Pyrroloquinoline Quinine Inhibits RANKL-mediated Expression Of NFATc1 In Part Via Suppression Of C-Fos In Mouse Bone Marrow Cells And Inhibits Wear Particle-induced Osteolysis In Mice

Aseptic loosening as a key problem which bothering the orthopedics doctor for a long time, and was considered to be induced many factors. Chronic bone resorption around exogenous implant devices until fixation is lost, and is considered as resulting from an innate immune response to wear-debris particles, with little contribution by components of the acquired immune system. There is extremely strong evidence that the biological response to particulate ultra-high molecular weight polyethylene(UHMWPE) wear debris generated primarily at the articulating interface is the key factor in the development of osteolysis. The UHMWPE wear particles enter the periprosthetic tissue where they are phagocytosed by macrophages. The macrophages then release an array of cytokines and other mediators of inflammation that lead to the development of an inflamed granulomatous tissue adjacent to the bone. Eventually, osteoclasts are recruited and/or activated to resorb the bone leading to osteolysis and finally loosening of the prosthesis. Osteoclasts are multinucleated giant cells that originate from the hematopoietic stem cell monocyte/macrophage lineage.Osteoclasts differentiate into multinucleated giant cells that attachto bone tissue and excrete various types of acids and enzymes.Osteoblasts and stromal cells express receptor activators of thenuclear factor-kB(NF-kB) ligand(RANKL) and macrophagecolony-stimulating factor(M-CSF). M-CSF provides survivalsignals to osteoclasts and their precursor cells. RANKL is amember of the tumor necrosis factor(TNF) family and binds to theRANK receptor expressed in osteoclast precursor cells. Thebinding of RANKL and RANK on osteoclast progenitor cellstriggers the activation of tumor necrosis factor receptor-associatedfactor 6(TRAF6) and subsequently the activation of NF-k B and mitogen-activated protein kinases(MAPKs), such as extracellularsignalregulated kinase 1/2(ERK1/2), p38 and stressactivatedprotein kinase/c-Jun N-terminal kinase(SAPK/JNK). Nuclear factor of activated T cells(NFATc1) is adownstream transcription factor in the RANKL/RANK signalpathway and as a key molecule of osteoclastogenesis, NFATc1 induces a series of osteoclast-specific genes, including cathepsin K, tartrate-resistant acid phosphatase(TRAP), calcitonin receptorand osteoclast-associated receptor. c-Fos is also an essentialtranscription factor for osteoclastogenesis and upregulates osteoclastogenesisvia NFATc1 activation. Nuclear factor of activated T cells(NFATc1) is a downstream transcription factor in the RANKL/RANK signal pathway and as a key molecule of osteoclastogenesis, NFATc1 induces a series of osteoclast-specific genes, including cathepsin K, tartrate-resistant acid phosphatase(TRAP), calcitonin receptor and osteoclast-associated receptor. c-Fos is also an essential transcription factor for osteoclastogenesis and upregulates osteoclastogenesis via NFATc1 activation.However, the effects of Pyrroloquinoline quinone(PQQ) on RANKL and M-CSF induced osteoclast differentiation remain unclear, and there is no report about the effects of pyrroloquinoline quinone(PQQ) on particle-induced osteolysis in vivo. In this study, we aimed to investigate the effects of pyrroloquinoline quinone(PQQ) on signaling pathways involved in osteoclast differentiation, activation, survival in vitro, and the role of PQQ in UHMWPE particle-induced osteolysis in vivo.RANKL and M-CSF were used to induce osteolysis in BMM cells. Pyrroloquinoline quinone(PQQ) was divided into different groups by the dose. The in vitro cell culture results showed no toxic side effects when co-culture murine macrophage cells with pyrroloquinoline quinone(PQQ), and the pyrroloquinoline quinone(PQQ) also inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages(BMMs) in a dose-dependent manner. The mRNA expression of c-Fos, NFATc1, and TRAP in RANKL-treated BMMs was inhibited by pyrroloquinoline quinone(PQQ) treatment. Moreover, RANKL-induced c-Fos and NFATc1 protein expression was suppressed by pyrroloquinoline quinone(PQQ). This study demonstrated that the pyrroloquinoline quinone(PQQ) could inhibits RANKL-mediated expression of NFATc1 in part via suppression of c-Fos in mouse bone marrow cells(BMMs).We further set up the UHMWPE induced-osteolysis model to study the effects of pyrroloquinoline quinone(PQQ) treating particle-induced osteoclastogenesis in vivo. When 30 mg of UHMWPE particleswere implanted in the calvariae of C57BL/6J mice, we observed apronounced inflammatory reaction with highly vascularized granulation containing macrophages, TRAP(+) multinuclear osteoclasts, and marked osteolysis located adjacent to these inflammatory reaction tissues. In the pyrroloquinoline quinone(PQQ) treated(1 and 10 mg/kg) groups, the inflammatory reaction, bone destruction, and resorption area were less than that of the UHMWPE group but were more pronounced than that of the PBS group.To evaluate whether PQQ would inhibit particle-induced osteolysis in the murine calvarial osteolysis model, we used Micro-CT. In the UHMWPE group, particle-induced osteolysis was apparent compared with the PBS group, where there was no pronounced osteolysis. In the pyrroloquinoline quinone(PQQ) treated(1 and 10 mg/kg) groups, the particle-induced osteolysis was reduced compared with the UHMWPE group, indicating that treatment with pyrroloquinoline quinone(PQQ) prevented the particle-induced effects on bone metabolism and bone microarchitecture in the murine skull. Compared with the PBS group, the UHMWPE group showed significant decrease in BMD, BVF, CMT, and Ct. The decreases in BMD, BVF, CMT, and Ct were associated with decreased bone formation and increased osteolysis according to previously study. In this study, treatment with pyrroloquinoline quinone(PQQ)(1 mg/kg) attenuated the UHMWPE particle-induced decrease in BMD, BVF, CMT and Ct. In a similar fashion, treatment with pyrroloquinoline quinone(PQQ)(10 mg/kg) significantly prevented the UHMWPE particle-induced decrease in BMD, BVF, CMT, and Ct. Treatment with pyrroloquinoline quinone(PQQ)(10 mg/kg) also resulted in the improvement in BMD, BVF, CMT, and Ct compared with the pyrroloquinoline quinone(PQQ)(1 mg/kg) treated group. All of these results showed that pyrroloquinoline quinone(PQQ) could inhibit wear particle-induced bone loosening.In conclusion, we demonstrated the inhibitory effects of pyrroloquinoline quinone(PQQ) on osteoclastogenesis in primary precursor cells in the present study. Further, the in vivo efficacy of pyrroloquinoline quinone(PQQ) was confirmed with a murine calvarial model of particle-induced osteolysis. Our findings suggest that pyrroloquinoline quinone(PQQ) may represent a promising agent for the prevention and treatment of bone erosion desease like osteolysis, through the regulating of osteoclastogenesis.