Correlation Between Intestinal Flora Imbalance And Experimental Colorectal Cancer In Rats

Objective:An experimental colorectal cancer mice model with intestinal dysbacteriosis was established.After antibiotic,probiotics and fecal bacteria transplantation were given to the model,the effects of antibiotics,probiotics and fecal bacteria transplantation on immune related indexes were observed.At the same time,the changes of intestinal flora in experimental colorectal cancer model of mice were detected,and the relationship among colorectal cancer,intestinal microbiota and immunity was discussed.Methods:C57BL/6 mice were randomly divided into 5 groups: blank control group,DMH group,antibiotic+DMH group antibiotic+DMH+probiotics group and antibiotic+DMH+fecal transplantation group.After a week of routine feeding,mice were allowed to drink ampicillin,neomycin,metronidazole and vancomycin for 2weeks.And then intraperitoneal injection was started with a dose of 30mg/kg1%DMH solution.The 1%DMH solution was injected continuously for 11 weeks,once a week.At the same time,probiotics and healthy fecal bacteria were transplanted into mice by gavage(2 times a week,200 ul each time).After the completion of modeling,mice were sacrificed for 24 weeks,and the general condition and tumor formation(tumor size,HE staining,etc.)of each group were observed.At the end of the experiment,the feces of mice in each group were collected,and the intestinal flora composition of the feces of mice in each group was detected by high-throughput sequencing technology.Immunohistochemical method was used to detect the expression of immune factor-related proteins in each group of mice.Meanwhile,the correlation heatmap was made between intestinal flora of mice feces and environmental factors(immune factors,antibiotics,probiotics,and fecal bacteria transplantation),and the correlation between intestinal flora and environmental factors was observed.Results:1.After the establishment of experimental colorectal cancer model,the antibiotic+DMH group of mice had the highest tumorigenesis rate,followed by DMHgroup,and finally the mice in antibiotic+DMH+fecal transplantation group and antibiotic+DMH+probiotics group.Probiotics and fecal bacteria transplantation could improved the inflammation of intestinal mucosa.2.The results of high-throughput sequencing showed that the Alpha diversity index curve of intestinal microflora in 5 groups showed that the depth of fecal sequencing had basically covered all the bacteria in fecal samples.There was no significant difference in the Alpha diversity of intestinal flora of mice faeces between the 5 groups treated with different methods(P > 0.05).3.The composition and structure of intestinal flora showed that the intestinal flora in fecal samples of five groups of mice was mainly composed of Bacteroidetes,Firmicutes,Actinobacteria,Verrucomicrobia and Proteobacteria,and the bacteria with higher relative abundance were Bacteroidetes,Firmicutes and Actinobacteria.4.The Beta diversity analysis showed that the intestinal flora of the five groups showed a trend of intra-group aggregation and inter-group separation.The results showed that there was significant difference in intestinal flora diversity among the five groups.Among them,there were significant differences in TM7,spirochaetes and cyanobacteria in 5 groups of mice by means of species difference analysis at the level of phylum.At the genus level,there were significant differences among F16,Butyricimonas,Treponema,Paraprevotella and YS2.At the same time,because of the use of probiotics and fecal bacteria transplantation,the abundance of TM7 and F16 increased significantly.5.LEfse multistage species difference discriminant analysis revealed that spirochaetes、TM7、paraprevotella and F16 were all different species of mice in the blank control group.Cyanobacteria、prevotellaceae、YS2 and Butyricimonas were all differential species of DMH group mice.6.Immunohistochemical results showed that the expression levels of CD8,PD-1and Foxp3 in the antibiotic+DMH+probiotics group were higher than those in the antibiotic+DMH+group.The expression level of PD-1 in the antibiotic+DMH+ fecal bacteria transplantation group was higher than that in the antibiotic +DMH group.Theexpression level of PD-L1 in the antibiotic +DMH group was higher than that in the antibiotic+DMH+probiotics group.These suggest that intestinal flora may alleviate intestinal inflammation through the high expression of CD8,PD-1 and Foxp3,and the low expression of PD-L1.7.The correlation between intestinal flora and environmental factors(Heatmap)showed that CD8 was negatively correlated with Elusimicrobia.PD-1,Foxp3 were positively correlated with spirochaetes,cyanobacteria and TM7.Vancomycin,neomycin,metronidazole,ampicillin were negatively correlated with spirochaetes,cyanobacteria,TM7,Prevotella,and Bacteroidaceae.Probiotics were positively correlated with Erysipelotrichaceae and Coriobacteriaceae,and negatively correlated with Prevotella and Bacteroidia.There was a negative correlation between the transplantation of healthy fecal bacteria and the genus Coriobacteriaceae.There was a significant negative correlation between body weight and Actinobacteria,and a significant positive correlation between body weight and Bacteroidetes.Conclusions:1.There was a certain correlation between the occurrence and development of experimental colorectal cancer and intestinal flora.2.Probiotics and fecal bacteria transplantation had significant alleviating effect on experimental colorectal cancer in rats with intestinal flora imbalance caused by antibiotics.Meanwhile,probiotics and fecal transplantation may alleviate intestinal inflammatory response through CD8-PD-1-PD-L1-Foxp3,thereby reducing the occurrence of tumors.3.External factors such as antibiotics,probiotics and fecal bacteria transplantation,and self-factors such as CD8,PD-1,PD-L1,Foxp3 and body weight had certain correlation with intestinal flora.