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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 osteoporosis1,2. 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 effective components from the granules5,6,7, the exact mechanisms and underlying pharmacological processes are still difficult to identify. This is because too many effective components from one granule may simultaneously exert similar or opposed effects4. Therefore, the development of one standard protocol to prepare the granules before delivering to the body not only would have a great impact on the therapeutic outcomes but is also required for both in vivo and in vitro assays.

Moreover, the curative effects of granules in the clinic are difficult to confirm and exactly identify using in vitro or ex vivo studies, which creates a challenge because the pharmacological mechanisms are too complex. To resolve this, the preparation of drug-containing serum was first proposed by Tashino in 1980s8. From then on, numerous researchers applied drug-containing serum to herbal medicine, including granules9,10,11. Currently, the choice of drug-containing serum for in vitro investigations is regarded as one strategy that closely mimics physiological conditions.

Gushukang (GSK) granules were developed to treat postmenopausal osteoporosis (POP) based on clinical practice in light of the theory of TCM. GSK granules prevent bone loss in ovariectomized (OVX) mice in vivo, inhibit osteoclastic bone resorption, and stimulate osteoblastic bone formation4. Consequently, Li et al.12 found that GSK granules have bone protective effects in OVX mice by enhancing the activities of calcium receptor to stimulate bone formation. To confirm the bone-protective effects as well as the pharmacological effects of GSK granules, the authors here provide a detailed procedure for preparation of working solutions and drug (GSK granule)-containing serum. Moreover, this article describes the application of GSK granules in an OVX-induced osteoporotic mouse model and GSK granule-containing serum for in vitro osteoclastogenesis/osteoblastogenesis.

GSK granules are composed of several herbs13,14 and can be completely dissolved in saline easily. Therefore, saline serves as the vehicle. Sham-operated mice (Sham) and OVX mice were administered the same volume of saline as the granule-administered mice. The equivalent doses of GSK granules for the mouse were calculated based on the Meeh-Rubner equation15. This equation not only has the advantage of obtaining safe dosages but also guarantees pharmacological effects15. The three dosages of GSK granules were generated as the follows: (1) GSKL: OVX + low-dose GSK granules, 2 g/kg/day. (2) GSKM: OVX + medium-dose GSK granules, 4 g/kg/day. (3) GSKH: OVX + high-dose GSK granules, 8 g/kg/day. Mice in the GSKL, GSKM and GSKH groups were intragastrically administered GSK granules. Calcium carbonate (600 mg/tablet) with vitamin D3 (125 international unit/tablet), for example, in a mature and marketed product (e.g., Caltrate [CAL]) for treating and preventing osteoporosis, was used as a positive control.

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 weight2/3)/1000, where the K values are 10.6 for human and 9.1 for mouse. Assuming a human body weight of 70 kg, then human body surface (m2) = 10.6 x (702/3)/1000 = 1.8 m2. Assuming a mouse body weight of 20 g (0.02 kg; e.g., 1 month old, female, C57/BL6), then mouse body surface (m2) = 9.1 x (0.022/3)/1000 = 0.0067 m2.
    2. Based on the calculated body surface, calculate the body transform ratio for human and mouse. Human: 70 kg/1.8 m2 = 39. Mouse: 0.02 kg/0.0067 m2 = 3. GSK granule = 20 g/70 kg x 39/3 = 3.72 g/kg ≈ 4 g/kg.
    3. Based on a body weight of 20 g per mouse, calculate the equivalent dosage for mouse: 4 g/kg x 0.02 kg = 0.08 g.
    4. Calculate three equivalent doses of GSK granules based on 20 mice per group and an intervention lasting for 3 months (90 days): (1) GSKL (OVX + low-dose GSK granules [2 g/kg/day]): 0.04 g mouse/day x 20 mice x 90 days = 72 g. (2) GSKM (OVX + medium-dose GSK granules [4 g/kg/day]): 0.08 g mouse/day x 20 mice x 90 days = 144 g. (3) GSKH (OVX + high-dose GSK granules [8 g/kg/day]): 0.12 g mouse/day x 20 mice x 90 days = 216 g.
      NOTE: Prepare an additional 20% of GSK granules in practice to offset the loss.
  2. Calculate the volume of GSK granule per mouse based on the body weight15: e.g., volume (V) = 0.24 mL/mouse/day.
    NOTE: The volume for intragastric administration for mouse is 0.12 mL/10 g.
  3. Weigh 10-days’ worth of three doses of GSK granules. Weigh 8 g, 16 g, and 24 g of GSK granules and serve as GSKL, GSKM, and GSKH, respectively.
  4. Calculate the equivalent dose of calcium carbonate with vitamin D3 (CAL) for mouse based on the Meeh-Rubner equation15 as in steps 1.1.1 and 1.1.2: CAL dosage = 2 tablet/70 kg x 39/3 = 0.372 tablet/kg ≈ 0.4 tablet/kg.
  5. Based on a body weight of 20 g per mouse (e.g., 1 month old, female, C57/BL6), calculate the equivalent dosage of CAL for mouse: 0.4 tablet/kg x 0.02 kg = 0.008 tablet. Then calculate the equivalent dose of CAL based on 20 mice per group and an intervention lasting for 3 months (90 days): 0.008 tablet x 20 x 90 = 14.4 tablets. Weigh 10-days’ worth of CAL (1.6 tablets).
  6. Dissolution
    1. Place 8 g of GSK granules into a 50 mL tube. Add 48 mL of saline and shake tube to completely dissolve.
      NOTE: The standard for complete dissolution is the absence of sediment. Complete dissolution can be further confirmed if a gavage needle can draw up the working solution and then expel it smoothly.
    2. Repeat step 1.5.1 with 16 g and 24 g of GSK granules.
    3. Place 1.6 tablets (10-days’ worth) of CAL into a 50 mL tube. Add 48 mL of saline and shake tube to completely dissolve.
      NOTE: The working solutions can be stored at -4 °C and prepared every 10 days.
  7. Intragastric administration
    1. Grasp the back of the mouse (1 month old, female, C57/BL6) with the mouse facing forward and ensure that it stays firmly in that position. Keep the mouse calm for 2−3 min before administration.
      NOTE: Ensure that the researcher can clearly see the front of the mouse. Wear gloves to prevent mouse bites, particularly for new researchers.
    2. Place the gavage needle (size: #12, 40 mm) in the working solution of GSK granules and draw 0.24 mL of the working solution.
    3. Put the gavage needle into the mouse through one side of its mouth until the gavage needle reaches the stomach.
      NOTE: To confirm the gavage needle has reached the stomach: (1) The gavage needle encounters the feeling of resistance. Meanwhile, the mouse shows the action of swallowing before the gavage needle passes the physical narrowing of the esophagus. (2) Inject approximately 0.5 mL of the working solution into the mouse and wait for 1 min. If there is no solution coming out of the mouse, this means that the gavage needle has reached the stomach.
    4. Inject the working solution of GSK granule (0.24 mL/mouse) into the stomach and then draw out the gavage needle. Return the mouse into its cage.
    5. Repeat step 1.6.4 with the CAL solution and inject 0.24 mL of CAL solution per mouse.
      NOTE: The volume of CAL solution is calculated as in step 1.2.

2. Micro-CT scanning

  1. Tibia harvesting and preparation
    1. Intraperitoneally anesthetize mouse with 300 mL/100 g of 80 mg/kg ketamine the day following the 90 day’s intervention. Use a needle pinch of the toes to confirm whether the mouse is completely anesthetized. No response indicates successful anesthesia. Then kill the mouse with cervical dislocation.
    2. Fix the mouse with both the arms and legs on foam with tacks.
    3. Cut off the skin with scissors (size: 8.5 cm) and tweezers (size: 10 cm) of the legs from the proximal to the distal end and then harvest tibias.
    4. Immediately put the tibias into 70% ethyl alcohol and wash for 3 times.
  2. Wrap the left tibia of the mouse with sponge foam and put it into a sample tube (35 mm diameter, 140 mm length).
    NOTE: The long axis of the specimen should be along with that of the sample tube. Ensure the proximal end of the tibia points upwards.
  3. Running the micro-CT 80 scan machine
    1. Start the micro-CT 80 scan machine at room temperature.
    2. Set the sample tube into micro-CT 80 and start cross-section scanning with the following scanning parameters: pixel size 15.6 μm, tube voltage 55 kV, tube current 72 μA, integration time 200 ms, spatial resolution 15.6 μm, pixel resolution 15.6 μm, and image matrix 2048 x 2048.
      NOTE: The cancellous bone is distinguished from the cortical bone by pre-scanning. The scan area of the tibia is defined as the cancellous bone area from 5 mm below the tibial plateau to the distal end.
  4. Quantification of bone parameter
    1. After completing cross-section scanning, obtain the images of the left tibias.
    2. Set the density threshold to 245−1000. Use the micro-CT evaluation program V6.6 to measure the following bone parameters: bone mineral densities (BMD), bone volume over total volume (BV/TV), trabecular bone number (Tb.N), trabecular bone thickness (Tb.Th), as well as bone trabecular bone separation (Tb.Sp).

3. Preparation of blood serum for in vitro experiments

  1. Calculation
    1. Based on a rat body weight of 0.2 kg (1 month old, female, Sprague-Dawley), calculate the dosage of GSK granule: human dosage/day x body weight of human x K/body weight of rat = 20 g/70 kg/day x 70 kg x K (K = 0.018) /0.2 kg = 2 g/kg/day.
      NOTE: K is the pharmacological transformation coefficient between human and mouse15 (K = 0.018).
    2. Repeat step 3.1.1 and calculate the following dosages.
      1. Calculate dosage of GSKL: 10 g/70 kg/day x 70 kg x K/0.2 kg = 1 g/kg/day.
      2. Calculate dosage of GSKM: 20 g/70 kg/day x 70 kg x K/0.2 kg = 2 g/kg/day.
      3. Calculate dosage of GSKL: 40 g/70 kg/day x 70 kg x K/0.2 kg = 4 g/kg/day.
      4. Calculate dosage of CAL: 2 tablet /70 kg/day x 70 kg x K/0.2 kg = 0.2 tablet/kg/day.
    3. Calculate the total dosage of GSK granule and CAL.
      1. Calculate the total dosage for GSKL: 1 g/kg/day x 0.2 kg x 6 rats x 3 days = 3.6 g.
      2. Calculate the total dosage for GSKM: 2 g/kg/day x 0.2 kg x 6 rats x 3 days = 7.2 g.
      3. Calculate the total dosage for GSKH: 4 g/kg/day x 0.2 kg x 6 rats x 3 days = 14.4 g.
      4. Calculate the CAL dosage = 0.2 tablet/kg/day x 0.2 kg x 6 rats x 3 days = 0.72 tablet.
        NOTE: A total of 10 mL of GSK granule-containing serum is needed to prepare 100 mL culture medium (20% GSK granule-containing serum). Each rat (6 rats/group) is expected to provide 1.5−2 mL of GSK granule-containing serum after centrifugation.
    4. Calculate the volume of GSK granules applied per rat based on body weight15: e.g., volume (V) = 2 mL/rat/day.
      NOTE: The volume for intragastric administration for rat is 0.1 mL/10 g.
  2. Weigh 3-days’ worth of three doses of GSK granules. Weigh 3.6 g, 7.2 g, and 14.4 g of GSK granules and serve as GSKL, GSKM, and GSKH, respectively. Weigh 0.72 tablet for the CAL group.
  3. Place 7.2 g of GSK granules into a 50 mL tube. Add 36 mL of saline and shake tube to completely dissolve. Repeat this with 3.6 g and 14.4 g of GSK granules.
  4. Repeat section 1.6 for intragastric administration with 2 mL of GSK working solution.
    NOTE: Administer the same volume of saline (2 mL per rat) to prepare serum and serves as a blank control group for in vitro assays.
  5. Preparation of the GSK-containing serum
    1. Intraperitoneally anesthetize the rats with 300 mL/100 g of 80 mg/kg ketamine 1 h after the last administration of GSK granules. Use a needle pinch of the toes to confirm whether the rat is completely anesthetized. No response indicates successful anesthesia.
    2. Expose the abdomen to the bottom of the thorax of rats using straight operating scissors after incising the skin and peritoneum.
      NOTE: The surgical instrument must be sterilized at high temperatures and high pressures prior to use. The surgical area must be sterilized with 70% ethanol during blood collection.
    3. Remove the connective tissue of the abdominal aorta with tissue paper to expose the vessel clearly.
    4. Draw blood from the abdominal aorta using a 10 mL, 22 G syringe. Then remove the needle and transfer the blood to a 15 mL sterile tube. Usually, 6−8 mL of blood can be obtained from one rat.
      NOTE: Each rat must be kept living when drawing blood. One indicator is that the abdominal aorta pulsates when the rat is alive. The rat is dead after blood draw.
    5. Keep the tube upright at room temperature for 30−60 min until the blood is clotted in the tube. Then centrifuge the tube at 500−600 x g for 20 min. Transfer all the supernatant (serum) from one group (6 rats) to one 50 mL sterile tube and shake to mix.
    6. Inactivate the serum by incubating in a 56 °C water bath for 30 min. Filter the serum using a 0.22-μm-pore-size hydrophilic polyethersulfone syringe filter. Store at -80 °C for long-term usage (less than 1 year).
      NOTE: The filtered serum can be used for in vitro osteoclastogenesis and osteoblastogenesis.
  6. Application
    1. In vitro osteoclastogenesis
      1. Dilute the three dosages of the GSK-containing serum (GSKL, GSKM, GSKH) at the ratio of 1:4 with minimum Eagle’s medium (α-MEM) containing L-glutamine, ribonucleosides, and deoxyribonucleosides.
        NOTE: Ensure that the final concentration of GSK-containing serum for in vitro osteoclastogenesis and osteoblastogenesis is 20%.
      2. Add the diluted GSK-containing serum (200 μL/well) from step 3.6.1.1 to bone marrow macrophages (BMMs) from 4−6 week old C57BL/6 mice for osteoclastogenesis and stimulate BMMs with macrophage colony-stimulating factor (M-CSF, 10 ng/mL) and receptor activator for nuclear factor-κB ligand (RANKL, 100 ng/mL) as previously described2.
    2. In vitro osteoblastogenesis
      1. Repeat step 3.6.1.1.
      2. Add the diluted GSK-containing serum (2 mL/well) to bone mesenchymal stem cells (BMSCs) from 4−6 week old C57BL/6 mice to generate osteoblast as previously described16.

Results

Micro-CT scanning results indicated that the OVX mice showed significant bone loss compared to saline control mice (Figure 1A). The intervention (90 days) of GSK granules greatly increased the BMD, particularly in the GSKM group (Figure 1B). The bone structure parameters, such as BMD, BV/TV, Tb.N, and Tb.Th, were quantified. GSK granule treatments led to increased BMD, BV/TV, Tb.N and Tb.Th but decreased Tb.Sp (

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...

Disclosures

The authors have nothing to disclose.

Acknowledgements

This study was supported by grants from the National Natural Science Foundation of China (81804116, 81673991, 81770107, 81603643, and 81330085), the program for Innovative Team, Ministry of Science and Technology of China (2015RA4002 to WYJ),  the program for Innovative Team, Ministry of Education of China (IRT1270 to WYJ), Shanghai TCM Medical Center of Chronic Disease (2017ZZ01010 to WYJ), Three Years Action to Accelerate the Development of Traditional Chinese Medicine Plan (ZY(2018-2020)-CCCX-3003 to WYJ), and national key research development projects (2018YFC1704302).

Materials

NameCompanyCatalog NumberComments
α-MEMHyclone
laboratories		SH30265.018For cell culture
β-GlycerophosphateSigmaG5422Osteoblastogenesis
Caltrate (CAL)WyethL96625Animal interventation
C57BL/6 miceSLAC Laboratory
Animal Co. Ltd.		RandomAinimal preparation
DexamethsomeSigmaD4902
Dimethyl sulfoxideSigmaD2438Cell frozen
Ethylene Diamine Tetraacetic Acid (EDTA)Sangon Biotech60-00-4Samples treatmnet
Fetal bovine serumGibcoFL-24562For cell culture
Gushukang granuleskangcheng companyin chinaZ20003255Herbal prescription
Light microscopeOlympus BX50Olympus BX50Images for osteoclastogenesis
L-Ascorbic acid 2-phosphate sequinagneium slat hyclrateSigmaA8960-5GOsteoblastogenesis
MicroscopeLeicaDMI300BOsteocast and osteoblast imagine
M-CSFPeprotechAF-300-25-10Osteoclastogenesis
Μicro-CTScanco
Medical AG		μCT80 radiograph microtomographBone Structural analsysis
RANKLPeprotech11682-HNCHFOsteoclastogenesis
Sprague DawleySLAC Laboratory
Animal Co. Ltd.		RandomBlood serum collection
Tartrate-Resistant Acid Phosphate (TRAP) KitSigma-Aldrich387A-1KTTRAP staining

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