Tussilagone Inhibits Osteoclastogenesis and Periprosthetic Osteolysis by Suppressing the NF-κB and P38 MAPK Signaling Pathways

Abstract

Background: Aseptic prosthetic loosening is one of the main factors causing poor prognosis of limb function after joint replacement and requires troublesome revisional surgery. It is featured by wear particle-induced periprosthetic osteolysis mediated by excessive osteoclasts activated in inflammatory cell context. Some natural compounds show antiosteoclast traits with high cost-efficiency and few side effects. Tussilagone (TUS), which is the main functional extract from Tussilago farfara generally used for relieving cough, asthma, and eliminating phlegm in traditional medicine has been proven to appease several RAW264.7-mediated inflammatory diseases via suppressing osteoclast-related signaling cascades. However, whether and how TUS can improve aseptic prosthetic loosening via modulating osteoclast-mediated bone resorption still needs to be answered.

Methods: We established a murine calvarial osteolysis model to detect the preventative effect of TUS on osteolysis in vivo. Micro-CT scanning and histomorphometric analysis were used to determine the variation of bone resorption and osteoclastogenesis. The anti-osteoclast-differentiation and anti-bone-resorption bioactivities of TUS in vitro were investigated using bone slice resorption pit evaluation, and interference caused by cytotoxicity of TUS was excluded according to the CCK-8 assay results. Quantitative polymerase chain reaction (qPCR) analysis was applied to prove the decreased expression of osteoclast-specific genes after TUS treatment. The inhibitory effect of TUS on NF-κB and p38 MAPK signaling pathways was testified by Western blot and NF-κB-linked luciferase reporter gene assay.

Results: TUS better protected bones against osteolysis in murine calvarial osteolysis model with reduced osteoclasts than those in the control group. In vitro studies also showed that TUS exerted antiosteoclastogenesis and anti-bone-resorption effects in both bone marrow macrophages (BMMs) and RAW264.7 cells, as evidenced by the decline of osteoclast-specific genes according to qPCR. Western blotting revealed that TUS treatment inhibited IκBα degradation and p38 phosphorylation.

Conclusions: Collectively, our studies proved for the first time that TUS inhibits osteoclastogenesis by suppressing the NF-κB and p38 MAPK signaling pathways, therefore serving as a potential natural compound to treat periprosthetic osteolysis-induced aseptic prosthetic loosening.

Keywords: MAPK; NF-κB; aseptic prosthetic loosening; osteoclast; p38; periprosthetic osteolysis; tussilagone.

Figure 1

TUS inhibited Ti particle-induced murine calvarium osteolysis. (A) Representative three-dimensional reconstructed images of calvarium of micro-computed tomography (micro-CT) from each group. (B) Bone volume against tissue volume (BV/TV), number of pores, and the percentage of total porosity of each sample was measured. (**: P < 0.01 versus control group).

Figure 2

(A) Inhibition of Ti particle-induced murine calvarial osteolysis by Tussilagone (TUS) as assessed using immunohistochemical staining analysis. Hematoxylin and eosin (HE) and tartrate-resistant acid phosphatase (TRAP) staining were performed on at least three sections per group. (B) The area and the number of TRAP positive cells of each sample was measured. (**: P < 0.01 versus control group).

Figure 3

Tussilagone (TUS) suppressed receptor activator of nuclear factor κB ligand (RANKL)–mediated osteoclastogenesis in a dose-dependent manner without cytotoxicity in the RAW264.7 cell line. (A) The structural formula of TUS. The microscopic images of RANKL-induced osteoclastogenesis, the area and number of tartrate-resistant acid phosphatase (TRAP)–positive osteoclasts after TUS treatments in (B) BMM and (C) RAW264.7 cells relative to that in the control group. Cell viability was determined in both types of preosteoclasts at 48 or 96 h respectively. (*: P < 0.05; **: P < 0.01 versus control group).

Figure 4

Tussilagone (TUS) treatment dose-dependently mitigated the bone resorption induced by osteoclast at noncytotoxic concentration. (A) Scanning electron microscope images of bone resorption pits of all groups. (B) Area of the bone resorption relative to control as quantified by ImageJ software. (*: P < 0.05; **: P < 0.01 versus control group).

Figure 5

Tussilagone (TUS) hindered transcription of osteoclast-specific genes, including Nfatc1, Calcr, Traf6, c-Fos, Dc-stamp, and Cathepsin K. Measured results were normalized to the expression of Gapdh. (*: P < 0.05; **: P < 0.01 versus control group).

Figure 6

(A) Luciferase activity of RAW264.7 cells stably transfected with an NF-κB luciferase reporter construct. (B) Tussilagone (TUS) inhibited receptor activator of nuclear factor κB ligand (RANKL)–induced NF-κB and p38-mediated MAPK signaling pathway activation as shown by Western blot of RAW264.7 cell lysates with specific antibodies against p38, p-p38, IκBα, p-IκBα, ERK, p-ERK, JNK, p-JNK, and β-actin. (C) Average ratio of IκBα relative to β-actin, p-p38 relative to p38. All experiments were performed at least thrice. (**: P < 0.01 versus control group).

Figure 7

A pattern diagram of Tussilagone (TUS) in downregulating osteoclastogenesis. By targeting IκBα degradation and p38 phosphorylation, TUS suppressed receptor activator of nuclear factor κB ligand (RANKL)–induced expression of osteoclast-specific genes to inhibit osteoclast formation and functions in vivo and in vitro.