The Mechanisms Of Varying The Ratios Of N-6/n-3 Polyunsaturated Fatty Acids On Atherogenesis In Apolipoprotein E Deficient Mice

Cardiovascular disease (including hyperlipidemia and atherosclerosis) continues to be the leading cause of morbidity and mortality in the industrialized world. According to the statistical result of the World Health Organization (WHO), the death toll by cardiovascular disease account 30 percent for total death toll in 2001. Moreover, the quantities will be more and more in future. Abnormal serum lipid (especially hyperlipidemia) is one of the main risk factors for atherosclerosis and cardiovascular disease. Improper dietary is the main factor for abnormal serum lipids. The present researches indicated that inflammation ran through all stages of atherosclerosis, which is authorized by researchers.Polyunsaturated fatty acids (PUFAs) which are not synthesized in vivo are the essential fatty acids for human growth and health. PUFAs can be classified in n-3 (omiga-3) fatty acids and n-6 (omiga-6) fatty acids. Both types of fatty acids are precursors of signaling molecules with opposing effects, that modulate membrane microdomain composition, receptor signaling and gene expression. The predominant n-6 fatty acids and its products are important regulators of cellular functions with inflammatory, atherogenic and prothrombotic effects. The n-3 fatty acids antagonize the pro-inflammatory effects of n-6 fatty acids. n-3 and n-6 fatty acids control various genes of inflammatory signaling and lipid metabolism. So, the mounts and the balance of the n-6 and n-3 fatty acids are important for health. The recommendation ratio of n-6/n-3 PUFAs was inconsistent in many countries at present, and there were some disputations. The regulations of lipid metabolism and inflammatory signaling in vivo by n-6 and n-3 fatty acids were unclear. As for the hyperlipidemia and atherosclerosis patients, dietary fatty acids influenced the progress of diseases. The mounts and the balance of n-6/n-3 PUFAs for above patients were also unclear.Apolipoprotein E deficient (apoE-/-) mice which spontaneously develop atherosclerosis with features similar to those observed in humans are the idea model of human atherosclerosis researches. It provides the advantage for the mechanism and intervention of atherosclerosis researches. Therefore, the present experiment observes the anti-atherogenesis effects by dietary different n-6/n-3 PUFAs in apoE-/- mice which similar similar features to those observed in humans type III familial hyperlipoproteinemia. The male mice were randomly assigned to four experimental groups (fed experimental diets) and one control group (fed control diet) at age of 8 wk old (n=12, respectively) for 6 wk and 13 wk. The experimental diets contained 5% (w/w) fat (11.5% total energy), were made by adding safflower oil and perilla seed oil based on the control diet. The n-6/n-3 PUFAs ratios of the experimental diets, for feeding groups 1-4 mice, were 1.28, 5.03, 9.98 and 68.26, respectively. At the end of the feeding period, all mice were sacrificed. The atherogenesis, lipid metabolism and inflammatory response would be analyzed and discussed. We looked forward to providing the dietary fat for humans.The present experiment analyzed the serum lipids, serum cytokines, aortic en face atherosclerotic lesions, liver lipids and explored the possible mechanisms of the apoB100 containing lipoprotein metabolism, high density lipoprotein cholesterol ester (HDL-C) metabolism by different n-6/n-3 PUFAs. The experiment performed using biochemical analysis, enzyme-linked immunosorbent assays (ELISA), gas chromatograph-mass spectrum (GC-MS), ultracentrifugation etc. analytic techniques; real-time RT-PCR, Western-Blot etc. molecular techniques; frozen section and staining etc. pathological morphology techniques. The results showed as follow:1. Different diets on atherosclerotic lesions.The result showed that no significant difference was observed in the growth of mice fed with different experimental diets. The results of atherosclerotic lesions in root and total en face aortic lesions area analysis indicated that the group 3 mice developed smaller area than other groups for 6 wk intervention. However, dietary low ratio of n-6/n-3 PUFAs inhibited significantly the atherosclerotic lesions in root and total en face aortic lesions area compared with the mice that dietary high ratio of n-6/n-3 PUFAs for 13 wk intervention. Moreover, as the dietary ratio of n-6/n-3 fatty acids ascended, so did the areas of aortic lesions.2. The mechanisms of varying the ratios of n-6/n-3 polyunsaturated fatty acids on atherogenesis(1) Different diets on lipid metabolism.①Different diets on serum lipids. After feeding the apoE-/- mice with the diets for 6 wk, no significant differences were found in the serum concentrations of free cholesterol (FC), total cholesterol (TC) and cholesterol ester (CE) among the apoE-/- mouse groups fed with different diets. After feeding the apoE-/- mice with the diets for 6 wk and 13 wk, the serum LDL-C level of group 3 mice was significant lower than those of other groups. The serum of triglyceride (TG) and HDL-C concentration of group 4 mice were significant higher than other groups.②Different diets on liver lipids. The results of liver lipids contents showed that after feeding with the diets for 6 wk, the TC and TG levels of group 1 mice were significant lower than other groups. Moreover, as the dietary ratio of n-6/n-3 fatty acids ascended, so did the TC and TG contents. Dietary low ratio of n-6/n-3 PUFAs significantly decreased TC and TG levels and lipids aggregation in the liver.③Different diets on serum and liver fatty acids. The results of serum and liver fatty acids analysis indicated that as the dietary ratio of n-6/n-3 fatty acids ascended, so did the concentrations of total n-6 fatty acids, arachidonic acid (AA) and 18: 2 n-6 fatty acids in the serum and the liver tissue. Moreover, as the dietary level of n-3 fatty acids declined, the concentration of total n-3 fatty acids and eicosapentaenoic acid (EPA) were reduced in the serum and the liver.④Different diets on apoB100 containing lipoprotein metabolism. After feeding the apoE-/- mice with the diets for 6 wk, the serum ApoB100 level and liverβ-hydroxy-β-methyl glutaryl CoA (HMG-CoA) reductase activity of group 1 mice were significant lower than other groups. Real-time RT-PCR results showed that the transcription levels of genes involved in apoB100 containing lipoprotein metabolism including fatty acid synthase (FAS), HMG-CoA reductase, acyl cholesterol acyl transferase 2 (ACAT2), low density lipoprotein receptor (LDLR), low density lipoprotein receptor related protein (LRP), sterol regulatory element binding protein (SREBP1c), liver X receptor alpha (LXRα) of group 1 and group 2 mice were inhibited significantly compared with the control mice. The hepatic apoB100 mRNA level was increased by the group 4 diet. These results suggested that dietary low n-6/n-3 PUFAs ratios inhibited the synthesis of fatty acids and cholesterol by decreased the enzymes involved in the synthesis of fatty acids and cholesterol. However, at the same time, the expression of LRP and LDLR were inhibited. Thus, it is not benefit to the uptake of VLDL-C and LDL-C.⑤Different diets on HDL-C metabolism. Varying ratios of n-6/n-3 PUFA on HDL-C concentration was assessed with respect to serum apolipoprotein (apo) A-I concentration, endogenous lecithin-cholesterol acyltransferase (LCAT) activities, and mRNA abundance of genes involved in HDL-C metabolism. The results indicated that the group 4 diet significantly increased the HDL-C and apo A-I concentrations in serum compared with the other groups. LCAT activity in serum increased with decreased ratios of n-6/n-3 PUFA. As the dietary ratio of n-6/n-3 fatty acids ascended, so did the mRNA levels of hepatic apo A-I, scavenger receptor B class-1 (SR-B1), LCAT, ATP binding cassette transporter A1 (ABCA1), ABCG1 and LXRα, however, apo A-II mRNA level had a tendency of decline. Group 4 diet up-regulated the apo A-I and ABCA1 and down-regulated the apo A-II transcriptional levels, whereas, group 1 diet down-regulated the mRNA expressions of apo A-I, LCAT, SR-B1 and ABCG1. Our results indicate that high ratio of n-6/n-3 PUFA increases the HDL-C concentration, possibly due to up-regulating the hepatic apo A-I and ABCA1 and down-regulating the apo A-II mRNA levels. Dietary low ratios of n-6/n-3 PUFAs significantly increased the expression of ABCA1 in aorta. Moreover, as the dietary ratio of n-6/n-3 fatty acids descended, the ABCA1 mRNA level increased. The result suggested that low ratios of n-6/n-3 PUFAs were benefit to the cholesterol efflux progress from the aorta.(2) Varying ratios of n-6/n-3 PUFAs diets on inflammatory cytokines in apoE-/- mice.①Serum concentrations of interleukin (IL) -1β, IL-6 and tumor necrosis factor (TNF)αdid not show any statistical difference among the mice fed the different diets. The results of real-time RT-PCR indicated that the group 1 diet inhibited significantly the transcription of pro-inflammatory cytokine IL-6 and TNFαin spleen and CRP in aorta, however, the group 4 diet increased significantly the expression of TNFα, MCP-1 and VCAM-1 in aorta. The expression of IL-4 in spleen and aorta did not show any significance.②In the present study of dietary varying of n-6/n-3 PUFAs ratios and hepatic CRP expression, we observed statistically significant low at mRNA and protein levels in apoE-/- mice fed the low n-6/n-3 PUFA ratios compared with that of the high n-6/n-3 PUFA ratio. Low ratios of n-6/n-3 PUFAs increased the hepatic PPARγmRNA level compared with the high ratio of n-6/n-3 PUFAs, which presumably inhibited the inflammatory status in the liver of mice fed low ratios of n-6/n-3 PUFAs.In summary, the present study showed that dietary varying the ratios of n-6/n-3 PUFAs for 6 and 13 wk influenced the AS development, lipid metabolism and inflammatory response in apoE-/- mice. After 6 wk intervention, dietary low ratio of n-6/n-3 PUFAs inhibited significantly the synthesis of fatty acids and cholesterol in liver and decreased the expression of hepatic apoB100. However, the expression of hepatic LDLR and LRP were also inhibited. Therefore, dietary low ratio of n-6/n-3 PUFAs did not show decreased serum LDL-C level. The present results indicated that there was the same tendency between serum LDL-C concentration and atherosclerotic lesion in root in different groups. The mice of group 3 showed the smallest lesion areas and the lowest LDL-C level. So it suggested that dietary ratio of n-6/n-3 PUFAs (9.98) could inhibited the early stage of AS development via decreasing the serum LDL-C level.However, the results of HDL-C metabolism showed that dietary high ratio of n-6/n-3 PUFAs (68.26) increased significantly HDL-C concentration and Apo A-I, but did not suppress the AS development, suggesting that HDL-C level did not show the anti-atherogenesis in apoE-/-. After 13 wk intervention, the group 3 diet also showed the lowest serum LDL-C, but did not show the lowest atherosclerotic lesions in root and the total en face lesions. On the contrary, the group 1 (low ratio of n-6/n-3 PUFAs) indicated the lowest lesion areas, suggesting that dietary low ratio of n-6/n-3 PUFAs inhibited the AS development independent on the serum lipids at the middle-late stage of AS. In the present study, we analyzed the expression of ABCA1 in aorta, which was responsible for the cholesterol efflux progress from the artery to the liver. The expression of genes involved in the inflammatory response in liver, spleen and artery tissues were also analyzed by real-time RT-PCR. We speculated on the anti-atherogenesis mechanisms that low ratio of n-6/n-3 PUFAs increased the cholesterol efflux progress from aorta and decreased the inflammatory responses and the local inflammatory responses in aorta on the basis of the present results.