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降糖三黄片干预2型糖尿病大鼠Ghrelin、α-glucosidase表达的研究
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摘要
第一章文献研究
     2型糖尿病(T2DM)由于胰岛素抵抗并胰岛素分泌不足所导致,以往称为非胰岛素依赖型糖尿病、成年发病型糖尿病。大量研究表明,胰岛素抵抗和β细胞功能障碍是2型糖尿病发病机制的两个主要环节。现代口服药物如双胍类、磺脲类等临床一线药物,其降糖效果较为明确,已在各国普遍运用。
     胃肠道激素在糖尿病的发病中起着重要作用。从胃肠道激素上出发,寻找更加安全、有效的药物,成为研发新型糖尿病治疗药物的一个重要方向。医学界对一些与糖尿病相关的胃肠道激素如Ghrelin、瘦素(Leptin).α-葡萄糖苷酶、GLP-1、GIP的关注和研究越来越多,并根据胃肠道激素的特点,逐渐研发了相关药物,如α-GI(α-葡萄糖苷酶抑制剂)、GLP-1、GLP-1类似物和DDP-Ⅳ抑制剂、高血糖素受体拮抗剂等,为2型糖尿病的治疗开辟了新的途径。
     2型糖尿病属于中医学消渴范畴。消渴是以多饮、多食、多尿、身体消瘦、或尿浊、尿有甜味为特征的病证。《黄帝内经》围绕“消”字记载了消瘅、膈消、肺消、消中等不同疾病。这以后,历代医家根据各自经验,在防治消渴病方面,撰写了大量珍贵的文献古籍。这些文献古籍对消渴病的病因病机、发生发展过程、治疗原则、方药都有专门的论述。
     其中,东汉张仲景所著的《伤寒杂病论》为后世治疗消渴提供了理论依据和临床指导。《伤寒杂病论》中详细记载了消渴的发生、症状、治疗方药等,并提出了消渴病以及消渴症两个概念。更为重要的是,《伤寒杂病论》提出的六经辨证思想及其记载的大量简单而实用的方剂,一直成为后世治疗消渴的重要理论和方法。近年来,中医界针对《伤寒杂病论》中六经辨证的研究成果不断:在伤寒经方的研究和开发上也取得了丰硕的成绩。
     桃核承气汤出自《伤寒杂病论》太阳病篇,历代有关桃核承气汤的医案不胜枚举,现代医家已经将桃核承气汤的功效发挥,运用在多种疾病的治疗上。进入21世纪,国内外学者对桃核承气汤的研究越来越深入。除了不断对桃核承气汤原方进行研究之外,更多的是对该方的拆方或加减方进行研究,从药理、药效等多个角度着手,引入细胞学、分子生物学等微观指标作为评价依据。上个世纪90年代,广州中医药大学伤寒教研室研究团队围绕加味桃核承气汤开展了一系列临床和动物实验。研究结果表明,本方对糖尿病有一定的降糖、降脂作用。教研室还围绕气阴不足、瘀血阻络的病机,在《伤寒论》攻下逐瘀法的理论指导下,以桃核承气汤加黄芪、生地、玄参、麦冬等药物制成了具有益气养阴、泄热逐瘀功效的降糖三黄片,并围绕该药的作用机理和作用靶点开展了一系列的研究。
     第二章实验研究
     目的:通过观察降糖三黄片干预2型糖尿病大鼠模型胃肠激素Ghrelin、α-glucosidase的表达,初步探讨降糖三黄片的作用机理。
     方法:①运用高脂饲料喂养加STZ注射造模,将实验大鼠分为正常组、对照组、降糖三黄片组(以下简称“三黄组”),以及拜糖苹组。其中降糖三黄片按787.5mg/kg/d的剂量进行灌胃,拜糖苹按20mg/kg/d的剂量进行灌胃,两药分别研磨成粉并调成水剂;正常组及模型组灌服等量蒸馏水。三个月后观察各组大鼠体重、空腹血糖、空腹胰岛素的变化。并观察各组大鼠肝脏、胰脏、胃、小肠等组织病理变化。②运用双抗夹心酶联免疫检测法检测各组大鼠血浆Ghrelin浓度;运用免疫组化法测量各组胃壁、小肠上段组织中Ghrelin光密度值,并用相关统计方法探讨各组大鼠胃肠组织中Ghrelin光密度值与空腹血糖、空腹胰岛素之间的关系。③对各组小肠组织进行匀浆,运用酶联免疫分析法检测并观察各组小肠组织中α-glucosidase含量变化。
     结果:①从各项指标统计分析表明,三黄组大鼠实验前后体重比较均有统计学意义(P<0.01),实验前后空腹血糖比较均有统计学意义(P<0.01),实验前后空腹胰岛素比较均有统计学意义(P<0.01)。实验后正常组、模型组、三黄组、拜糖苹组体重、空腹血糖、空腹胰岛素等各项指标的组间比较均无统计学意义(P>0.05)。从病理检验来看,降糖三黄片对实验大鼠肝脏组织轻度脂肪变性可能起到一定作用。同时,与拜糖苹相比,降糖三黄片对实验大鼠小肠的炎症样改变可能略轻。从实验过程来看,降糖三黄片能有效改善实验大鼠模型整体情况,经降糖三黄片干预后的大鼠,其精神状况、皮毛色泽等均与正常组相似。②正常组、模型组、三黄组、拜糖苹组各组血浆Ghrelin含量组间比较无统计学意义(P>0.05)。模型组、三黄组、拜糖苹组胃组织中Ghrelin平均光密度值均较正常组显著增加,有统计学意义(P<0.01);而模型组、三黄组、拜糖苹组三组胃组织中Ghrelin平均光密度值组间比较无统计学意义(P>0.05)。正常组、模型组、三黄组、拜糖苹组小肠组织Ghrelin平均光密度值组间比较无统计学差异(P>0.05)。此外,以实验后胃组织Ghrenlin光密度值为白变量,以实验前后空腹血糖差值、空腹胰岛素差值分别作为应变量建立线性回归方程,结果显示:胃组织Ghrenlin光密度值与实验前后空腹胰岛素差值不存在统计学意义(P>0.05)。胃组.织Ghrenlin光密度值与实验前后空腹血糖差值存在统计学意义(P<0.01)。Ghrelin在胃和小肠组织中均有表达,但在胃中表达要高于小肠。③正常组、模型组、降糖三黄片组、拜糖苹组各组组间小肠α-glucosidase含量比较无统计学意义(P>0.05)。
     结论:①本研究未能说明降糖三黄片对糖尿病大鼠体重、空腹血糖、空腹胰岛素产生影响。但在研究中发现了降糖三黄片能够改善糖尿病大鼠整体状态,与西药拜糖苹相比有一定优势。②本研究观察了正常组、模型组、三黄组、拜糖苹组各组的肝脏、胰脏、胃、小肠等器官组织的病理变化。提示降糖三黄片对实验大鼠肝组织脂肪变性有一定改善作用;且与拜糖苹相比,降糖三黄片对糖尿病大鼠小肠的炎症样改变稍轻,有待于进一步验证。③本研究未能说明降糖三黄片对实验大鼠血浆Ghrelin含量产生影响。但发现了实验大鼠空腹血糖随着胃组织Ghrelin的升高而降低,提示胃组织Ghrelin可能参与了2型糖尿病的发生发展过程。本研究还进一步证明,Ghrelin在胃和小肠组织中均有表达,在胃中的表达要高于小肠。④本研究未能说明降糖三黄片对实验大鼠肠组织α-glucosidase含量产生影响。
First Part:Literature study
     The type 2 diabetes mellitus (T2DM) is caused by insulin resistance or low level of insulin. It was named non-insulin-dependent diabetes mellitus or maturity onset diabetes of the young. Though the T2DM is a genetic disease, but the mechanisms of it are not clear. Many studies have indicated that insulin resistance and dysfunction ofβcells are two important pathogenic factors of the T2DM. Sulfonylurea and metformin are orally drugs widely used in clinical cure in recent years.
     The gastrointestinal tract hormone plays an important role in diabetes. Researchers have carried on studies about gastrointestinal tract hormone to find a medicine which is safer and more effective. It is a new direction to in the research and development of new diabetes drug. More and more diabetes drugs have appeared, for exampleα-grape indicant enzyme inhibitors (α-GI), GLP-1, GLP-1 analogue and DDP-inhibitors, glucagons receptor antagonist. These drugs are designed to inhibit the effect of gastrointestinal tract hormone. It is a good way to cure T2DM.
     T2DM belongs to quench category in TCM, which is with more eat and drink, and urine than normal, and urine has a sweet taste. It is the characteristics of diabetic syndrome. The Medical Classic of the Yellow Emperor records varies type of diabetes. After that, there's thesis on it from many doctors in generations according to their experience they have made many discusses on the etiology, pathogenesis, syndrome differentiation and therapeutic principle for diabetes.
     Among that,《Treatise on Febrile and Miscellaneous Diseases》of Zhangzhong Jing in the Eastern Han Dynasty provides the theoretical basis and clinical guidance to cure diabetes. The monograph records the etiology, pathogenesis, syndrome differentiation and therapeutic principle for diabetes. The analyzing and differentiating of febrile diseases in accordance with the theory of the six pair of channels; differentiation of six channels and various prescriptions become to play important role in cure diabetes. In these years, a lot of researchers have done plenty studies on it and achieved remarkable
     progresses.
     Tao He Cheng Qi Tang is from《Treatise on Febrile and Miscellaneous Diseases》, and it has been used in plenty of cases to cure the sickness and save the patients. In recent years, researchers have made plentiful works for using Tao He Cheng Qi Tang. Some decomposed recipes analyses have made progresses in diabetes mellitus treatment. We have carried out Series of clinical and animal experiments to explore how it works. These studies turned out that Tao He Cheng Qi Tang is a useful prescription to reduce blood lipid and glucose. After that, we do research on Jiang Tang San Huang Pian, which is modified Tao He Cheng Qi Tang, and have proved it can supplement Qi for reinforce Yin and expel Heat to remove Blood stasis.
     Second Part:Experimental Study
     Objective:We studied the affection of Jiang Tang San Huang Pian on the expression level of Ghrelin、α-glycosidase. to primary discuss on mechanism. And to thorough research the underlying molecular mechanism。
     Method:Rats were divided into four groups randomly:normal control group, model control group, acarbose tablets group, and Jiang Tang San Huang Pian group. Type 2 diabetic rat models were established by feeding with high-lipid diet and intraperitoneal injection of small-dose streptozotocin (STZ) once. After 4 weeks treating by physiological saline, caboose tablets (20mg/kg/d), and San Huang Jiang Tang Pian (787.5mg/kg/d). By the end of 12th week, stopping given drugs for 24 hours, the blood were collected from femoral artery,Ⅰ.and then animal weight, free blood sugar(FBS), fasting insulin(FINS) were tested.And tissues such as liver, insulin, stomach and intestine were taken off from narcotized dead rats, and pathological lesions were analyzed.Ⅱ. At the same time, the tissue samples, including gastric, small intestine were collected, Ghrelin were inspected by immunohistochemical method; Ghrelin in blood plasma were inspected by ELSIA;Ⅲ. And small intestine were homogenated,α-glycosidase were inspected by ELISA.
     Result:Ⅰ. The blood glucose, weight and insulin level of Jiang Tang San Huang Pian group were higher than themselves before models were established (P<0.05). And there was no difference between the four treatment group (P> 0.05). But comparing, with the normal rats, Jiang Tang San Huang Pian can made some affection on lesion the fatty degeneration of rats'liver and inflammation in small intestine, but statistically insignificant. And mental symptoms and fur colors of rats in Jiang Tang San Huang Pian group was no differences with normal group.Ⅱ. Compared with the blank group, the level of Ghrelin in blood plasma was no difference (P all>0.05). Compared with the blank group, the expression of Ghrelin in stomach of other three groups significantly increased (P<0.01), but there were no differences among three groups. (P>0.05) Compared with the blank group, the expression of Ghrelin in small intestine were no noticeable distinction among these four groups (P>0.05). Furthermore, the expression of Ghrelin in stomach decline with the rise in FBS (P<0.01). Ghrelin expressed in stomach wall and small intestine, and the level in stomach wall was higher than that in small intestine.Ⅲ. There was no noticeable distinction among the four groups ofα-glycosidase.
     Conclusions:Ⅰ.Our study failed to illustrate the affection of Jiang Tang San Huang Pian on weight, blood sugar and insulin, but we found Jiang Tang San Huang Pian can greatly improve health status, and better than caboose tablets.Ⅱ. Jiang Tang San Huang Pian can make some affection on lesion the fatty degeneration of rats'liver and inflammation in small intestine,Ⅲ. Our study failed to illustrate the affection of Jiang Tang San Huang Pian on Ghrelin level of stomach wall, but the expression of Ghrelin in stomach of other three groups significantly increased. And the expression of Ghrelin in stomach can make affection on free blood sugar(FBS).In addition, Ghrelin expressed both in stomach wall and small intestine, and the level in stomach wall was higher than that in small intestine. IV. Our study failed to illustrate the affection of Jiang Tang San Huang Pian on content ofα-glycosidase in small intestine.
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