The Effects Of High Fish Oil Diet On Liver Inflammation、Complement And Intestinal Microflora

Object: A plenty of studies reveal that high intake of saturated fatty acids and n-6 polyunsaturated fatty acids(n-6 PUFAs)can promote the occurrence and development of chronic inflammation and related diseases,such as cardiovascular disease.Reports suggest that fish oil is rich in n-3 polyunsaturated fatty acids,which has a great anti-inflammatory effect.Intake of low level n-3 PUFAs can help prevent the generation and development of high-fat-diet induced chronic diseases.Unfortunately,the exact role of high intake of n-3 PUFAs in complement activation,liver and the microbiota community in intestine is still unknown.In this study,we used mice models to explore fish oil diet on the complement activation,inflammation in liver and the microbiota community in intestine,and expect to investigate the effects of high intake of n-3 PUFAs on body.Methods:(1)Adult C57BL/6 mice were randomly divided into two groups,and fed with low-fat-diet(LFD)or high n-3 PUFAs rich fish oil diet(FO)respectively.Four weeks later,mice were sacrificed to analyze body weight,spleen weight,liver weight.General observations of mesenteric lymph nodes(MLNs)were taken by photos.(2)Livers from LFD or FO diet fed mice were collected to do immune cell population,histology,and inflammatory cytokine profiles analysis.Nonparenchymal hepatic cells(NPCs)were prepared from liver,and the percentage of CD4+ or CD8+ T cells and CD11b+Gr-1+cells in livers were analyzed using flow cytometer.The m RNA levels of IL-1β、IL-6 and MCP-1 in liver tissues were quantified by q-PCR.The histology of livers was examined by H&E staining.(3)Serum and liver tissues were collected from LFD and FO fed mice.The 50% haemolytic complement activities(CH50)and the levels of membrane attack complex(MAC)of complement in serum samples were detected with ELISA.The protein levels of C3 in serum and the m RNA levels of C3 in liver tissues were analyzed with ELISA and q-PCR respectively.The m RNA levels of C3、C4b、 C1qb、B factor、H factor、mannose-binding protein-associated serine protease(MASP)in were detected using q-PCR.Single cell samples were prepared from blood,MLN,spleen and liver tissues.The fluorescence intensities of CR3 or CR4 on these single cells were detected using flow cytometer.(4)After four weeks feeding,the feces were collected from mice and the bacterial composition which includes OTUs cluster analysis and Alpha diversity,were analyzed by using Miseq high-throughput sequencing and statistical methods.Results:(1)FO diet feeding had no obvious effects on the body weight,but dramatically increased the weights and sizes of spleens,and then pharyngalgia and congestion in spleen from FO diet fed mice was obversed macroscopiclly.Although,the data shown that there were no difference in the weights of livers in LFD and FO diet fed mice,but the congestion can be found in livers with FO diet fed cohort by using visual inspection.Similarly,the changes can be found in the secondary lymphoid organs of MLNs of FO fed mice showed greasier,more adhesive and light yellow color,and then were obviously different from MLNs in LFD cohort,which with pearly white,smooth and transparent surface.(2)Interestingly,after FO diet feeding,the percentage of CD4+ T cells dramatically increased,and the ratio of CD4+ T cells to CD8+ T cells was converted.We found that both populations of CD11b+Gr-1hi and CD11b+Gr-1int myeloid cells were significantly increased in FO diet cohorts in liver,and liver histology shows more inflammatory immune cells infiltration in FO diet fed mice.The m RNA levels of pro-inflammatory cytokine IL-1β,IL-6 and MCP-1 in liver tissue of FO mice were higher than LFD cohort.(3)Higher total complement activity(CH50)and MAC production were found in liver of FO diet fed mice,meanwhile higher level of C3 protein and C3 m RNA were also detected in FO cohort.Although there was no difference in the level of MBL associated serine protease(MASP1),one specific component of MBL pathway,the m RNA levels of C4 b and C1 qb which were two other important activation components in the complement classical activation pathway(CP)and MBL pathway in liver were increased in FO diet fed mice.Furthermore,higher m RNA level of complement factor B(B factor),one component in alternative pathway,was higer in liver compared with LFD cohort.Simultaneously,complement factor H(H Factor),an inhibitory complement regulatory protein in AP,was lower in mice with FO diet.The expression of CR3 was obviously increased in both MLNs and spleens,but only slightly up-regulated in liver and blood.However,no obvious changes of the expressions of CR4 were observed.(4)The population diversity of micobiota in FO group was significantly decreased compared with LFD control.Interestingly,after FO feeding,the proportion of Bacteroidetes was reduced obviously,and one new dominant bacterial community Akkermansia was detected.Conclusions: High fish oil diet feeding promotes liver inflammation and increases complement activation in liver.Furthermore,FO diet feeding significantly influences the microbiota community in intestine and changes the diversity of intestinal flora.