| BackgroundAtherosclerosis (AS), which is a pathological process based on endothelial dysfunction and abnormal lipid metabolism, characterized by vascular chronic inflammation, can lead to myocardial infarction, stroke and other diseases of serious harm to human health and life. Oxidative stress and inflammation plays a pivotal role in all stages of atherosclerosis from endothelial dysfunction and plaque formation to plaque destabilization and disruption. Oxidative modification of low density lipoprotein (LDL) is considered as a key event in the development of atherosclerosis. Oxidized LDL (oxLDL) is apparently involved in the atherogenic process of the vascular wall such as endothelial dysfunction, macrophage adherence and production of inflammatory cytokines. Therefore, as well as decreasing cholesterol levels, anti-inflammatory and anti-oxidation is very important for the anti-atherosclerotic process.Studies have demonstrated that high-density lipoprotein (HDL) is considered as an anti-atherogenic lipoprotein. In addition to the ability of promoting cholesterol efflux, the anti-oxidant and anti-inflammatory properties of high-density lipoprotein (HDL) are important as well. To improve its anti-inflammatory and antioxidant functions is important for slow the progression of atherosclerosis. Paraoxonase-1(PON1), which is a HDL-associated enzyme, was capable of inhibiting LDL from oxidative modification, preventing the accumulation of lipid hydroperoxides in LDL, which play an important role in anti-atherosclerosis. Myeloperoxidase (myeloperoxidase, MPO), which is a strong oxidative enzyme, can impair ability of HDL to promote cholesterol efflux, anti-oxidation and shift HDL from an anti-inflammatory state to a dysfunctional and proinflammatory state. Apolipoprotein AI (apoA-Ⅰ) is the main protein in HDL, accounting for about70%of the protein content, and play a key role in promoting reverse cholesterol transport (RCT), as well as anti-inflammatory, antioxidant, anti thrombosis and endothelial protection function. High-density lipoprotein inflammatory index(HII) is an important markers that inflect the the inflammatory/anti-inflammatory properties of HDL. Improving the anti-inflammatory properties of HDL maybe play a more significant role than only increasing HDL-C levels. Furthermore, in recent years it has become increasingly apparent that measures of the quality and novel functions of HDL might provide an improved means of identifying subjects at increased risk for atherosclerotic events, compared with only measuring HDL-cholesterol levels.Statins, currently used in the clinic for lipid-lowering, could alleviate atherosclerosis and maintain the stabilizations of atherosclerotic plaque, reduce CV morbidity and mortality. The major target of statins in anti-atherosclerosis is lowering low density lipoprotein-cholesterol(LDL-C) levels, and with mildly elevated high density lipoprotein-cholesterol(HDL-C). Nonetheless, cardiovascular disease remains the number one cause of death. Accordingly, considerable efforts have focused on development of novel therapeutic agents designed to address residual cardiovascular risk. Because HDL is considered as an anti-atherogenic lipoprotein, its quality and function are also attractive targets for emerging therapies. ApoA-I mimetic peptide L-4F, a peptide with only18amino acid residues that lacked sequence homology with apoA-I but contained a class A amphipathic helix, like that found in apoA-I. L-4F had no significant effect on plasma lipid levels, but it can promote reverse cholesterol transport from macrophages, markedly reduce atherosclerosis, inhibition secretion of inflammatory cytokines and improve diastolic function of blood vessels in patients with hyperlipidemia. However, there are few studies about observing on the effects of simvastatin combination with L-4F on the anti-oxidant and anti-inflammatory properties of HDL and the progression of atherosclerosis.Therefore, these studies were designed to determine that the effects of simvastatin, L-4F and the combination of simvastatin with L-4F on the anti-oxidant and anti-inflammatory properties of HDL and the progression of atherosclerosis.ObjectivesApoE knockout mice fed with a high-fat diet were chosen as a AS model to identify that the combination of simvastatin with L-4F provides a signifioantly better improvement in the anti-oxidant and anti-inflammatory function of HDL and a more markedly reduction in atherosclerosis than monotherapy with either in apoE-/-mice and to explore a particularly potent treatment strategy to render the anti-oxidant and anti-inflammatory properties of HDL and reduce atherosclerosis.Methods1. Animal studiesEighteen8weeks old C57BL/6J mice were purchased from Southern Medical University. Fifty-four apoE-/-mice on a C57BL/6J background,8-weeks age, were purchased from Peking University (Beijing, China). C57BL/6J mice were fed with normal diet as a control group, and apoE-/-mice were fed a high-fat Western-type diet (21%fat and0.15%cholesterol).4weeks later,6mice were sacrificed in the two kinds of mice. Then the rest of apoE-/-mice fed the original high-fat diet were randomly divided into four groups:atherosclerosis(AS) group, simvastatin group(5mg/kg-d), L-4F group(1mg/kg-d) and the combination group. In the8and16weekend,6mice were sacrificed in each group.2. Plasma lipid and lipoprotein analysis After4,8or16weeks, mice were anaesthetized by ethyl ether. Blood samples were obtained by cardiac puncture. Plasma lipid and lipoprotein levels were determined using an automated clinical chemistry analyzer(OLYMPUS AU5421).3. Assessment of the anti-oxidant and anti-inflammatory function of HDLAfter4,8or16weeks, blood samples were obtained. Serum PON1activity was measured by using phenylacetate as a substrate. Serum MPO activity were tested by chromatometry kit according to the manufacturer’s instructions. High-density lipoprotein inflammatory index (HII) were determined by the CFA assay.4. Measurement of serum hs-CRP levelsAfter4,8or16weeks, blood samples were obtained. Serum hs-CRP levels were measured using enzyme-linked immunosorbent assay (ELISA) kits (Cusabio Biotech Co.,Ltd, China) according to the manufacturer’s instruction.5. PON1mRNA and protein expression in liverAfter4,8or16weeks, liver tissues of mice were obtained. Quantitative real-time PCR was performed to detected PON1mRNA and protein expression in liver. Data analysis was performed using the2-AACT method, where β-actin was used as a reference gene. The protein expression of PON1in liver were detected by immunohistochemistry. The ratio of positive cells was analyzed by Image-pro plus6.0software.6. Aortic atherosclerosis for histological examination and quantitationAfter4,8or16weeks, the entire aorta tissues of mice were obtained. The aortic intima-media thickness were examinated by HE staining. Plaque size of the aorta were displayed by oil red O staining.7. Statistical analysisData are presented as mean±SD, unless otherwise specified. SPSS for Windows (version13.0), was used for statistical analysis. The normal distribution of our data had been checked by SPSS program. For comparison of two groups, a two-tailed Student’s t-test was used as appropriate. Intergroup differences were analyzed by one-way ANOVA. Correlations between groups were determined by Spearman’s correlation test. In all cases, differences of P<0.05were considered to be statistically significant.Results1. Serum lipid levels in experimental miceComparison within each group at different time points:In control group, TC、TG、LDL-C and HDL-C were not changed at4th,8th, and16th weekend(all P>0.05).In AS group:compared with that at the4th weekend, all of TC, TG and LDL-C at the8th and16th weekend were increased(P<0.05or P<0.01);TC, TG and LDL-C at the16th weekend were higher than that at the8th weekend(all P<0.01). There was no significant statistical difference in HDL-C between the4th and8th weekend(P>0.05). Compared with its the8th weekend, HDL-C at the16th weekend was significantly decreased(P<0.01).In simvastatin group, compared with that at the8th weekend, there were significantly decreased TC and TG(P<0.05and P<0.01) but increased HDL-C(P<0.01) at the16th weekend; there was no significant statistical difference in LDL-C(P>0.05).In L-4F group, compared with that at the8th weekend, there were significantly increased TC, TG and LDL-C(all P<0.01) but decreased HDL-C at the16th weekend(P<0.01).In simvastatin+L-4F group, compared with that at the8th weekend, there was no significant statistical difference in TC, TG, LDL-C and HDL-C(all P>0.05).Comparison among different groups at the same time point:Compared with control group, TC, TG and LDL-C of AS group increased at the4th,8th and16th weekend(P<0.05or P<0.01), HDL-C was not changed(P>0.05). Compared with AS group, TC and TG of simvastatin group decreased at8th weekend(P<0.05or P<0.01), but LDL-C and HDL-C were not changed(P>0.05); TC, TG and LDL-C of the simvastatin group were decreased(P<0.05or P<0.01) but were higher than those of control group (all P<0.01). Compared with concurrent AS group, HDL-C of the simvastatin group at the16th weekend was increased(P<0.05), but there was no difference in HDL-C between simvastatin group and control group at the16th weekend(P>0.05).Compared with AS group, TC, TG, LDL-C and HDL-C of L-4F group were not changed at8th and16th weekend(P>0.05).Compared with control group, TC, TG, LDL-C and HDL-C of simvastatin+L-4F group were increased at the8th weekend(all P<0.01); Compared with the AS group, TC and LDL-C of simvastatin+L-4F group were decreased at the8th weekend(P<0.05or P<0.01); Compared with the simvastatin group, TC, TG, LDL-C and HDL-C of the combination group were not changed at8th weekend(P>0.05); Compared with the L-4F group, TC and LDL-C of simvastatin+L-4F group were decreased at the8th weekend(P<0.01, P<0.05).Compared with control group, TC, TG and LDL-C of simvastatin+L-4F group were increased at16th weekend(all P<0.01), but HDL-C was not changed(P>0.05); Compared with AS group, TC, TG and LDL-C of simvastatin+L-4F group decreased at16th weekend(P<0.05or P<0.01); Compared with simvastatin group, TC, TG, LDL-C and HDL-C of the combination group were not changed at16th weekend(P>0.05); Compared with L-4F group, TC and LDL-C of simvastatin+L-4F group decreased at16th weekend(P<0.01, P<0.05).2. Effect of simvastatin, L-4F and the combination therapy on the anti-oxidant and anti-inflammatory function of HDL2.1Comparison on serum PON1activities among the five groups Comparison within each group at different time points;In control group:there was no significant difference in serum PON1activity between the4th weekend and the8th weekend(P>0.05); serum PON1activities at the16th weekend was significantly lower than that at the4th and8th weekend(P<0.01). In AS group, with the progress of time, serum PON1activities was gradually decreased(all P<0.01). In simvastatin group, L-4F group and simvastatin+L-4F group, serum PON1activities at the16th weekend was significantly lower than that at the8th weekend(all P<0.01).Comparison among different groups at the same time point:There was no significant difference in serum PON1activity between control group and AS group at the4th weekend(P>0.05). But at the8th andl6th weekend, serum PON1activity in AS group were significantly lower than concurrent control group(P<0.01).Compared with concurrent AS group, Serum PON1activity in simvastatin group were increased at the8th and16th weekend(P<0.01), but still lower than the concurrent control group (P<0.01).Compared with concurrent AS group, serum PON1activity in L-4F group was increased (P<0.05), but still lower than the concurrent control group (P<0.05). There was no significant difference between L-4F group and concurrent simvastatin group(P>0.05).At the8th weekend, PON1activity in simvastatin+L-4F group was significantly higher than concurrent AS group (P<0.01), and there was no significant difference between simvastatin+L-4F group and all of the concurrent control group, simvastatin group and L-4F group(P>0.05). At the16th weekend, PON1activity in Simvastatin+L-4F group was significantly lower than the concurrent control group (P <0.01), but significantly higher than concurrent AS group, simvastatin group and L-4F group (P<0.01, P<0.05and P<0.05).2.2Comparison on serum MPO activities among the five groupsComparison within each group at different time points:In control group, there was no difference in serum MPO activity among the three time points(P>0.05). In AS group, with the progress of time, serum MPO activities was gradually increased(P<0.01). In simvastatin group, L-4F group and simvastatin+L-4F group, there was no difference in serum MPO activity between the8th weekend and the16th weekend (all P>0.05).Comparison among different groups at the same time point:The difference in Serum MPO activity between AS group and concurrent control group at4th weekend was not statistically significant(P>0.05). But at8th and16th weekend, Serum MPO activity in AS group were significantly increased compared with control group (P<0.01).Serum MPO activity in simvastatin group at the8th and16th weekend was significantly reduced compared to concurrent AS group(P<0.01), but still higher than the concurrent control group(P<0.01).Serum MPO activity in L-4F group at the8th and16th weekend was decreased compared with concurrent AS group(P<0.01), but still higher than concurrent control group (P<0.05). Compared with concurrent simvastatin group, the difference was not statistically significant (P>0.05).At the8th weekend, MPO activity in simvastatin+L-4F group was significantly lower than concurrent AS group and L-4F group(P<0.05), but still higher than concurrent control group(P<0.01). Compared with concurrent simvastatin group, the difference was not statistically significant (P>0.05). At the16th weekend, MPO activity in Simvastatin+L-4F group was significantly lower than concurrent AS group, simvastatin group and L-4F group (P<0.01, P<0.05and P<0.01), but still significantly higher than the concurrent control group (P<0.01).2.3Comparison on serum HII among the five groupsComparison within each group at different time points:In control group:there was no significant difference in serum HII between the4th weekend and the8th weekend(P>0.05); serum HII at the16th weekend was significantly higher than that at the4th and8th weekend(P<0.01). In AS group, with the progress of time, serum HII was gradually increased(all P<0.01). In simvastatin group and L-4F group, compared with that at the8th weekend, there were significantly decreased HII(P<0.05and P<0.01) at the16th weekend. In simvastatin+L-4F group, compared with that at the8th weekend, there was no significant statistical difference in HII at the16th weekend(P>0.05).Comparison among different groups at the same time point:The difference in Serum HII between AS group and concurrent control group at4th weekend was not statistically significant(P>0.05). But at8th and16th weekend, Serum HII in AS group were significantly increased compared with control group (P<0.01).Serum HII in simvastatin group at the8th and16th weekend was significantly reduced compared to concurrent AS group(P<0.01), but still higher than the concurrent control group(P<0.01).Serum HII in L-4F group at the8th and16th weekend was decreased compared with concurrent AS group(P<0.01and P<0.05), but still higher than concurrent control group(P<0.05). Compared with concurrent simvastatin group, the difference was not statistically significant (P>0.05).At the8th weekend, HII in simvastatin+L-4F group was significantly higher than concurrent control group (P<0.05), but still lower than concurrent AS group (P<0.01). Compared with concurrent either simvastatin group or L-4F group, the difference was not statistically significant(P>0.05). At the16th weekend, HII in Simvastatin+L-4F group was significantly lower than concurrent AS group, simvastatin group and L-4F group (P<0.01, P<0.05and P<0.01). Compared with concurrent control group, the difference was not statistically significant (P>0.05).3. Effect of simvastatin, L-4F and the combination therapy on serum hs-CRP levelsComparison within each group at different time points:In control group:there was no significant difference in serum hs-CRP level between the4th weekend and the8th weekend(P>0.05); serum hs-CRP level at the16th weekend was significantly higher than that at the4th and8th weekend(P<0.01and P<0.05). In AS group, with the progress of time, serum hs-CRP level was gradually increased(all P<0.01). In simvastatin group, L-4F group and simvastatin+L-4F group, compared with that at the8th weekend, there were significantly decreased hs-CRP levels at the16th weekend(all P<0.01).Comparison among different groups at the same time point:The difference in serum hs-CRP levels between AS group and concurrent control group at4th weekend was not statistically significant(P＞0.05). But at8th and16th weekend, Serum hs-CRP levels in AS group were significantly increased compared with control group (P<0.01).Serum hs-CRP levels in simvastatin group at the8th and16th weekend was significantly reduced compared to concurrent AS group(P<0.01), but still higher than control group at the16th weekend(P<0.01). Compared with concurrent control group, the difference at the8th weekend was not statistically significant (P>0.05).Serum hs-CRP levels in L-4F group at the8th and16th weekend was decreased compared with concurrent AS group(P<0.05). Compared with either control group or simvastatin group at the8th weekend, the difference was not statistically significant (P>0.05). At the16th weekend, Serum hs-CRP levels in L-4F group still higher than concurrent control group(P<0.01). Compared with concurrent simvastatin group, the difference at the16th weekend was not statistically significant (P>0.05).At the8th weekend, Serum hs-CRP levels in simvastatin+L-4F group was significantly lower than concurrent AS group (P<0.01). Compared with concurrent control group, simvastatin group or L-4F group, the difference was not statistically significant(P>0.05). At the16th weekend, serum hs-CRP levels in Simvastatin+L-4F group was lower than concurrent AS group, simvastatin group or L-4F group (P<0.01, P<0.01and P<0.05), but still significantly higher than concurrent control group(P<0.01). 4. Effect of simvastatin, L-4F and the combination therapy on PON1mRNA expression in liverComparison within each group at different time points:In control group, there was no significant difference in PON1mRNA expression in liver among the three time points(all P>0.05). In AS group, PON1mRNA expression in liver at the8th and16th weekend was significantly higher than that at the4th weekend(all P<0.01); however, there was no significant difference between the8th and16th weekend(P>0.05). In simvastatin group, PON1mRNA expression at the16th weekend was significantly decreased compared with the8th weekend(P<0.05). In L-4F group and simvastatin+L-4F group, there was no significant statistical difference in PON1mRNA expression between the8th and16th weekend (P>0.05).Comparison among different groups at the same time point:Compared with concurrent control group, PON1mRNA expression in AS group at the4th weekend had no significant difference. But that at8th and16th weekend was significantly decreased(all P<0.01).Compared with concurrent AS group, PON1mRNA expression in simvastatin group at the8th and16th weekend was significantly increased(all P<0.01). However, compared with concurrent control group, that in simvastatin group was still decreased at the8th and16th weekend(P<0.01and P<0.05).Compared with concurrent AS group, PON1mRNA expression in L-4F group at the8th and16th weekend was significantly increased(all P<0.01), but that was decreased compared with concurrent control group(all P<0.01). Compared with concurrent simvastatin group, the difference at both the8th and16th weekend was not statistically significant (P>0.05).At the8th weekend, PON1mRNA expression in simvastatin+L-4F group was significantly higher than concurrent AS group(P<0.01) but lower than concurrent control group(P<0.05). Compared with concurrent simvastatin group or L-4F group, the difference was not statistically significant(P>0.05). At the16th weekend, that in Simvastatin+L-4F group was higher than concurrent AS group, simvastatin group and L-4F group(P<0.01, P<0.05and P<0.01) but lower than concurrent control group(P<0.01).5. Effect of simvastatin, L-4F and the combination therapy on PON1protein expression in liverComparison within each group at different time points:In control group, there was no significant difference in PON1protein expression in liver among the three time points(all P>0.05). In AS group, PON1protein expression in liver at the4th weekend did not differ from that at the8th weekend(P>0.05). But that at the16th weekend was significantly higher than that at the4th and8th weekend(all P<0.01); however, there was no significant difference between the8th and16th weekend(P>0.05). In simvastatin group, L-4F group and simvastatin+L-4F group, PON1protein expression at the16th weekend was significantly decreased compared with the8th weekend(all P<0.01).Comparison among different groups at the same time point:Compared with concurrent control group, PON1protein expression in AS group at the4th weekend had no significant difference. But that at8th and16th weekend was significantly decreased(all P<0.01).Compared with concurrent AS group, PON1protein expression in simvastatin group at the8th and16th weekend was significantly increased(P<0.05and P<0.01). However, compared with concurrent control group, that in simvastatin group was still decreased at the8th and16th weekend(all P<0.01).At the8th weekend, PON1protein expression in L-4F group did not differ from concurrent AS group(all P>0.05), but that was significantly decreased compared with concurrent control group(P<0.01). At the16th weekend, that in L-4F group at the16th weekend was significantly increased(P<0.01) compared with concurrent AS group but significantly decreased compared with concurrent control group(P<0.01). Compared with simvastatin group,the difference at both the8th and16th weekend was not statistically significant (P>0.05).At the8th weekend, PON1protein expression in simvastatin+L-4F group was significantly higher than concurrent AS group(P<0.01) but lower than concurrent control group(P<0.05). Compared with concurrent simvastatin group or L-4F group, the difference was not statistically significant(P>0.05). At the16th weekend, that in Simvastatin+L-4F group was higher than concurrent AS group, simvastatin group and L-4F group(P<0.01, P<0.05and P<0.01) but lower than concurrent control group(P<0.01).6. Effect of simvastatin, L-4F and the combination therapy on IMT of the aorta Comparison within each group at different time points:Within control group, there were no difference in IMT of the aorta among the three time points(all P>0.05).Within AS group, IMT of the aorta at the8th and16th weekend was significantly higher than that at the4th weekend(P<0.01); there was no significant difference in IMT between the8th weekend and the16th weekend(P>0.05).Within simvastatin group, L-4F group and simvastatin+L-4F group, there was no difference in IMT between the8th weekend and the16th weekend (all P>0.05).Comparison among different groups at the same time point:IMT of the aorta in AS group were significantly increased at4th,8th and16th weekend compared with concurrent control group (all P<0.01).IMT of the aorta in simvastatin group at the8th and16th weekend were significantly reduced compared to concurrent AS group(P<0.01), but still higher than concurrent control group(P<0.01).IMT of the aorta in L-4F group at the8th and16th weekend was decreased compared with concurrent AS group(P<0.05and P<0.01), but still higher than concurrent control group (P<0.01). Compared with concurrent simvastatin group, the difference was not statistically significant (P>0.05).At the8th weekend, IMT of the aorta in simvastatin+L-4F group was significantly lower than concurrent AS group and L-4F group(P<0.01and P<0.05), but still higher than concurrent control group (P<0.01). Compared with concurrent simvastatin group, the difference was not statistically significant (P>0.05). At the16th weekend, IMT of the aorta in simvastatin+L-4F group was significantly higher than the concurrent control group (P<0.01), but significantly lower than concurrent AS group(P<0.01); compared with either concurrent simvastatin group or L-4F group, the difference was not statistically significant (P>0.05).7. Effect of simvastatin, L-4F and the combination therapy on Plaque area of the aortaComparison within each group at different time points:Within control group, there were no difference in Plaque size of the aorta among the three time points(all P>0.05).Within AS group, percentage of plaque area at the8th and16th weekend was significantly higher than that at the4th weekend(P<0.01); there was no significant difference in percentage of plaque area between the8th weekend and the16th weekend(P>0.05).Within simvastatin group and L-4F group, compared with that at the8th weekend, there were significantly decreased percentage of plaque area(P<0.01) at the16th weekend.Within simvastatin+L-4F group, compared with that at the8th weekend, there was no significant statistical difference in percentage of plaque area at the16th weekend(P>0.05).Comparison among different groups at the same time point:Percentage of plaque area in AS group was significantly increased compared with concurrent control group at4,8, and16th weekend(all P<0.01).Percentage of plaque area in simvastatin group at the8th and16th weekend was significantly reduced compared to concurrent AS group(P<0.01and P<0.05), but still higher than concurrent control group(P<0.01).Percentage of plaque area in L-4F group at the8th and16th weekend was significantly reduced compared to concurrent AS group(P<0.01and P<0.05); the difference was not statistically significant compared with concurrent simvastatin group (P>0.05); but still higher than concurrent control group(P<0.01).Percentage of plaque in simvastatin+L-4F group at the8th weekend was significantly lower than concurrent AS group(P<0.01), but still higher than concurrent control group(P<0.01); compared with either concurrent simvastatin group or L-4F group, the difference was not statistically significant (P>0.05). That in simvastatin+L-4F group at the16th weekend was significantly lower than concurrent AS group, simvastatin group and L-4F group(P<0.01, P<0.01and P<0.05), but still higher than concurrent control group(P<0.01).8. Spearman’s Correlation AnalysisThere was a negtive correlation between serum PON1activity and any one of hs-CRP, IMT and Plaque area of the aorta(r=-0.803,-0.615and-0.669, all P=0.000). Whereas either MPO activity or HII values had a positive correlation with any one of hs-CRP, IMT and Plaque area of the aorta(MPO:r=0.657,0.838and0.882, all P=0.000; HII:r=0.730,0.746and0.798, all P=0.000).ConclusionsSimvastatin could lower density lipoprotein-cholesterol(LDL-C) levels and with mildly elevated high density lipoprotein-cholesterol(HDL-C). Whereas L-4F had no significant effect on plasma lipid levels.Either simvastatin or L-4F could reduce the formation of aortic plaques and had effect of improving the anti-oxidant and anti-inflammatory properties of HDL through not only increased serum PON1activity, PON1gene and protein expression in liver but also decreased MPO activity, HII and serum hs-CRP levels to play the role of anti-atherosclerosis.Furthermore, the combination therapy may be a particularly potent treatment strategy to render the anti-oxidant and anti-inflammatory properties of HDL and reduce atherosclerosis. |
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