Bushen huoxue decoction inhibits RANKL-stimulated osteoclastogenesis and glucocorticoid-induced bone loss by modulating the NF-κB, ERK, and JNK signaling pathways

Abstract

Glucocorticoid-induced osteoporosis (GIOP) is the most common form of secondary osteoporosis, which is caused by a disorder in bone metabolism due to excessive activation of osteoclasts. Bushen Huoxue decoction (BHD) is an herbal formula with multiple pharmacological effects, including anti-inflammatory, antioxidant activity and stem cell migration promotion. However, the effect of BHD on osteoclastogenesis has not been reported. In this study, we aimed to elucidate the effect of BHD on RANKL-stimulated osteoclastogenesis and explored its underlying mechanisms of action in vitro. Our results show that BHD had no effect on BMMs and RAW264.7 cells viability, but inhibited RANKL-induced osteoclast formation in vitro. Furthermore, BHD attenuated RANKL-induced NF-κB, ERK, and JNK signaling. The attenuation of NF-κB, ERK, and JNK activation were enough to impede downstream expression of c-fos and NFATc1 and related specific genes. Meanwhile, we investigated the therapeutic effect of BHD on glucocorticoid-induced osteoporosis (GIOP) mice. The result indicated that BHD prevents glucocorticoid-induced osteoporosis and preserves bone volume by repressing osteoclast activity. Collectively, BHD shows significant osteoclast inhibition and holds great promise in the treatment of osteoporosis.

Keywords: ERK pathway; JNK pathway; NF-κB pathway; bushen huoxue decoction; osteoporosis.

FIGURE 1

BHD suppressed osteoclastogenesis of mature osteoclasts in RAW264.7cells and BMMs (A,B) Mouse RAW264.7 cells (5 × 103cells) incubated with RANKL at the concentration of 50 ng/ml for 5 days with the indicated doses of BHD (50 and 200 μg/ml). Mouse BMMs (5 × 103 cells/well) incubated with RANKL and M-CSF at the concentration of 50 ng/ml for 7 days with the indicated doses of BHD (50 and 200 μg/ml). All Cells were stained using trap staining tool. Count trap-positive MNCs with 3 or more nuclei (C,D) The cell viability of BMMs and Raw264.7cells after pretreatment with indicated concentrations of BHD for 5 days or 7 days examined by cck-8. (E) The Raw264.7cells (5 × 103cells) were cultured in a-MEM containing 50 ng/ml RANKL pretreated with or without BHD for 5 days, and then the F-actin belts staining was performed. The data are shown as mean ± SD, (n = 3, three independent experiments),***p<0.001. Scalebars, 100 µm BHD: Bushen Huoxue decoction.

FIGURE 2

BHD suppressed RANKL-stimulated ERK, JNK and NF-κB activation (A–E) The protein expression levels of p-p65, p65, IκBα, p-JNK, JNK, p-ERK, ERK were examined utilizing the western blot as-says for Raw 264.7 cells pretreated with BHD at indicated concentrations. BHD inhibited RANKL-stimulated degradation of IκBα and phosphorylation of p65,ERK, and JNK. All bar charts are presented as mean ± SD, (n = 3, three independent experiments). *p<0.05,**p<0.01,***p<0.001 vs. group only treated with RANKL.(F) Representative immunofluorescence images of the translocation of p65. BHD suppressed RANKL-stimulated p65 translocation. Scale bars, 20 µm.

FIGURE 3

BHD suppressed RANKL-stimulated osteoclastogenesis-related protein and gene expression (A–D) The protein levels of NFATc1, c-fos and Ctsk of Raw 264.7cells pretreated with or without BHD for 0,1,3,5 days. BHD suppressed RANKL-stimulated protein expression levels increase in a time-dependent manner. (E–H) The mRNA levels of NFATc1, Ctsk, V-ATPase-d2 and Acp5 of Raw 264.7cells pretreated with or without BHD. BHD suppressed RANKL-stimulated mRNA expression levels increase of these genes. Columns in the charts were presented in a manner of mean ± SD. (n = 3, three independent experiments). *p<0.05, **p<0.01, ***p<0.001.

FIGURE 4

BHD prevents glucocorticoid -induced bone mass loss in vivo. All mice were randomly divided into three groups: sham group (n = 6), DEX group (n = 6), and DEX + BHD group (n = 6). The mice in the sham group were injected with 5% ethanol intramuscularly (the same amount as Dexamethasone); the mice in the other groups were injected with Dexamethasone (5 mg/kg, twice a week for 6 weeks) to establish the GIOP model. then an intraperitoneal injection of BHD (16 mg/kg, every 2 days for 6 weeks) was delivered for DEX + BHD group. The sham and DEX group mice were intraperitoneally injected with PBS as a vehicle control (A) Representative Micro-CT images of 2D and 3D demonstrating that glucocorticoid -induced bone loss was prevented by BHD treatment. (B) Quantitative analyses of parameters regarding to bone architecture, including BMD, BV/TV, Tb.N, Tb.Th (n = 6) (C) Representative images of H&E staining of decalcified bone sections, Scale bar = 200 μm or 100um. (D) trap staining of decalcified bone sections, Scale bar = 100 μm or 20um. All bar charts are presented as mean ± SD, *p<0.05, **p<0.01, ***p<0.001. BMD, bone mineral density; BV/TV, bone volume per tissue volume; Tb.N, trabecular number; Tb.Th, trabecular thickness.

FIGURE 5

BHD affects osteoclast formation and differentiation by suppressing the expression of the NF-κB signaling pathway in vivo. (A–E) Representative images of Immunohistochemical staining of decalcified bone sections. Scale bar = 20 μm.

FIGURE 6

A mode of operation for the inhibition of osteoclastogenesis by BHD is proposed. ERK, JNK and NF-κB pathways are activated upon binding of RANKL to RANK. Activation of TRAF6, in turn, results in the degradation of IκBα and the upregulation of osteoclast-specific genes. Our results indicate that BHD can inhibit osteoclast formation and bone resorption by attenuating the activities of ERK, JNK, and NF-κB signaling pathways, then reduce the expression of osteoclast-related genes Acp5, V-ATPase-d2, Ctsk.