| objective: Colorectal cancer(CRC)is one of the high incidence cancers all over the world,its incidence ranks third and fatality rate is fourth.The diagnosis of colorectal cancer is mostly advanced,which seriously affects the treatment effect and quality of life.Therefore,for the health of human beings,the early screening and treatment of colorectal cancer are emphasized.It is necessary to explore whether there is a change in the relevant biological indicators in predicting the occurrence of colorectal cancer before the stage of colorectal cancer.Aberrant crypt foci(ACF)has been shown to be a precancerous lesion of colorectal cancer in chemical carcinogen 1,2-dimethylhydrazine(DMH)injected rats,precedes adenoma and follows the colorectal adenoma-carcinoma pathway of colorectal cancer.In recent years,it has been found that the change of structure and function of intestinal microbiota is closely related to the damage of human health and the development of disease.Therefore,we explore the colon precancerous lesions of ACF formation in DMH injected rats,whether the structure and metabolism of the gut microbiota have changed,as a biological indicator to predict the screening of colorectal cancer.Methods: Sample collection:After the acclimatized for 1 week,forty-two 4-week-old SD rats weredivided into two groups:the experimental group(n=30)and the control group(n=12).Rats in the experimental group were injected with DMH once a week for 2 consecutive weeks,while the control group intraperitoneally injected with EDTA–normal saline as controls.After modeling every 1 weeks,collected every rat's blood(1ml)and fecal sample(3-4 grains,about 1g),and at the same time,randomly sacrificed4 rats(experimental group,n=3;control group,n=1),until 17 weeks all the remaining rats were killed.2.Experimental detection: ?ACF observation and count: The entire colon was surgically removed and opened longitudinally from the sacrificed rat.Mucosal surface was stained with 0.2% methylene blue.Changes in the number of ACF in the colon was observed under light microscope.Paraffin section HE staining to observe intestinal epithelium change of ACF.?Blood samples were collected after centrifugation(3000rpm,10min),and serum ELISA was used to detect the concentrations of IL-6,IL-8 and TNF-?.? Fecal microbiota analysis: The fecal DNA extraction kit was used to extract DNA from fecal samples,and the 16 S rRNA V4 area of the extracted DNA was amplified by PCR.The amplified and purified products were sequenced by Illumina MiSeq platform to detect the structure and metabolism of fecal microbiota.Detection of microbiota structure includes Alpha diversity and Beta diversity analysis,to detect significant changes of microbiota structure in the experimental group and controlgroup;The metabolism of microbiota was analyzed by using PICRUSt software,and the samples were predicted according to the category of microbial metabolic function in KEGG database.Results: The number of ACF in the experimental group increased gradually with the prolongation of the time after DMH injected.The number of ACF in 1 week after DMH injected is zero(3w,n=4.7±1.5;5w,n=34.3±6.0;7w,n=67.7±11.5;9w,n=68.3±12.7;11w,n=100.7±33.3;13w,n=114.0±24;15w,n=117.0±14.5;17w,n=122.2±25.7).At third week,the number of crypts per ACF was 1 or 2,and the number of crypts per ACF was 3~5 from 5week to 17 week.From the third week after modeling,HE staining of paraffin sections showed that intestinal epithelial cells in ACF were accompanied by hyperplasia or atypical hyperplasia.2.In the ACF period of colorectal precancerous lesions,with the prolongation of time after DMH modeling,the concentration of proinflammatory cytokines IL-6,IL-8 and TNF-? in serum of SD rats increased.In addition to first weeks after modeling,the IL-6,IL-8 and TNF-? concentrations in the experimental group were higher than those in the control group in the remaining time points(P<0.05).3.In colorectal precancerous lesions ACF period,Beta diversity analysis showed that the microbiota structure between the two groups had no significant changes at first week,but at fifth week,ninth week,thirteenth week and seventeenth week had significant changes.With the extension of DMH modeling time,thestructural differences between the two groups are getting bigger and bigger.Metastats statistical method to analysis the two groups' samples at the same time: the Bacteroides abundance increased in the experimental group at ninth week(P=0.018);the Lactobacillus abundance reduced at the seventeenth week(P=0.004).4.In the precancerous lesions of colorectal cancer,PICRUSt software for analysis of changes of the bacterial metabolism function: at first weeks,5 weeks,9 weeks and 13 weeks between the two groups of bacteria metabolism did not change significantly,the carbohydrate metabolism function of the experimental group decreased at seventeenth week after modeling,and the function of amino acid metabolism increased.Conclusion: 1.Carcinogen DMH induced ACF in the early stage of precancerous lesions of CRC in SD rats,which had no significant effect on their health status.Therefore,the structural change of intestinal microbiota and the change of metabolic function can be used as an important biological marker for predicting the precancerous lesions of colorectal cancer.2.In the ACF stage of precancerous lesions,the increase of inflammatory factors IL-6,IL-8 and TNF-?,suggesting that inflammation may be involved in the formation of precancerous lesions of colorectal cancer. |
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