新疆哈族传统发酵乳酪的乳清对实验性动脉粥样硬化的作用及其机制
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摘要
目的:探讨新疆哈萨克族传统发酵乳酪的乳清(简称乳酪乳清,TFCW)抗实验性动脉粥样硬化作用的物质基础及作用机制。方法:依据《中华人民共和国国家标准》颁布要求的测定方法,测定TFCW中蛋白质、灰分、水分、氨基酸、维生素、脂肪酸、矿物质和微量元素的含量。药效学研究:通过1)采用三种不同的动脉粥样硬化动物模型:高胆固醇饲料喂养大鼠注射大剂量维生素D3(VitD3)致内皮损伤模型、灌胃高脂乳剂大鼠注射大剂量VitD3致内皮损伤模型及高胆固醇饲料喂养新西兰兔致动脉粥样硬化模型模型。视各模型组动物血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL)和C-反应蛋白(CRP)水平升高,以及显微镜下冠状动脉和主动脉出现动脉粥样硬化病理改变,为建立动脉粥样硬化模型成功的标准。以辛伐他汀(20 mg·Kg-1)为阳性对照,TFCW(25,50,100 mg·Kg-1)给药4周后,分别测定血清总TC、TG、LDL、高密度脂蛋白(HDL)、肝脂、CRP、内皮素(ET)、血栓烷素B2(TXB2)、肿瘤坏死因子(TNF-α)、白细胞介素-8(IL-8)、血管细胞黏附分子(VCAM-1)和血管细胞黏附分子(ICAM-1)等指标,观察TFCW对内皮的影响及冠状动脉,主动脉病理形态学的改变;同时应用免疫组化方法检测VCAM-1及ICAM-1表达。2)采用三种不同的炎症模型:角叉菜胶致小鼠足肿胀急性炎症模型、棉球植入大鼠腋下致肉芽肿慢性炎症模型及角叉菜胶致大鼠背部气囊滑膜炎模型,分别以阿司匹林和布洛芬为阳性对照,测定小鼠足肿胀的抑制率及肉芽肿的重量,角叉菜胶致大鼠背部气囊滑膜炎模型中渗出液含量,白细胞(WBC)计数及血清中CRP、丙二醛(MDA)含量和血清总氧化能力,观察TFCW 3个剂量组对急、慢性炎症的影响及抗氧化作用。3)利用酵母双杂交方法,从分子水平观察TFCW和TFCW醇提取物对大鼠载脂蛋白APO-AI及清道夫受体SR-BI间相互作用的影响,揭示其升高HDL的机制。结果:TFCW中总蛋白含量为1.72%,灰分为1.16%,水分为88.9%;18种氨基酸中,9种人体必需的氨基酸(含婴幼儿必需的组氨酸)含量为0.30%,占总氨基酸的比例40.33%;各氨基酸中以丝氨酸的含量最高,为0.47g/100g,其次为谷氨酸,为0.14 g/100g;饱和脂肪酸(棕榈酸、硬脂酸)含量为38.7%,不饱和脂肪酸含量为61.3%,其中单不饱和脂肪酸高达46.0%,多不饱和脂肪酸为15.3%;乳糖含量为5.58%;矿物质中钾、钙、镁含量较高,分别为246.3 mg/100g、200.2 mg/100g和18.7 mg/100g;维生素B2、C的含量分别为0.23 g/100g和0.30 g/100g。TFCW 3个剂量组均显著降低动脉粥样硬化大鼠血清总TC、LDL和TG以及肝脏匀浆中TG水平(P<0.05),显著降低血清CRP(P<0.05),并降低血浆ET和血清TNF-α及IL-8水平(P<0.05)。病理结果显示:除正常组外,其余各组动脉均有不同程度的动脉粥样硬化改变。模型组冠状动脉形成典型动脉粥样斑块,内膜有堆积分布的泡沫状细胞病变区,平滑肌细胞增殖,内膜增厚,结缔组织增生以及胆固醇结晶核等出现,病变评分为1.23±0.51,心肌组织脂质沉积较严重。TFCW 25、100 mg·Kg-1组大多形成脂斑,未见到动脉粥样斑块,病变评分分别为0.82±0.24和0.67±0.33。辛伐他汀组病变评分为0.73±0.23。在新西兰兔动脉粥样硬化模型组中,TFCW 25,50 mg·Kg-1组显著降低CRP(P<0.01)。TFCW 25 mg·Kg-1组显著降低ICAM-1水平。在免疫组化检测中,ICAM-1在正常状态下呈低表达,动粥模型组内皮内均有ICAM-1表达,而TFCW 25,50 mg·Kg-1组ICAM-1表达减少。TFCW对角叉菜胶致小鼠足肿胀和棉球致大鼠肉芽肿形成均有显著的抑制作用,其能明显减少角叉菜胶致大鼠背部气囊滑膜炎模型渗出液体量,降低灌洗液中WBC数量和血清中CRP、MDA含量,100 mg·Kg-1组还可以提高角叉菜胶致大鼠气囊滑膜炎的血清中总抗氧化能力,并且可以降低MDA含量。在酵母双杂交系统,不同浓度的TFCW及TFCW醇提取物分别使报告基因活力平均上升了22.09%、41.84%和15.62%,即可提高大鼠载脂蛋白APO-AI及清道夫受体SR-BI之间相互作用。结论:哈萨克族传统发酵的TFCW具有抗动脉粥样硬化作用。物质基础为:TFCW总蛋白含量为1.72%,18种氨基酸中,9种人体必需的氨基酸(含婴幼儿必需的组氨酸)含量为0.30%,占总氨基酸的比例40.33%;其中以丝氨酸的含量最高,富含单不饱和脂肪酸及镁、钙、钾等元素。各剂量组可能是通过调节血脂—降低血清总TC、LDL、TG水平、升高HDL水平,降低细胞因子、CRP的含量,减少血清黏附因子水平及在血管内皮表达,以及具有抗炎抗氧化作用而发挥抗实验性动脉粥样硬化作用。其中血清HDL水平的升高及与其清道夫SR-BI受体相互作用的增强,以及显著降低CRP可能是TFCW抗动脉粥样硬化作用的重要环节。
Object: To study the effects and mechanisms of the traditional fermented cheese whey (TFCW) , a kind of the traditional Kazakh medicine, At first, to determine the main nutritional contents of TFCW ingredients according to national standards of P.R China, then to establish three different experimental atherosclerotic models in rats and rabbits, to determine the anti-atherosclerotic of TFCW. Methods: 120 rats were administrated with intragastric high-fat diet and injection of high-dose of vitamin D3, 55 male New Zealand white rabbits were fed with a normal diet or a high cholesterol diet for 12 weeks to induce injury of endothelial cells and atherosclerotic formation, then animals were treated with 25, 50 and 100 mg·Kg-1 TFCW, and 20mg·Kg-1 simvastatin or placebo for 4 weeks. Then plasma lipids, hepatic lipids, and serum C-reaction protein (CRP) , endothelin (ET) , TXB2, TNF-α, IL-8 and the serum levels of intercellular adhesion molecule-1 (ICAM-1) , vascular adhesion molecule-1 (VCAM-1) were determined, and the expression of VCAM-1 were detected by immunohistochemistry. The aorta and coronary were determined by computerized image analysis. Also, using 3 different animals model-an acute inflammatory reaction model employing as carrageenan inflammagen to induce mouse foot edema and swelling, a chronic inflammatory reaction model placing cottons into axillal cavity of rats to induce granuloma and an acute inflammatory model injecting carrageenan into back air-pouch of rats to induce synovitis were set up to test anti-inflammatory activity of TFCW. Then determined the amount of efflusion, WBC (white blood cell) count, and CRP, MDA content of blood serum. Results: In TFCW, total protein content is 1.72%, ash content is 1.16%and moisture content is 88.9%. Among the 18 kinds of amino acids, nine essential amino acids (including infant’s essential histidine) contents share 0.308%, accounting for about 40.33%. Serine content is the highest content (0.47g/100g) in all amino acids. Saturated fatty acid (palmitic acid, stearic acid) content is 38.7%, while unsaturated fatty acid content is 61.3%, among which monounsaturated fatty acid content is as high as 46.0%, and polyunsaturated fatty acid content is 15.3%. Lactose content is 5.58%. In minerals, potassium, calcium and magnesium, respectively are 246.3 mg/100g, 200.2 mg/100g and 18.7mg/100g; in vitamins, vitamin B2 and C content respectively are 0.23 g/100g and 0.30 g/100g. Rabbits and rats fed with cholesterol-rich diet showed higher serum lipid levels compared with the atherosclerotic control. Serum total cholesterol, low density lipoprotein cholesterol, triglyceride in plasma decreased compared with those of the model group. L area of lipid deposition on the intimae surface of the aorta and coronary arteries was reduced and the ultrastructulia injuries of endothelial cells were milder in dosage groups. The IL-8, ET, TNF-αand CRP in serum were dose-dependently decreased in the TFCW groups. TFCW groups also showed significant inhibitions of inflammatory activities compared with models, and could reduce the amount of efflusion, decrease WBC count, and CRP, MDA content of blood serum. The mechanisms of anti-inflammatory action of TFCW are related to reducing of inflammatory mediator and CRP, MDA contents. In decreasing the levels of inflammatory factors, the action of 25mg·Kg-1 TFWC is more powerful than the positive control group. TFCW decreased the levels of VCAM-1,ICAM-1and CRP. Conclusion: TFCW has anti-atherosclerotic effect, material constituents: total protein content is 1.72%. Among the 18 kinds of amino acids, nine essential amino acids (including infants essential histidine) contents share 0.308%, accounting for about 40.33%, Serine content is the highest content, higher constants of unsaturated fatty acid content, potassium, calcium and magnesium, the effect of anti-inflammatory action and anti-oxidation to experimental rabbits and rats, and it is suggested TFCW exert the significant effects of anti-atherosclerosis, TFCW decreasing the level of CRP, increasing the level of HDL in serum and intensifying mutual effect with SR-BI receptor are the main factor for anti-atherosclerosis effect.
Object: To study the effects and mechanisms of the traditional fermented cheese whey (TFCW) , a kind of the traditional Kazakh medicine, At first, to determine the main nutritional contents of TFCW ingredients according to national standards of P.R China, then to establish three different experimental atherosclerotic models in rats and rabbits, to determine the anti-atherosclerotic of TFCW. Methods: 120 rats were administrated with intragastric high-fat diet and injection of high-dose of vitamin D3, 55 male New Zealand white rabbits were fed with a normal diet or a high cholesterol diet for 12 weeks to induce injury of endothelial cells and atherosclerotic formation, then animals were treated with 25, 50 and 100 mg·Kg-1 TFCW, and 20mg·Kg-1 simvastatin or placebo for 4 weeks. Then plasma lipids, hepatic lipids, and serum C-reaction protein (CRP) , endothelin (ET) , TXB2, TNF-α, IL-8 and the serum levels of intercellular adhesion molecule-1 (ICAM-1) , vascular adhesion molecule-1 (VCAM-1) were determined, and the expression of VCAM-1 were detected by immunohistochemistry. The aorta and coronary were determined by computerized image analysis. Also, using 3 different animals model-an acute inflammatory reaction model employing as carrageenan inflammagen to induce mouse foot edema and swelling, a chronic inflammatory reaction model placing cottons into axillal cavity of rats to induce granuloma and an acute inflammatory model injecting carrageenan into back air-pouch of rats to induce synovitis were set up to test anti-inflammatory activity of TFCW. Then determined the amount of efflusion, WBC (white blood cell) count, and CRP, MDA content of blood serum. Results: In TFCW, total protein content is 1.72%, ash content is 1.16%and moisture content is 88.9%. Among the 18 kinds of amino acids, nine essential amino acids (including infant’s essential histidine) contents share 0.308%, accounting for about 40.33%. Serine content is the highest content (0.47g/100g) in all amino acids. Saturated fatty acid (palmitic acid, stearic acid) content is 38.7%, while unsaturated fatty acid content is 61.3%, among which monounsaturated fatty acid content is as high as 46.0%, and polyunsaturated fatty acid content is 15.3%. Lactose content is 5.58%. In minerals, potassium, calcium and magnesium, respectively are 246.3 mg/100g, 200.2 mg/100g and 18.7mg/100g; in vitamins, vitamin B2 and C content respectively are 0.23 g/100g and 0.30 g/100g. Rabbits and rats fed with cholesterol-rich diet showed higher serum lipid levels compared with the atherosclerotic control. Serum total cholesterol, low density lipoprotein cholesterol, triglyceride in plasma decreased compared with those of the model group. L area of lipid deposition on the intimae surface of the aorta and coronary arteries was reduced and the ultrastructulia injuries of endothelial cells were milder in dosage groups. The IL-8, ET, TNF-αand CRP in serum were dose-dependently decreased in the TFCW groups. TFCW groups also showed significant inhibitions of inflammatory activities compared with models, and could reduce the amount of efflusion, decrease WBC count, and CRP, MDA content of blood serum. The mechanisms of anti-inflammatory action of TFCW are related to reducing of inflammatory mediator and CRP, MDA contents. In decreasing the levels of inflammatory factors, the action of 25mg·Kg-1 TFWC is more powerful than the positive control group. TFCW decreased the levels of VCAM-1,ICAM-1and CRP. Conclusion: TFCW has anti-atherosclerotic effect, material constituents: total protein content is 1.72%. Among the 18 kinds of amino acids, nine essential amino acids (including infants essential histidine) contents share 0.308%, accounting for about 40.33%, Serine content is the highest content, higher constants of unsaturated fatty acid content, potassium, calcium and magnesium, the effect of anti-inflammatory action and anti-oxidation to experimental rabbits and rats, and it is suggested TFCW exert the significant effects of anti-atherosclerosis, TFCW decreasing the level of CRP, increasing the level of HDL in serum and intensifying mutual effect with SR-BI receptor are the main factor for anti-atherosclerosis effect.
引文
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