Study Of The Mechanism Of Berberine Regulates Macrophage Polarization In Familial Adenomatous Polyposis

Objective FAP as an autosomal dominant inherited disease has high canceration rate,so early intervention is of great significance.Our previous research has shown that Berberine has chemopreventive potential against FAP,and the latest study found the regulation of macrophages polarization can inhibit the development of FAP.we will firstly analyze the phenotype of macrophages after using Berberine in ApcMin/+ mouse and peritoneal macrophages,and then identify the mechanisms of inhibing gowth of colon cancer cell line by Berberine using transwell cocultur.Hopefully we will uncover a new intervention way for the reversal of colon cancer.Methods Part 1 Four weeks old ApcMin/+ mice were equally divided into control group(without treatment)and the Berberine group.After 12 weeks,all the mice were sacrificed.The number,size and distribution of mice intestinal and colonic polyps were measured to evaluate intestinal tumor development.Immunohistochemistry,Realtime-PCR and immunofluorescence double staining were used to detect the alteration of the phenotype of macrophage in stroma of intestinal polyps.Western blot analysis and immunostaining were performed to detect COX-2 expression in the intestinal polyps.Part 2 Mouse peritoneal macrophages were treated with cytokines in the presence or absence of Berberine.Realtime-PCR was performed to detect the alteration of the phenotype of macrophages after the intervention fo Berberine.Part 3 To investigate the invasiveness of colorectal cancer cells which underwent non-contacted co-culture with the M2 type tumor associated macrophage which intervented by Berberine.Results Part 1 1.Berberine was well tolerated,and all mice grew slowly without significant difference.2.Compared with the control group,the total numbers of polyps in the small intestine and the colon were reduced by 62%.In berberine group,all sizes of polyps were significantly reduced,and the numbers of polyps were significantly reduced in proximal,middle and distal portions of small intestine.3.The percentage of F4/80 positive expression cells in stroma of intestinal polyps of Berberine group decreased by 45%compared with the control group(P<0.05).The percentage of iNOS positive expression cells in stroma of intestinal polyps of Berberine group increased by 56%compared with the control group(P<0.001).But the percentage of MR positive expression cells in stroma of intestinal polyps of berberine group decreased by 23%compared with the control group(P<0.05).3.The The percentage of COX-2 positive cells of Berberine group decreased by 56%compared with the control group(P<0.001).The expression of COX-2 at protein level of Berberine group was lower than that of control group(P<0.05).4.The expression of mRNA of iNOS and IFN-γ from Berberine group was significantly higher than those from control group(P<0.05);the expression of mRNA of MR and IL-10 from berberine group was significantly lower than those from control group(P<0.05).Part 2 1.The expression of mRNA of MR and Arg-1 from Berberine group was significantly lower than those from IL-10/IL-4 group in mouse peritoneal macrophages(P<0.05),when the expression of mRNA of iNOS and CXCL-10 from Berberine group was not significantly higher than those from IL-10/IL-4group(P>0.05).2.The expression of mRNA of iNOS and CXCL-10 from IFN-γ group was significantly higher than those from IL-10 group(P<0.05),and the expression of mRNA of MR and Arg-1 from IFN-γ group was significantly lower than those from IL-10 group(P<0.05).Part 3 1.The RAW264.7 cells were treated with 20ng/ml IL-10 for 24h,after the treatment the expression of mRNA of MR was significantly increased.2.Berberine could inhibit the activity of HT-29 cells significantly(P<0.05).3.The detection of migration and invasiveness in experimental groups via wound-healing/transwell shows that HT-29 which were co-cultrued with the M2 type tumor associated macrophage which intervented by Berberine has the lower migration and invasiveness capability(P<0.05).Conclusions Long term use of Berberine is safe,and can significantly prevent intestinal polyposis in FAP models.Berberine can inhibit the growth of intestinal polyps in ApcMin/+ mice through altering the phenotype of macrophages.Moreover,Berberine can infulence the motility and invasion ability of colorectal tumor cells in the vitro experimental conditions through altering the phenotype of macrophages.