The Study Of Varying The Ratios Of DHA/EPA On Atherosclerosis In Apolipoprotein E Deficient Mice

Objective:To investigate the protective roles and potential mechanisms of different ratios of DHA/EPA against atherosclerosis in Apo E-/- mice.Methods:1. 90 male Apo E-/- mice(6 weeks of age,18 ±3 g) were divided randomly into six groups of fifteen animals each group as follows:Normal diet group(ND),High fat diet group(HFD), HFD+ALA 1mg/g bw(ALA), HFD+DHA/EPA=2 ∶ 1 1mg/g bw(DHA/EPA=2 ∶ 1), HFD+DHA/EPA=1 ∶ 1 1mg/g bw(DHA/EPA=1 ∶ 1), HFD+DHA/EPA=1∶2 1mg/g bw(DHA/EPA=1∶2).15 wildtype(CT,C57BL/6J) mice were considered to be the control group.High fat diet was comprised of 1.25% cholesterol and 21% fat.Mice in all groups were given a diet for 12 weeks.2. After 12 weeks of SPF animal room feeding,all mice were killed by drawing the blood from eyeball.The blood samples were collected for serum lipid and antioxidant capacity detection.Serum lipid levels(TC,TG,HDL-C and LDL-C),antioxidant indexs(MDA,SOD,GSH and T-AOC),inflammatory cytokines(TNF-α and IL-1β) and ox-LDL levels were measured in mice.The level of ROS in aortic tissue of mice was determined by DHE staining.The levels of Mac-3 and ox-LDL in aortic tissue of mice were determined by immunohistochemistry.Besides,the m RNA levels of inflammatory cytokines(IL-6,IL-10,IL-1β,TNF-α,MCP-1 and VCAM-1),scavenger receptors(CD36,MSR-1 and LOX-1) and reverse cholesterol transport-related genes(ABCA-1,ABCG-1,ACAT-1 and LAL) in liver and aortic tissue of mice were quantified with RT-PCR methodology.The protein expression levels of scavenger receptors(CD36,MSR- 1 and LOX-1) in liver and aortic tissue of mice were detected by western blot.Result:1. The intact aorta of mice stained with Oil Red O showed that compared with the CT group and the ND group,the aortic plaque area in HFD mice were significantly increased;compared with the HFD group,different ratios of DHA/EPA intervention could significantly reduce the AS plaques induced by high fat diets(P<0.01).The aortic arch stained with Oil Red O and hematoxylin and eosin(H&E) showed that different ratios of DHA/EPA intervention can relieve AS symptoms in mice. Immunohistochemistry showed that different ratios of DHA/EPA intervention can reduce the expression levels of plaque macrophages(Mac-3) and oxidized low-density lipoprotein(ox-LDL). TEM results showed that different ratios of DHA/EPA intervention can significantly reduce the number of lipid droplets in macrophages.2. Compared with the ND group,the levels of TC, TG,LDL-C and MDA in serum in HFD group were significantly increased(P<0.01);compared with the HFD group,different ratios of DHA/EPA intervention can alleviate HFD-induced rise of them(P<0.05).Compared with the HFD group,different ratios of DHA/EPA intervention group can significantly increase the levels of HDL-C,SOD,GSH and T-AOC in serum(P<0.05).Different ratios of DHA/EPA intervention can inhibit the expression of ROS in aorta(Derected by ROS probe DHE)(P<0.01).3. Compared with the HFD group,different ratios of DHA/EPA intervention can significantly reduce the expression levels of inflammatory cytokines(TNF-α and IL-1β) in serum(P<0.01).The results of RT-PCR showed that:different ratios of DHA/EPA intervention can effectively inhibit the m RNA expression of the proinflammatory cytokines(IL-6,IL-1β and TNF-α) and monocyte chemotactic protein(MCP-1) and vascular cell adhesion molecules(VCAM-1) in liver and aorta tissue(P<0.05),while promote the m RNA expression of anti-inflammatory cytokine(IL-10)(P<0.05).4. Compared with the ND group,the m RNA and protein expression of scavenger receptors(CD36,MSR-1 and LOX-1) were both significantly increased in the liver and aorta tissue in HFD group(P<0.05).Compared with the HFD group,different ratios of DHA/EPA intervention can inhibit the increased gene and protein expression levels of CD36,MSR-1 and LOX induced by high fat diet(P<0.05);and different ratios of DHA/EPA intervention can promote the m RNA expression of reverse cholesterol transport-related genes ABCA-1,ABCG-1,ACAT-1 and LAL in liver and aortic tissue(P<0.05).Conclusion:Different ratios of DHA/EPA may play protective role on the high fat-induced AS through the lipid metabolism,oxidative damage,inflammation cytokines and the regulation of scavenger receptors.In which,DHA/EPA= 2∶1 group may primarily play a protective effect on AS by regulating lipid metabolism, oxidative damage and inflammatory pathways;DHA/EPA = 1∶1 group may primarily play a protective effect on AS by adjusting the scavenger receptors.