Depression Effects On Reverse Cholesterol Transport And Related Molecular Mechanisms

ObjectivesTo establish an experimental rat model of co-occurring atherosclerosis and depression symptomatologic by chronic unpredictable mild stress (UCMS) combined with a high-fat diet, and observative the expression of reverse cholesterol transport(RCT) related gene and protein using the method of Real-time PCR and Western blot.Using liquid chromatography-mass spectrometry (HPLC-MS) determination of each group in rat plasma homocysteine (Hcy) levels. Preliminary discussion on the molecular mechanisms of depression is associated with atherosclerosis.Methods1. The establishment of the modelThe rats were randomized into the control group, UCMS group, high-fat diet group (HFD) and Model group (n=10in each group). Rats in the control group and UCMS group were fed a standard diet, formulated for the maintenance of adult rodents. The HFD and Model group received a modified high-fat but not high-sugar diet, based on a purified moderately kept in standard laboratory conditions and gavage for vitamin D3according to the700000IU·Kg-1three days prior to the experiment. The rats of UCMS group and Model group were housed individually and exposed to the following stressors in a random order every day between9:30and12:00a.m. for8weeks:24h of food deprivation and24h of water deprivation, swimming in4℃cold water for5min or swimming in45℃for5min, spin the tail for lmin, hang the tail for5min, tail clip for30min, strain for8h. No animals died in the whole process.2. Behavioral observationBefore the end of the experiment, to observe the behaviour including food consumption, sucrose preference and open-field test.3. Materials and processingIntraperitoneal injection of10%chloral hydrate anesthesia (3.5ml· kg-1), recumbent position fixed rats and laparotomy, using10ml syringe blood from the abdominal aorta. After blood collection liver tissue was immediately removed and weighed. Apart of liver was snap-frozen in liquid nitrogen and stored an-80℃for RNA extraction and Western blot analysis;the remaining part and aorta were fixed in a4%poly-formaldehyde solution and processed for paraffin embedding and sectioning.4. Biochemical analysisBlood samples were collected under chloral hydrate anesthesia and centrifuged at3500rpm for15minutes by a refrigerated centrifuge, and the serum was transferred into a separate vial and stored at4℃. Serum concentrations of total cholesterol (TC), total triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT), aspertate aminotransferase (AST), alkaline phosphatase (ALP), albumin (ALB) and globulin (GLB) were measured by enzymatic assays using an automated biochemical analyzer. According to the kit instruction steps, detection of superoxide dismutase (SOD) and malondialdehyde (MDA) content.5. Histology of abdominal aorta and liverAfter fixed in a4%poly-formaldehyde solution embedded in paraffin wax, aorta and liver were cut into5-μm-thick transverse sections and stained with hematoxylin-eosin (HE).The measurements of intima thickness, media thickness, total intima-media thickness (IMT), and wall thickness were made in HE stained sections of abdominal aorta under a microscope using Image Pro Plus6.0software.6. LXRa protein expression in liver tissueReference SP immunohistochemical kit instructions, immune liver biopsy for dewaxing to water, closed, serum antibody, antigen repairing, nuclei are complex and dehydration, random determination of five horizons, under a microscope with Image-Pro Plus analysis system to determine the average optical density (IOD) value.7. Reverse cholesterol transport related gene and protein expressionRespectively, the Real-time PCR and Western blot test technology, detection of rat liver LXRa、SR-B I、ABCG5. ABCG8、LCAI\CPY7A1mRNA and protein expression.8. Plasma homocysteine content determinationThe liquid chromatography-mass spectrometry (HPLC-MS) technology in the determination of plasma homocysteine (Hcy) levels. Conditions of chromatographic spectrometry using gradient elution, conditions of mass spectrometry using electrospray ion source (ESI), positive ion scanning,3,4-2hydroxy benzyl amine (DHBA) as internal standard.9. Statistical analysisThe results are presented as mean±SEM and the application of SPSS13.0statistical software for statistical analysis. Statistical significance was determined by ANOVA, a p value of less than0.05was considered statistically significant.Results1. Behavioural indicators observationsCompared with control group, UCMS group, HFD group and Model group significantly reduced food consumption; UCMS group and Model group rats sucrose preference decreased significantly, HFD group of sucrose preference is decreased, but the result is no statistical significance;Stressed rats from the UCMS and Model groups exhibited significantly less horizontal and vertical scores compared with controls, and rats in Model group got much less scores both in horizontal and vertical motion than in UCMS group.2. Biochemical analysisThe level of TC, TG and LDL in serum of both HFD and Model group is remarkably higher than Control group, while HDL is much lower, UCMS alone showed a little effect on lipid level. Compared with Control group, Model group showed a higher level of ALT and AST,UCMS and HFD groups of ALT and AST are increased, but compared with control group, no significant difference; Specifically, compared with the concentrations of ALP, ALB and GLB in control group, respectively, rats had significantly higher levels of ALP, ALB and GLB in HFD group and Model group are both increase. What’s more, rats in HFD group and Model group had a lower level of SOD in serum, while UCMS group and Model group had a higher level of MDA compared with Control group.3. Histological changes of abdominal aorta and liverChronic unpredictable mild stress combined with a high-fat diet promotes the formation of unstable aortic plaques and liver fat deposits.4. LXRa protein expression in liver tissueCompared with control group, HFD, UCMS and Model groups protein expression of LXRa were decreased significantly, the model group decreased more significantly.5. Reverse cholesterol transport related gene and protein expressionCompared with control group, UCMS enhanced mRNA expressions of ABCG8, ABCG5, SR-B I, and LXRa, and showed little effect on CYP7A1and LCAT while HFD reduced all these tested genes’expression. We also found that Model group seemed to neutralize the effects of UCMS and HFD, Model group had up-regulated mRNA expression of SR-B I and LXRa and down-regulated mRNA expression of ABCG8, ABCG5, CYP7A1and LCAT. These results were confirmed by western blotting.6. Plasma homocysteine content determinationUCMS group compared with Control group, in rat plasma homocysteine was increased, but the degree of increase is not obvious; HFD group in rat plasma homocysteine significantly decreased, while the model group was significantly higher.ConclusionThis study used chronic unpredictable mild stress (UCMS) combined with a high-fat diet to establish an experimental rat model, body weight, food intake and sucrose preference significantly less than normal control group, and open-field test shows that rats activity decreased, and curiosity about the outside world is abate, artery of rats appeared early symptoms of hardening of the arteries, the endothelial cells swelled, lining appear different degree of thickening, elastic fiber structure is not clear, show that successfully established an experimental rat model of co-occurring atherosclerosis and depression symtomatology.Reverse cholesterol transport related gene and protein expression in rats which feeding high-fat diet, compared with the simple chronic stress rats decreased significantly, chronic stress can significantly affect the liver lipid metabolism in rats only after joint on a high-fat diet.Chronic stress joint on a high-fat diet can make the model in rat plasma homocysteine levels increased significantly, just give a chronic unpredictable mild stress in rats plasma homocysteine levels in fall.