| Study BackgroundAtherosclerosis is pathophysiology of angina, myocardial infarction, sudden cardiac death and cerebral infarction, cerebral hemorrhage and other cerebrovascular disease. Low-density lipoprotein cholesterol level is the most important pathological changes of atherosclerotic (AS) risk factor. We found that patients'LDL-C level receiver desired level can reduce cardiovascular events by 30%, but remain 70% cardiovascular events would be happened. So we believe that high density lipoprotein (HDL) was a therapy target in atherosclerosis prevention.Almost all epidemiological studies had demonstrate an inverse relationship between plasma levels of HDL-C and cardiovascular atherosclerotic disease. However, several lines of evidence indicate that the relationship between HDL and CAD risk is more complex and extends beyond the serum HDL-C levels. Torcetrapib, a potent CETP inhibitor, markedly increased the plasma concentration of HDL-C, but the risks of deaths and cardiac events in patients receiving tocetrapib had been increased simultaneously. The Milano people who carry the apolipoprotein A-ⅠMilano mutant have very low serum HDL-C level while show very low incidence of CAD. Our results support the theory that not all HDL possess atheroprotective properties. Some HDL is dysfunctional or pro-inflammatory. Surem HDL-C level is not equivalent to HDL function.The proposed atheroprotective properties of HDL are multifaceted, including Reverse Cholesterol Transport and cholesterol efflux capacity, anti-oxidative and anti-inflammatory activities. The most important theory revolves around the role of HDL in macrophage reverse cholesterol rransport (RCT), in which excess cholesterol is effluxed to HDL and ultimately returned to the liver for metabolism by receptor of ATP binding cassette transporter A1 (ABCA1) and Scavenger receptor class B type I (SR-BI).HDL has anti-inflammatory and anti-antioxidant property, which play an important role in AS protecting. HDL plays an important role in protecting against LDL oxidative modification, and the enzyme PON1 contributes the key role to the antioxidative effects of HDL. MPO binding occurs via specific interactions with apoA-Ⅰ, the predominant protein of HDL, and carboxy-terminal deletions in apoA-I may prevent the lipidation of apoA-I and thus may reduce property of anti-inflammatory and anti-antioxidant in HDL.Probucol, once employed clinically for hypercholesterolemia, is also potent anti-oxidant and anti-inflammatory properties that can reduce atherosclerosis. Probucol is a unique hypolipidemic agent that down-regulate HDL-C level while significantly inhibiting the progression of atherosclerosis. But the mechanism is still unclear.ObjectivesProbucol decreases HDL-C level while showing greatly controlled progression of atherosclerosis. We speculate that probucol may improve HDL functions. This study was designed to evaluate the effects of probucol on HDL functions. We will investigate the change of the expression of ABCA1 and SR-B1 and the anti-inflammative and anti-oxidative function of HDL in AS rabbit's models to found the mechanism by which probucol affects HDL function.Methods1. Animal modelEighteen male New Zealand white rabbits (3 months,2.0±0.11kg) were purchased from Southern Medical University and individually housed in air-conditioned room. They were randomly assigned to three groups, including control group (n=6), atherosclerotic group (n=6) and probucol group (n=6). Control group was fed with normal diet, atherosclerotic group and probucol group received a diet containing 1% cholesterol,8% fat and 0.05% bile salts with or without probucol (400mg/kg/day) for 12 weeks.2. Serum lipid analysisBlood lipid analysis was performed at 0 week and 12 weeks end of the experiment. Serum triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (HDL-C) concentrations were measured by using a automated biochemical analyzer.3. Assessment of the anti-oxidantive function of HDLSerum PON1 activity was assayed using synthetic substrates. MPO activity was determined by a MPO activity kit according to the manufacturer's instructions.4. Aortic atherosclerosis for histological examination and quantificationAt the end of 12 weeks, after sacrifice of rabbits, the entire aorta was moved, fixed in 10% neutral buffered formaldehyde solution for 48 hours. All segments were embeded in paraffin stained with hematoxylin and eosin (HE) for histological examination. The percentage plaque area, which was defined as surface area of plaque/surface area of whole intima, and the aortic intima-media thickness were calculated for evaluating the degree of aortic atherosclerosis.5. Detection of ABCA1 and SR-B1 mRNA in liver tissues(1) The total RNA was isolated from rabbit liver by a Trizol reagent. The quantitative analyses of ABCA1 and SR-B1 mRNA level on rabbit liver tissues were performed by real-time quantitative polymerase chain reaction (RTQ-PCR).(2) For microscopic evaluation of ABCA1 and SR-B1 protein expression in the lesions of aorta, serial paraffin sections of the aortic arch were immunohistochemically stained with the following antibodies against ABCA1 and SR-B1. The expression of ABCA1 and SR-B1 (including percentage of ABCA1 or SR-B1 protein positive area and staining intensity in the lesions by immunohistochemical staining) were measured with Image pro plus 6.0 specially image analysis software.Results1. There were no differences in TC(F=0.921, P=0.420), TG(F=0.453, P=0.644), LDL-C(F=0.003, P=0.997) and HDL-C(F=0.338, P=0.718) among three groups at the baseline. After 12 weeks of experiment, there were significant differences in TC (F=1261.325, P=0.000), TG (F=16.902, P=0.000), LDL-C (F=155.323, P=0.000) and HDL-C (F=92.845, P=0.000) among three groups. Compare between groups: Compared with control group, serum TC (23.26±3.30 vsl.79±0.21, P=0.000), TG (1.58±0.25 vs 1.02±0.15, P=0.000), LDL-C (18.09±4.04 vs 1.09±0.23, P=0.000) and HDL-C (1.11±0.09 vs 0.45±0.12, P=0.000) in AS group were significantly increased; Compared with AS group, the levels of serum TC (16.70±0.70 vs 23.25±3.30P=0.000), LDL-C (13.79±2.01 vs 18.09±4.04, P=0.012) and HDL-C (0.51±0.06 vs 1.11±0.09, P=0.000) were significantly reduced in Probucol group. However, the serum triglyceride (1.60±0.17 vs 1.58±0.25, P=0.826) concentration was unaffected by probucol.Compare Within each group, There were no differences in TC (1.85±0.12 vs 1.79±0.21, t=0.538, P=0.614), TG (1.04±0.19 vs 1.02±0.15, t=0.162, P=0.878), LDL-C (1.00±0.20 vs 1.09±0.23, t=0.566, P=0.0.596), HDL-C (0.47±0.12 vs 0.45±0.12, t=0.369, P=0.0.727) before and after intervention in control group; There were significant differences in TC (1.77±0.15 vs 23.26±3.30, t=16.603, P=0.000), TG (1.10±0.14 vs 1.58±0.25,1=4.498, P=0.006), LDL-C (1.00±0.20 vs 18.09±4.04, t=10.251, P=0.000), HDL-C (0.48±0.12 vs 1.11±0.09, t=14.615, P=0.000) before and after intervention in AS group; There were significant differences in TC (1.76±0.11 vs 16.70±0.70, t=48.680, P=0.000), TG (1.02±0.15 vs 1.60±0.17, t=6.956, P=0.001), LDL-C (1.00±0.15 vs 13.79±2.01, t=16.244, t=16.244, P=0.000) before and after intervention in probucol group, but there was no difference in HDL (0.43±0.10 vs 0.51±0.06,t=1.612,P=0.168).2. Serum PON1 activity (F=29.24, P=0.000) was significant difference in three groups. Conpared with control group (96.77±5.58 U/ml), serum PON1 activity was significantly inhibited in atherosclerosis group (72.26±12.03, P=0.001). Conpared with AS group, serum PON1 activity was markedly elavated in probucol group (118.20±12.21 U/ml, P=0.000).Serum MPO activity (F=46.994, P=0.000) was significant difference in three groups. Conpared with control group (14.94±6.36 U/L), Serum MPO activity was substantially higher in atherosclerosis group (85.67±17.92 U/L, P=0.001). Conpared with AS group, Serum MPO activity was significantly inhibited in probucol group (44.11±10.65 U/L,P=0.000).3. At the 12th treatment week end, Compared with the control group, expression of ABCA1mRNA (0.61±0.12 vs 1.00±0.03, P=0.000) and SR-B1 mRNA (0.63±0.11 vs 1.00±0.02, P=0.000) in hepatocytes were both significantly lower in atherosclerosis group rabbits than in control groups rabbits. Compared with the atherosclerosis group, the probucol group showed a significantly higher level of expression of ABCA1 (0.87±0.08 vs 0.61±0.12, P=0.000) and SR-B1 (1.34±0.13 vs 0.63±0.11, P=0.000) in hepatocytes at the mRNA levels.4. There were no lesion, plaque and immunohistochemical staining in aortic wall in control group, Immunohistochemical staining revealed substantial ABCA1 and SR-B1 protein infiltration in the aortic plaques in both atherosclerosis group and probucol group. Compared to atherosclerosis group, ABCA1 (45.25±11.22% vs 24.13±9.85%, t=3.464, P=0.006) and SR-BI (46.22±11.56% vs 18.81±7.58%, t=4.858, P=0.001) protein expression was significantly increased at percentage of protein positive area in the Probucol group; Compared to atherosclerosis group, SR-BI (0.25±0.04 vs O.18±0.03, t=3.286, P=0.001) protein expression was significantly increased at staining intensity in the probucol group too. However, there was no significant difference in ABCA1 protein expression in staining intensity (0.19±0.03 vs 0.17±0.03, t=1.405, P=0.190) between Probucol group and atherosclerosis group.5. The histomorphometric analysis of atherosclerotic lesion was performed by means of HE at the 12th experimental week end:Compared with control group (203.20±30.61 um), the aortic intima-media thickness (IMT) (P=0.000) was significantly increased in AS group (527.41±85.16 um). Compared with AS group, the IMT (239.83±50.51, P=0.000) and the percentage plaque area (5.92±3.38% vs 27.77±12.01%, P=0.002) significantly decreased in probucol group.Conclusions1. Probucol decreased serum TC,LDL-C,HDL-C concerntration.2. Probucol up-regulated ABCA1 and SR-B1 in hepatic cells and Plaque cells of aortic wall for promoting RCT.3. Probucol promoted the role of HDL in anti-inflammation and anti-oxidation by decreasing serum myeloperoxidase and increasing paraoxonase 1 activity in atherosclerotic rabbits.4. Probucol reduced intima-media thickness (IMT) and the percentage plaque area in atherosclerotic models.5. Serum HDL-C level cannot accurately represent HDL function. Probucol decreases HDL-C level, but also reduces AS plaque formation and improves HDL functon by increasing the expression of ABCA1 and SR-B1 in hepatic cells and Plaque cells, elevating PON1 activity and reducing MPO activity. |
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