Cholic Acid Induced Intestinal Adenoma-Adenocarcinoma Sequence By Altering Phenotype Of Tumor Associated Macrophage In Apc^min/+ Mice

Objective Colorectal cancer?CRC?,is a malignant tumor of the large intestine mucosa,which is one of the most common cancer in the digestive cancer.In recent years,the incidence of CRC is increasing in most countries in the world,which is related to the change of lifestyle,diet and obesity.Studies have shown that high fat diet can increase the level of bile acids in the intestinal tract,especially cholic acid,in order to promote the metabolism of lipids and cholesterol.Cholic acid can be transformed into deoxycholic acid by the metabolism of intestinal flora,and then promote the development of CRC,however,the specific mechanism of promoting cancer is not clear.In recent years,more and more evidence shows that tumor associated macrophages?TAMs?play an key role in tumor growth,proliferation,invasion and metastasis.In most solid tumors,TAMs play a role in promoting tumor development,but TAMs have pro tumor and anti-tumor activity of CRC,therefore,a variety of biological behavior intervention in the evolution process of TAMs in CRC may be a new strategy for the prevention and treatment of CRC at present.Apc^min/+ mice spontaneously develop adenomas,a good model for the study of intestinal tumorigenesis and drug intervention.The purpose of this study was to investigate the role of TAMs phenotype transformation in the carcinogenesis of intestinal adenoma induced by cholic acid in Apc^min/+ mice,and to provide a theoretical basis for the prevention and treatment of CRC.Methods 1.Four-week-old Apc^min/+ mice were divided into two groups randomly: control group?regular diet?and CA group?0.4% CA in regular diet?,each group has ten mice.The state of every mice was observed every day and the weight of every mice was weighted.All mice were sacrificed after 12 weeks.The number,size and location of intestinal adenomas were observed.The pathology was evaluated via hematcxylin-eosin?HE?staining.2.Ki-67 expression was detected by immunohistochemistry?IHC?to evaluate the cell proliferation.3.Intestinal inflammatory factors IL-1?,IL-6 and TNF-? were measured by real-time PCR.4.Monocyte chemotactic protein 1?MCP-1?was detected by real-time PCR and IHC.5.the expression of total macrophages surface molecule F4/80 and the surface molecules of M1 and M2 macrophages detected by Realtime-PCR.Immunofluorescence double staining was used to detect the expression of total macrophages surface molecule F4/80,i NOS and MR.Result 1.CA was generally tolerated,and mild bloody stool was found at the end of the experiment.All mice grew slowly without significant difference.2.Compared with control group?22.80 ± 0.93?,CA increased the number?33.40±1.17,P=0.002?and carcinogenesis of the intestinal tumors.3.The elevated positive cells of Ki-67 were observed in Apc^min/+ CA group?70.60 ± 5.45?compared with that of Apc^min/+ control group?29.80 ± 1.66,P < 0.0001?.4.Although there was no inflammatory infiltration according to HE staining,the relative m RNA expression levels of IL-1??14.14 ± 2.78?,IL-6?3.40 ± 0.11?and TNF-??3.99 ± 0.44?dramatically increased in CA group,compared with control group?1.24 ± 0.14,P = 0.0099?,?1.45 ± 0.29,P =0.0018?and?1.42 ± 0.24,P =0.0071?.5.And the relative m RNA expression levels of MCP-1 increased in CA group?13.25 ± 1.55?compared with control group?1.96 ± 0.68,P =0.0026?.Based on IHC,the protein levels of MCP-1 in stroma of intestinal polyps in CA group?86.67 ± 2.03?significantly increased compared with control group?43.99 ± 4.73,P = 0.0011?.6.Realtime-PCR showed CA down-regulates of CXCL-10 and i NOS,and up-regulates of Arg-1,MR and CCL17.The immunofluorescence double staining results showed that F4/80 and MR positive cells increased in the CA group than the control group,while the i NOS positive cells decreased,prompting that CA may promote the phenotypic transformation of macrophages from M1 to M2.Conclusion CA induced low-grade intestinal inflammation and upregulated MCP-1,and then increased the total macrophage and shifted macrophage phenotype in tumor microenvironment from M1 to M2 type,finally promoted the carcinogenesis of intestinal adenoma in Apc^min/+ mice.