Effects Of Immunonutrition And Intestinal Microbiota On The Treatment Of Colorectal Cancer

Part.l Effects of immunonutritional intervention on chemotherapy and gut microbiota deviation in mice colon cancer modelBackground:The most common side effect of radiochemotherapy for colorectal cancer is gastrointestinal complications,which are mainly manifested as gastrointestinal motility and intestinal flora disorder,together with gut barrier injure.Immune nutrients play an important role in nutritional support,immune regulation,intestinal flora and intestinal mucosal barrier function maintenance.Many studies have shown that immune nutrients combined with chemotherapy could contribute to the treatment of cancer.Immune nutrients supplementation can improve intestinal flora and gut barrier function in inflammatory bowel disease and partial hepatectomy animals.However,few studies have reported such results in chemotherapy-induced intestinal barrier injury,and its role in the treatment of colorectal cancer needs to be further studied.Objective:To explore the effect of different immune nutrients supplementation on the chemotherapeutic efficacy and the intestinal flora structure of tumor-bearing mice by feeding chemotherapeutic mouse models with glutamine,n-3 polyunsaturated fatty acids,and prebiotics,which would lay a foundation for the application of immune nutrients in the clinical treatment of colorectal cancer.Methods:Thirty female BALB/c mice were subcutaneously injected with CT26 mouse colon cancer cells to construct a tumor-bearing mouse model.The tumor-bearing mice were randomly divided into five groups:Group 1 no chemotherapy intervention(Control,CON,n=6);Group 2 chemotherapy + saline group(Normal saline,NS,n=6);Group 3 chemotherapy+ glutamine intervention group(Glutamine,GLN,n=6);Group 4 chemotherapy+ n-3 polyunsaturated fatty acid intervention group(PUFA,n=6);Group 5 chemotherapy+ prebiotics intervention group(Prebiotics,PRE,n=6).Before(Baseline,BAS)and 2 weeks after intervention in each group,fresh stools were taken for DNA extraction and intestinal fecal microbiota was detected by 16S rRNA pyrosequencing.The tumor tissues were taken and tumor volume of mice was compared between the groups.Results:The levels of Firmicutes and Lactobacillus in feces of NS group mice receiving chemotherapy were significantly higher than those at baseline(BAS)and CON group,while Bacteroidetes and Bacteroides were significantly lower than baseline(BAS)and CON group mice(P<0.05).The level of Bacteroidetes in feces of PUFA group was significantly higher than that of NS group,while Firmicutes and Actinobacteria were significantly lower than those of NS group(P<0.05).There was no significant difference in fecal microbiota between the GLN group or PRE group and the NS group(P>0.05).The tumor volume of mice in NS group,GLN group,PUFA group,and PRE group receiving chemotherapy intervention was significantly smaller than that of CON group without chemotherapy intervention(P<0.05).In mice receiving immune nutrients supplementation,tumor volume of mice in PUFA group was significantly smaller than that of NS group(P<0.05)and there was no significant difference in tumor volume between GLN group or PRE group and NS group(P>0.05).Western blot results showed that chemotherapy or chemotherapy combined with immunonutrition significantly inhibited the expression of MMP2,COX2,and CyclinD1 in tumor tissues(P<0.05).Conclusion:5-Fu chemotherapy causes changes in intestinal flora of mice,reduced Bacteroidetes levels together with elevated Firmicutes levels.The n-3PUFAs supplementation can repair the imbalance of intestinal flora caused by chemotherapy,make the content of Bacteroides increased and the content of Streptomyces and Actinobacteria reduced,and increase the effect of chemotherapy.Part.2 Effect of changes in intestinal microenvironment on intestinal function in postoperative patients with colorectal cancerBackground:The patients often suffer from gastrointestinal function related complications after gastrointestinal surgery,and it is often manifested as diarrhea or paralysis in patients with colorectal cancer radical operation.Many studies have shown that these complications are related to surgical stress,perioperative bowel preparation,postoperative fasting,and postoperative intestinal dysbacteriosis.However,there are few studies on the relationship between intestinal microflora structure changes and surgical complications after colorectal cancer surgery.The mechanism in the recovery of intestinal function after surgery is worthy to further study.Objective:To analyze the differences of fecal microbiota structure before and after surgery in patients with colorectal cancer,and to explore the role of intestinal microbiota changes in the recovery of intestinal function after colorectal cancer radical operation.Methods:The patients receieving elective surgery for colorectal cancer from Peking Union Medical College Hospital were assigned to normal postoperative intestinal function group(normal group)and postoperative diarrhea/enteric paralysis group(diarrhea group)according to the postoperative intestinal function recovery of patients.DNA was collected from fresh feces before operation and 5-7 days after operation,and fecal intestinal flora was detected by 16S rRNA pyrosequencing.Fasting blood was collected on the first postoperative day(POD1)and third postoperative day(POD3).The levels of serum inflammatory cytokines(TNF-a,IL-6,IL-8)were detected by ELISA.The differences of intestinal microflora structure and postoperative plasma inflammatory factors between the normal group and the diarrhea group before and after operation were compared,and the relationship between intestinal microbiota structure changes and postoperative intestinal function was analyzed.Results:A total of 38 patients undergoing elective colorectal cancer surgery were included,with 27 in the normal group and 11 in the diarrhea group.There was no significant difference in the abundance of fecal dominant bacteria between the two groups before surgery,including Finnicutes(67.05%vs 58.14%,P>0.05),Bacteroidetes(21.01%vs 11.75%,P>0.05),Proteobacteria(5.36%vs 18.39%,P>0.05)and Actinobacteria(5.65%vs 10.03%,P>0.05).Postoperative fecal microbiota testing results showed that the levels of Bacteroidetes(34.20%vs 14.47%,P<0.05),Bacteroides(21.40%VS 8.68%,P<0.05)and Parabacteroides(21.40%VS 8.68%,P<0.05)in the normal group were significantly higher than that of the diarrhea group,while the level of Proteobacteria(19.19%VS 43.14%,P<0.05)was significantly lower than that of diarrhea group.There were no significant differences between the two groups in the abundance of Firmicutes(41.88%VS 39.52%,P>0.05)and Fusobacteria(2.03%vs 0.88%,P>0.05).The levels of inflammatory cytokines(IL-6)on POD1 in the diarrhea group were significantly higher than those in the normal group,but there was no significant difference between the two groups on POD3.Conclusion:The post-surgical significant difference in the fecal microbiota structure between the normal group and the diarrhea group was not seen pre-surgically.The ratio of Bacteroidetes and Bacteroides in the fecal flora of patients with colorectal cancer was decreased,and the proportion of Proteobacteria increased may be related to postoperative complications such as diarrhea/intestinal paralysis.The early detection of serum inflammatory factors can play an early warning role in the complications of postoperative diarrhea.The proportion of Bacteroidetes and Bacteroides in the fecal flora of patients with colorectal cancer was decreased and the proportion of Proteobacteria was increased,which may be related to postoperative complications such as diarrhea/intestinal paralysis.The early detection of serum inflammatory factors after surgery could predict the occurrence of postoperative diarrhea.Part.3 Study on changes of intestinal barrier and intestinal microenvironment in patients receiving colorectal surgery under the concept of enhanced recovery after surgeryBackground:In the past decades,the enhanced recovery after surgery(ERAS)has been rapidly promoted and applied in major developed countries all over the world,especially in colorectal cancer.Compared with traditional surgery procedures,there are many optimized measures in colorectal surgery under ERAS mode,such as simplifying preoperative bowel preparation,early postoperative recovery diet,reducing traumatic stress of intestinal surgery,early recovery of intestinal function,and rapid recovery.However,the existing evidences supporting the ERAS concept are derived from clinical observational studies.Few studies reported the ERAS model to protect intestinal barrier function and to maintain the intestinal microenvironment in patients undergoing colorectal surgery.Therefore,it is necessary to carry out relevant research to provide basic theoretical support for the promotion of ERAS.Objective:To compare the changes of intestinal flora in patients undergoing elective colorectal surgery between conventional treatment model and ERAS,and to explore the mechanism of postoperative accelerated recovery and improvement of intestinal function in patients with ERAS.Methods:Patients receieving elective radical surgery for colorectal cancer at the Department of General Surgery of Peking Union Medical College Hospital were enrolled.The subjects were assigned into conventional treatment group(control group)and enhanced recovery after surgery group(ERAS group)according to different preoperative bowel preparation methods and postoperative nutritional support measures.DNA was collected from fresh feces before operation and 5-7 days after surgery,and fecal intestinal flora was detected by 16S rRNA pyrosequencing.Fasting blood was collected on the first postoperative day(POD1)and third postoperative day(POD3).The levels of serum inflammatory cytokines(TNF-?,IL-6,IL-8)were detected by ELISA.The differences of intestinal microflora structure and postoperative plasma inflammatory factors between the normal group and the ERAS group before and after operation were compared.Results:A total of 32 patients undergoing elective colorectal cancer surgery were included,with 22 in the control group and 10 in the ERAS group.There was no significant difference in the abundance of fecal dominant bacteria between the two groups before surgery,including Firmicutes(64.21%vs 64.11%,P>0.05),Bacteroidetes(20.14%vs 18.14%,P>0.05),Proteobacteria(8.23%VS 9.26%,P>0.05)and Actinobacteria(6.71%VS 7.67%,P>0.05).Postoperative fecal microbiota test results showed that there were no significant differences in the abundance of Firmicutes(44.23%vs 46.37%,P>0.05),Proteobacteria(26.98%vs 16.52%,P>0.05),Bacteroidetes(25.11%VS 30.77%,P>0.05),Fusobacteria(1.84%vs 1.37%,P>0.05)and Actinobacteria(1.25%VS 1.22%,P>0.05).However,the ratio of Bacteroidetes and Proteobacteria in the control was significantly lower than that of ERAS group(0.93 VS 1.88,P<0.05).The levels of inflammatory cytokines(TNF-a?IL-6?IL8)on POD 1 and POD3 in the control group were significantly higher than those in the ERAS group.Conclusion:Compared with the traditional model,the ERAS process can increase the proportion of Bacteroidetes in feces and reduce the abundance of Proteobacteria in patients undergoing colorectal surgery,which may improve intestinal flora and accelerate the recovery of postoperative intestinal function.