The Mechanism Of Swimming Inhibition On The Growth Of Colorectal Cancer Graft Tumor In Mice Based On Gut Microbiota

Objective:To explore the effects of swimming on tumor growth and exercise capability in mice bearing colorectal cancer cells,as well as the underlying mechanisms,using various omics techniques such as RNA sequencing,metabonomics,and intestinal flora sequencing.Methods:1.To produce a Xenograft mouse model,CT-26 cells were subcutaneously injected in the right flank area of mice.Mice were randomly divided into Model and Swimming groups(n=10 for each group),and another 10 mice without injection of CT-26 cells were considered as Control group.Mice in the Swimming group were subjected to physical training to swim twice per day,six days in a week,for a total of four weeks.Tumor growth were measured using a vernier caliper to monitor tumor volume and an electronic scale were used to determine tumor weight.Autonomous activity of mice was determined by motion detector.The muscle tension of mice was detected using tension detector.HE staining and electron microscopy were performed to assess the pathological and microstructure changes of muscle tissues.2.The Xenograft mouse model was constructed in the same way as part 1 and divided into Model group and Swimming group(n=6 for each group).Physical training to swim in Swimming group was conducted for 3 weeks,followed by collecting mouse feces.The effect of swimming on the composition of intestinal flora in CT-26 bearing mice was assessed using 16 S r RNA sequencing and OTU cluster analysis.The functional enrichment of microbiota in transplanted tumor mice was evaluated using PICRUSt.3.The model construction,grouping and physical training of swimming were the same as part 2.Tumor growth were assessed by monitor of tumor volume using vernier caliper and determination of tumor weight using electronic scale.Differential expressed transcripts(DETs)was identified using RNA sequencing and pathway enrichment analysis was performed based on databases of Kyoto Encyclopedia of Genes and Genomes(KEGG)and PANTHER.ELISA was performed to determine level of IL-1β,IL-10 and TNF-α in serum of mice.Immunohistochemistry(IHC)was used to detect the expression of TNF-α and IL-6 in NF-κB signaling pathway,as well as CD31,HIF-1α,VEGF-A and VEGF-R2 in HIF-1α/VEGF-A signaling pathway.Metabolomics was performed to identify the different expression of metabolic products in part 1 muscle tissue.TUNEL assay was used to detect the cell apoptosis in muscle tissues.Western-blotting and IHC were performed to determine the expression of Bax and Bcl-2 in muscle tissues.Results:1.The effects swimming on CRC tumor growth and muscular atrophy in tumor-bearing mice: Swimming significantly reduced the volume of transplanted tumor and increased the body weight of transplanted tumor mice(subtracted tumor weight).Swimming obviously improves the capability of autonomous movement,muscle tension,muscle loss,pathological and microstructural changes in muscle tissues.2.Effect of swimming on gut microbiota: Swimming significantly affected the diversity of gut microbiome,improved the structure of microbiota and reduced the ratio of intestinal F/B in CT-26 bearing mice.The content of some beneficial bacteria,such as Prevotellaceae,Alloprevotella and Roseburia,were increased while the abundance of Streptococcus and Peptostreptococcus were reduced.Further analysis of KEGG metabolic pathway enrichment of the intestinal flora shown that there were 19 metabolic pathways different between the Model group and the Swimming group(P < 0.05).Gene functions in the Swimming group were mainly involved in immune system,transportation and storage,biosynthesis of other secondary metabolites,energy metabolism and so forth.3.The effects and underlying mechanisms of swimming on CRC tumor growth: Swimming significantly affected the expression of 1344 genes in colorectal carcinoma xenograft(715 up-regulated genes and 629 down-regulated transcript,all P<0.05,fold changes≥2).Pathway enrichment analysis based on KEGG and PANTHER databases identified multiple signaling pathways,including T cell activation,oxidative stress response,B cell activation,angiogenesis,hypoxia response via HIF activation,VEGF signaling pathway and so on.Moreover,IHC analysis indicated that swimming obviously inhibited the activation of NF-κB signaling pathway and down-regulated the expression of TNF-α,IL-6 in tumor tissues.ELISA analysis revealed that swimming significantly increased the levels of IL-10,while decreased the levels of IL-1β and TNF-α in serum of mice.IHC analysis also revealed that swimming significantly inhibited the activation of HIF-1α/VEGF-A signaling pathway,down-regulating CD31,HIF-1α,VEGF-A,and VEGF-R2 protein expression in tumor tissues.Analysis of metabolomics found that swimming significantly increased 222(43 anions and 179 cations)and decreased 184(151 cations and 33 anions)metabolites,and the taurine and hypotaurine metabolism pathways in muscle were significantly enriched.The levels of multiple metabolites involved in the enriched signaling pathway were increased in muscle tissues,including taurochenodeoxycholic acid,taurocholic acid,ascorbic acid and eicosapentaenoic acid.Western-blotting and IHC detection showed that swimming intervention significantly inhibited the activation of NF-κB signaling pathway in muscle,reduced the apoptosis of muscle cells,down-regulated the expression of Bax and up-regulated the expression of Bcl-2.Conclusion:1.Swimming intervention can significantly delay the growth of CT-26 colorectal cancer transplanted tumors,improve the autonomous activity,muscle tension and motor ability of transplanted tumor mice,and significantly improve their quality of life.2.Swimming can significantly improve the abundance and structure of intestinal microflora of transplanted tumor mice,increase the content of beneficial bacteria,reverse the ratio of Firmicutes and Bacteroidetes,Improve body immunity,reduce body inflammation level.3.Swimming can improve intestinal flora to regulate NF-κB,HIF-1α/VEGF-A signaling pathway mediated inflammation and angiogenesis,Improves muscle metabolism,which may be one of the mechanisms that swimming interferes with intestinal flora to delay the growth of transplanted tumor and improve the quality of life.