复方中药对高脂饮食大鼠胰岛素抵抗的影响
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摘要
目的: 胰岛素抵抗(insulin resistance，IR)不仅是2型糖尿病的主要病理生理特征，也是高血压、冠心病、肥胖、血脂异常、动脉粥样硬化发生的独立危险因素。近年来，关于2型糖尿病发病机制，炎症学说备受重视，越来越多的证据表明炎症在2型糖尿病的发生、发展和预后过程中起重要的作用。多种炎症因子与2型糖尿病的关系是糖尿病研究领域的一个热点，揭示炎症与2型糖尿病的关系有重要临床意义。胰岛素发挥作用必须首先与细胞膜上的胰岛素受体结合。二者结合后诱导细胞内发生一系列代谢变化，发挥胰岛素降低血糖，增加组织细胞对葡萄糖摄取的作用。因此胰岛素受体的数目及功能的缺陷，均会导致胰岛素作用的减弱。而肝脏细胞是胰岛素作用的经典靶器官之一，观察肝细胞表面胰岛素受体结合动力变化，有助于了解IR的发病机制；血浆纤溶酶原激活物抑制剂-1（plasminogen activator inhibitor type 1，PAI-1）水平的升高会促进动脉粥样硬化形成，与糖尿病大血管并发症密切相关，而且血浆PAI-1活性高低与胰岛素抵抗程度有关。对于IR，中医学有肾虚血瘀理论解释其病因，并认为IR属于消渴症范畴。本研究在用于治疗“消渴症”的中药复方----玉液汤的基础上，对其中的成分进行了加减，制备成新的中药复方----玉液汤化裁。本方剂具有益气滋阴，固肾消渴，疏血通络之功效。因此本研究旨在: 1 通过高脂
饮食复制大鼠胰岛素抵抗模型； 2 探讨炎症因子TNF-α等在胰岛素抵抗发病中的作用，观察胰岛素抵抗大鼠肝细胞膜胰岛素受体的数目、结合力和PAI-1mRNA在肝组织表达的变化; 3 观察复方中药（玉液汤化裁）对高脂饮食大鼠血浆炎症因子、肝细胞膜胰岛素受体数目、结合力及PAI-1mRNA在肝组织表达的影响，探讨复方中药对IR的防治作用，开发一种新的预防胰岛素抵抗有效的中药配方，为临床治疗胰岛素抵抗及糖尿病提供新方法。
方法: 健康SD雄性大鼠90只， 体重208±20ｇ，由河北省实验动物中心提供。随机分为6组(每组15只):普通饲料组(第1组，正常对照组)，高脂饲料组(2－6组:含20%熟猪油，2%胆固醇，0.5%胆酸盐)， 第2组为单纯高脂饮食组（疾病组），第3组为二甲双胍干预组，第4、5、6组为低、中、高剂量复方中药干预组。分笼饲养，每笼5只，共6个月。
每组大鼠均于实验开始、结束时称量体重、取血。禁食12h后，晨起眼内眦取血4.5ml，分离血清，保存于－20℃，用于各指标的检测（测定指标时均使用同一批试剂盒）。实验结束时留取肝脏组织保存在液氮中，用于肝细胞膜胰岛素受体测定及RT-PCR。
1 血糖、胰岛素的测定: 空腹血糖（FPG）用葡萄糖氧化酶法测定；空腹胰岛素（FINS）用放射免疫法测定。采用稳态模型（Homeoestasis Model Assessment， HOMA）及胰岛素敏感指数(Insulin Sensitivity Index， ISI) 评价胰岛素抵抗的程度。HOMA-IR=(FBG×FINS)/22.5表示胰岛素抵抗的程度； ISI=1/(FINS×FBG)，表示胰岛素敏感性。均使用自然对数表
示。
2 血脂:甘油三脂(triglyceride，TG)、总胆固醇(total cholesterole，TC)、高密度脂蛋白胆固醇(highdensity lipoprotein –cholesterole，HDL-C)用比色法测定，低密度脂蛋白胆固醇（lowerdensity lipoprotein–cholesterole ， LDL-C）由Friedewald公式（LDL-C=TC－TG/2.2）求出。
3 炎症相关因子测定: 放射免疫法测定TNF-α含量，免疫沉淀法测定C反应蛋白（CRP）含量，Cu2+比色法测定游离脂肪酸（FFA）含量。
4 肝细胞膜胰岛素受体测定: 4.1 肝细胞膜的分离；4.2取少量细胞膜悬液测蛋白浓度，Lowry蛋白定量；4.3肝细胞膜胰岛素受体放射配基竞争结合；4.4 Scatchard 分析，计算胰岛素受体数目及亲和力。
5 肝组织PAI-1mRNA的表达: 异硫氰酸胍一步法抽提RNA， 反转录-聚合酶链反应（RT-PCR）分别扩增PAI-1和β- actin mRNA，扩增产物经琼脂糖电泳、摄片和计算机图像扫描。RT-PCR 产物半定量: 因β- actin在组织中均匀表达， PAI-1基因的表达水平用靶基因与β- actin的比值表示。
6 统计学处理
所有数据用SPSS10.0软件处理，计量资料以均数±标准差（±S）表示，组间差异用两样本均数的 t检验进行比较，两组以上比较采用单因素方差分析。胰岛素受体数据用Scatchard 分析。检验的显著性用P值表示，P＜0.05为差异有显著性，P＜0.01为差异有极显著性。
结果
1 高脂饮食对各组体重的影响
实验初，大鼠随机分配，各组体重无统计学差别。实验末，单纯高脂饮食组体重(492.5±46.5g)明显高于正常对照组(433.8±25g)，有显著差异（P＜0.01）。中药中剂量(按公斤体重计，为临床用量的5.4倍)组体重(437.5±25.5g)和二甲双胍组体重(440±36g)与正常对照组相比，无显著差异，但均明显低于单纯高脂组体重（P＜0.01）。
2 高脂喂养和复方中药对血糖、胰岛素和HOMA-IR、ISI的影响
不同饲料喂养大鼠6个月后，各组间血糖无显著差别，单纯高脂组的空腹胰岛素水平(33.3±8.7μIU/L)与正常对照组(18.1±2.4μIU/L)相比明显升高（P＜0.01），单纯高脂组HOMA-IR(8.9±3.0)明显高于正常对照组(4.1±0.8)，ISI(－5.25±0.37)明显低于正常对照组(－4.53±0.21)，差异有显
Objective: Insulin resistance (IR) is not only the main pathophysioloy characteristic of type 2 diabetes mellitus (DM)，but also the independent risk factor of hypertension 、coronary heart disease(CHD)、obesity、dyslipidemia and atherosclerosis (AS). Hypothesis of inflammation in the pathogenesis of DM is paid attention to in recent years. And many lines of evidence had shown that inflammation plays an important role in the occurrence 、development and prognosis of DM. The relation between inflammatory factors and DM is the hot spot in the field of DM research. It has the important clinical significance to reveal the relation between them. Insulin should be fast bound to insulin receptors on the cell surface membrane, which induce a series cascade reaction to decrease blood glucose and increase the cell uptake of glucose. Therefore, the decreased number or affinity can weaken the role of insulin. The liver is one of the classic targets of insulin. So it is helpful to understand IR by observing the change of binding of insulin with it’s receptor. The increased plasminogen activator inhibitor type 1(PAI-1) can accelerate the formation of AS. The level of PAI-1 has the close relation with the large vessels complication . Moreover, it
relates with IR. We improved the traditional compound herbal medicine----“yu ye tang” which was used in treating “xiao ke zheng”,and got a new compound herbal medicine prescription that has the efficacy of “ yi qi zi yin, gu shen xiao ke, shu xue tong luo”. So the aim of the study were as follows: 1 Through the determination of HOMA-IR and Insulin sensitive index (ISI), to observe the amelioration of IR. 2 Through observing changes of inflammatory factors, the number and binding capacity of insulin receptor, the expression of PAI-1 mRNA in liver to explore the effect of compound herbal medicine on IR. 3 Through the observation of effect of compound herbal medicine on IR, to develop a new prescription of traditional Chinese medicine which has the effect therapy for IR and DM . So providing a new method for the clinic to intervene IR and diabetes.
Methods: Ninety healthy SD male rat, which weigh 208±20g, were provided by HeBei province experimental animal center. They were randomly divided into 6 groups and 15 rats in each group. Group 1 were the normal control group which were fed with the common feed. From group 2 to group 6 were fed with the high fat feed (which include 20% ripe porcine fat and 2% cholesterol, 0.5% cholalic salt). In the meantime, Group 3 were given with metformin. Group 4,5,6 were intervened with compound herbal medicine(yu ye tang hua cai) at various doses. Five rats were fed in each cage for 6 months.
Rats were weighed and their blood samples were taken at
the beginning and the end of the experiment. After an overnight fast, 4.5ml blood samples were taken from the medial canthus of eyes. Serum was stored at –20℃ until used. Liver tissue were taken and stored in liquid nitrogen for the determination of insulin receptor and PAI-I mRNA.
1 Measurements of glucose and insulin: method of glucose oxidase for determining FBG and radio-immunity for FINS. HOMA-IR and insulin sensitivity index (ISI) were calculated to evaluate the resistance and sensitivity of insulin. And they were express by logarithm.
2 TG 、TC 、HDL were measured by the ratio of chrom. LDL were calculated by the Friedewald (LDL-C=TC-TG/2.2)
3 The measure of inflammatory factors: TNF-α，CRP, FFA respectively measured by radio-immunity, immunoprecipitation, the ratio of Cu2+ chrom.
4 The determination of insulin receptor in liver cells: 4.1 the membrane of the rat liver cells were separated 4.2 measure the protein concentration by some liquor of membrane 4.3 the competitive binding of radio-ligand with insulin receptors 4.4 analysis with Scatchard
5 Expression of PAI-I mRNA: Rat liver tissues mRNA was extracted by the quanidinum thiocyanate. Then PAI-1 and β-actin mRNA were respectively amplified by RT-PCR. The RT-PCR products were observed by agarose
饮食复制大鼠胰岛素抵抗模型； 2 探讨炎症因子TNF-α等在胰岛素抵抗发病中的作用，观察胰岛素抵抗大鼠肝细胞膜胰岛素受体的数目、结合力和PAI-1mRNA在肝组织表达的变化; 3 观察复方中药（玉液汤化裁）对高脂饮食大鼠血浆炎症因子、肝细胞膜胰岛素受体数目、结合力及PAI-1mRNA在肝组织表达的影响，探讨复方中药对IR的防治作用，开发一种新的预防胰岛素抵抗有效的中药配方，为临床治疗胰岛素抵抗及糖尿病提供新方法。
方法: 健康SD雄性大鼠90只， 体重208±20ｇ，由河北省实验动物中心提供。随机分为6组(每组15只):普通饲料组(第1组，正常对照组)，高脂饲料组(2－6组:含20%熟猪油，2%胆固醇，0.5%胆酸盐)， 第2组为单纯高脂饮食组（疾病组），第3组为二甲双胍干预组，第4、5、6组为低、中、高剂量复方中药干预组。分笼饲养，每笼5只，共6个月。
每组大鼠均于实验开始、结束时称量体重、取血。禁食12h后，晨起眼内眦取血4.5ml，分离血清，保存于－20℃，用于各指标的检测（测定指标时均使用同一批试剂盒）。实验结束时留取肝脏组织保存在液氮中，用于肝细胞膜胰岛素受体测定及RT-PCR。
1 血糖、胰岛素的测定: 空腹血糖（FPG）用葡萄糖氧化酶法测定；空腹胰岛素（FINS）用放射免疫法测定。采用稳态模型（Homeoestasis Model Assessment， HOMA）及胰岛素敏感指数(Insulin Sensitivity Index， ISI) 评价胰岛素抵抗的程度。HOMA-IR=(FBG×FINS)/22.5表示胰岛素抵抗的程度； ISI=1/(FINS×FBG)，表示胰岛素敏感性。均使用自然对数表
示。
2 血脂:甘油三脂(triglyceride，TG)、总胆固醇(total cholesterole，TC)、高密度脂蛋白胆固醇(highdensity lipoprotein –cholesterole，HDL-C)用比色法测定，低密度脂蛋白胆固醇（lowerdensity lipoprotein–cholesterole ， LDL-C）由Friedewald公式（LDL-C=TC－TG/2.2）求出。
3 炎症相关因子测定: 放射免疫法测定TNF-α含量，免疫沉淀法测定C反应蛋白（CRP）含量，Cu2+比色法测定游离脂肪酸（FFA）含量。
4 肝细胞膜胰岛素受体测定: 4.1 肝细胞膜的分离；4.2取少量细胞膜悬液测蛋白浓度，Lowry蛋白定量；4.3肝细胞膜胰岛素受体放射配基竞争结合；4.4 Scatchard 分析，计算胰岛素受体数目及亲和力。
5 肝组织PAI-1mRNA的表达: 异硫氰酸胍一步法抽提RNA， 反转录-聚合酶链反应（RT-PCR）分别扩增PAI-1和β- actin mRNA，扩增产物经琼脂糖电泳、摄片和计算机图像扫描。RT-PCR 产物半定量: 因β- actin在组织中均匀表达， PAI-1基因的表达水平用靶基因与β- actin的比值表示。
6 统计学处理
所有数据用SPSS10.0软件处理，计量资料以均数±标准差（±S）表示，组间差异用两样本均数的 t检验进行比较，两组以上比较采用单因素方差分析。胰岛素受体数据用Scatchard 分析。检验的显著性用P值表示，P＜0.05为差异有显著性，P＜0.01为差异有极显著性。
结果
1 高脂饮食对各组体重的影响
实验初，大鼠随机分配，各组体重无统计学差别。实验末，单纯高脂饮食组体重(492.5±46.5g)明显高于正常对照组(433.8±25g)，有显著差异（P＜0.01）。中药中剂量(按公斤体重计，为临床用量的5.4倍)组体重(437.5±25.5g)和二甲双胍组体重(440±36g)与正常对照组相比，无显著差异，但均明显低于单纯高脂组体重（P＜0.01）。
2 高脂喂养和复方中药对血糖、胰岛素和HOMA-IR、ISI的影响
不同饲料喂养大鼠6个月后，各组间血糖无显著差别，单纯高脂组的空腹胰岛素水平(33.3±8.7μIU/L)与正常对照组(18.1±2.4μIU/L)相比明显升高（P＜0.01），单纯高脂组HOMA-IR(8.9±3.0)明显高于正常对照组(4.1±0.8)，ISI(－5.25±0.37)明显低于正常对照组(－4.53±0.21)，差异有显
Objective: Insulin resistance (IR) is not only the main pathophysioloy characteristic of type 2 diabetes mellitus (DM)，but also the independent risk factor of hypertension 、coronary heart disease(CHD)、obesity、dyslipidemia and atherosclerosis (AS). Hypothesis of inflammation in the pathogenesis of DM is paid attention to in recent years. And many lines of evidence had shown that inflammation plays an important role in the occurrence 、development and prognosis of DM. The relation between inflammatory factors and DM is the hot spot in the field of DM research. It has the important clinical significance to reveal the relation between them. Insulin should be fast bound to insulin receptors on the cell surface membrane, which induce a series cascade reaction to decrease blood glucose and increase the cell uptake of glucose. Therefore, the decreased number or affinity can weaken the role of insulin. The liver is one of the classic targets of insulin. So it is helpful to understand IR by observing the change of binding of insulin with it’s receptor. The increased plasminogen activator inhibitor type 1(PAI-1) can accelerate the formation of AS. The level of PAI-1 has the close relation with the large vessels complication . Moreover, it
relates with IR. We improved the traditional compound herbal medicine----“yu ye tang” which was used in treating “xiao ke zheng”,and got a new compound herbal medicine prescription that has the efficacy of “ yi qi zi yin, gu shen xiao ke, shu xue tong luo”. So the aim of the study were as follows: 1 Through the determination of HOMA-IR and Insulin sensitive index (ISI), to observe the amelioration of IR. 2 Through observing changes of inflammatory factors, the number and binding capacity of insulin receptor, the expression of PAI-1 mRNA in liver to explore the effect of compound herbal medicine on IR. 3 Through the observation of effect of compound herbal medicine on IR, to develop a new prescription of traditional Chinese medicine which has the effect therapy for IR and DM . So providing a new method for the clinic to intervene IR and diabetes.
Methods: Ninety healthy SD male rat, which weigh 208±20g, were provided by HeBei province experimental animal center. They were randomly divided into 6 groups and 15 rats in each group. Group 1 were the normal control group which were fed with the common feed. From group 2 to group 6 were fed with the high fat feed (which include 20% ripe porcine fat and 2% cholesterol, 0.5% cholalic salt). In the meantime, Group 3 were given with metformin. Group 4,5,6 were intervened with compound herbal medicine(yu ye tang hua cai) at various doses. Five rats were fed in each cage for 6 months.
Rats were weighed and their blood samples were taken at
the beginning and the end of the experiment. After an overnight fast, 4.5ml blood samples were taken from the medial canthus of eyes. Serum was stored at –20℃ until used. Liver tissue were taken and stored in liquid nitrogen for the determination of insulin receptor and PAI-I mRNA.
1 Measurements of glucose and insulin: method of glucose oxidase for determining FBG and radio-immunity for FINS. HOMA-IR and insulin sensitivity index (ISI) were calculated to evaluate the resistance and sensitivity of insulin. And they were express by logarithm.
2 TG 、TC 、HDL were measured by the ratio of chrom. LDL were calculated by the Friedewald (LDL-C=TC-TG/2.2)
3 The measure of inflammatory factors: TNF-α，CRP, FFA respectively measured by radio-immunity, immunoprecipitation, the ratio of Cu2+ chrom.
4 The determination of insulin receptor in liver cells: 4.1 the membrane of the rat liver cells were separated 4.2 measure the protein concentration by some liquor of membrane 4.3 the competitive binding of radio-ligand with insulin receptors 4.4 analysis with Scatchard
5 Expression of PAI-I mRNA: Rat liver tissues mRNA was extracted by the quanidinum thiocyanate. Then PAI-1 and β-actin mRNA were respectively amplified by RT-PCR. The RT-PCR products were observed by agarose
引文
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31 熊曼琪. 加味桃核承气汤治疗Ⅱ型糖尿病的临床核实验
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26 Damdona P，Aljada A. A rational approach to pathogensis and treatment of type 2 diabetes mellitus，insulin resistance，inflammation and atherosclerosis. Am J Cardiol，2002，90，27G～33G
27 胡世荣. 中草药治疗糖尿病经验的整理与分析. 山东中医学院学报， 1982，6（2）: 57
28 刘永玉. 实验性NIDDM大鼠的胰岛素抗性. 中国病理生理杂志，1991，7（4）: 422
29 于志清. 纯单黄酮甙131碘－胰岛素与脂肪细胞结合的影响. 广西医学院学报，1980，（1）: 22
30 梁晓春. 金芪降糖片治疗气阴两虚火旺型糖尿病临床及实验研究. 中国中西医结合杂志，1993，13（10）: 587
31 熊曼琪. 加味桃核承气汤治疗Ⅱ型糖尿病的临床核实验
研究. 中国中西医结合杂志，1992，12（2）: 74