Study On The Mechanisms Of Circadian Disorders And Smoking Cause Atherosclerosis

Atherosclerosis (AS) is the most common and most important one in a group known as arteriosclerosis vascular disease. It can affects the all arteries of body, with large and medium artery are the most common. Coronary heart disease is the most common type of atherosclerosis leading to organ disease. It has become mortality of the disease in the first place around the world and is far more than the cancer threat to humans. Therefore, how to effectively inhibit the progress of atherosclerosis to prevent coronary heart disease, has become an urgent problem. At present, by the research of Framingham and large-scale epidemiological survey, many risk factors has made clear of the coronary heart disease, such as smoking, obesity, hypertension, hyperlipidemia, diabetes, age, lifestyle and resting, hyperuricemia and high homocysteine, and the circadian disorders effects on coronary heart disease has taken more and more attention. It is said that abnormal circadian may directly affect the heart rate, blood pressure, hormone level, autonomic nerve function and the blood components and cause a variety of cardiovascular diseases and decreased cardiac function, while produce great harm to health. Metabolic syndrome (MS) is a aggregation onset clinical syndrome of obesity, hyperglycemia, dyslipidemia and hypertension, and is a pathological state that a variety of cardiovascular diseases (CVD) risk factors gather in the same individual. Our research showed that, cardiovascular risk in patients with metabolic syndrome in 10 years increased 1.85 times than that in nonmetabolic syndrome. The normal circadian rhythms has important clinical significance in the regulation of metabolism, and chronic circadian disorders, such as shift work, is closely related to the high prevalence of insulin resistance, obesity, metabolic syndrome and diabetes. Bmall(brain and muscle Arnt-like protein) gene, also known as MOP3 or ARNT3, one of the biological clock gene, which is the key gene to maintaining the circadian of oscillation. A time-resolved and genome-wide map of Bmall binding in mouse liver, derived from ChIP-seq analyses, has shown that carbohydrate metabolism is a major output of the circadian clock in this tissue. Although skeletal muscle is a primary site of insulin-dependent glucose disposal, the specific role the muscle clock plays in regulating glucose uptake and metabolism is not known. This study is through skeletal muscle-specific Bmall knockout mice to observe their effects on glucose and fat metabolism in muscle and the whole body, and to investigate the mechanism of the circadian disorders lead to AS in the metabolism.Research showed that smoking is the major risk factor for cardiovascular disease. It is showed that, the coronary vascular endothelial cells were damaged after 20min smoking, and early coronary atherosclerosis occurs mainly in the coronary artery endothelial cells. But the mechanism of smoking on vascular endothelial injury remains to be further studied. As atherosclerosis lesion the damaged vascular endothelial cells showed increased expression of cell adhesion molecules such as ICAM-1 and VCAM-1. The impact of p38MAPK cell signaling pathway on endothelial cell surface expression of adhesion molecules has not been explored, although there were conclusive evidence showed that p38MAPK plays a key role in the inflammatory response to various stimuli. This study observed the effects of nicotine on the expression of ICAM-1 and VCAM-1 in mouse cardiac vascular endothelial cells, to further clarify the pathogenesis of coronary heart disease by smoking, and explore the role of p38 mitogen activated protein kinase signal pathway acting on the expression of cell adhesion molecules.Previous research indicated, smoking can effect HRV and the secretion rhythm of cortisol, this shows that smoking is associated with circadian rhythms, can affects circadian rhythms, leading to a series of changes in the body. Smoking also has close relationship with metabolic syndrome. Existing data demonstrate that p38MAPK participated in the clock genes expression and input and output of circadian rhythm.Thus it can be seen, circadian disorders and smoking and metabolic syndrome are very closely related, they can effect each other and play synergistic interaction.While p38MAPK is probably one of the key signaling pathways.Therefore, this study is to observe circadian disorders and smoking cause pathogenic mechanism of atherosclerosis, at the same time, further explore the mutual influence and the inner link between the two, so as to clarify their causes.Part 1 Effect of skeletal muscle intrinsic clock Bmall ablation on muscle metabolismObjectives:To observe the effect of selective knockout of Bmall in skeletal muscle of muscle glucose and lipid metabolism and the changes of related gene and enzyme, while to further explore the mechanism.Methods:Mice 8-12week old, selective deletion of Bmall in skeletal muscle as objective, Bmall-/-mice (B6.129-Arnt1tm1Bra/J) provided by the Jackson laboratory, as the experimental group, denoted as KO group (MCK-Cre+/BMfl/fl), the control group denoted as WT group (MCK-Cre-/BMfl/fl). According to the different feeding condition,the mice were divided into Fasting group and refed group in the two group.5-6 mice per group, treated with continuous 12:12 light dark cycle, light start at 7:00a.m, denoted by ZTO, for 10 week. Isolated TA, Soleus and GN of hindlimb muscles, liver and hypothalamus of all the mice, with qRT-PCR, Western blotting, immunofluorescence to observe and compare the expression of gene and protein mentioned below:1、the biological clock genes Bmall、Rev-erba; 2、glucose and lipid metabolism related genes MCAD、CPT-1b、LPL、FAS、 Glut4、PDK4. Isolated and cultured primary muscle cells from 4-week growth mice hindlimb muscles in the WT group and the KO group, and compared the expression of the genes Mck、Myf5、Myogenin、MLCland Myh3 by cell immunofluorescence staining and qRT-PCR. Evaluate the quality between the two groups of muscles at 10 weeks with magnetic resonance imaging. Isolated TA and GN of hindlimb muscles, Determined the rate of fatty acid oxidation and glucose oxidation rate of TA and EDL through immunohistochemical staining and Western blot, compare the expression of MyHC IIA and MyHC IIB.Results:The differentiation of primary muscle cell, the expression of the myoblast related genes was not affected and the total body weight accounted for the percentage of muscle and body weight accounted for the percentage of single muscle were not affected. In normal mice, Glut4 protein, Bmall protein expression in soleus muscle and liver increased significantly while feeding after fasting state. In the fasting state, the expression of Rev-erba protein and PDK4 protein in the tibialis anterior muscle and soleus muscle were all increased. In the fed state after fasting, the expression of PDK4 in TA and Soleus in the KO group were significantly increased, but that of the Glut4 protein was decreased; and in the fasting state, the expression of PDK4 and Glut4 protein between the two groups had no significant difference. qRT-PCR analysis of PDK4, MCAD, CPT-lb also showed the same trend (P<0.05). The oxidation of glucose level in skeletal muscle in the KO group decreased but fatty acid oxidation (P<0.05) level increased. Test the expression level of the genes in TA related glucose and fatty acid metabolism at 9AM (ZT2) and 5PM (ZT10) by qRT-PCR analysis of the two groups, the results showed that MCAD and CPT-lb genes related to lipid metabolism at 9AM in the KO group was significantly increased, but PDK4 at 9AM and 5PM were both significantly increased. Immunohistochemical staining and Western blot analysis showed that, muscle fibers MyHC IIA in tibialis anterior (TA) in the KO group increased, but MyHC IIB decreased.Part 2 Effect of skeletal muscle intrinsic clock Bmall ablation on whole body metabolismObjectives:To observe the effect of skeletal muscle selective knockdown Bmall on glucose and lipid metabolism in the whole body, and to further explore the mechanism of obesity.Methods:Mice 8-12week old, selective deletion of Bmall in skeletal muscle as objective, Bmall-/-mice (B6.129-Arntm1Bra/J) provided by the Jackson laboratory, as the experimental group, denoted as KO group (MCK-Cre+/BMfl/fl), the control group denoted as WT group (MCK-Cre-/BMfl/fl). According to the type of diet the KO group and the WT group was divided into normal diet feeding group (Chow) and high-fat diet group (HFD).5-6 mice per group, treated with continuous 12:12 light dark cycle, light start at 7:00a.m, denoted by ZTO. Got the eyeball Venous blood of the WT group and the KO group, and compare the level of serum free fatty acid (FFA), glycerol (Glycerol), triglyceride (Triglyceride) of blood glucose Glucose), cholesterol (Cholestorel) in the two groups in both refed and fasting stain. Isolate the liver of mice, in including refed group, Fasting group, Chow group and HFD group, hepatic triglyceride content determination and extraction of oil-red-O staining, then to compare liver fat content under different conditions of the WT group and the KO group. Compare the Changes of body weight between the WT group and KO group in Chow and HFD for 4-12 week. Compare the Changes of body fat content in the WT group and the KO group for 0,12,24,36 hours after fasting. Test the respiratory exchange ratio, oxygen consumption, the overall level of activity of two groups by Comprehensive Lab Animal Monitoring System (CLAMS).Results:The results showed that in the fasted state (Fasted), the circulating free fatty acids (FFA), glycerol (Glycerol) and triglyceride (Triglyceride) levels in the KO group were significantly decreased, respectively P<0.01, P<0.05, P<0.01; but blood glucose levels were significantly increased, P<0.01; In the fed state after fasting (Refed,4hrs), no significant difference between the two groups above index. Triglyceride content decreased significantly in the fasting state, in the liver in the KO group, P<0.01. Oil-red-O staining of liver section images showed that in the fasting state, liver fat droplets increased significantly of the WT group, while that of the KO group was significantly decreased. Whether it is general food fed or high fat fed, at 9AM, the liver triglyceride (TG) content of the KO group were significantly reduced (P<0.01), Oil-red-O staining of liver section images also showed the same trend. When was high fat fed, weight of the KO group at 12 week decreased significantly, P<0.01; body fat content was significantly decreased at 4 and 12 week, P<0.05, P<0.01 respectively. The body fat amount of KO group is lower than WT group when fasted for 12 hours,24 hours,36 hours, P<0.01, P<0.05, P<0.05 respectively. While there were no significant difference between the two groups in the respiratory exchange ratio, daily food intake, every hour of oxygen consumption, the overall level of activity.Part 3 Nicotine-induced ICAM-1 and VCAM-1 Expression in Mouse Cardiac Vascular Endothelial Cell via p38 MAPK Signaling PathwayObjectives:To explore the effects of smoking on vascular endothelial inflammatory markers ICAM-1 and VCAM-1 and the role of p38 MAPK signaling pathway in which.Methods:Mouse cardiac vascular endothelial cells were isolated and cultured. The passage 3-5 cells were used for the nicotine exposure. Three different concentrations of nicotine,10-5,10-6, and 10-7 M, were used in our research, which demonstrated peak activation of ICAM-1 and VCAM-1 when mouse cardiac vascular endothelial cells were exposed to 10-6 M nicotine for 8 hours. Therefore, a concentration of 10-6 M nicotine was used in subsequent experiments. The cultured mouse cardiac vascular endothelial cells were divided into two groups. One group cells were exposed to 10-6 M nicotine for 6,8,12hours. The other group cells were pretreated for 30 minutes with 10 uM of the p38 MAPK inhibitor (SB203580), then were treated with 10-6 M nicotine like the first group cells. RNA and protein were extracted from these cells, the expression level of ICAM-1 and VCAM-1 in two groups cells were compared by qPCR and Western Blotting.Results:Our results indicate that nicotine can enhance the expression of CAM-1 and VCAM-1 on mouse cardiac vascular endothelial cell via p38 MAPK signaling pathway, resulting in increased expression of the cellular adhesion molecules ICAM-1 and VCAM-1.Conclusions:The study found that genetic ablation of Bmall in muscle causes a metabolic shitt toward FA Oxidation, and reduced uptake and utilization of glucose of muscles, causing insulin resistance, lead to the lack a normal physiological phenomenon that fat metabolism is given priority in rest/fasting phase switch to glucose metabolism is given priority in the activity/feeding phase under normal condition, and lead the changing composition of muscle fiber type. The enhanced fat oxidation in muscle in turn prevents lipid accumulation in liver while promoting fat mobilization from adipose tissue. Due to the excess glycerol supply to liver for gluconeogenesis together with reduced disposal in muscle, glucose level rises in circulation while triglyceride and free fatty acids level drops, and then inhibit the obesity induced by high fat diet.So we can infer that enhanced Bmall function will make the opposite result, that the increase in insulin sensitivity, which led to the increased use of sugar, inhibiting fatty acid oxidation, and body fat excessively accumulation resulted in the formation of fatty liver and obesity.We demonstrate that 10-6 M nicotine maximally enhances mouse cardiac vascular endothelial cell expression of ICAM-1 and VCAM-1 at 8 hours. Our results provide a putative mechanism by which nicotine stimulates expression of these adhesion molecules via p38MAPK signaling pathway.In summary, the lack of Bmall function will lead to insulin resistance and elevate the levels of circulating blood sugar, the enhancing of Bmall function may inhibit fat oxidation and result in increased levels of circulating lipids and body fat accumulation, so whether Bmall function is reduced or enhanced, it can cause abnormal glucose and lipid metabolism to promote the occurrence of atherosclerosis. Nicotine stimulates expression of adhesion molecules via p38MAPK signaling pathway in cardiac vascular endothelial cells, thereby promoting the vascular endothelial cell inflammatory response, leading to atherosclerosis. In addition, we conclude that there may be some connection among the circadian disorders, smoking and metabolic syndrome.That smoke cause inflammation of vascular endothelial, at the same time will affect the body’s metabolism. In this study, the circadian disorders has been proven to have the exactly influence to metabolism. In this study confirmed that smoking can cause adverse reaction via p38MAPK signaling pathways, and previous studies confirmed that p38MAPK signaling pathways can affect the circadian, so smoking may affect the body’s circadian expression via p38MAPK, in turn, affect the body’s metabolism. But this remains to be established.