药学学报  2015, Vol. 50 Issue (4): 453-458   PDF    
仙人掌多糖对大鼠动脉粥样硬化的治疗作用及其机制
王玉春 , 齐占朋, 刘振中, 李涛, 崔红霞, 王宝庆, 迟娜    
齐齐哈尔医学院, 黑龙江 齐齐哈尔 161006
摘要:本研究旨在探索仙人掌多糖(Opuntia dillenii Haw polysaccharide,OPS)治疗大鼠动脉粥样硬化的作用及其机制.利用高脂高钙饮食构建大鼠动脉粥样硬化模型,于造模第30 天开始同时给予低剂量(0.2g·kg-1·d-1)、高剂量(0.4 g·kg-1·d-1)仙人掌多糖腹腔注射,连续治疗60 天.治疗结束后,分离胸主动脉血管,利用血管环技术检测不同组大鼠胸主动脉的舒张功能; 采集血液及肝脏组织,利用全自动生化分析仪检测大鼠血浆中总胆固醇(total cholesterol,TC)、甘油三酯(triglyceride,TG)及低密度脂蛋白(low density lipoprotein,LDL)的含量; Western Blot 技术检测肝脏载脂蛋白B(apolipoprotein,ApoB)及二酰基甘油酰基转移酶(diglycerideacyltransferase,Dgat1)蛋白表达变化.结果显示,模型对照组大鼠胸主动脉血管的舒张功能与正常对照组比较明显降低,不同浓度的卡巴胆碱(carbamylcholine chloride,Carb)引起的舒张率与模型对照组和正常对照组之间均存在显著统计学差异(均P<0.01);仙人掌多糖治疗后,血管舒张功能明显改善,其中低剂量仙人掌多糖组,Carb浓度为5 和10 μmol·L-1时胸主动脉的血管舒张率分别为0.34 ± 0.08 和0.62 ± 0.15; 高剂量仙人掌多糖组,Carb 浓度为1和5 μmol·L-1时血管舒张率分别为0.54 ± 0.08和0.98 ± 0.02,与模型组比较均存在显著统计学差异(均P<0.01); 低、高剂量仙人掌多糖治疗后,大鼠血浆TC、TG 和LDL 含量较模型组显著降低(均P<0.01); 高剂量仙人掌多糖组大鼠肝脏ApoB 含量及Dgat1 表达量与模型对照组比较明显降低(均P<0.01).结果表明,仙人掌多糖能够明显改善动脉粥样硬化大鼠的血管舒张功能,有显著的抗动脉粥样硬化作用; 抑制肝脏ApoB 蛋白及Dgat1 蛋白的表达,进而降低血浆TC、LDL 及TG 含量,是仙人掌多糖治疗动脉粥样硬化的分子机制之一.
关键词仙人掌     多糖     动脉粥样硬化    
Therapeutic effects and mechanisms of Opuntia dillenii Haw on atherosclerosis of rats
WANG Yu-chun , QI Zhan-peng, LIU Zhen-zhong, LI Tao, CUI Hong-xia, WANG Bao-qing, CHI Na    
Qiqihar Medical University, Qiqihar 161006, China
Abstract: The research aimed to investigate the therapeutic effects and mechanisms of Opuntia dillenii Haw polysaccharide (OPS) on atherosclerosis of rats. First atherosclerotic rat models were established by high-fat and high-calcium diet. Thirty days later, the rats were treated with low dosage of OPS (0.2 g ·kg-1·d-1) or high dosage of OPS (0.4 g·kg-1·d-1) by intraperitoneal injection for 60 days continuously. At the end of treatment, thoracic aorta rings were prepared and vasorelaxation of rat thoracic aorta in different experiment groups were determined by using 620M multi wire myograph system in vitro. Blood and livers of rats were collected. Then plasma levels of total cholesterol (TC), triglyceride (TG) and low density lipoprotein (LDL) of rats were separately determined using whole automatic biochemical analyzer; protein level of hepatic apolipoprotein B (ApoB) and that of hepatic diglyceride acyltransferase (Dgat1) were measured by Western Blot technique. Results showed that the ability of rat thoracic aorta to relax decreased markedly in the model group compared with that in the normal group, and significant differences existed in vasorelaxation ratios induced by different concentrations of carbamylcholine chloride (Carb) between these two groups (P<0.01). After OPS treatment, the ability of rat thoracic aorta to relax improved markedly, the vasorelaxation ratios induced by Carb at 5 and 10 μmol·L-1 were respectively 0.34 ± 0.08 and 0.62 ± 0.15 in the group treated with low dosage of OPS, while the ratios induced by Carb at 1 and 5 μmol·L-1 were respectively 0.54 ± 0.08 and 0.98 ± 0.02 in the group treated with high dosage of OPS, which were all significantly different with those in the model group (P<0.01). Plasma contents of TC, TG and LDL reduced significantly by the treatments both with low and high dosages of OPS compared with those in the model group (P<0.01). Protein level of hepatic ApoB and that of hepatic Dgat1 decreased significantly after the treatment with high dosage of OPS compared with those in the model group (P<0.01). These results indicate that OPS can markedly improve the vasorelaxation of thoracic aorta of atherosclerotic rats and has significant anti-atherosclerotic effect; inhibiting the expression of ApoB and Dgat1 and thus decreasing the amounts of TC, LDL and TG serving as one of the molecular mechanisms of its antiatherosclerosis effect.
Key words: Opuntia dillenii Haw     polysaccharide     atherosclerosis    

近年来,随着人们经济水平的提高以及生活方式的转变,动脉粥样硬化血管病变疾病的发病率及死亡率逐年上升,严重危害人类的健康[1]。其发生发展的主要原因是巨噬细胞结合并吞噬变性低密度脂蛋白 (LDL),造成细胞内脂质蓄积、泡沫化细胞的形成,继而在血管内壁堆积形成脂质纹理[2,3]以及导致动脉血管壁持续的炎症浸润[4]。目前首选的调脂药物是他汀类药物,这类药通过抑制羟甲基戊二酰辅酶A (HMG-CoA) 还原酶降低低密度脂蛋白胆固醇以及改善内皮细胞功能、抑制平滑肌细胞增殖等作用,对动脉粥样硬化有显著治疗作用[5],但这类药物在预防或治疗动脉粥样硬化的同时,存在潜在的导致肌炎、糖尿病、肾病、白内障和勃起功能障碍等疾病风险[6]。因此,研发有效预防及治疗动脉粥样硬化的药物具有重要意义。

研究资料表明,仙人掌多糖具有显著的降血脂、抗炎和增加患者机体免疫力等多种作用[7,8,9],由于血脂异常及炎症等因素是促进动脉粥样硬化形成的主要因素,因此,推测仙人掌多糖可能具有抗动脉粥样硬化的作用,故设计实验考察其作用及可能机制。

材料与方法 实验动物

成年SD大鼠,雄性,250~300g,购自哈尔滨医科大学附属第二医院动物实验中心。自由进食和饮水。

药品和试剂

仙人掌多糖由齐齐哈尔大学提供; 重酒石酸去甲肾上腺素 (NA,天津金耀氨基酸有限公司); 卡巴胆碱 (Carb,日本和光纯药工业株式会社); 兔抗ApoB多克隆一抗 (Sc-25542) 购自Santa Cruz公司; 兔抗Dgat1多克隆一抗 (NB110-41487) 购自Novus Biologicals公司; GADPH一抗购自上海康成生物公司; 驴抗兔荧光二抗购于美国Invitrogen公司。

仪器

Olympus 5400全自动生化分析仪,Odyssey红外荧光扫描仪成像系统 (美国LI-COR公司); 蛋白电泳仪 (BIO-RAD公司); DMT620微血管张力测定仪 (丹麦DMT公司); 倒置显微镜 (日本Nikon公司)。

仙人掌多糖的制备

按照文献[10]提取仙人掌多糖,纯度为77.3%,3种水溶性多糖重均分子量分别为2.32×106、1.24×106和7.92×106

模型的建立及给药

实验动物随机分为正常对照组、模型对照组、仙人掌多糖低剂量组 (0.2 g·kg-1·d-1) 和高剂量 (0.4 g·kg-1·d-1),每组10只。正常对照组给予普通大鼠饲料,其余3组动物均按照文献[11]方法建立动脉粥样硬化大鼠模型,即每日给予大鼠高脂饲料 (2% 胆固醇、0.5% 胆酸钠、3% 猪油、0.2% 丙基硫氧嘧啶和94.3% 的基础饲料) 加维生素D3粉剂 (1.25×106 u·kg-1),实验开始时于右下肢肌肉注射维生素D3针剂 (3×105 u·kg-1体重),每隔30天重复1次,连续造模90天。从造模第30天开始,高剂量组和低剂量组腹腔注射仙人掌多糖,至造模第90天结束,对照组给予同体积生理盐水。

min后取上清液

样本采集与处理 给药结束次日取样,于取样前12 h禁食不禁水。眼球取血,肝素抗凝,以3 000 r·min-1离心,10 ,于 -20 ℃保存备用; 颈椎脱臼处死大鼠后,迅速开胸,取胸主动脉,制备主动脉环; 取肝脏组织,于 -80 ℃冻存备用。

胸主动脉环的制备

将取出的胸主动脉置于通混合气体 (95% O2 + 5% CO2) 的生理盐溶液 (PSS,4 ℃) 中,成分为 (mmol·L-1): NaCl 130、KCl 4.7、KH2PO4 1.18、MgSO47H2O 1.17、NaHCO3 14.9、葡萄糖5.5、EDTA 0.026和CaCl2 1.60。分离血管周围结缔组织,剪约2~3 mm的动脉环,通过2个L型钢针悬挂于血管张力仪浴糟中 (37 ℃,pH 7.4),让血管环在浴槽中静置20 min后,调节血管张力至最适被动张力值,继续静置40 min,每20 min更换一次PSS。最后利用NA及KCl对血管环执行唤醒程序,以唤醒血管。

血管舒张功能的检测

向浴槽中加入终浓度为1 μmol·L-1的NA诱导胸主动脉预收缩,待血管张力达平稳状态后,分别给予终浓度为1、5和10 μmol·L-1的Carb溶液,记录血管张力变化曲线,并以NA诱导的血管收缩最高峰为对照,计算加入不同浓度的Carb后主动脉的舒张率。

血浆TCTGLDL含量测定

利用Olympus 5400全自动生化分析仪检测血浆中总胆固醇 (TC)、甘油三酯 (TG) 和LDL含量。

肝脏ApoBDgat1蛋白含量检测

采用Western Blot方法检测。 取肝脏提取的总蛋白50 μg与5×loading buffer充分混合,经100 ℃变性5 min后,分别经聚 丙烯酰胺凝胶电泳 (SDS-PAGE) (ApoB: 6%; Dgat: 12%)、转膜及5% 脱脂牛奶室温封闭2 h,然后加入一抗4 ℃孵育过夜 (ApoB: 1∶200; Dgat: 1∶500),次日使用1∶10 000稀释的红外荧光标记二抗室温避光孵育60 min,应用Odyssey红外荧光扫描成像系统对NC膜上蛋白进行成像及结果分析。

统计方法

利用SPSS12.0软件进行分析,计量资料组间比较进行完全随机设计单因素方差分析,组内比较进行Dunnett-t检验。

结果 1 仙人掌多糖对动脉粥样硬化大鼠胸主动脉舒张功能的影响

在正常对照组内,NA诱导主动脉预收缩后,加入1和5 μmol·L-1 Carb均引起大鼠主动脉明显舒张,舒张率分别为0.77 ± 0.10和0.98 ± 0.02,如图 1A及表 1所示; 在动脉粥样硬化模型组内,1和5 μmol·L-1 Carb均未引起大鼠主动脉显著舒张,舒张率分别为0.07 ± 0.03和0.14 ± 0.04,与正常对照组相应浓度Carb引起的舒张率比较存在显著性差异 (均P < 0.01),表明模型大鼠血管舒张功能与正常对照组比较明显降低; 将Carb浓度增至终浓度为10 μmol·L-1时,仍未引起显著舒张,如图 1B及表 1所示。在仙人掌多糖低剂量组,1、5和10 μmol·L-1 Carb引起的舒张率分别为0.09 ± 0.08,0.34 ± 0.08和0.62 ± 0.15,其中5及10 μmol·L-1 Carb引起的舒张率与模型对照组相应浓度的舒张率比较有显著性差异 (均P < 0.01),如图 1C及表 1所示; 在仙人掌多糖高剂量组,1及5 μmol·L-1 Carb引起的舒张率分别为0.54 ± 0.08,0.98 ± 0.02,与模型对照组相应浓度的舒张率比较有显著性差异 (均P < 0.01),如图 1D及表 1。结果表明: 仙人掌多糖治疗后,大鼠胸主动脉的血管舒张功能明显改善。

Figure 1 Recording of vasorelaxation of rat thoracic aorta in different experimental groups. A: Normal group; B: Atherosclerosis rat model group; C: Model group with low dosage of Opuntia dillenii Haw polysaccharide (OPS); D: Model group with high dosage of OPS. NA: Noradrenaline bitartrate; Carb: Carbamylcholine chloride

Table 1 Effects of OPS on the vasorelaxation of thoracic aorta of atherosclerotic rats. n = 6,x± s. CTL: Control group; AM: Atherosclerosis model group; H-OPS: High dosage of OPS; L-OPS: Low dosage of OPS. **P < 0.01 vs CTL; ##P < 0.01 vs AM
2 血浆TCTGLDL含量变化

动脉粥样硬化模型对照组血浆TC、TG及LDL与正常对照组比较明显增加,低、高剂量仙人掌多糖治疗后,血浆TC、TG及LDL水平与模型对照组比较明显降低 (均P < 0.01),见表 2

Table 2 Effects of OPS on plasma contents of TC,TG and LDL. n = 10,x± s. TC: Total cholesterol; TG: Triglyceride; LDL: Low density lipoprotein. **P < 0.01 vs CTL; ##P < 0.01 vs AM
3 肝脏ApoB蛋白表达变化

模型组大鼠肝脏ApoB蛋白水平与正常对照组比较明显增高 (P < 0.01),而高剂量仙人掌多糖治疗后ApoB蛋白水平明显降低,与模型对照组比较差异显著 (P < 0.01,图 2A及2B),但低剂量仙人掌多糖治疗后,大鼠肝脏ApoB蛋白水平未见明显改变 (P > 0.05),见图 2C及2D。

Figure 2 Effects of OPS on protein level of hepatic ApoB of atherosclerotic rats. A: High dosage of OPS inhibited protein level of hepatic ApoB of atherosclerotic rats; B: Standardized intensity of ApoB on Western blot in normal,atherosclerotic model and high dosage of OPS groups were respectively presented as mean ± SEM; C: Low dosage of OPS showed no effect on protein level of hepatic ApoB of atherosclerotic rats; D: Standardized intensity of ApoB on Western blot in normal,atherosclerotic model and low dosage of OPS groups were respectively presented as mean ± SEM. n = 5. ApoB: Apolipoprotein B; AS: Atherosclerosis. **P < 0.01 vs control group; ##P < 0.01 vs AM
4 肝脏Dgat1蛋白表达变化

模型组大鼠肝脏Dgat1蛋白水平与正常对照组比较明显增高 (P < 0.01),而高剂量仙人掌多糖治疗后Dgat1蛋白水平明显降低,与模型对照组比较差异显著 (P < 0.01,图 3A及3B),但低剂量仙人掌多糖治疗后,大鼠肝脏Dgat1蛋白水平未见明显改变 (P > 0.05),见图 3C及3D。

Figure 3 Effects of OPS onprotein level of hepatic Dgat1 of atherosclerotic rats. A: High dosage of OPS inhibited protein level of hepatic Dgat1 of atherosclerotic rats; B: Standardized intensity of Dgat1 on Western blot in normal,atherosclerotic model and high dosage of OPS groups were respectively presented as mean ± SEM; C: Low dosage of OPS showed no effect on protein level of hepatic Dgat1 of atherosclerotic rats; D: Standardized intensity of Dgat1 on Western blot in normal,atherosclerotic model and low dosage of OPS groups were respectively presented as mean ± SEM. n = 5. Dgat1: Diglyceride acyltransferase. **P < 0.01 vs control group; ##P < 0.01 vs AM
讨论

在动脉粥样硬化时,由于血管内一氧化氮 (nitric oxide,NO) 和内皮素 (endothelin,ET) 的平衡失调等原因,普遍存在内皮功能障碍,后者是导致或加重该动脉粥样硬化的重要因素之一[12]。本研究结果显示,模型组大鼠胸主动脉的舒张功能明显降低,仙人掌多糖治疗后血管舒张功能明显改善,说明仙人掌多糖对动脉粥样硬化血管功能有显著的改善作用。本实验结果也从侧面证明此研究中实验动物模型建立成功。

动脉粥样硬化疾病与血浆胆固醇水平直接相关,TC或LDL-CHOL水平是国内及国际诊断和治疗动脉粥样硬化的依据[13,14]。本研究结果显示,模型组大鼠血浆TC、TG及LDL与正常对照组比较明显增加,证明动脉粥样硬化模型建立成功; 仙人掌多糖能够显著降低动脉粥样硬化大鼠的血浆总胆固醇水平及低密度脂蛋白胆固醇的水平,说明仙人掌多糖具有显著的抗动脉粥样硬化作用。

ApoB是由肝脏合成的LDL-CHOL的主要结构蛋白,约占LDL-CHOL总蛋白含量的97%。研究表明,ApoB被动脉壁细胞捕获后可始动并维持动脉粥样硬化的形成过程[15],因此对ApoB蛋白表达水平的抑制直接影响动脉粥样硬化的形成,即减少ApoB蛋白水平可减少动脉粥样硬化斑块的形成[16]。本研究结果显示,高剂量仙人掌多糖组大鼠肝脏ApoB的水平较模型组明显降低,说明仙人掌多糖可通过抑制ApoB蛋白表达水平发挥抗动脉粥样硬化的作用。

前期研究结果[16]表明,降低ApoB蛋白水平会引起Dgat1蛋白表达水平显著降低,后者是促进脂肪酸合成甘油三酯的关键酶,而降低Dgat1的表达能降低脂质诱导的炎症及巨噬细胞迁移,具有预防动脉粥样硬化形成的作用[17,18],因此本研究进一步检测了肝脏Dgat1的表达水平。结果显示,高剂量仙人掌多糖组大鼠肝脏Dgat1水平及血浆TG水平均较模型组明显降低,说明抑制Dgat1蛋白表达水平的增高是仙人掌多糖抗动脉粥样硬化的另一重要机制。

本研究中尽管低剂量仙人掌多糖对动脉粥样硬化大鼠的血管功能及血脂异常有显著的改善,但未见对肝脏ApoB及Dgat1表达量的显著影响,提示仙人掌多糖治疗动脉粥样硬化的作用尚存在其他重要机制,有待进一步研究。

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