Effect Of Per1/Per2 Double Gene Knockout On Bile Acid Metabolism In Normal And High-fat Fed Mice

The destruction of circadian rhythm will cause abnormal metabolism of bile acid,causing disorder of lipid metabolism.High-fat diet is an environmental condition that causes circadian rhythm disturbances,meanwhile,it is also an important factor that causes disturbances in bile acid metabolism and lipid metabolism.This study explored the circadian rhythm disorder and the circadian rhythm of intestinal bile acid metabolism caused by the knockout of Per1 / Per2 gene under normal and high-fat diet conditions.Male C57 BL / 6 mice were randomly divided into 4 groups,namely wild type control group(WTCON),wild type high fat group(WTHFD),Per1 / Per2 gene knockout control group(DKOCON)and Per1 / Per2 gene knockout high fat The group(DKOHFD)was fed normal feed and high-fat feed separately for 16 weeks.At the 16 th week of the experiment,the animals were randomly divided into two time point groups: ZT0(7:00 am,n=10)and ZT12(7:00 pm,n=10),mice were sacrificed at two time points,ZT0 and ZT12 respectively.Fasting blood glucose,blood lipid and other biochemical analysis,liver tissue oil red O staining,intestinal tissue H & E staining were performed after sacrifice.The contents of the intestinal tract were taken,and bile acid among it was quantitatively determined using LC-MS,the abundance of intestinal flora was determined using lllumina Mi Seq sequencing.Take intestinal tissues and determine the contents of bile acid regulating proteins FXR,SHP,FGF15 and circadian protein CLOCK and its output factor REV-ERBα by western blot.The results showed that the liver biochemical indexes and blood biochemical indexes of HFD mice were significantly higher than that of CON mice(P <0.05).Oil red O stain liver tissue and H & E stain intestinal tissue showed lipid accumulation and inflammatory infiltration appeared in HFD mice.Quantitative determination of intestinal bile acid by bile acid-targeted metabolomics results showed that no matter day or night,the contend of total bile acid,conjugated bile acid and unconjugated bile acid in DKO mice on normal or high-fat diet were significantly reduced compared with WT mice(P <0.05).The correlation analysis of bile acid-intestinal flora showed that in Firmicutes,Acetatifactor abundance was significantly negatively correlated with β-TMCA content(P <0.05),and positively correlated with β-MCA content;Lactobacillus abundance and CA content were Significant negative correlation(P <0.05),positive correlation with DCA;Ruminococcaceae abundance was significantly positively correlated with DCA content(P <0.05),negative correlation with CA content.Western blot detection of intestinal bile acid-regulated protein showed that the expression trends of FXR,SHP and FGF15 in WT mice and DKO mice were basically the same.Taking SHP as an example,on the whole,the expression of DKO mice is significantly lower than that of WT mice;the expression of both mice at ZT12 is significantly higher than that at ZT0(a difference of 2-3 times between day and night);high-fat feeding also significantly increases the expression of SHP(increased by 1.6-3 times).Western blot detection of the core rhythm gene CLOCK and its regulatory factor REV-ERBα in the circadian rhythm pathway of the intestine showed that in the intestine groups,the expression of CLOCK and REV-ERBαwere basically the same.Under normal dietary conditions,There was no significant difference in the expression of CLOCK and REV-ERBα in mice(except for the significant increase in the expression of CLOCK in DKO mice during ZT12).But under high-fat diet conditions,the expression of these two proteins showed significant changes: at ZT12,The expression of CLOCK and REV-ERBα in WT mice was significantly lower than that in ZT0(P <0.01),while it is opposite in DKO mice: the expression of these two proteins was significantly higher in ZT12 than that in ZT0(P <0.01).Conclusion: The knockout of Per1 / Per2 gene reduced the synthesis and secretion of bile acids in the liver.The total amount of bile acids and conjugated bile acids in the intestine were significantly reduced,which affected the digestion,absorption and growth metabolism of the mouse intestine.related.The intestinal bile acid receptor signaling pathway and the circadian rhythm of FGF15 expression have an important effect on the diurnal variation of hepatic bile acid synthesis.At the same time,high-fat diet and other factors have an obvious effect on the expression of FGF15,which may play a role in the regulation of bile acid synthesis.Knocking out of Per1 / Per2 gene can change the circadian rhythm of the intestine,and make the expression of CLOCK and REV-ERBα show abnormal expression of circadian rhythm reversal.