激动β3肾上腺素受体对动脉粥样硬化的影响
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摘要
背景
动脉粥样硬化(atherosclerosis，As)所致的心血管疾病成为威胁人类生命的重要疾病之一，目前已知的主要危险因素有：1、高脂血症，2、高血压，3、糖尿病，4、吸烟等。其中血脂异常主要表现为低密度脂蛋白胆固醇（LDL-C）、甘油三酯（TG）水平升高和高密度脂蛋白胆固醇（HDL-C）水平下降。,调节血脂是预防和治疗动脉粥样硬化性疾病的主要手段。目前，流行病学研究显示，LDL-C单一达标只能降低30％～45％主要冠状动脉事件，不能忽略的是，与高LDL-C水平一样，低HDL-C水平同样具有高心血管事件风险，HDL-C每升高1%，心血管事件发生率可降低2％～3％。HDL抗动脉硬化的作用主要与HDL功能有关，HDL通过参与胆固醇逆向转运，抑制胆固醇酯在巨噬细胞内聚集，阻止动脉内壁泡沫细胞的形成，将斑块中泡沫细胞内的胆固醇酯转运至肝脏，起到了延缓或逆转斑块的作用。此外，HDL还具有抗炎、抗氧化、抗凝和改善内皮功能等有益的作用。近年来，人们正在积极研发新的升高HDL水平并能增强其功能的药物。
胆固醇逆转运环节中，HDL与细胞膜表面的B类1型清道夫受体(SR-B1)特异性结合后介导外周胆固醇转运至肝组织,以胆汁酸或游离胆固醇形式随胆汁和粪便排出体外，起到延缓或逆转粥样斑块的作用。其中，载脂蛋白A-I（ApoA-I）是HDL与SR-B1结合的基本结构区,只有适当定位和含量的ApoA-I才能有效发挥胆固醇逆转运作用，才能将外周过剩的胆固醇转运至肝脏。
过氧化物酶增殖物激活受体(PPARs)也具有抗动脉粥样硬化的重要作用，PPARs属于核受体超家族成员，其中PPARα通过调节脂肪酸代谢，PPARγ通过影响脂肪细胞分化、脂肪贮存以及胰岛素的作用，参与脂代谢、糖尿病、炎症等过程，进而干预动脉粥样硬化的发生发展。
β_3肾上腺素受体（β_3-AR）发现于20世纪80年代，主要分布在脂肪组织，通过Gs蛋白偶联激活腺苷酸环化酶，增加胞内环磷酸腺苷水平，激活蛋白激酶A，调节白色和棕色脂肪组织的脂肪分解和产热作用。有研究显示，β_3-AR具有调节糖、脂代谢的作用，能够降低TC和TG，升高HDL-C水平，但目前有关β_3-AR激动剂对动脉粥样硬化性疾病影响的研究甚少。
目的
本研究通过观察β_3-AR激动剂和拮抗剂对老龄ApoE~(-/-)小鼠血脂、血糖、胰岛素、脂肪组织β_3-AR mRNA和PPARγ mRNA水平、肝组织ApoA、SR-B1和PPARα水平及胸主动脉粥样硬化病变形成的影响，初步探讨激动β_3-AR在抗动脉粥样硬化中的作用及其可能机制。
方法
选用10周龄C57BL/6J10只作为正常对照组，给予普通饲料饲养，10周龄ApoE~(-/-)小鼠50只给予高脂饲料饲养。36周龄时ApoE~(-/-)小鼠随机分为ApoE~(-/-)高脂模型组（n=10）、阿托伐他汀组（n=10）、β_3-AR激动剂小剂量组（n=10）、β_3-AR激动剂大剂量组（n=10）、β_3-AR拮抗剂组（n=10），给予各组小鼠生理盐水或药物干预12周。48周龄时，观察各组小鼠血脂、血糖（Glu）和胰岛素(Ins)水平，Western blot法测定肝组织ApoA-I和SR-B1蛋白的表达，实时定量RT-PCR测定脂肪组织β_3-AR mRNA和PPARγ mRNA、肝组织ApoA-I mRNA、ApoA-IImRNA、SR-B1mRNA和PPARα mRNA表达水平，并取胸主动脉常规HE染色、Masson染色和油红O染色后行组织病理形态学观察，测定干预后胸主动脉粥样硬化斑块的改变。
结果
1.48周龄时，与正常对照组相比，Apo E~(-/-)高脂模型组小鼠血脂水平、Glu及Ins水平升高（P<0.01)，HDL-C/TC比值下降（P<0.01)，病理可见胸主动脉粥样硬化斑块形成（P<0.01)，脂肪组织β_3-AR mRNA水平下调（P<0.01)，肝脏组织ApoA-I、ApoA-II和PPARα水平上调（P<0.01)，SR-BI水平下调（P<0.01)，脂肪组织PPARγ mRNA水平无明显变化（P＞0.05）。
2.与Apo E~(-/-)高脂模型组相比，阿托伐他汀组小鼠血TC、VLDL/LDL-C、HDL-C和Ins水平均明显降低（P<0.01)，TG和Glu水平无明显变化（P＞0.05），胸主动脉粥样硬化斑块面积减小（P<0.01)，肝组织ApoA-I、ApoA–II、SR-BI和PPARα表达水平上调（P<0.01)，脂肪组织PPARγ mRNA表达水平上调（P<0.01)，脂肪组织β_3-AR mRNA表达水平无明显改变（P＞0.05）。
3.与Apo E~(-/-)高脂模型组相比，β_3-AR激动剂小剂量和大剂量组小鼠血TC、VLDL/LDL-C、TG、Glu和Ins水平均明显降低（P<0.01)，HDL-C/TC比值升高（P<0.01)，胸主动脉粥样硬化斑块面积减小（P<0.01)，脂肪组织β_3-AR mRNA水平、肝组织ApoA-I和SR-BI表达水平上调（P<0.01),肝组织ApoA-II和PPARαmRNA水平下调（P<0.01)，脂肪组织PPARγ mRNA水平无明显改变（P＞0.05），但β_3-AR激动剂大剂量组作用明显优于小剂量组（P<0.01)。
4.β_3-AR激动剂大剂量组减小胸主动脉粥样硬化斑块面积的作用与阿托伐他汀组相似（P>0.05），升高HDL-C、HDL-C/TC、降低Ins水平的作用则显著优于阿托伐他汀（P<0.01)，大剂量β_3-AR激动剂促进脂肪组织β_3-AR mRNA表达水平上调（P<0.01)，肝组织ApoA-I、ApoA-II、SR-BI、PPARα和脂肪组织PPARγmRNA表达水平明显下调（P＜0.01）。
5.与Apo E~(-/-)高脂模型组相比，β_3-AR拮抗剂组血脂、Glu、Ins水平及胸主动脉粥样硬化斑块面积无明显变化（P>0.05），脂肪组织PPARγ mRNA水平明显上调（P<0.01)，脂肪组织β_3-AR、肝组织ApoA-I、ApoA-II、SR-BI和PPARα mRNA表达水平均无明显变化（P>0.05）。结论
1.激动β_3-AR显著改善老年Apo E~(-/-)小鼠血脂代谢，降低TC、VLDL/LDL-C和TG，升高HDL；
2.激动β_3-AR显著改善老年Apo E~(-/-)小鼠血糖代谢，降低Glu和Ins水平；
3.激动β_3-AR明显上调老年Apo E~(-/-)小鼠脂肪组织β_3-AR mRNA、肝组织ApoA-I和SR-BI表达水平；
4.激动β_3-AR显著下调老年Apo E~(-/-)小鼠肝组织ApoA-II和PPARα mRNA的表达水平；
5.激动β_3-AR对老年Apo E~(-/-)小鼠脂肪组织PPARγ mRNA表达水平无显著影响；
6.激动β_3-AR明显缩小老年Apo E~(-/-)小鼠胸主动脉动脉粥样硬化斑块面积，提示其具有抗动脉粥样硬化的作用。
Background
Atherosclerotic (AS) disease is the leading cause of morbidity and mortality inthe western world and developing countries. It is associated with a metabolic andvascular cluster of disorders. The major risk factors of atherosclerosis includehyperlipidemia, glucose intolerance, hypertension, smoking and aging.Hyperlipidemia is summarized as raised trigIyceride (TG), total cholesterol (TC) andlow density lipoprotein-cholesteroI (LDL-C) levels, reduced (high densitylipoprotein-cholesteroI) HDL-C level. Studies indicate that reducing LDL-C levelsand/or increasing HDL-C levels can decrease efficiently the incidence of AS disease.Recently, the epidemiology showed that the single standard of LDL-C level can onlyreduce the30%to45%of major coronary events. AS the raised LDL-C level, thereduced HDL-C levels also have high cardiovascular event risk. The increase ofHDL-C level with every1%would reduce the incidence of cardiovascular eventswith2%to3%. HDL involves in reverse cholesterol transport (RCT) which HDLinhibites the aggregation of cholesteryl ester in macrophages and prevents theformation of foam cells in the plaque. In addition, HDL also has anti-inflammatory,antioxidant, anticoagulant and improves the endothelial function. In recent years, thenew medcines are actively developed which could elevated HDL levels and enhanceits functionality.
As the most important anti—atherosclerotic mechanism in vivo RCT，is aprocess which dynamically transport of excess cholesterol from the periphery into theliver, followed by excretion in the feces or the bile．Scavenger receptor class B type I (SR-BI) had already proved in molecular level as the sole HDL receptor whichlocates on celluar membrane．As the HDL specific receptor, SR-BI plays an importantrole in the HDL metabolism and the process of RCT. Apolipoprotein A-I (ApoA-I) isthe basic structure area of HDL combinated with SR-B1. It is essential that theappropriate position and content of ApoA-I in order to work effectively in the processof RCT. In addition, peroxisome proliferator-activated receptors (PPARs) recently areone of the main mechanisms of anti-atherosclerotic. PPARs belong to the members ofthe nuclear receptor superfamily including PPARα, PPARβand PPARγ. PPARαregulates fatty acid metabolism. PPARγ involve in lipid metabolism, glucosemetabolism and inflammation process by affecting fat cells differentiation andstorage.Thus PPARs interfere with the development of atherosclerosis.
During the1980s, the classification of β-adrenoceptor (β-AR) was modifiedfollowing the discovery of a third β-AR primarily in rat adipose tissue. β_3-ARmediates the major effects of adrenaline and noradrenaline in adipose tissues, such aslipolysis in white adipose tissue and thermogenesis in brown adipose tissue. Itbelongs to the G-protein coupled receptor family which involves the activation ofadenylyl cyclase, cAMP/protein kinase A (PKA) pathway and recruits a PI3-kinasepathway. Previous studies have shown that chronic treatments of β_3-AR agonists wereeffective in improving glycaemic control, insulin sensitivity and reducing plasma TG,free fatty acid levels and body weight in obese diabetic animals. However the effectsof β_3-AR on atherosclerosis disease are largely unknown.
Objective
To study the effects of β_3-adrenoceptor (β_3-AR) activation on lipid, glucosemetabolism, the β_3-AR mRNAexpressions in withe adipose tissue, ApoA-I, ApoA-II,SR-BI expressions in liver tissue and atherosclerotic plaque development in agedapolipoprotein E-deficient (ApoE~(-/-)) mice. The study discussed β_3-AR possible mechanisms and provided the evidenceand which its function of HDL metabolism，the RCT process and atherosderosis．
Methods
Ten10-week-old wild-type C57BL/6J mice (A group) were fed normal chow.Fifty10-week-old ApoE~(-/-)mice were fed high-fat diet. At36-week-old, ApoE~(-/-)micewere randomly given normal saline (B group), atorvastatin (10mg kg-1d-1, C group),β_3-AR agonist BRL37344(1.65μg kg-1, twice a week, D group), BRL37344(3.30μg kg-1, twice a week, E group) and β_3-AR antagonist SR52390A (50μg kg-1,twice a week, F group) for12weeks. The serum lipid, glucose and insulin levels weremeasured. The protein expressions of ApoA-I, SR-BI in the liver tissue by westernblot．Real-time quantitative PCR was used to determine the expressions of ApoA-ImRNA, ApoA-II mRNA, SR-BI mRNAand PPARα mRNA in the liver tissue and theexpression of β_3-AR mRNA and PPARγ mRNA in withe adipose tissue of mice. Theatherosclerotic plaque area in the thoracic aortic was determined withHematoxy-Eosin stain, and level of fibrosis in the plaque was assessed with Massonstain.
Results
1. Compared with wild-type C57BL/6J mice, ApoE~(-/-)mice showed a significanthyperlipidemia, insulin résistance, the downregulation expression of β_3-AR mRNAand SR-BI (P <0.01), the upregulation expression of ApoA-I, ApoA-II and PPARαmRNA (P <0.01), PPARγ mRNA levels in adipose tissue was no significant changes(P＞0.05), atherosclerotic plaque formation with obvious fibrosis in the thoracicaortic (P <0.01).
2. Compared withApoE~(-/-)control mice with saline, atorvastatin obviouslydecreased the serum levels ofTC, VLDL/LDL-C, HDL-C and Ins (P <0.01), but hadno effect on TG and Glu (P＞0.05). In additionally, atorvastatin significiantly upregulated the expression ofApoA-I, ApoA–II, SR-BI and PPARα in liver tissueand the expression of PPARγ mRNA in adipose tissue (P <0.01). β_3-AR mRNAlevelsof adipose tissue did not change significantly (P>0.05).Atorvastatin could decreasethe atherosclerotic plaque size (P <0.01).
3. Compared with ApoE~(-/-)control mice with saline, low or high dose β_3-ARagonist significantly decreased the serum levels of TG, Glu and Ins, and obviouslyelevated HDL-C level and HDL-C/TC ratio. Simultaneously, β_3-AR agonist coulddistinctly up-regulate β_3-AR mRNA, ApoA-I and SR-BI levels, down-regulateApoA-II and PPARα mRNA levels and decrease the atherosclerotic plaque size（P<0.01). PPARγ mRNA levels of adipose tissue did not change significantly (P>0.05). Effect of high dose β_3-AR agonist was significantly superior to low dose（P<0.01).
4. Compared with atorvastatin group, the effects of high dose β_3-AR agonistwere similar in decreasing the atherosclerotic plaque size（P>0.05）, but high doseβ_3-AR agonist was better than atorvastatin in increasing levels of HDL-C, HDL-C/TCand in reducing levels of insulin（P<0.01). β_3-AR agonist obviously up-regulated theβ_3-AR expression and down-regulated the expression ofApoA-I, ApoA-II, SR-BI,PPARα and PPARγ (P<0.01).
5. Compared with ApoE~(-/-)control mice，β_3-AR antagonist had no effect on lipids、Glu、Ins and the atherosclerotic plaque size (P>0.05). PPARγ mRNA levels wasincreased significiantly in β_3-AR antagonist group（P<0.01). There was no obviouschange about ApoA-I, ApoA-II, SR-BI, β_3-AR and PPARα expression (P>0.05).
Conclusion
1. β_3-AR activation can improve the aged ApoE~(-/-)mice lipid metabolism,reducing levels of TC, VLDL/LDL-C and TG，increasing level of HDL；
2. β_3-AR activation can improve the aged ApoE~(-/-)mice glucose metabolism,reducing levels of glucose and insulin；
3. β_3-AR activation can up-regulate levels of β_3-AR mRNAexpression in whiteadipose tissue, ApoA-I and SR-BI expression in the liver tissue；
4. β_3-AR activation can down-regulate levels of ApoA-II and PPARα mRNAexpression in the liver tissue；
5. β_3-AR activation had no obvious effect on PPARγ mRNA expression of theaged ApoE~(-/-)mice；
6. β_3-AR activation can significantly decrease the atherosclerotic plaque sizeand play a role of anti-atherosclerosis.
动脉粥样硬化(atherosclerosis，As)所致的心血管疾病成为威胁人类生命的重要疾病之一，目前已知的主要危险因素有：1、高脂血症，2、高血压，3、糖尿病，4、吸烟等。其中血脂异常主要表现为低密度脂蛋白胆固醇（LDL-C）、甘油三酯（TG）水平升高和高密度脂蛋白胆固醇（HDL-C）水平下降。,调节血脂是预防和治疗动脉粥样硬化性疾病的主要手段。目前，流行病学研究显示，LDL-C单一达标只能降低30％～45％主要冠状动脉事件，不能忽略的是，与高LDL-C水平一样，低HDL-C水平同样具有高心血管事件风险，HDL-C每升高1%，心血管事件发生率可降低2％～3％。HDL抗动脉硬化的作用主要与HDL功能有关，HDL通过参与胆固醇逆向转运，抑制胆固醇酯在巨噬细胞内聚集，阻止动脉内壁泡沫细胞的形成，将斑块中泡沫细胞内的胆固醇酯转运至肝脏，起到了延缓或逆转斑块的作用。此外，HDL还具有抗炎、抗氧化、抗凝和改善内皮功能等有益的作用。近年来，人们正在积极研发新的升高HDL水平并能增强其功能的药物。
胆固醇逆转运环节中，HDL与细胞膜表面的B类1型清道夫受体(SR-B1)特异性结合后介导外周胆固醇转运至肝组织,以胆汁酸或游离胆固醇形式随胆汁和粪便排出体外，起到延缓或逆转粥样斑块的作用。其中，载脂蛋白A-I（ApoA-I）是HDL与SR-B1结合的基本结构区,只有适当定位和含量的ApoA-I才能有效发挥胆固醇逆转运作用，才能将外周过剩的胆固醇转运至肝脏。
过氧化物酶增殖物激活受体(PPARs)也具有抗动脉粥样硬化的重要作用，PPARs属于核受体超家族成员，其中PPARα通过调节脂肪酸代谢，PPARγ通过影响脂肪细胞分化、脂肪贮存以及胰岛素的作用，参与脂代谢、糖尿病、炎症等过程，进而干预动脉粥样硬化的发生发展。
β_3肾上腺素受体（β_3-AR）发现于20世纪80年代，主要分布在脂肪组织，通过Gs蛋白偶联激活腺苷酸环化酶，增加胞内环磷酸腺苷水平，激活蛋白激酶A，调节白色和棕色脂肪组织的脂肪分解和产热作用。有研究显示，β_3-AR具有调节糖、脂代谢的作用，能够降低TC和TG，升高HDL-C水平，但目前有关β_3-AR激动剂对动脉粥样硬化性疾病影响的研究甚少。
目的
本研究通过观察β_3-AR激动剂和拮抗剂对老龄ApoE~(-/-)小鼠血脂、血糖、胰岛素、脂肪组织β_3-AR mRNA和PPARγ mRNA水平、肝组织ApoA、SR-B1和PPARα水平及胸主动脉粥样硬化病变形成的影响，初步探讨激动β_3-AR在抗动脉粥样硬化中的作用及其可能机制。
方法
选用10周龄C57BL/6J10只作为正常对照组，给予普通饲料饲养，10周龄ApoE~(-/-)小鼠50只给予高脂饲料饲养。36周龄时ApoE~(-/-)小鼠随机分为ApoE~(-/-)高脂模型组（n=10）、阿托伐他汀组（n=10）、β_3-AR激动剂小剂量组（n=10）、β_3-AR激动剂大剂量组（n=10）、β_3-AR拮抗剂组（n=10），给予各组小鼠生理盐水或药物干预12周。48周龄时，观察各组小鼠血脂、血糖（Glu）和胰岛素(Ins)水平，Western blot法测定肝组织ApoA-I和SR-B1蛋白的表达，实时定量RT-PCR测定脂肪组织β_3-AR mRNA和PPARγ mRNA、肝组织ApoA-I mRNA、ApoA-IImRNA、SR-B1mRNA和PPARα mRNA表达水平，并取胸主动脉常规HE染色、Masson染色和油红O染色后行组织病理形态学观察，测定干预后胸主动脉粥样硬化斑块的改变。
结果
1.48周龄时，与正常对照组相比，Apo E~(-/-)高脂模型组小鼠血脂水平、Glu及Ins水平升高（P<0.01)，HDL-C/TC比值下降（P<0.01)，病理可见胸主动脉粥样硬化斑块形成（P<0.01)，脂肪组织β_3-AR mRNA水平下调（P<0.01)，肝脏组织ApoA-I、ApoA-II和PPARα水平上调（P<0.01)，SR-BI水平下调（P<0.01)，脂肪组织PPARγ mRNA水平无明显变化（P＞0.05）。
2.与Apo E~(-/-)高脂模型组相比，阿托伐他汀组小鼠血TC、VLDL/LDL-C、HDL-C和Ins水平均明显降低（P<0.01)，TG和Glu水平无明显变化（P＞0.05），胸主动脉粥样硬化斑块面积减小（P<0.01)，肝组织ApoA-I、ApoA–II、SR-BI和PPARα表达水平上调（P<0.01)，脂肪组织PPARγ mRNA表达水平上调（P<0.01)，脂肪组织β_3-AR mRNA表达水平无明显改变（P＞0.05）。
3.与Apo E~(-/-)高脂模型组相比，β_3-AR激动剂小剂量和大剂量组小鼠血TC、VLDL/LDL-C、TG、Glu和Ins水平均明显降低（P<0.01)，HDL-C/TC比值升高（P<0.01)，胸主动脉粥样硬化斑块面积减小（P<0.01)，脂肪组织β_3-AR mRNA水平、肝组织ApoA-I和SR-BI表达水平上调（P<0.01),肝组织ApoA-II和PPARαmRNA水平下调（P<0.01)，脂肪组织PPARγ mRNA水平无明显改变（P＞0.05），但β_3-AR激动剂大剂量组作用明显优于小剂量组（P<0.01)。
4.β_3-AR激动剂大剂量组减小胸主动脉粥样硬化斑块面积的作用与阿托伐他汀组相似（P>0.05），升高HDL-C、HDL-C/TC、降低Ins水平的作用则显著优于阿托伐他汀（P<0.01)，大剂量β_3-AR激动剂促进脂肪组织β_3-AR mRNA表达水平上调（P<0.01)，肝组织ApoA-I、ApoA-II、SR-BI、PPARα和脂肪组织PPARγmRNA表达水平明显下调（P＜0.01）。
5.与Apo E~(-/-)高脂模型组相比，β_3-AR拮抗剂组血脂、Glu、Ins水平及胸主动脉粥样硬化斑块面积无明显变化（P>0.05），脂肪组织PPARγ mRNA水平明显上调（P<0.01)，脂肪组织β_3-AR、肝组织ApoA-I、ApoA-II、SR-BI和PPARα mRNA表达水平均无明显变化（P>0.05）。结论
1.激动β_3-AR显著改善老年Apo E~(-/-)小鼠血脂代谢，降低TC、VLDL/LDL-C和TG，升高HDL；
2.激动β_3-AR显著改善老年Apo E~(-/-)小鼠血糖代谢，降低Glu和Ins水平；
3.激动β_3-AR明显上调老年Apo E~(-/-)小鼠脂肪组织β_3-AR mRNA、肝组织ApoA-I和SR-BI表达水平；
4.激动β_3-AR显著下调老年Apo E~(-/-)小鼠肝组织ApoA-II和PPARα mRNA的表达水平；
5.激动β_3-AR对老年Apo E~(-/-)小鼠脂肪组织PPARγ mRNA表达水平无显著影响；
6.激动β_3-AR明显缩小老年Apo E~(-/-)小鼠胸主动脉动脉粥样硬化斑块面积，提示其具有抗动脉粥样硬化的作用。
Background
Atherosclerotic (AS) disease is the leading cause of morbidity and mortality inthe western world and developing countries. It is associated with a metabolic andvascular cluster of disorders. The major risk factors of atherosclerosis includehyperlipidemia, glucose intolerance, hypertension, smoking and aging.Hyperlipidemia is summarized as raised trigIyceride (TG), total cholesterol (TC) andlow density lipoprotein-cholesteroI (LDL-C) levels, reduced (high densitylipoprotein-cholesteroI) HDL-C level. Studies indicate that reducing LDL-C levelsand/or increasing HDL-C levels can decrease efficiently the incidence of AS disease.Recently, the epidemiology showed that the single standard of LDL-C level can onlyreduce the30%to45%of major coronary events. AS the raised LDL-C level, thereduced HDL-C levels also have high cardiovascular event risk. The increase ofHDL-C level with every1%would reduce the incidence of cardiovascular eventswith2%to3%. HDL involves in reverse cholesterol transport (RCT) which HDLinhibites the aggregation of cholesteryl ester in macrophages and prevents theformation of foam cells in the plaque. In addition, HDL also has anti-inflammatory,antioxidant, anticoagulant and improves the endothelial function. In recent years, thenew medcines are actively developed which could elevated HDL levels and enhanceits functionality.
As the most important anti—atherosclerotic mechanism in vivo RCT，is aprocess which dynamically transport of excess cholesterol from the periphery into theliver, followed by excretion in the feces or the bile．Scavenger receptor class B type I (SR-BI) had already proved in molecular level as the sole HDL receptor whichlocates on celluar membrane．As the HDL specific receptor, SR-BI plays an importantrole in the HDL metabolism and the process of RCT. Apolipoprotein A-I (ApoA-I) isthe basic structure area of HDL combinated with SR-B1. It is essential that theappropriate position and content of ApoA-I in order to work effectively in the processof RCT. In addition, peroxisome proliferator-activated receptors (PPARs) recently areone of the main mechanisms of anti-atherosclerotic. PPARs belong to the members ofthe nuclear receptor superfamily including PPARα, PPARβand PPARγ. PPARαregulates fatty acid metabolism. PPARγ involve in lipid metabolism, glucosemetabolism and inflammation process by affecting fat cells differentiation andstorage.Thus PPARs interfere with the development of atherosclerosis.
During the1980s, the classification of β-adrenoceptor (β-AR) was modifiedfollowing the discovery of a third β-AR primarily in rat adipose tissue. β_3-ARmediates the major effects of adrenaline and noradrenaline in adipose tissues, such aslipolysis in white adipose tissue and thermogenesis in brown adipose tissue. Itbelongs to the G-protein coupled receptor family which involves the activation ofadenylyl cyclase, cAMP/protein kinase A (PKA) pathway and recruits a PI3-kinasepathway. Previous studies have shown that chronic treatments of β_3-AR agonists wereeffective in improving glycaemic control, insulin sensitivity and reducing plasma TG,free fatty acid levels and body weight in obese diabetic animals. However the effectsof β_3-AR on atherosclerosis disease are largely unknown.
Objective
To study the effects of β_3-adrenoceptor (β_3-AR) activation on lipid, glucosemetabolism, the β_3-AR mRNAexpressions in withe adipose tissue, ApoA-I, ApoA-II,SR-BI expressions in liver tissue and atherosclerotic plaque development in agedapolipoprotein E-deficient (ApoE~(-/-)) mice. The study discussed β_3-AR possible mechanisms and provided the evidenceand which its function of HDL metabolism，the RCT process and atherosderosis．
Methods
Ten10-week-old wild-type C57BL/6J mice (A group) were fed normal chow.Fifty10-week-old ApoE~(-/-)mice were fed high-fat diet. At36-week-old, ApoE~(-/-)micewere randomly given normal saline (B group), atorvastatin (10mg kg-1d-1, C group),β_3-AR agonist BRL37344(1.65μg kg-1, twice a week, D group), BRL37344(3.30μg kg-1, twice a week, E group) and β_3-AR antagonist SR52390A (50μg kg-1,twice a week, F group) for12weeks. The serum lipid, glucose and insulin levels weremeasured. The protein expressions of ApoA-I, SR-BI in the liver tissue by westernblot．Real-time quantitative PCR was used to determine the expressions of ApoA-ImRNA, ApoA-II mRNA, SR-BI mRNAand PPARα mRNA in the liver tissue and theexpression of β_3-AR mRNA and PPARγ mRNA in withe adipose tissue of mice. Theatherosclerotic plaque area in the thoracic aortic was determined withHematoxy-Eosin stain, and level of fibrosis in the plaque was assessed with Massonstain.
Results
1. Compared with wild-type C57BL/6J mice, ApoE~(-/-)mice showed a significanthyperlipidemia, insulin résistance, the downregulation expression of β_3-AR mRNAand SR-BI (P <0.01), the upregulation expression of ApoA-I, ApoA-II and PPARαmRNA (P <0.01), PPARγ mRNA levels in adipose tissue was no significant changes(P＞0.05), atherosclerotic plaque formation with obvious fibrosis in the thoracicaortic (P <0.01).
2. Compared withApoE~(-/-)control mice with saline, atorvastatin obviouslydecreased the serum levels ofTC, VLDL/LDL-C, HDL-C and Ins (P <0.01), but hadno effect on TG and Glu (P＞0.05). In additionally, atorvastatin significiantly upregulated the expression ofApoA-I, ApoA–II, SR-BI and PPARα in liver tissueand the expression of PPARγ mRNA in adipose tissue (P <0.01). β_3-AR mRNAlevelsof adipose tissue did not change significantly (P>0.05).Atorvastatin could decreasethe atherosclerotic plaque size (P <0.01).
3. Compared with ApoE~(-/-)control mice with saline, low or high dose β_3-ARagonist significantly decreased the serum levels of TG, Glu and Ins, and obviouslyelevated HDL-C level and HDL-C/TC ratio. Simultaneously, β_3-AR agonist coulddistinctly up-regulate β_3-AR mRNA, ApoA-I and SR-BI levels, down-regulateApoA-II and PPARα mRNA levels and decrease the atherosclerotic plaque size（P<0.01). PPARγ mRNA levels of adipose tissue did not change significantly (P>0.05). Effect of high dose β_3-AR agonist was significantly superior to low dose（P<0.01).
4. Compared with atorvastatin group, the effects of high dose β_3-AR agonistwere similar in decreasing the atherosclerotic plaque size（P>0.05）, but high doseβ_3-AR agonist was better than atorvastatin in increasing levels of HDL-C, HDL-C/TCand in reducing levels of insulin（P<0.01). β_3-AR agonist obviously up-regulated theβ_3-AR expression and down-regulated the expression ofApoA-I, ApoA-II, SR-BI,PPARα and PPARγ (P<0.01).
5. Compared with ApoE~(-/-)control mice，β_3-AR antagonist had no effect on lipids、Glu、Ins and the atherosclerotic plaque size (P>0.05). PPARγ mRNA levels wasincreased significiantly in β_3-AR antagonist group（P<0.01). There was no obviouschange about ApoA-I, ApoA-II, SR-BI, β_3-AR and PPARα expression (P>0.05).
Conclusion
1. β_3-AR activation can improve the aged ApoE~(-/-)mice lipid metabolism,reducing levels of TC, VLDL/LDL-C and TG，increasing level of HDL；
2. β_3-AR activation can improve the aged ApoE~(-/-)mice glucose metabolism,reducing levels of glucose and insulin；
3. β_3-AR activation can up-regulate levels of β_3-AR mRNAexpression in whiteadipose tissue, ApoA-I and SR-BI expression in the liver tissue；
4. β_3-AR activation can down-regulate levels of ApoA-II and PPARα mRNAexpression in the liver tissue；
5. β_3-AR activation had no obvious effect on PPARγ mRNA expression of theaged ApoE~(-/-)mice；
6. β_3-AR activation can significantly decrease the atherosclerotic plaque sizeand play a role of anti-atherosclerosis.
引文
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