Study On The Changes Of Clock Genes, Lipid Metabolism-related Genes In Atherosclerotic Mouse With Circadian Rhythm Disorder

To adapt the changes of environmen, all species on the earthfrom prokaryotes to humans, have a life cycle synchronized approximately with the circadian rhythm of the planet. The circadian rhythm is achieved by the transcription and translation feedback loop formed by circadian clock genes.Clock genes dominate many physical functions by regulating the clock control genes(CCGs).This domination is not only reflected in the performance of arousal, feeding and other behaviors,but also shown in the immunization, blood cell count, body temperature regulation, and ather basic physical activities in protein and gene levels.If this biological circadian rhythm was disrupted by some reasons, individual will get sick and even be eliminated. Long-term shift work or irregular sleep might result in circadian rhythm and metabolic disorders, lead to increased incidence and occurrence of many diseases represented by a variety of cardiovascular diseases.Although recent years more and more research on energy metabolism related diseases, biological clock and the impact of light on circadian clock gene expression in experimental animals. However, studies on relationship between lipid metabolism and the circadian clock in circadian rythm disorder model of atherosclerosis have not yet reported. Therefore, by changing the external light environment we established a atherosclerosis model with circadian clock disorder in mice.We detected the24hours expression of5circadian genes,4clock controlled, lipid metabolism related genes. NAD-dependent deacetylase sirtuin-1(SIRT1)on the one hand enhance gluconeogenesis,and lipolysis,regulate differentiation of adipocyte,promote insulin secretion,enhance tissue sensitivity to insulin.On the other hand, it inhibite endothelium-dependent vascular contraction; is involved in cell cycle, senescence, apoptosis and metabolism;by NF-kB signaling pathway reduce expression of pro-inflammatory factors to play anti-inflammatory role; reduce macrophage uptake of oxidized low density lipoprotein;promote antiport of cholesterol from macrophages in cholesterol plaque;play an antithrombotic role by inhibite the expression of endothelial tissue factor (coagulation factor Ⅲ).Recent studies have reported that SIRT1regulate the function of CLOCK-BMAL1heterodimer through deacetylation to mediate energy metabolism and circadian clock.Therefore, we detected expression of sirt1gene in four different tissues of mice by Real-time PCR, and by observing the dynamic changes of sirt1gene expression in24hours in mice with atherosclerosis to clarify expression rhythm changes of sirt1gene in lipid metabolism and the development of atherosclerosis.We also explored the effect of H2S on the circadian rhythm of mouse liver cells, so as to reveal the links between changes in energy metabolism disorder and biological clock in mice.Part Ⅰ Rhythm changes of clock genes, lipid metabolism-related genes in atherosclerotic mouse with circadian rythm disorderPurpose:Observe changes of associated genes in atherosclerosis mice with circadian clock disorder to explore the possible mechanismDesign:Establish circadian clock disorder model of atherosclerosis, using apoE-/-mice and C57mice by changing the environmental light.24male apoE-/-mice and24male C57BL/6J mice were maintained on a light-dark(LD) cycle(12h light,12h dark) for2weeks, a dark-light(DL) cycle for2weeks and then a light-dark(LD) cycle for2weeks. apoE-/-mice were divided into two groups:mice of one group were fed normal chow,mice of the other group were fed with a western type diet (containing0.15%cholesterol and21%fat).C57BL/6J mice were given the same treatment. According to Zeitgeber time(ZT; ZT0is defined as lights-on time and ZT12as lights-off time), mice were sacrificed at different time points including ZT0, ZT4, ZT8, ZT12, ZT16and ZT20. The serum was prepared for glucose, total cholesterol, triglyceride,LDL, and HDL detection. The arterial arcades were prepared for frozen sections. Lillie-Ashburnes oil red O staining method was used to detect atherosclerotic plagues. The heart,liver, brain and fat tissue were removed, frozen in liquid nitrogen and stored at-70℃.The expression of clock genes mClock, mPer2, mBmall, mRev-erba and mCryl and sirtl which mediated circadian clock genes and energy metabolism genes in four tissues was detected by Real-time PCR. And the expression of lipid metabolism-related genes ppar-α, ppar-γ, ror-α, rxr-γ was also detected by Real-time PCR.Results:The results suggested that after six weeks of light stimulation,atherosclerotic vulnerable plaque formed in arterial arcades in apoE-/-mice fed with western type diet. Body weight, serum glucose, total cholesterol and low density lipoprotein were higher than normal diet feeding group.Atherosclerotic plaques in apoE-/-mice fed with normal chow also have been formed. However in C57BL/6J mice fed with western type diet only a limited number of subendothelial lipid deposited and foam cell formed. Body weight, serum glucose, total cholesterol and low density lipoprotein were also higher than normal diet feeding group. No significant pathological changes was detected in intravascular of C57BL/6J mice fed with normal chow. Real-time PCR showed that mClock gene which expresses constantly in normal mice presented a higher expression level in liver and hypothalamus of apoE knockout mouse than in C57BL/6J group.The expression of other clock genes mBmal1、mPer2、mCry1、mRev-erb a show circadian rhythm.However, the amplitude and phase between the rhythm of apoE-/-mice and C57BL/6J mice is obviously different. In addition to the amplitude of mCryl in the heart of the C57BL/6J mice is higher than of apoE-/-mice, Other genes in all4tissues presented a haigher amplitude in apoE-/-mice than in C57BL/6J mice. The expression of lipid metabolism-related genes also showed circadian rhythm. In4tissues of apoE-/-mice, total express level and circadian rhythm amplitude of ppar-a was higher than that of C57BL/6J mice. Ppar-γ showed no significant difference in the hypothalamus among the groups. While apoE-/-mice showed a higher amplitude of circadian rhythm and total expression level in heart and liver than C57BL/6J mice.well the adipose tissue is in the opposite. Expression of ror a, rxr Y and ppar a showed many similarities. Expression of sirtl genes only showed circadian rhythm in peripheral tissues of apoE-/-mice and the hypothalamus of C57BL/6J mice. It is worth noting that the disorder of expression of some central and peripheral clock gene is closely related to rhythm changes of lipid metabolism-related genes in circadian clock disorder model of atherosclerosis.Part II effect of H2S on the circadian rhythm of mouse hepatocytePurpose:Observe the effect of H2S on the circadian rhythm of hepatocyte in vitroDesign:By liver perfusion of collagenase Ⅳto obtain liver cells of8-week-old C57BL/6J mice. Cells were divided into H2S and control groups and then were cultured in vitro. Every4hours after serum shock, collected cells into Trizol for extraction of total mRNA. Observed continuously for3days. Detect expression rhythm changes by Real-time PCR method of clock genes mClock, mBmall, mPer2, mRev-erb aResults:In control group, expression of mClock did not show rhythm and relative mRNA level decreased with time goes by. The other three genes have shown circadian rhythm for more than60hours. In H2S group,4genes’ total expression levels are higher than in control group and the amplitude was significantly greater than the control group.Conclusion:1、We established a atherosclerosis model with circadian clock disorder in mice by changing the external light environment. We found that in liver and brain of atherosclerosis mice with circadian clock disorder, total expression of mClock gene in24hours rised, while the opposite is in the heart.In adipose tissue the expression peak shifted and amplitude increaseed. In addition, the expression phase of mBmall. mPer2and mRev-erb a genes shifted significantly.And these changes of clock genes influent the expression level, phase and amplitude of ppars, rxr-γ, ror-agenes. This is why long-term shift work and jet lag so easily lead to metabolic syndrome. In the cardiovascular system of atherosclerosis mice, translocation of the peak expression of sirtl,particularly the reduction of sirtl expression in early morning maybe one of the reasons for high incidence of acute cardiovascular events in early morning.In the hypothalamus of control group, expression of sirtl gene showed significant volatility,lower in the day and higher at night, yet, the amplitude of this phenomenon significantly reduced and the reduce has a positive correlation with the degree of disease.2、We established hepatocytes model with circadian rhythm by Serum shock method. In control group,four clock genes showed china circadian rhythm and the expression level and the peak decreases gradually with time gose by. H2S can maintain expression and amplitude of mClock,mPer2,mBmalland mRev-erb a gene within48hours. This shows that exogenous reducing agent H2S can maintain the circadian rhythm of clock gene in isolated liver cells.We consider that H2S has changed NAD+/NADH in liver cell and thus enhanced the activity of SIRT1protein.The specific mechanism of this protective effect remains to be further studied.