The Study On The Effect Of ω-3 PUFA Regulates Circadian Rhythm Of Gut Microbiota In High-fat Mice

Objective: To study the regulation of omega-3 PUFA on lipid metabolism and circadian rhythm of intestinal flora in high fat mice.Methods: Male C57BL/6 mice were fed a high-fat diet for 12 weeks to construct a high-fat animal model.The experiment was divided into four groups(20 animals in each group): normal group(CON group);high-fat group(HFD group);Fish oil group(FO group),adding fish oil on the basis of high fat feed;algae oil group(AO group),adding algae oil on the basis of high fat feed.After 12 weeks of feeding,the mice were randomly divided into two groups(ZT0 and ZT12)with 10 mice at each time point according to the interval of 12 hours.Plasma,cecal and colon contents were collected and liver,colon,perirenal fat and epididymal fat were removed and weighed.The blood lipid levels of TC,TG,LDL-C and HDL-C were determined by colorimetry,and the activities of ALT and AST were detected by enzymatic method.Liver tissue paraffin sections,HE staining.The bacterial genomic DNA of the cecal contents was extracted,and 16 S r RNA gene sequencing was performed to analyze the intestinal flora changes of each group of mice.The content of short-chain fatty acids(SCFAs)in colonic contents was rapidly and quantitatively analyzed by gas chromatography-mass spectrometry(GC/MS).RT-qPCR was used to detect the expression of GPR41 and GPR43,the expression of proinflammatory cytokines such as IL-1β,TNF-α and IL-6,and the expression of tight junction proteins of occludin,claudin-1 and zo-1.Results: Compared with the normal diet,the high-fat group showed a significant increase in physiological indicators.The FO group and the AO group rich in omega-3 PUFA significantly reduced the liver index.Compared with the CON group,the HFD group showed an opposite change in plasma lipid content.Compared with the HFD group,TC,TG,LDL-C,ALT and AST decreased significantly in the FO group;TC,LDL-C,ALT and AST decreased significantly in the AO group,and the HDL-C content increased significantly.The HFD group induced steatosis,and there were more macrovesicular lipid droplets in the hepatocytes,showing a diffuse distribution.The FO and AO groups improved HFD-induced hepatic steatosis,and markedly decreased lipid droplets in hepatocytes,with only sparse lipid droplets scattered around the liver cells.The abundance and diversity of intestinal flora in normal mice showed obvious early(ZT0)high-late(ZT12)low circadian rhythm.In the high-fat diet,the intestinal flora of the mice was disordered,the circadian oscillation of the flora was lost,and the number of bacteria with significant difference between ZT0 and ZT12 decreased.The abundance of beneficial bacteria decreased at night compared with CON group,and the abundance of harmful bacteria increased in both morning and evening compared with CON.Fish oil and algae oil had no significant effect on the abundance and diversity of high-fat mice,but could increase the number and abundance of beneficial bacteria,regulate the rhythm disorder of intestinal flora in high-fat mice.(a)The number of genus with significant difference between morning and evening in FO group and AO group was more than that in HFD group.(b)The relative abundance of Eubacterium coprostanoligenes in the FO group and the AO group increased.(c)The relative abundance of Lactobacillus was increased in the morning and relatively abundant Oscillibacter was increased in the evening in the AO group.(d)In FO and AO group,Butyricimonas and some genera of Lachnospiraceae returned to normal circadian rhythm.(e)In the FO group,Streptococcus,Erysipelatoclostridium and Sutterella returned to normal circadian rhythm.(f)In the AO group,Roseburia,which produces SCFAs,returned to normal circadian rhythm.The high-fat diet mice had the lowest content of short-chain fatty acids in the intestine.In the FO and AO groups the content increased,and GPR41 and GPR43 activated,and the diurnal trend returned to normal.This may be due to the higher content of SCFAs-producing Butyricimonas in the morning than in the evening in the ω-3 PUFA intervention group.The FO group and AO group rich in omega-3 PUFA significantly decreased the expression of pro-inflammatory factors Il-1β,TNF-α and IL-6 in colon tissue compared with HFD group,significantly increasing tight junction protein claudin-1,occludin and zo-1 expression.The tight junction proteins claudin-1 and occludin in the HFD group did not have circadian rhythm expression in the colon,while the AO group partially restored the tight adhesion protein rhythm.Conclusion: DHA/EPA-rich fish oil and DHA-rich algae oil can alleviate the lipid metabolism disorder caused by high-fat diet,increase the number and abundance of beneficial bacteria in the intestine,increase the number of genus of circadian rhythms,restoring the circadian rhythm of the genus to normal,and improve the circadian rhythm of intestinal bacterial metabolite SCFAs and intestinal permeability.