Andrographolide

Kinase experiment:

In vitro osteoclastogenesis assays are preformed to examine the effects of Andrographolide on osteoclast differentiation. Bone marrow macrophages (BMM) cells are prepared. Briefly, cells extracted from the femur and tibiae of a 6-week-old C57/BL6 mouse are incubated in complete cell culture media and 30 ng/mL M-CSF in a T-75 cm2 flask for proliferation. When changing the medium, the cells are washed in order to deplete residual stromal cells. After reaching 90% confluence, cells are washed with PBS three times and trypsinized for 30 min to harvest BMMs. Cells adhering to the bottom of the dish are classified as BMMs; these BMMs are plated in 96-well plates at a density of 8×103 cells per well in triplicate and incubated in a humidified incubator containing 5% CO2 at 37°C for 24 h. The cells are then treated with various concentrations of Andrographolide (0, 2.5, 5, or 10 μM) plus M-CSF (30 ng/mL) and RANKL (50 ng/mL). After 5 days, cells are fixed and stained for tartrate-resistant acid phosphatase (TRAP) activity. TRAP-positive multinucleated cells with more than five nuclei are counted as osteoclasts[1].

Cell experiment:

Effects of Andrographolide on cell proliferation are determined with a CCK-8. BMMs are plated in 96-well plates at a density of 3×103 cells per well in triplicate. Twenty-four hours later, the cells are treated with increasing concentrations of Andrographolide (0, 2.5, 5, 10 or 20 μM) for 2 days. Next, 10 μL CCK-8 is added to each well, and the plates are then incubated at 37°C for an additional 2 h. The optical density (OD) is then measured with an ELX800 absorbance microplate reader at a wavelength of 450 nm (650 nm reference). The cell viability is calculated[1].

Animal experiment:

Mice[1]C57BL/6 mice (8 weeks old) are divided into four groups of seven mice each. Mice are injected i.p. with Andrographolide (5 or 30 mg/kg body weight) or PBS as a control 1 day before injection of LPS (5 μg/g body weight). Andrographolide or PBS is injected intraperitoneally every other day for 8 days. LPS is injected intraperitoneally on days one and four. All mice are killed 8 days after the initial LPS injection, and the left femurs of all animals are scanned with a high-resolution micro-CT at a resolution of 9 μm.

References:

[1]. Zhai ZJ, et al. Andrographolide suppresses RANKL-induced osteoclastogenesis in vitro and prevents inflammatory bone loss in vivo. Br J Pharmacol. 2014 Feb;171(3):663-75.