Most recently, nearly 2.5 million patients who suffer from joint dysfunction caused by diseases such as hip or knee osteoarthritis receive arthroplasty surgery each year around the world 25. Artificial joint replacement has gained ground as one of the most effective treatment for articular diseases for which conservative approaches failed. This number will continue to rise in the next 20 to 30 years with younger population who prone to higher load of exercise. Moreover, about 5% of patients undergo revision surgery due to ALP during the first 15 years after primary arthroplasty 1. Thus, ALP secondary to periprosthetic osteolysis causes increasingly problems in this the worldwide.
ALP is one of the most common long-term complication of arthroplasty. It is widely hold that wear-particle-induced osteolysis is the leading cause of ALP after arthroplasty 26. Specifically, wear particles were phagocytosed by accumulated macrophages and then triggered the over-expression of inflammatory mediators such as IL-1, IL-6, IL-17, TNF-α, M-CSF, MCP-1 (monocyte chemoattractant factor 1), MIP-1α (macrophage inflammatory protein 1 alpha), etc, which promote the differentiation from macrophages to osteoclasts 27; 28. RANKL, a specifc receptor activator for NF-κB ligand expressed by osteoblasts and osteocytes, serve as another key molecular in osteoclastogenesis 1. Both of these two manners could cause periprosthetic osteolysis and subsequent ALP 26; 29.
Given the continuous improvement in the material and manufacturing technology though, the ALP seems to be inevitable. To date, nearly all types of debris worn from different interfaces of prosthesis system, including metal, polymethyl methacrylic (PMMA), polyethylene (PE) and ceramics, were reported as causes of periprosthetic osteolysis at varying severity 28; 30. Therefore, pharmacotherapies targeting osteoclast have drawn high attention.
In the current study, the CIM demonstrated inhibitory effect on osteoclastogenesis in vitro. Noncytotoxic CIM repressed osteoclast differentiation in RAW264.7 and BMMs, and alleviated subsequent bone resorption. The impaired formation F-acting ring also confirmed the findings above. Moreover, we discovered that CIM downregulated expression of specific genes in osteoclast, including those involved in regulation of downstream genes expression (Nfatc1, c-Fos and Traf6) 31, bone resorption function (Cathepsin K and Acp5) 32, calcium homeostasis (Calcr) 33, and cell fusion of precursors (Dc-stamp) 34. Then, we further proved that CIM could protect bone from Ti particle-induced osteolysis in vivo. This therapeutic effect for potential ALP treatment were supported by results of both micro-CT scanning and immunohistochemical assay that CIM dose-dependently mitigated the bone erosion and osteoclast accumulation.
After confirming the anti-osteoclastogenetic property of CIM, we elucidated potential molecular mechanism of it. RANKL specifically bind to the RANK on cytomembrane of osteoclast precursor and then trigger the recruitment of TRAFs and TAK1 binding protein 2 in cytoplasm 35. The RANKL/RANK/TRAFs complex then activates the phosphorylation of TAK1 (TGF-b-activated kinase 1), which in turn initiates both NF-kB and MAPKs signaling by phosphorylating both IKK complex and MKKs 35; 36. For one, phosphorylated IKK complex causes cleavage of NF-kB from IkB and the following degradation of IkB 37. For another, phosphorylated MKKs activated the phosphorylation of JNK, ERK and p38 38. The activation of all these signaling pathways contributed to the upregulation of osteoclast specific genes expression. In the current study, we found that CIM inhibits osteoclastogenesis by downregulating the phosphorylation of IkBa in NF-kB signaling pathway, as is shown in Fig. 7.
CIM is kind of chromone first discovered in Cimicifuga Racemosa but mainly prepared from the root of Saposhnikovia Divaricata, both of which are used in traditional Chinese medicine to treat upper respiratory infection and skin inflammatory diseases with a long history. Previous studies prove that CIM has potential to multiple inflammatory diseases. Liu et al. 14 reported that CIM could mitigate imiquimod-induced psoriasis in murine by oxidative stress and inhibiting inflammation and repress NF-κB and MAPKs (JNK, ERK and p38) signaling in HaCaT cells, in which CIM may inhibit the phosphorylation of IkB, JNK, ERK and p38. Han et al. 12 concluded that CIM could alleviate Lipopolysaccharide-induced Inflammatory Responses of RAW264.7 as a rheumatoid arthritis model via inhibiting the phosphorylation of IkB, ERK and p38 (without detecting JNK) in NF-κB and MAPKs signaling pathways.
In the present study, we demonstrated that CIM inhibited RANKL-induced NF-κB signaling via blocking the phosphorylation of IκBα without affecting JNK, ERK and p38 activation of MAPKs signaling during the osteoclastogenesis. In comparison, previous studies on the bioactivity of CIM suggested different conclusions. For one, Liu et al. 14 reported that CIM ameliorates imiquimod-mediated phosphorylation of NF-κB (IκB and p65) and MAPK (JNK, ERK, and p38) signaling in psoriasis models. For another, Han et al. 12 indicated that CIM exerts inhibitory effect on lipopolysaccharide-induced inflammatory response in RAW264.7 cell by downregulating the generation of p-IκBα, p-p65, p-ERK, and p-p38 of NF-κB and MAPK signaling pathways without detecting the level of JNK. The discrepancy between may result from the diverse cell and animal models and limited exploration of molecular mechanism. Imiquimod and lipopolysaccharide were adopted to established experimental models in studies of Liu et al. and Han et al., which shares identical pathway in the downstream of TRAF6 with RANKL-induced signaling but not in the upstream of it. CIM treatment may alter regulation pattern in the upstream of TRAF6 and in NF-κB pathway simultaneously, which causes different conclusion between studies. In addition, time-dependent changes of NF-κB and MAPKs signaling proteins are absent in studies of Liu et al. and Han et al. Moreover, all these three studies including the present one only investigate the level of signaling proteins in limited number, so it seems impossible to come to an agreement for now.
There are several limitations existing in this study. First, we only investigate the expression of IkB, JNK, ERK, p38 and their phosphorylation forms as key factors in NF-κB and MAPKs signaling pathways. To unravel exact target protein in the cascade and acting site of specific protein, more experiment including Western blotting for upstream signaling proteins, “rescue” experiment and molecular docking assay may be required. Second, homeostasis of bone metabolism inevitably involves opposing bone resorption induced by osteoclast and bone formation induced by osteoblast. M-CSF, RANKL and OPG (osteoprotegerin) that serve as critical modulator of osteoclast formation in bone metabolism were all expressed by osteoblast. Thus, the effect of CIM on osteoblast-induced bone formation is supposed to be explored in future studies. Finally, the adopted Ti particle-induced murine calvarial osteolysis model cannot perfectly simulate ALP in patient. From metal and UHMWPE (ultrahigh-molecular-weight polyethylene) to ceramic and PEEK (polyether-ether-ketone), the myriad of particles worn from interfaces of any kind could contributed the periprosthetic osteolysis of varying severity 39. With structure of thin cortical bone and little stress bearing, murine calvarium only serve as a defective analogue of ALP in hip or knee joint of human 9. Even though, this model demonstrates similar pathological change compared with polyethylene particle-induced model and widely used in studies of ALP 36; 40. Therefore, it is reasonable to establish Ti particle-induced murine calvarial osteolysis model for ALP simulation in vivo.
Conclusively, for the first time we demonstrated that CIM could alleviate RANKL-induced osteoclastogenesis and Ti particles-induced osteolysis in vitro and in vivo via inhibiting NF-κB signaling pathway. The current findings suggests that CIM could serve as a potential drug to treat ALP as well as other osteopathies mediated by excessive osteoclasts, therefore broadening the spectrum of bone-protective natural compounds.