secondly,AOM and DSS significantly reduced the number of OTU,made structure of intestinal flora toward the direction of disease,and ASA could inhibit the effect and stabilize structure of intestinal flora to maintain its diversity,even slow down the changes of flora.Thirdly,according to analysis of the results of flora composition,at the level of phylum,CAC group was enriched with Verrucomicrobia and Proteobacteria;while after Aspirin intervention it was enriched with Firmicute.At the level of genus,CAC group was enriched with Akkermansia,while it was enriched with Lactobacillus after ASA intervention,and they were both lack of Alloprevotella genus.2 Immune cell changes In the C57 BL/6N colorectal cancer mice model of aspirin intervention,the proportion of CD11c + MHC? + DC cells in aspirin high dose intervention group was significantly higher,suggesting that aspirin affected CD11c + MHC ? in different mouse models + The number and function of DC.The expression of CD19 + GL-7 + T cells was significantly increased in aspirin standard dose group and CD19 + GL-7 + T cells in Aspirin high dose intervention group.Suggesting that aspirin affects the number and function of CD19 + GL-7 + T in different mouse models.There was no significant difference in CD4 + T-bet + Th1,CD4 + IL4 + Th2 and CD4 + CXCR5 + Tfh cells in the aspirin-treated group.However,the aspirin intervention group,CD4 + IL17 + Th17 cells in the aspirin-treated group were significantly higher than those in the untreated group?P <0.05?.The dose of aspirin was significantly higher than that of the untreated group CD4 + CD25 + T cells were significantly increased in the C57 BL/6N colorectal cancer mice model,suggesting that aspirin affects the number and function of CD4 + IL17 + Th17 and CD4 + CD25 + T in C57 BL/6N colorectal cancer mouse model.3 Cytokine changes In the C57 BL/6N colorectal cancer mice model treated with aspirin,the expression level of GM-CSF in the normal control group was significantly higher than that in the untreated group at 16 th weeks.In the normal control group,compared with 0 week,Week GM-CSF expression levels were significantly reduced.At week 0,the expression level of IL-2 was significantly lower in the aspirin untreated group,aspirin standard dose group and aspirin high dose group compared with the normal control group.At 0 week,the expression level of IL-6 in the aspirin standard dose group was significantly The expression level of IL-6 in the high dose group was significantly higher than that in the normal control group and the aspirin standard dose group at 4 weeks.At the 16 th weeks,the normal control group were significantly higher than those in the aspirin group.The expression level of IL-6 was significantly higher in aspirin than the intervention group,standard dose of aspirin group and high dose of aspirin group,suggesting that aspirin affects the expression of IL-6 in different time periods.At the 4th week,the expression level of IL-10 in standard dose of aspirin group was significantly higher than that in normal control group,suggesting that aspirin affects the expression of IL-10 in different time periods in different models.The expression level of IL-15 in aspirin standard dose group was significantly higher than that in normal control group and aspirin group at 0 weeks.In the normal control group,the expression level of IL-15 at week 4 was significantly higher than that of 0 weeks,suggesting that aspirin Affecting the expression of IL-15 in different time periods in different models.At 4 weeks,the expression level of IL-17 A in aspirin standard dose group was significantly higher than that in normal control group,suggesting that aspirin affected the expression of IL-17 A in different time periods in different models.At 16 weeks,the expression level of IL-23 in the normal control group was significantly higher than that in the aspirin untreated group and the aspirin standard dose group,suggesting that aspirin affects the expression of IL-23 in different time periods in different models.At 4 weeks,the expression level of IL-21 in aspirin high dose group was significantly higher than that in normal control group,suggesting that different doses of aspirin could affect the expression of IL-21.CONCLUSION: 1.In different dose of aspirin at various time-points there are different rules of changes of intestinal flora in three mice modle,Aspirin is to prevent the occurrence and development of colorectal cancer by affecting the changes of intestinal flora.2.In different dose of aspirin there are different rules of changes of immune cells in peripheral immune organ in three mice modle,Aspirin can prevent the occurrence and development of colorectal cancer by regulating cellular immunity.3.In different dose of aspirin there are different rules of changes of serum cytokine levels in three mice modle,Aspirin prevents the development of colorectal cancer by regulating the dynamic changes of cytokines. |
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