Melatonin In Rat Blood Sugar, Blood Lipid Metabolism And Atherosclerosis Related Factors And Mechanism Research

Background:Melatonin (Mel) is an indole-like hormone that synthesized and secreted by pineal gland, with a secretion rhythm controlled by the light-dark cycle (intensity of the light). Researches have found that Mel has a variety of cardiovascular relevant effects, such as the inhibition of oxidants and regulation of blood lipid and glucose metabolism. It has been reported that the decrease of Mel concentration is prominently related with the development of atherosclerotic diseases, and with the increase of the blood lipid and glucose-associated disorders. However, it is unclear whether the alternation of in vivo Mel concentration will lead to such dysmetabolisms. Endothelial inflammation participates in the development of cardiovascular diseases including atherosclerosis (As). Several publications presented a correlation between Mel and vascular inflammation and dysfunction. Nevertheless, mechanism of the effect of Mel on endothelial inflammation remains to be elucidated. Atherosclerotic lesions are typically infiltrated by macrophage-derived foam cells, in which cholesterol esters are overloaded. ATP-binding cassette transporter Al (ABCA1), which expressed on macrophages, plays critical roles in mediating the reverse cholesterol efflux to maintain intracellular cholesterol homeostasis. Studies indicated that the dysfunction of ABCA1mediated cholesterol efflux to apolipoprotein A1was closely associated with As. ATP-binding cassette transporter G1(ABCGl) mediated cholesterol efflux shown to be coordinated to ABCA1in removal of excessive cellular cholesterol, which also plays a role in the development of As. The mechanisms of Mel deficiency on regulating ABCA1and ABCG1expression and their mediated cholesterol efflux function, however, are still unknown. Therefore, we created melatonin-deficiency animal models and performed a study consist of three parts to answer those questions above.Part I. Creation of Melatonin-Deficiency Rats and the Metabolic Characteristics of their Serum Lipid and Glucose ProfilesObjective:1. To create melatonin-deficiency animal models in rats.2. To analyse the metabolic characteristics of their serum lipid and glucose profiles in melatonin-deficiency rats. Methods:1. Thirty-six Wistar rats, male,10-week old were included. Melatonin-deficiency (Px) rats were created by pinealectomy. Nocturnal levels of melatonin before and after surgery were measured and compared with those levels in sham-operated (Con) rats to evaluate the success of surgery.2. Serum lipid and glucose profiles in Px rats were measured at the0week (baseline), and the4th,8th,16th week after surgery respectively, and compared with those profiles in Con rats at each time-point.3. The contents of total cholesterol and triglyceride in liver tissue were measured at the16th week after surgery respectively, and compared with those in Con rats.Results:1. Nocturnal serum level of melatonin in Px group (N=16) was significantly diminished after surgery, compared with that in Con group (N=14)(8.19±2.05ng/L vs.39.98±5.91ng/L, P<0.001, respectively), suggesting a melatonin deficiency state in pinealectomized rats.2. Comparison of serum lipid and glucose metabolic profiles between Px and Con rats at the0week(baseline), and the4th,8th,16th week after surgery were listed below:1. Lipid metabolic profile:No significant differences of serum lipid levels at the baseline were observed between the Px (N=16) and Con (N-14) groups. At the4th week after surgical operation, serum TG, VLDL-C and FFA in Px group were all significantly increased compared to those in Con group at4th week(all P<0.05), and continued to be elevated until the16th week (all P<0.05). The changes of serum TG, VLDL-C and FFA levels from baseline at the16th week in Px group were all significantly higher than those in control group (all P<0.05for the between-group comparison).2. Glucose metabolic profile:No significant differences of serum glucose-relevant parameters at the baseline were observed between the Px (N=8) and Con (N=7) groups. The serum glucose level at the16th week was significantly elevated, with the significant decrease of ISI index in Px group, compared with that in Con group (P<0.05). However, the INS level in Px group had no significant difference from that in Con group. The changes of serum glucose levels, INS and ISI from baseline at the16th week in Px group were all significantly higher than those in Con group (all P<0.05for the between-group comparison).3. The average content of triglyceride in the liver tissue of the Px group at16th week was significantly higher than that in Con group (P<0.01). However, The average content of total cholesterol in the liver tissue in Px group had no significant difference from that in Con group.Part II. Changes of Inflammatory Cytokines in Pinealectomized Melatonin-Deficiency Rats and the Underlying MechanismsObjective:1. To investigate the effect of melatonin on in-vivo inflammation and expression of aortic inflammatory cytokines in melatonin-deficiency rats.2. To study the possible mechanisms of melatonin in regulating the expression of inflammatory cytokines on endothelial cells.Methods:1. Wistar rats, male,10-week old were included. Eight melatonin-deficiency (Px) rats were created and compared with seven sham-operated (Con) rats (See Part I).2. Serum oxidative stress and inflammatory biomarkers MDA, oxLDL, TNF-a, IL-6and CRP in Px rats were measured at the0week and16th week after surgery respectively, and compared with those levels in Con rats. IHC stain of the aorta in each group was performed and quantified to analyse the expression of inflammatory cytokines MCP-1, ICAM-1、VCAM-1and MMP-9in endothelium.3. Rat aortic endothelial cell lines (RAECs) were pre-incubated with oxLDL and treated by melatonin in vitro. The protein expression of inflammatory cytokines and phosphorylation levels of relevant signal pathways including NFκB, P38-MAPK, ERK and JNK were determined.Results:1. Serum levels of MDA, oxLDL, TNF-α, IL-6and CRP in Px group (N=8) at the16th week were all significantly increased, compared with those in Con group (N=7) respectively (all P<0.01). The change from baseline at the16th week for each above-mentioned parameter in Px group was significantly higher than that in Con group (all P<0.05for the between-group comparison). 2. The IOD values of MCP-1, VCAM-1and MMP-9in Px group (N=8) were all significantly elevated compared to those in Con group (N=7)(all P<0.05). However, ICAM-1expression was not significantly changed compared to that in Con group.3. Melatonin dose-dependently attenuated oxLDL-induced MCP-1, VCAM-1and MMP-9expressions on RAECs (all P<0.05). It also dose-dependently decreased NFκB and P38-MAPK protein phosphorylation levels (all P<0.05), but the phosphorylation levels of ERK1/2and JNK were not affected significantly by melatonin.Part Ⅲ. Alternations of Cholesterol Efflux of Peritoneal Macrophages in Pinealectomized Melatonin-Deficiency Rats and the Underlying MechanismsObjective:1. To investigate the function of intercellular cholesterol efflux in macrophages from melatonin-deficiency rats.2. To study the expressions of ABCA1and ABCG1on macrophages from melatonin-deficiency rats.3. To study the mechanisms of melatonin in regulating the expression of ABCA1on macrophages.Methods:1. Wistar rats, male,10-week old were included. Sixteen melatonin-deficiency (Px) rats were created and compared with sixteen sham-operated (Con) rats (See Part Ⅰ).2. Peritoneal macrophages were collected from Px and Con rats at0week and16th week respectively, and cultured in vitro.3. Their macrophage cholesterol efflux rates of Px and Con groups at0week and16th week were measured respectively.4. Their ABCA1, ABCG1mRNA and protein expression levels of the two groups at the16th week were determined respectively.5. THP-1derived macrophages were cultured and treated by melatonin in vitro. The mechanism of melatonin in regulating ABCA1protein expression was evaluated using different signal pathway/receptor antagonists.Results:4. No differences of cholesterol efflux rates of peritoneal macrophages at the baseline were observed between the Px (N=6) and Con (N=6) groups. The differences of serum-induced cholesterol efflux rates (-7.29±5.91%vs.-3.80±4.79%, P<0.01) and ABCA1-induced cholesterol efflux rates (-4.21±4.82%vs.-1.20±2.12%, P<0.01) between0week and16th week in Px group were both significantly lowered, compared to those in Con group respectively. However, those differences of ABCG1-induced cholesterol efflux rates showed no significance between the two groups.5. The ABCA1mRNA expression level of the peritoneal macrophages was significantly decreased in Px group (N=6) at16th week, compared to that in Con (N=6) group (P<0.001). However, the protein expression level of ABCA1showed no significant differences between the two groups.6. Neither the mRNA expression level (N=6) nor the protein expression level of ABCG1on peritoneal macrophages showed significant differences between the two groups at the16th week.7. Melatonin dose-dependently augmented ABCA1and LXRa expressions on the cultured THP-1derived macrophages (all P<0.05). The use of PPARy and LXRa antagonists both significantly attenuated melatonin-induced ABCA1expression. However, the use of melatonin membrane receptor antagonists showed no significant influences on melatonin-induced ABCA1expression on macrophages.Conclusions:Melatonin-deficiency rats were successfully created by pinealectomy. Melatonin deficiency initiates blood lipid and glucose-associated disorders, augments in vivo inflammatory state and the expression of aortic inflammatory cytokines in pinealectomized rats. Melatonin regulates these inflammatory cytokines expressions probably via NF-κB and P38-MAPK involved pathways. Melatonin deficiency inhibits the function of cholesterol efflux and ABCAl gene expression of peritoneal macrophage from pinealectomized rats. The PPARy-LXRa pathways were involved in the regulatory effect of melatonin on ABCA1protein expression, with a mechanism independent of melatonin membrane receptors.