古树康 (GSK) 颗粒的制备,用于 Vivo 和体外实验
1Longhua Hospital,Shanghai University of Traditional Chinese Medicine, 2Spine Institute,Shanghai University of Traditional Chinese Medicine, 3Key Laboratory of Theory and Therapy of Muscles and Bones, Ministry of Education, 4School of Rehabilitation Science,Shanghai University of Traditional Chinese Medicine
Summary
本文为制备古树康颗粒的工作溶液和用于体外实验的含有血清的GSK颗粒提供了详细的方案。该协议可应用于草药的药理学研究以及体内和体外实验的处方。
Abstract
中草药在治疗许多疾病(如绝经后骨质疏松症)方面起着替代作用。古树康(GSK)颗粒,在中国销售的处方,在治疗POP具有骨保护作用。在给药到身体之前,通常需要一个标准的制备程序,其目的是促进从生草药中释放活性成分,增强药理作用以及治疗效果。本研究提出了在体内和体外实验测定中使用葛兰素素梅粒的详细方案。作者首先提供了一个详细的协议,以计算动物适合剂量的颗粒进行体内调查:称重,溶解,储存和给药。其次,本文介绍了微CT扫描和骨骼参数测量方案。评估了样品制备、运行微CT机的规程和骨参数的定量。第三,制备含血清的GSK颗粒,提取含药物血清进行体外骨细胞生成和骨细胞生成。葛兰素史克颗粒连续三天每天对大鼠进行两次胃内施用。然后收集血液,离心,灭活,并过滤。最后,对血清进行稀释,用于实施骨细胞生成和骨细胞生成。此处描述的协议可被视为草药处方药(如颗粒)的药理学研究的参考。
Introduction
中医是治疗骨质疏松症的重要补充和替代方法之一。水净化是公式3的基本和最常用的形式。然而,缺点也存在:味道不好,运输不便,保质期短,协议不一致,限制了使用以及治疗效果。为了避免上述缺点,以及追求更好的效果,颗粒被开发,并已被广泛应用4。虽然许多研究已经探索了一个或多个有效成分的药理机制从颗粒5,6,7,确切的机制和潜在的药理过程仍然是难以识别。这是因为一个颗粒中过多的有效成分可能同时产生类似或相反的效果4。因此,开发一个标准方案,在输送到人体之前制备颗粒,不仅对治疗效果有很大的影响,而且对体内和体外检测也是必需的。
此外,在临床颗粒的疗效难以确认和准确识别使用体外或体外研究,这带来了挑战,因为药理机制过于复杂。为了解决这个问题,Tashino于20世纪80年代首次提出制备含药物血清。从那时起,许多研究人员将含药物血清应用于草药,包括颗粒9,10,11。目前,选择含药物血清进行体外调查被认为是一种与生理条件密切相关的策略。
根据中医理论,在临床实践的基础上,研制出用于治疗绝经后骨质疏松症(POP)的古树康(GSK)颗粒。葛兰素史克颗粒可防止体内卵泡(OVX)小鼠的骨质流失,抑制骨细胞骨吸收,刺激成骨骨形成4。因此,Li等人12日发现,GSK颗粒通过增强钙受体的活性来刺激骨骼形成,对OVX小鼠具有骨保护作用。为了确认GSK颗粒的骨保护作用和药理作用,作者在这里提供了一个详细的程序,用于制备工作溶液和含有药物(GSK颗粒)的血清。此外,本文还介绍了GSK颗粒在OVX诱导的骨化小鼠模型和GSK含颗粒血清中的应用,用于体外骨细胞形成/骨细胞发生。
GSK颗粒由几种草药13、14组成,很容易完全溶解在盐水中。因此,盐水作为车辆。沙姆操作小鼠(Sham)和OVX小鼠施用的盐水量与颗粒施用小鼠相同。根据Meeh-Rubner方程15计算了小鼠的葛兰素史克颗粒的等效剂量。该方程不仅具有获得安全剂量的优点,而且保证了药理作用15。葛兰素史克颗粒的三种剂量如下: (1) GSKL: OVX = 低剂量葛兰素史克颗粒, 2 g/kg/天.(2) GSKM:OVX = 中剂量葛兰素史克颗粒,4 克/千克/天。(3) GSKH:OVX = 高剂量葛兰素史克颗粒,8克/千克/天。GSKL、GSKM和GSKH组的小鼠是胃内管理的葛兰素史克颗粒。碳酸钙(600毫克/片)与维生素D3(125国际单位/片剂),例如,在成熟和上市的产品(例如,钙酸盐[CAL])用于治疗和预防骨质疏松症,被用作积极的控制。
Protocol
所有实验程序均经上海中医药大学动物护理与使用委员会(SZY201604005)批准。 1. 葛兰素史克工作解决方案的准备和管理 计算小鼠的葛兰素史克颗粒的等效剂量。 基于 Meeh-Rubner 方程15计算身体表面 :身体表面 = K x (体重2/3) /1000,其中 K 值为 10.6,用于人类,9.1 表示鼠标。假设人体重量为70公斤,则人体表面(m 2)= 10.6 x (702/3)/1000 = 1.8 m2</sup…
Representative Results
微CT扫描结果表明,与盐水对照小鼠相比,OVX小鼠骨质流失显著(图1A)。葛兰素史克颗粒的介入(90天)大大提高了BMD,特别是在GSKM组(图1B)。骨骼结构参数(如 BMD、BV/TV、Tb.N 和Tb.Th)进行了量化。GSK颗粒处理导致BMD、BV/TV、Tb.N和Tb.Th增加,但Tb.Sp(图1C)减少。 与对照小鼠相?…
Discussion
中药制剂的颗粒已成为配方或处方的常见选择之一。葛兰素史克颗粒由几种基于临床经验或中医理论的草药组成,具有较好的疗效,副作用少4。与水解相比,颗粒具有以下优点:口感好、交货方便、长期储存、标准方案、疗效一致、生产效率高。目前,颗粒是中医中最常用的药学形态之一。然而,药理作用的基本机制仍然很少被研究。有必要确定制备颗粒的关键步骤,以研究潜在的药理?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了中国国家自然科学基金(81804116、81673991、81770107、81603643和81330085)的资助,该项目由中国科技部创新团队项目(2015RA4002至WYJ)资助。中国教育部创新团队(IRT1270至WYJ)、上海中医慢性病医学中心(2017ZZ01010至WYJ)、《三年行动加快中医药发展计划》(ZY(ZY(2018-2020)-CCCX-3003至WYJ)和国家重点研究开发项目(2018YFC1704302)。
Materials
| α-MEM | Hyclone laboratories |
SH30265.018 | For cell culture |
| β-Glycerophosphate | Sigma | G5422 | Osteoblastogenesis |
| Caltrate (CAL) | Wyeth | L96625 | Animal interventation |
| C57BL/6 mice | SLAC Laboratory Animal Co. Ltd. |
Random | Ainimal preparation |
| Dexamethsome | Sigma | D4902 | |
| Dimethyl sulfoxide | Sigma | D2438 | Cell frozen |
| Ethylene Diamine Tetraacetic Acid (EDTA) | Sangon Biotech | 60-00-4 | Samples treatmnet |
| Fetal bovine serum | Gibco | FL-24562 | For cell culture |
| Gushukang granules | kangcheng companyin china | Z20003255 | Herbal prescription |
| Light microscope | Olympus BX50 | Olympus BX50 | Images for osteoclastogenesis |
| L-Ascorbic acid 2-phosphate sequinagneium slat hyclrate | Sigma | A8960-5G | Osteoblastogenesis |
| Microscope | Leica | DMI300B | Osteocast and osteoblast imagine |
| M-CSF | Peprotech | AF-300-25-10 | Osteoclastogenesis |
| Μicro-CT | Scanco Medical AG |
μCT80 radiograph microtomograph | Bone Structural analsysis |
| RANKL | Peprotech | 11682-HNCHF | Osteoclastogenesis |
| Sprague Dawley | SLAC Laboratory Animal Co. Ltd. |
Random | Blood serum collection |
| Tartrate-Resistant Acid Phosphate (TRAP) Kit | Sigma-Aldrich | 387A-1KT | TRAP staining |
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Cite This Article
Zhao, Y., Wang, Q., Liu, S., Wang, Y., Shu, B., Zhao, D. Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments. J. Vis. Exp. (147), e59171, doi:10.3791/59171 (2019).