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餐附注射液对糖尿病大鼠心肌超微结构的保护
时间：2010-08-24 09:09:46 来源： 作者：
| 方法：实验于2003－12/2005－01在重庆医科大学生理教研室完成。①实验分组：30只Wistar大鼠随机分为正常对照组10只、糖尿病组20只。②实验方法：所有大鼠禁食12 h 后，糖尿病组大鼠以链脲佐菌素(以0.1 mol/L，pH=4.2的枸橼酸缓冲液溶解) 按60 mg/kg经腹腔注射，正常对照组以等量的枸橼酸缓冲液腹腔注射。72 h 后，大鼠剪尾取静脉血测定空腹血糖，当空腹血糖> 16.67 mmol/L并有多饮、多食、多尿者，即为1型糖尿病造模成功。糖尿病组大鼠又按分层随机法分为单纯糖尿病组和参附注射液处理组，每组10只，参附注射液组以参附注射液 10 mL/kg腹腔注射，1次/d。正常对照组和单纯糖尿病组大鼠均以生理盐水10 mL/kg腹腔注射。所有大鼠均以普通食物喂养，自由饮水和单笼饲养20周。③实验评估：20周后测定各组血糖、血脂、血清胰岛素、血清与心肌超氧化物歧化酶活性和丙二醛含量；免疫组化法检测心肌细胞膜葡萄糖转运蛋白4 mRNA蛋白表达；观察各组大鼠胰岛细胞的病理结构以及心肌超微结构的变化。 结果：30只大鼠均进入结果分析。①空腹血糖、血清胰岛素水平：造模后20 周，参附注射液组的空腹血糖明显低于造模3 d时水平（P < 0.05），明显低于单纯糖尿病组，但高于正常对照组（P < 0.01）；单纯糖尿病组空腹血糖明显高于造模3 d时水平 （P < 0.01）。造模后20 周，参附注射液组血清胰岛素水平明显低于正常对照组，但显著高于单纯糖尿病组(P < 0.05)。②血脂水平：参附注射液组和单纯糖尿病组的总三酰甘油、总胆固醇和低密度脂蛋白胆固醇水平明显高于正常对照组（P < 0.05），参附注射液组各值明显低于单纯糖尿病组（P < 0.05）；单纯糖尿病组和参附注射液组高密度脂蛋白胆固醇水平明显低于正常对照组(P < 0.05)，参附注射液组高密度脂蛋白胆固醇水平明显高于单纯糖尿病组(P < 0.05)。③血清和心肌超氧化物歧化酶活性及丙二醛含量：超氧化物歧化酶活性：参附注射液组和单纯糖尿病组明显低于正常对照组(P < 0.01)，参附注射液组明显高于单纯糖尿病组(P < 0.05)；血清丙二醛的含量：参附注射液组和单纯糖尿病组明显高于正常对照组(P < 0.05)，参附注射液组明显低于单纯糖尿病组(P < 0.05)。④葡萄糖转运蛋白4 mRNA阳性表达：参附注射液组吸光度显著低于正常对照组（P < 0.01），明显高于单纯糖尿病组（P < 0.01），而参附注射液组的面密度值显著低于正常对照组（P < 0.01），明显高于单纯糖尿病组（P < 0.01）。⑤光镜观察：参附注射液组大鼠胰岛细胞水肿或空泡样变比单纯糖尿病组数量少而且更轻；透视电镜观察参附注射液组其心肌细胞线粒体和心肌毛细血管内皮细胞的水肿较单纯糖尿病组程度更轻。 结论：参附注射液对糖尿病大鼠心肌超微结构具有保护作用，这与其清除氧自由基和抗脂质过氧化物有关；参附注射液对糖尿病大鼠健存胰岛细胞的保护，提高了糖尿病大鼠的血清胰岛素水平，降低了血糖、血脂，对糖尿病大鼠心肌内微血管内皮细胞和心肌细胞线粒体的保护具有促进作用。 关键词：糖尿病；心肌病；胰岛细胞；超微结构；参附注射液 Protection of myocardium ultrastructure by Shenfu injection in diabetes mellitus rats Ａｂｓｔｒａｃｔ ＡＩＭ: To investigate the effect of Shenfu injection (SFI) on the ultrastructure of myocardium in diabetes mellitus (DM) rats, and explore the possible mechanism. ＭＥＴＨＯＤＳ: The experiments were carried out in the Department of Physiology at Chongqing Medical University from December 2003 to January 2005.①Thirty Wisatr rats were randomly divided into normal control group (n =10) and DM group (n =20).②All the rats were fasted for 12 hours, then those of DM group were treated with 60 mg/kg streptozotocin (dissolved in 0.1 mol/L citric acid buffer, pH=4.2) by intraperitoneal injection, while those of normal control group were injected with equal volume of citric acid buffer solution. And 72 hours later, venous blood was sampled from rat tails to detect fasting blood glucose (FBG). Those rats appearing polydipsia, polyphagia and polyuria, whose FBG were more than 16.7 mmol/L indicated successful animal model of type 1 DM. According to stratified random, DM group was assigned to the diabetic control group (C-DM group) and the SFI treated group (S-DM group), 10 rats in each. The rats in S-DM group were injected with SFI (10 mL/kg) into peritoneal cavity once a day. The rats in control group and C-DM group were injected with 10 mL/kg saline by peritoneal cavity. All the rats were fed routinely for 20 weeks.③At the end of week 20, the FBG, blood fat, serum insulin, superoxide dismutase (SOD) and malondialdehyde (MDA) in serum and myocardium were assayed. The expressions of glucose transport protein 4 mRNA on the myocardial cytomembrane were detected by immunohistochemical assay; the pathologic changes of islets of pancreas and myocardial ultrastructure of rats in three groups were also observed. ＲＥＳＵＬＴＳ: Thirty rats were all involved in the result analysis.①Levels of FBG and serum insulin: The FBG of rats in S-DM group was remarkably lower than that of C-DM group but much higher than that of control group (P < 0.01) at the end of 20 weeks. FBG of rats at the end of 3 weeks was obviously higher than that at the end of 20 weeks in S-DM group (P < 0.05) but obviously lower in C-DM group (P < 0.01). The serum insulin of rats in S-DM group was obviously higher than that of C-DM group but significantly lower than that of control group (P < 0.05).②Blood fat level: The levels of total triglyceride, cholesterol, and low-density lipoprotein cholesterol were obviously higher in S-DM group and C-DM group than those of control group (P < 0.05), and all obviously lower in S-DM group than those of C-DM group (P < 0.05); The levels of high-density lipoprotein cholesterol in C-DM group and S-DM group were obviously lower than that of control group (P < 0.05), and the level was significantly higher in S-DM group than in C-DM group (P < 0.05).③Contents of SOD and MDA in serum and myocardium: SOD in S-DM group was much more than that of C-DM group (P < 0.05), but much less than that of control group (P < 0.01) while MDA was much less than that of C-DM group (P < 0.05), but much more than that of control group (P < 0.05).④Expressions of glucose transport protein 4 mRNA on cardiac cytomembrane of rats in S-DM group were significantly less than those of control group (P < 0.01) while obviously more than those of C-DM group (P < 0.01).⑤Under optic microscope, there was much less cellular edema or vacuole of pancreatic islets in rats from S-MD group compared with that of C-DM group. Under transmission electron microscope, the edema on myocardial mitochondria and endothelial cells of cardiac capillary in C-DM group was much more serious than that of S-DM group. ＣＯＮＣＬＵＳＩＯＮ: SFI can protect the ultrastructure of myocardium in DM rats and remove oxygen-derived free radicals and resist lipid oxidation, moreover, it has the protective effect on healthy islets of pancreas in diabetic rats, which does facilitate the protection on endothelial cells of cardiac capillary and the mitochondria of myocardium, because of the increase in serum insulin and decrease in blood glucose and fat. 引言 糖尿病心肌病是一种特征性心肌病变，其病理改变有别于高血压性、动脉粥样硬化性、瓣膜性以及肺源性心肌病变，主要是因高血糖引起糖、脂代谢异常，导致心肌内微血管病变，微血管通透性增加，血管、血管周围膜及心肌间质纤维化等病变，即糖尿病性心肌病变。 参附注射液其主要成分为人参皂苷和附子，通过改善微循环，抗氧自由基和抗脂质氧化作用，扩张冠状动脉[1－3]，具有钙离子通道阻滞样作用，防止细胞内钙超载[4,5]，增强心肌的收缩力，改善心脏的舒缩功能。本实验旨在观察参附注射液对糖尿病大鼠血糖、血脂及糖尿病鼠胰岛细胞的病理改变和心肌超微结构的变化并探索其可能机制。 材料和方法 设计：完全随机对照动物实验。 单位：广西医科大学第五附属医院，重庆医科大学附属第一医院。 材料：实验于2003－12/2005－01在重庆医科大学生理教研室完成。健康成年雌性Wister大鼠30只，清洁级，体质量200～250 g，由重庆医科大学动物实验中心提供（许可证号：SYXK(渝)20020007）。链脲佐菌素(购自美国Sigma公司)、参附注射液（购自雅安三九药业有限公司，批号030402）、考马斯亮蓝、超氧化物歧化酶及丙二醛试剂盒（购自南京建成生物工程公司）、血清胰岛素（购自中国原子能同位素研究所）、葡萄糖转运蛋白4（购自美国Santa Cruz 公司）、One Touch II血糖仪及试纸（德国生产），低温离心机（上海生产），其他化学试剂均为国产分析纯等。 设计、实施、评估者：设计、实验和评估均由第一、二作者完成，第三、四作者参与实验资料的整理。 方法：建立1型糖尿病动物模型与分组：所有动物适应性观察一周后，随机分为正常对照组10只和糖尿病组20只。所有大鼠禁食12 h 后，糖尿病组大鼠以链脲佐菌素(以0.1 mol/L，pH=4.2的枸橼酸缓冲液溶解) 按60 mg/kg经腹腔注射，正常对照组以等量的枸橼酸缓冲液腹腔注射。72 h 后，所有大鼠剪尾取静脉血测定空腹血糖，当空腹血糖> 16.67 mmol/L并有多饮、多食、多尿者，即为1型糖尿病造模成功。糖尿病组大鼠又按分层随机法分为单纯糖尿病组和参附注射液处理组，每组10只，参附注射液组以参附注射液 10 mL/kg腹腔注射，1次/d。正常对照组和单纯糖尿病组大鼠均以生理盐水10 mL/kg腹腔注射。所有大鼠均以普通食物喂养，自由饮水，饲养环境：温度20～25 ℃，湿度40%～60%，单笼饲养20周。取材前大鼠禁食12 h，自由饮水。取眼眶静脉血测定空腹血糖、取3×1.0 mL静脉血以2 000 r/min离心5 min，取血清置 －20 ℃冰箱备用以测定血清胰岛素、总三酰甘油、总胆固醇和低密度脂蛋白胆固醇、高密度脂蛋白胆固醇以及血清超氧化物歧化酶和丙二醛。拉颈处死动物，迅速开胸取出心脏于4 ℃冰盐水中，剪开左心室壁，取2 mm×2 mm×2 mm 左心室壁数块置于4 ℃ 2.5%戊二醛备电镜切片。取左心室壁0.5 cm×0.5 cm×0.5 cm置4%多聚甲醛固定，石蜡包埋备组织切片，以同样的方法开腹取胰腺组织备组织切片。取左心肌组织200 mg加1.8 mL冰生理盐水高速匀浆，制备成10%心肌匀浆，低温离心机（3 000 r/min）离心10 min，取上清液－20 ℃冰箱备用。 血糖采用邻甲苯胺微量法测定，总三酰甘油和总胆固醇采用氧化酶法测定，高密度和低密度脂蛋白胆固醇均采用化学直接法测定，血清胰岛素采用放免法测定，超氧化物歧化酶和丙二醛分别采用邻苯三酚自氧化法和硫代巴比妥酸法测定。 心肌免疫组化及结果判断：心肌石蜡切片常规二甲苯及梯度乙醇脱蜡至水，采用S-P免疫组化法检测心肌细胞膜葡萄糖转运蛋白4 mRNA蛋白表达，其阳性表达利用图像分析系统(Image-Pro-Plug 5.02.9，美国)，以其吸光度和面密度表示，吸光度和面密度值越高，表示其阳性表达也越多。 Nuttall SL, Dunne F, Kendall MJ, et al.Age-independent oxidative stress in elderly patients with non-insulin-dependent diabetes mellitus. QJM 1999;92(1):33-38 Esposito K, Nappo F, Marfella R, et al.Inflammatory cytokine concentrations are acutely increased by hyperglycemia in humans: role of oxidative stress. Circulation 2002;106(16):2067-2072 Mahgoub MA, Abd-Elfattah AS.Diabetes mellitus and cardiac function.Mol Cell Biochem 1998;180(1-2):59-64 Zhao XY, Hu SJ, Li J, et al.Decreased cardiac sarcoplasmic reticulum Ca2+ -ATPase activity contributes to cardiac dysfunction in streptozotocin-induced diabetic rats. J Physiol Biochem 2006;62(1):1-8 Singh J, Chonkar A, Bracken N, et al.Effect of streptozotocin-induced type 1 diabetes mellitus on contraction, calcium transient, and cation contents in the isolated rat heart. Ann N Y Acad Sci 2006;1084:178-190 Sakata S, Lebeche D, Sakata Y, et al.Transcoronary gene transfer of SERCA2a increases coronary blood flow and decreases cardiomyocyte size in a type 2 diabetic rat model. Am J Physiol Heart Circ Physiol 2007;292(2):H1204-H1207 Iwakura K, Ito H, Ikushima M, et al.Association between hyperglycemia and the no-reflow phenomenon in patients with acute myocardial infarction. J Am Coll Cardiol 2003;41(1):1-7 Coskun O, Kanter M, Korkmaz A, et al.Quercetin, a flavonoid antioxidant, prevents and protects streptozotocin-induced oxidative stress and beta-cell damage in rat pancreas. Pharmacol Res 2005;51(2):117-123 Coskun O, Ocakci A, Bayraktaroglu T, et al.Exercise training prevents and protects streptozotocin-induced oxidative stress and beta-cell damage in rat pancreas. Tohoku J Exp Med 2004;203(3):145-154 Zorzano A, Palacin M, Guma A.Mechanisms regulating GLUT4 glucose transporter expression and glucose transport in skeletal muscle.Acta Physiol Scand 2005;183(1):43-58 Luiken JJ, Coort SL, Koonen DP, et al.Regulation of cardiac long-chain fatty acid and glucose uptake by translocation of substrate transporters.Pflugers Arch 2004;448(1):1-15 Kaul N, Siveski-Iliskovic N, Hill M, et al.Probucol treatment reverses antioxidant and functional deficit in diabetic cardiomyopathy.Mol Cell Biochem 1996;160-161:283-288 Chen Y, Saari JT, Kang YJ. Weak antioxidant defenses make the heart a target for damage in copper-deficient rats. Free Radic Biol Med 1994;17(6):529-536 Ren J, Bode AM. Altered cardiac excitation-contraction coupling in ventricular myocytes from spontaneously diabetic BB rats. Am J Physiol Heart Circ Physiol 2000;279(1):H238-H244 |
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