The Mechanism Of Per1/Per2 Double Knockout On The Abnormal Circadian Rhythm Of Hepatic Lipid Metabolism In Normal And High Fat Fed Mice And The Intervention Of DHA

Objective: The loss of clock gene function and high-fat diet(HFD)can disturb the homeostasis of lipid metabolism,and may promote the development of various diseases.The aim of this study is to explore the effects of core clock genes Per1/Per2 double knockout(DKO)on hepatic lipid metabolism in mice with normal and high-fat diets patterns and the intervention effects of DHA substitution in HFD on lipid metabolism disorder and non-alcoholic fatty liver disease(NAFLD)in Per1/Per2 DKO mice through mouse liver transcriptomics.Methods: 7-week-old wild-type(WT)and Per1/Per2 DKO male C57BL/6 mice were housed in an environment with a 12 h light/dark cycle,after one week of adaptive feeding,they were randomly divided into 6 groups,24 in each group.Among them,the normal diet groups(WTCON group,DKOCON group)were fed with standard control feed;the high-fat diet groups(WTHFD group,DKOHFD group)were fed with high-fat and high-cholesterol diet;the high-fat algal oil groups(WTAO group,DKOAO group)were fed high-fat and high-cholesterol diet supplemented with algal oil.After 15 weeks,the animals were killed and sampled at the early and late time interval of 12 hours,i.e.Zeitgeber time(ZT)0(n = 12)and ZT12(n = 12),plasma TG,TC,ALT,AST,and liver TG,TC indicators were detected by a microplate reader colorimetry.H&E staining and oil red O staining of liver tissue were used for histological examination.Trizol method was used to extract the total RNA of liver tissue,transcriptome sequencing analysis compares the expression of differential genes at different genotypes or at different time points or between different diet groups,Go function annotation and KEGG pathway were used to analyze the different metabolic pathways and mechanisms in each group,qPCR was used to detect the expression of clock genes and genes related to lipid metabolism in liver tissues.Results: Per1/Per2-deficient animals were lighter in weight.Blood biochemistry,liver lipid testing,liver tissue H&E staining,and oil red O staining showed that long-term intake of a western-style high-fat diet caused varying degrees and types of elevated blood lipids,liver damage,and liver lipid accumulation in WT and DKO mice;the above indicators were significantly improved in the AO group of mice with DHA.Principal component analysis and venn analysis showed that the diurnal metabolism difference of mice liver in each diet group decreased significantly after the deletion of Per1/Per2.Statistical analysis of differential genes and heat map analysis showed that almost all circadian differentially expressed genes in the livers of mice in each diet group showed diminished circadian rhythm fluctuations after the deletion of Per1/Per2.The analysis of GO and KEGG showed that DKO mice exhibited multiple dysfunctions in circadian rhythm,drug metabolism,cell cycle,cancer pathway and lipid metabolism.For lipid metabolism,on the one hand,the two high-fat diets(HFD,AO)weakened or even eliminated the expression differences of steroids biosynthesis genes(Msmo1,Lss,Sqle,Cyp51,Dhcr7,Nsdhl,Dhcr24,Sc5d),cholesterol biosynthesis genes(Nsdhl,Fdps,Cyp51,Lss,Pmvk,Insig1)and cholesterol metabolism related genes(Pcsk9,Ldlr,Abcg5)between DKO and WT mice at ZT0,the expression of these genes in DKO mice fed with CON diet was significantly higher than that in WT mice.And compared with WT mice,the two high-fat diets were more likely to affect the expression of cholesterol biosynthesis genes in DKO mice.On the other hand,at ZT0,the expression levels of PPAR signaling pathways and fatty acidβ-oxidation-related genes(p-value<0.05)in the liver of DKO mice with three diet patterns were reduced compared with those in WT mice;while at ZT12,DKO mice in the CON group up-regulated the expression of DEGs in the PPAR signaling pathway compared to WT mice,but this difference between WT and DKO mice was attenuated in the HFD and AO groups.In addition,DHA mainly protects the liver of WT mice and DKO mice from NAFLD by improving liver fatty acid metabolism,such as promoting fatty acid uptake genes CD36,Slc27a1,Slc27a2,inhibiting fatty acid synthesis genes Srebf1,Scd1,Fasn,Elov16,Fads1,Fads2,and stimulating fatty acid oxidation genes Ehhadh,Acaa1 b,Acox1,etc.Conclusion: Per1/Per2 plays an important role in regulating the circadian rhythm of hepatic lipid metabolism in mice.After the deletion of Per1/Per2,a large number of genes in the liver of mice show a decreased circadian rhythm fluctuation.DKO mice seem to be more sensitive to high-fat diet,especially compared with WT mice,high-fat diet is more likely to affect the expression of cholesterol biosynthesis genes in the liver of DKO mice.A HFD with DHA substitution can alleviate the abnormal lipid metabolism and NAFLD induced by high-fat diet in WT mice and DKO mice by improving fatty acid metabolism in the liver.