| ObjectiveThe occurrence of colorectal cancer（CRC）is affected by genetic factors and environmental factors.The majority of colorectal cancer patients have mutations in the tumor suppressor gene Apc gene,and the Wnt pathway is activated abnormally.Gut microbiota can affect the progression of CRC through multiple pathways.Clinical studies have shown that the abundance of producing-butyrate bacteria and the concentration of short chain fatty acids（SCFAs）in patients with CRC are significantly lower than those in healthy people.Among them,the reduction of fecal butyrate levels not only serves as a predictor of the risk of CRC,but also reflects the extent and severity of CRC progression.Butyrate acts as a histone deacetylase inhibitor,which stabilizes tumor-associated miRNA clusters and maintains post-transcriptional expression of target genes.Dietary factors are essential to maintain the microecological balance of the intestine.In the high-fat diet,the population of Lactobacillus in the colon decreased and the abundance of C.subtilis XIVa increased.The IL-10 gene-deficient mice with a high-fat diet have a marked increase in the abundance of B.cholerae in the colon.This bacterium can use taurine-containing bile acids to release hydrogen sulfide,which mediates acute inflammation and increases the risk of CRC.The purpose of this study was to explore how producing-butyrate bacteria regulate the dysbiosis of the Gut microbiota,thereby inhibiting the occurrence and mechanism of high-fat diet-induced CRC.Methods1.Animal experimental methods:Thirty 4-week-old Apcmin/+mice that spontaneously developintestinaladenomasweregivendifferenttreatmentsoflive producing-butyrate bacteria+high-fat diet,PBS+high-fat diet,and PBS+normal diet.At the 12th week of the experiment,fresh feces were collected from each mouse.Feces were collected and the mice were killed.The liver,spleen,small intestine and colon were isolated.The distal small intestine and colon were embedded in paraffin and partially frozen in liquid nitrogen.（1）The size,number and tumor burden of intestinal adenoma in each group were counted.HE staining of paraffin sections of intestinal adenomas in each group was used to evaluate pathological type and degree of malignancy;（2）Immunohistochemical staining of Ki-67 and TUNEL method were used to detect the proliferation and apoptosis of tumor cells in each group;（3）The contents of various short-chain fatty acids in the ileocecal contents of mice in each group were detected by gas chromatography,and the expression level of butyric acid recognizing receptors in each group of mice was detected by Realtime-PCR;（4）16S DNA high-throughput sequencing was used to detect the gut microbiota in each group of mice.Immunohistochemical staining was used to detect the signal activation of Wnt signaling pathway related tumors in mice with colon adenomas.2.Cell experiment methods:The human colon cancer cell lines Caco-2 and HCT116were selected as subjects to be treated with producing-butyrate bacteria supernatant solution,sodium butyrate solution and medium to treat cells.The treatment conditions included different time gradients and different concentration gradients.（1）After different treatments,CCK-8 kit was used to detect the proliferation of colon cancer cells.（2）Realtime-PCR was used to detect the expression of TLR/MyD88 in the rectal cancer cell butyric acid recognition receptor and tumor-related signaling molecules.（3）Western blot was used to detect the expression of Wnt-related signaling pathway related molecules in colorectal cancer cells.Results1.Animal experiment results:（1）Compared with mice fed with high-fat diet alone,the number of colonic adenomas in mice treated with butyrate was significantly reduced,the proportion of large adenomas>3 mm in diameter was significantly reduced,and the burden of intestinal tumors was significantly reduced.（2）Compared with mice fed with high-fat diet alone,the percentage of Ki-67 positive staining of intestinal adenoma cells in the intervention group of butyrate-producing bacteria was significantly lower,and the percentage of positive staining of apoptotic cells was significantly increased;（3）The contents of acetic acid,propionic acid,butyric acid and isovaleric acid in the ileocecal intervention group were significantly higher than those in the high-fat diet group alone.Butyric acid recognition receptors were also found in colon tissues.The expression of GPR43 and GPR109A increased significantly,while the expression of GPR41 increased,but there was no statistical significance.（4）Compared with the mice in the high-fat diet alone,the abundance of the pathogenic bacteria Desulfovibrio and Clostridium in the intervention group of butyric acid-producing bacteria was significantly reduced.The abundance of XIVa,IV,and XI in the bacterial subgroups increased significantly,and the abundance of Lactobacillus also increased significantly.（5）The ectopic expression ofβ-catenin in the intestinal adenoma cells was significantly reduced in the butyrate-producing intervention group compared with the mice in the high-fat diet alone group,and the proportion of the downstream target molecule cyclin D1 positive cells was significantly decreased.2.Cell experiment results:（1）Compared with the pure medium,20%of the supernatant of Clostridium butyricum had significant growth inhibitory effect on the two kinds of colorectal cancer cells.The main metabolite of Clostridium butyricum,sodium butyrate,significantly inhibited the growth of colorectal cancer cells.,and showed a significant concentration dependence,but the time dependence is not yet clear;（2）After stimulation with C.butyricum supernatant and sodium butyrate solution,the expression level of GPR43 receptor was increased by 4.5-fold and 4.2-fold,respectively,in the control group,and GPR109A was 2.6-fold and 7.0-fold higher,respectively,in the control group.The expression level of butyric acid recognition receptor GPR41 gene was only 52.9%and 33.5%of the control group after the intervention of C.butyricum and sodium butyrate solution.（3）The expression level of TLR4 gene was 9.1%and 17.6%of the control group after treatment with producing-butyrate bacteria supernatant solution and sodium butyrate solution,and the MyD88 level was 23.3%and 58.6%of the control group,respectively.Compared with the control group,the expression of cyclin D1 and c-Myc in colon cancer cells after treatment was significantly lower,and the ectopic expression ofβ-catenin in the nucleus was significantly decreased.ConclusionProducing-butyrate bacteria can inhibit the malignant transformation of intestinal adenoma induced by a high-fat diet by remodeling intestinal flora and down-regulating Wnt/β-catenin signaling pathway. |
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