Effect Of N-3 Polyunsaturated Fatty Acids On Colon Tumorgenesis In Mice Fed With High-fat Diet

Objective Fat-1 transgenic mice endogenously producing n-3 polyunsaturated fatty acids(n-3 PUFAs)and wild-type C57BL/6J mice were used in the study.Mice were intraperitoneally injected with azoxymethane(AOM)to induce colon tumor.Compare the tumor loads of wild-type mice fed with high-fat diet(HFD)and that of wild-type mice fed with normal fat diet(NFD)to verify whether HFD can promote the colon tumorgenesis of mice induced by AOM.Compare the tumor loads of wild-type mice fed with HFD and that of fat-1 mice fed with HFD to clarify whether n-3 PUFAs can inhibit the colon tumorgenesis of mice.The expression of inflammationrelated genes in colon of mice and the effects of n-3 PUFAs(DHA)on macrophage phenotype and inflammation-related signaling pathway in human colon HT-29 cells were examined to clarify the mechanism for the inhibitory effect of n-3 PUFAs on colon tumorgenesis of mice.Method(1)Mice were divided into three groups: WT mice assigned to NFD containing 10% safflower oil(WT+NFD),WT mice assigned to HFD containing 10% safflower oil and 20% lard(WT+HFD),fat-1 mice fed with HFD(fat-1+HFD).fat-1 transgenic mice with fat-1 gene could endogenously produce n-3 PUFAs from n-6 PUFAs provided by safflower oil of diet.Mice were administrated intraperitoneally to AOM at a dose of 10 mg/kg?bw one time per week for 6 weeks,keeping on their diet for 22 weeks to induce colon carcinogenesis.Record the number of tumor in colon of mice and calculate the incidence of tumor and tumor burden.Histopathological analysis of distal colonic tissue was performed.Fatty acid content was determined by gas chromatography.The expression level of PCNA was detected by immunohistochemistry to identify the proliferation of colon cells.The mRNA of TNF-?,IL-6,IL-1?,and oncogene c-myc and macrophage marker F4/80 in colonic tissue were detected by quantitative Real-time RT-PCR(qRT-PCR).Western blotting was used to detect the expression of tumor suppressor gene p53,apoptosis related protein caspase 3 and cleaved caspase 3 as well as the levels of p-GSK3?,?-catenin,NF-?B,p-NF-?B,STAT3,p-STAT3 and NLRP3.(2)THP1 cells were induced to differentiate into macrophages(M0)by 100ng/ml phorbol 12-myristate 13-acetate(PMA).M0 macrophages were pre-incubated for 24 hours by different concentrations of DHA,and then exposed to 100 ng/ml lipopolysaccharide(LPS)for 18 h.The mRNA levels of TNF-?,IL-1? and IL-6 in macrophages were determined by qRT-PCR.M0 macrophages were incubated with 100ng/ml LPS combined with 50ng/ml IFN-? for 48 hours to be differentiated into M1 phenotype,subsequently were treated with different concentrations of DHA.The mRNA levels of marker genes of M1 macrophages(including IL-6,TNF-?,IL-1?,CCR7)and that of marker genes of M2 macrophages(including CCL4,CD206)were detected using qRTPCR to uncover the effects of DHA on macrophage phenotype.(3)HT-29 cells were pre-incubated with different concentrations of DHA(0-50?M)for 24 h,and then treated with TNF-? for 1 h.Western blot and qRT-PCR were used to detect the expression of IL-1?,IL-8 respectively.The levels of NF-?B and p-NF-?B in cytoplasm and nucleus were detected by Western blot to investigate the effects of DHA on NF-?B activation in HT-29 cells induced by TNF-?.The levels of Akt,ERK1/2,p38 MAPK,JNK,p-Akt,p-ERK1/2 and p-p38 MAPK were detected by Western blot.Results(1)The tumor burden of WT-HFD group was higher than WT-NFD group,while the tumor burden of fat-1+HFD group was lower than that of WT+HFD.According to the results of gas chromatography,the ratio of n-6 / n-3 PUFAs in colon tissue of WT mice was significantly higher than that of fat-1 mice.qRT-PCR analysis showed that the mRNA levels of IL-6,TNF-?,IL-1? in WT+HFD group were significantly higher than those in WT+NFD and fat-1+HFD groups.Western blot showed that the expression of p-GSK3?,?-catenin,NF-?B,p-NF-?B,p-STAT3 and NLRP3 in WT+HFD group were significantly higher than that in WT+NFD group and fat-1+HFD group.Immunohistochemical staining showed that the expression of PCNA in WT+HFD group was higher than that in WT+NFD group and fat-1+HFD group.Western blot analysis showed that cleaved caspase 3 in the WT + HFD group was significantly lower than that in WT+NFD and fat-1+HFD groups.qRT-PCR results showed that the expression of c-myc mRNA in WT+HFD mice was significantly higher than that in WT + NFD mice and fat-1 + HFD mice.(2)DHA was found to reduce the expression of TNF-?,IL-1? and IL-6 in macrophages,and also inhibited the expression of marker genes of M1 macrophage including TNF-?,IL-1?,IL-6,CCR7 and enhanced the level of CCL4 mRNA,a marker gene of M2 macrophage.(3)DHA inhibited the expression of IL-1?,L-8 as well as the levels of ERK1/2,p-ERK 1/2,JNK,p-JNK,Akt,p-Akt,also inhibited the activation of NF-?B in colon HT-29 cells pre-treated with TNF-?.Conclusions(1)HFD enhanced AOM-induced colon tumorgenesis through up-regulating TNF-?/NF-?B,IL-6/STAT3 and GSK3?/?-catenin/c-myc signaling pathway.(2)n-3 PUFAs blocked the AOM-induced colon tumorgenesis through reducing TNF-?/NF-?B,IL-6/STAT3 signaling pathway,and inhibiting NLRP3 formation.(3)n-3 PUFA(DHA)inhibited the production of inflammatory factors in macrophage and induced phenotype transition of macrophage into M2 type.(4)n-3 PUFA(DHA)inhibited NF-?B activation in TNF-? stimulated HT-29 cells via downregulating Akt and MAPK signaling pathway.In summary,the present study has demonstrated that n-3 PUFAs functioned to inhibit the colonic tumorgenesis by inhibiting inflammatory signaling pathways and regulation of macrophage phenotypes.