Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Summary

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

Abstract

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Introduction

Traditional Chinese medicine (TCM) is one of the important complementary and alternative approaches to treat osteoporosis^1,2. Water decoction is the basic and most commonly used form of the formula³. However, drawbacks also exist: bad taste, inconvenience for carriage, short shelf life and inconsistent protocols, limiting the uses as well as the curative effects. To avoid the above disadvantages as well as to pursue better effects, granules were developed and have been widely used⁴. Although many studies have explored the pharmacological mechanisms of one or more....

Protocol

All of the experimental procedures were performed with the approval of Institutional Animal Care and Use Committee of the Shanghai University of TCM (SZY201604005).

1. Preparation and administration of GSK working solution

1. Calculate the equivalent doses of GSK granules for mouse.
   1. Calculate body surface based on the Meeh-Rubner equation¹⁵: body surface = K x (body weight^2/3)/1000, where the K values are 10.6 for human and 9.1 for mouse. Assuming a h.......

Discussion

Granules of TCM agents have become one of the common choices for formulations or prescriptions. GSK granules are composed of several herbal medicines based on clinical experiences or the TCM theory, and they exert better curative effects with fewer side effects⁴. Compared with water decoction, the granules have these advantages: good taste, convenience of delivery, long-term storage, standard protocol and consistent curative effects, as well as higher productivity. Currently, granules are one of t.......

Materials

Name	Company	Catalog Number	Comments
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