芪蓝糖脂宁胶囊复方再优化研究及其糖脂双调的机理探讨

【作者】 欧丽娜；

【导师】 高学敏；

【作者基本信息】 北京中医药大学 ， 临床中药学， 2007， 博士

【摘要】 糖尿病(Diabetes Mellitus, DM)是一种常见的内分泌代谢性疾病,其基本病理特点为胰岛素分泌绝对或相对不足,或外周组织对胰岛素不敏感,引起以糖代谢紊乱为主,包括脂肪、蛋白质代谢紊乱的一种全身性疾病。因此,单纯控制血糖不能完全消除糖尿病患者冠心病等大血管并发症,往往同时进行调脂治疗。中医传统复方注重整体调节,标本兼治,近年来,在治疗糖尿病合并高脂血症方面表现了巨大的潜力。芪蓝糖脂宁胶囊是导师高学敏教授针对糖尿病合并高脂血症的病因病机,在中医基础理论指导下,结合现代药理学研究成果创制的科研处方。前期张德芹博士已对其进行了文献、理论和药效学实验研究,证明其有显著的糖脂双调的效果,是治疗糖尿病合并高脂血症的有效良方。但是,该方含有黄芪、绞股兰、红曲等九味中药,药味较复杂,为生产、质控和服用都带来了不便,因此,本课题以此方为研究对象,采用直接实验设计法,对其进行复方再优化研究,然后,对再优化所得最优方进行糖、脂双降的药效和机理探讨。1文献研究1.1糖尿病合并高脂血症与胰岛素抵抗全面查阅了近年来国内外糖尿病合并高脂血症、胰岛素抵抗及理论的相关研究文献资料,阐明了胰岛素抵抗为糖、脂代谢紊乱的共同病因,并对糖尿病合并高脂血症胰岛素抵抗的发病机理及研究方法进行了系统的总结和综述。1.2中药复方再优化研究进展查阅整理了目前应用于复方再优化的主要实验设计方法及数据优化处理方法,掌握了相关项目科研的研究动态。2.实验研究2.1芪蓝糖脂宁复方再优化试验运用直接实验设计法安排药效实验,获得实验信息,在前期复方再优化理论研究的基础上,结合专业知识,将药味间的交互作用引入变量筛选过程,利用SAS软件进行多元线性回归分析,得到针对不同效应指标的回归方程,并预测出各指标相应的优化方。依据传统中医药理论及各优化方中药味和药量出现的频次,拟合出最优方――芪曲糖脂宁胶囊(QQTZN)。筛选所得最优方QQTZN由黄芪、葛根等五味中药组成,药味精良,且符合医理药理,功能益气健脾,生津止渴,化瘀降浊。2.2最优方药效实验验证采用高糖高脂饲料合并小剂量链脲佐菌素注射的方法复制2型糖尿病合并高脂血症模型。在此基础上观察最优方对模型大鼠糖、脂代谢以及胰腺和肝脏病理形态学改变。2.2.1对糖代谢血清生化学指标的影响给药6周后,二甲双胍组、最优方高、中、低剂量组血糖水平均较模型组有显著的下降,而反证方组降糖作用明显不如最优方。大鼠糖耐量实验结果表明,与模型组相比,最优方高、中、低剂量组可不同程度的降低糖耐量曲线下面积,疗效优于反证方。2.2.2对脂代谢血清生化学指标的影响给药6周后,模型组大鼠血清TC、TG与给药前相比也有所下降,分析与给药期间停止高脂饮食及大鼠自身的脂质清除能力较强有关。各给药组血清TC、TG和血清LDL-C水平与模型组相比均明显下降。同时,血清HDL-C水平均较模型组有显著上升。最优方降低血清TC和TG的作用优于反证方。2.2.3胰腺、肝脏组织病理形态学改变胰腺、肝脏HE染色结果显示,最优方高、中、低剂量组可不同程度的改善胰腺、肝脏病理损害,效果优于反证方。综上所述,最优方药少力专,糖脂双调的药效学作用确切,明显优于反证方,与理论预测结果一致,进一步证实了复方再优化方法对确有疗效的中药复方的筛选和精练处方的作用。结合现代医学知识和中医传统理论,分析反证方的组方特点,可以看出数据筛选剔除了部分滋阴及降脂作用相对较强而补气和降糖作用相对较弱的中药,从而达到精练处方的目的,使最优方更突出了补气健脾,化瘀降浊,进而糖脂双调的特点。2.3机理探讨2.3.1对胰岛素受体前水平影响采用放射免疫技术研究了最优方对模型大鼠血清胰岛素含量的影响,结果表明,QQTZN高剂量组与模型组相比,血清胰岛素水平明显降低,胰岛素敏感性指数明显升高。中、低剂量组与模型组相比,血清胰岛素水平无明显变化,但胰岛素敏感性指数明显升高。采用免疫组化技术研究了最优方对模型大鼠胰岛α、β细胞胰岛素、胰高糖素表达的影响。结果显示,QQTZN高、中、低剂量组β细胞数量比模型组增多,胰岛素表达较模型组明显增强;α细胞数量比模型组减少,胰高血糖素表达较模型组明显降低。上述结果表明,QQTZN可以通过增加胰岛素敏感性,修复链脲佐菌素(STZ)造成的胰岛β细胞损伤,以及调节胰岛α细胞分泌胰高糖素水平来影响胰岛素发挥正常的生物效应。2.3.2对胰岛素受体后PI-3K信号传导途径的影响采用免疫组化技术研究了最优方对肝脏组织IRS-2及PI-3K表达的影响。结果表明,最优方高、中、低剂量组肝脏组织IRS-2表达较模型组明显提高。QQTZN低剂量组与模型组相比PI-3K的表达有明显提高,QQTZN高、中剂量组PI-3K表达与模型组相比有升高的趋势,但无显著差异。采用RT-PCR产物半定量技术对实验大鼠骨骼肌GluT4mRNA进行了检测,结果表明,QQTZN高、低剂量组GluT4 mRNA表达与模型组相比有显著升高。从上述结果可以看出,QQTZN可以使IRS-2、PI-3K以及GluT4的表达有不同程度的提高,表明该药可以从多个位点改善胰岛素的信号传导障碍,使胰岛素得以发挥其正常的生理功能,调节糖、脂代谢。2.3.3对PPARγ调控及其它脂肪源性细胞因子的影响采用免疫组化技术研究了最优方对肝脏组织PPARγ表达的影响。结果表明,与模型组相比,高、中剂量组有不同程度的增加模型大鼠肝脏PPARγ表达的作用。采用ELISA法检测了最优方对脂肪组织TNF-α表达的影响。结果显示,QQTZN高、中剂量组有显著降低脂肪中的TNF-α浓度的作用。提示最优方可通过降低TNF-α水平,解除TNF-α对脂肪和肌肉GluT4表达抑制,刺激脂肪分解等作用提高胰岛素敏感性。采用比色法研究了最优方对血清中FFA含量的影响。结果表明,QQTZN高、中、低剂量组均可显著降低血清中FFA的含量。提示最优方可以通过降低脂毒性对机体的损害而达到调节糖、脂代谢的作用。上述结果表明,QQTZN可增加PPARγ表达,抑制TNF-α及FFA的释放,调节脂代谢,而且可以通过抑制TNF-α、FFA的释放,及改善胰岛素信号传导通路障碍等多条途径增加胰岛素的敏感性,从而对糖代谢进行调节。本课题的创新点在于:(1)在张学中教授的指导下,将直接实验设计法用于中药有效复方的再优化研究,在考察单味药对药效贡献度的基础上,根据中药复方用药的特点,考虑了多味药交互作用对药效的贡献度,更加符合中医基础理论及用药规律。(2)本课题研究的是中药复方对糖尿病合并高脂血症的影响,发病机理存在糖代谢紊乱和脂代谢紊乱并存的现象,因此在机理探讨方面,从二者的共同致病因素胰岛素信号传导通路障碍入手,同时考察了对脂肪源性细胞因子的影响,更好的解决了糖、脂共调的问题,为中药干预糖、脂代谢的作用机理研究提供了新的思路。综上所述,通过本实验研究,在原有效复方芪蓝糖脂宁的基础上凝炼出了药味精良的新复方QQTZN,并在复方优化过程中考察中药交互作用的贡献度,丰富了复方优化的方法。随着数理统计学的发展,将不断有新的方法产生,不断探索更符合中医理论特点的数据处理方法是我们努力的目标。在机理探讨的过程中,通过对胰岛素信号传导通路以及脂源性细胞因子的研究,表明QQTZN从多环节、多靶点影响胰岛素信号传导通路,从而实现糖、脂双调的效果,为中药复方治疗糖尿病合并高脂血症提供了科学证据,具有一定的理论和实践价值。更多还原

【Abstract】 Diabetes Mellitus (DM) is a complex disease, not only relates to glycometabolism, but also to lipid metabolism disorder, and the lipid metabolism disorder is not totally secondary with glycometabolism. So just control the level of serum glucose can not eliminate the Complication and von Willebrand Factor in Type 2 Diabetic Patients completely, and we always treat it with regulate the lipid metabolism. The traditional Chinese multi-prescription focus on integral regulation of metabolism, and treat branch and root, so it present a large potential in treatment of diabetes mellitus with hyperlipemia.Qilan Tangzhining capsule have the effect of regulating both glycometabolism and lipid metabolism, consist of radix astragali, Gold Theragran, red rice, etc.The ingredients is complex, and take inconvenient of production and quality controlling. So we make this prescription as an object, further optimized it with direct experimental design, and then research the mechanism of regulating glycometabolism and lipid metabolism of the optimized prescription.1 literature research1.1 Insulin resistant and diabetes with hyperlipemia We have reviewed DM-related literature in the past 10 years home and abroad, interpreting that the insulin resistant is the common disease causes of glycometabolism and lipid metabolism disorder, and having a good hand of the present study of insulin resistant, DM and hyperlipoidemia.1.2 Study advancement of Further optimization of prescription(FOP) we have reviewed FOP-related literature, such as experiment design and data optimizing methods, mastered the research development of related project.2 Laboratory experiments2.1 further optimization of Qilan Tangzhining prescription Use the direct experimental design to arrange the animal experiment, obtain the information, and based on FOP theory and expertise, consider the interaction of drugs into variable screen, doing multiple linear regression analysis with SAS software, and obtain the regression equation correspond to different effect index, then predict the best optimized prescription. According to the frequency of ingredients and dosages in different optimized prescription, recombine the best prescription—Tangzhining capsule.The best prescription—Tangzhining capsule consist of Radix Astragalus, Radix Puerariae, and so on, according to traditional Chinese medicine theory and with excellent prescription ingredient, with indications of nourishing qi to invigorate the spleen, promoting the production of body fluid to quench thirst, and remove stasis and transform turbid.2.2 The best prescription’s Efficacy Study Diabetic with Hyperlipoidemia model was induced by a high fat diet with intraperitoneal injection of streptozotic into male Wistar rats at a low dose. The effects of Qilan Tangzhining capsule on glycometabolism, lipid metabolism, and pathomorphological changes of liver and pancreas were observed among modeled rats.2.2.1Serum biochemics tests of glycometabolism After treatment for 6 weeks, the diformin group, the medium-dose and low-dose Tangzhining groups significantly lower down the blood-fasting sugar level of model rats, while the effect of disproof group is not as good as Tangzhining groups. Sugar tolerance test in rat indicated that the high dose, medium-dose and low-dose Tangzhining groups significantly lower down the area under the sugar tolerance curve, and do better than that of disproof groups.2.2.2 Serum biochemics tests of lipid metabolism After treatment for 6 weeks, serum level of cholesterol and triglyceride of model group are decreased, we think it relate to the ceasing of high fat diet and the rat ability of removing lipid.The result shows that Tangzhining groups of all doses have the significant effect of cutting down serum level of cholesterol, triglyceride, LDL-cholesterol, and elevating serum level of HDL-cholesterol.2.2.3 HE stain of pancreas and liver tissues of rats The result indicates that Tangzhining groups of all doses can improve pathological impairment of pancreas and liver. And the effects are better than that of disproof group.In general, Primary pharmacodynamics proves that FOP theory is scientific and feasible; Tangzhining capsule is well-formulated and effective in treating DM with hyperlipoidemia.2.3 Mechanism approaches2.3.1 The effect of before insulin receptor levelUse of radio-immunity method in studying the effects of Tangzhining capsule on serum insulin in experimental DM rats with hyperlipoidemia. The result shows that high-dose Tangzhining capsule significantly decreases the serum insulin level and improve the insulin sensitive. The medium-dose and low-dose Tangzhining capsule groups have no significant effect on serum insulin but can improve insulin sensitive significantly.Use of immunohistochemical method in studying the effects of Tangzhining capsule on pathomorphological changes of pancreaticβcells andαcells in experimental DM rats with hyperlipoidemia. The result shows that Tangzhining capsule increases the number of insulin positiveβcells and positive reactions while glucagons positiveαcells decrease in number and positive reactions are less compared with model group. It suggests that Tangzhining capsule can repair pancreaticβcell impairment, inhibit the secretion of glucagons fromαcells and lower down blood sugar level in DM rats with hyperlipoidemia.2.3.2 The effect of PI-3K signal conduction path (after insulin receptor level) Use of immunohistochemical method in studying the effects of Tangzhining capsule on IRS-2 and PI-3K expression of the liver. The result shows that Tangzhining capsule dosages groups improve the expression of IRS-2 compare with model group. The low-dose Tangzhining group improve the expression of PI-3K while the medium-dose and high-dose Tangzhining groups improve the expression of PI-3K with no significant deviation.Use of RT-PCR semiquantitive analysis in studying the effects of Tangzhining capsule on the expression of GluT4 mRNA of skeletal muscle on experimental DM rats with hyperlipoidemia. The result shows that low-dose and high-dose Tangzhining groups increase the expressions of GluT4 mRNA.The above result indicates that Qilan Tangzhining capsule may enhancing the expressions of IRS-2, PI-3K and GluT4 mRNA. Therefore, it can improve the insulin signal conduction from multi-site, and help the insulin play the normal role in regulating glycometabolism and lipid metabolism.2.3.3 The effect of PPARγand adipocytokinesUse of immunohistochemical method in studying the effects of Tangzhining capsule on PPARγexpression of the liver. The result shows that high-dose and medium-dose Tangzhining groups improve the expression of PPARγcompare with model group. PPARγnot only can regulate the lipid metabolism, but also can inhibit the release of TNF-αand FFA and regulate glycometabolism. So the result indicated that Tangzhining capsule can improve insulin sensitive by activating the PPARγ.Use of ELISA method in studying the effects of Tangzhining capsule on tumor mecrosis factor (TNF-α) expression in adipocyte. The result shows that the TNF-αdensity in high-dose and medium-dose are significently lower down than that of model group. It indicate that Tangzhining capsule can improve insulin sensitive by low down the level of TNF-α, and then remove the inhibition of the GluT4 in adepocyte and skeletal, and stimulate the lipoclasis.Use of chromatometry method in studying the effects of Tangzhining capsule on serum FFA level. The result shows that Tangzhining capsule dosages groups have the effect of lowing down the serum FFA level. Tangzhining capsule can regulate glycometabolism and lipid metabolism by cutting down the impairment of FFA.In a word, the further optimization prescription theory is scientific and feasibility. And the best prescription which screened have the effect of decreasing the blood sugar level and lipid level. And the optimized prescription regulate glycometabolism and lipid metabolism with affecting of the insulin signal conduction by the multi-angle and multi-target.更多还原