Effects Of Per1/Per2 Gene Knockout On Circadian Rhythm Of Intestinal Flora In Normal And High-fat Diet Fed Mice

Objective: To investigate the effect of high-fat diet on the intestinal flora with Per1/Per2 gene deficiency,and to study the mechanism and rhythm of intestinal damage.Methods: Laboratory-bred C57 BL / 6(DKO)and wild C57 BL / 6(WT)male mice with Per1/Per2 gene deficiency were divided into three groups,with 24 mice in each group,fed with normal diet(CON),High-fat diet(HFD).The mice were sacrificed at two-time points,7 am(ZT0)and 7 pm(ZT12),with 12 mice at each time.Blood glucose was detected,plasma and cecum contents were collected,and intestinal tissues,perirenal fat,and sexual fat were collected.The plasma lipid and ALT and AST activity were measured with specific kit.H&E staining of intestinal tissue was performed for histological observation,genomic DNA of cecum contents was extracted to 16 S r RNA gene sequencing,comparison and analysis were performed,and intestinal flora composition and abundance of each group were analyzed.Compare the changes in groups.Western Blotting was used to detect the change of claudin-1 in colon tissue.RT-q PCR was used to detect the expression of bacterial recognition receptors tlr2 and tlr4,the expression of Il-1β,Il-10,Il-22,Il-6,Il-22 R,and the expression of tight junction proteins Occludin-1,Claudin-1 and Zo-1 in colon tissues.Results: In the normal feeding group,the weight gain curve and final weight of DKO mice were lower than WT mice(p <0.05),while under high-fat diet conditions,the obesity index of DKO mice was not significantly different from that of WT mice.High-fat diet significantly increased TC levels,and liver injury indicators AST and ALT levels.Plasma TC levels of DKO mice fed a normal diet were lower than those of WT mice at ZT0,and other indicators of DKO mice were not significantly different from WT mice.Intestinal tissue H & E results showed that there was no significant difference between DKO and WT mice in the normal group,and high-fat diet had obvious damage to intestinal tissue: intestinal mucosa was damaged,villi were shed,and glands were damaged.Intestinal tissue molecular analysis results showed that the expression of intestinal tight junction protein was significantly different between DKO mice and WT mice.The protein expression of tight junction protein claudin-1 in wild mice on a normal diet showed ZT0 was significantly higher than the circadian rhythm expression pattern of ZT12(that is morning high and night low),while this rhythm completely disappeared in DKO mice,and the expression was significantly higher in WT mice fed normal diet(p <0.05).But the expression gap between DKO mice and WT mice on a high-fat diet completely disappeared.RT-q PCR results of m RNA expression showed that under normal diet feeding conditions,the expressions of Tlr2,Il-10,Il-22,and Occludin-1 were different in WT mice,and the circadian rhythm pattern dissapeared in DKO.Il-1β and Claudin-1 showed differences in morning and evening expression in WT mice,and their diurnal expression patterns were reversed in DKO mice.There was no difference in the expression of Il-6 between ZT0 and ZT12 in WT mice,but in DKO mice.Under high-fat diet conditions,the expression of these molecules was not significantly different in DKO mice and WT mice.16S r RNA gene sequencing of the intestinal microbial genome showed that: 1.Under normal diet conditions,the abundance and diversity of the intestinal flora of DKO mice was significantly lower than that of WT mice(P <0.05).Under high-fat diet feeding conditions,there was no significant difference in abundance and diversity between DKO mice and WT mice(P> 0.05).2.The diurnal pattern of the intestinal flora of DKO mice changed:(1)There was a diurnal difference in Bacteroides of DKO mice fed with normal diet,and in Verrucomicrobia of WT mice fed with high-fat diet.(2)The normal circadian rhythem of bacteria related to host energy production and metabolism(short-chain fatty acid-producing bacteria: unidentified＿Ruminococcaceae,succinic acid-producing(Parasutterella),vitamin metabolism-related bacteria(Bifidobacterium),lipid metabolism-related bacteria(Lactobacillus,Lachnoclostridium)disappeared in both DKO mice and the high-fat diet groups.3.In terms of microbial composition,DKO mice are significantly different from WT mice.DKO mice have fewer biomarker bacteria and are mainly related to inflammation and metabolism.Under normal diet condition,the relative abundance of most bacteria with significant differences in DKO mice is lower than that of WT mice.Under high-fat diet conditions,the relative abundance of intestinal bacteria related to amino acid,bile acid metabolism,and inflammation decreased in DKO mice,and the relative abundance of intestinal bacteria related to short-chain fatty acid metabolism increased,compared with that of WT mice.4.On intestinal function-related microorganisms,compared with WT mice,DKO mice have significantly improved host cell membrane transport function,replication and repair functions,and amino acid metabolism,lipid metabolism,vitamin metabolism and other metabolic functions have been significantly reduced.While under high-fat diet,differences in functions such as cell membrane transport and amino acid metabolism still exist,and are consistent with changes under normal diet conditions.Conclusion: The loss of the circadian gene Per1 / Per2 significantly affects the physiology and biochemistry of mice,and appears thinner under normal diet conditions.The composition and abundance of the intestinal flora are affected to varying degrees.The expression of the factor is abnormal,and the expression of tight junction protein is reduced.The effect of high-fat diet on intestinal flora is more significant than that of Per1 / Per2 knockout,which can mask abnormalities caused by genetic defects.