Molecular Mechanisms Of Gambogic Acid On Osteoclast Related Osteolytic Diseases

Osteoporosis is one of the main factors that endanger the health of the elderly, and has become the fourth major factor in the death of the population. With the acceleration of the aging population, China’s osteoporosis patients will reach 286 million until year 2020. Osteoporosis has rapidly developed into a public health problem in our country. It is urgent for the correct understanding and early intervention. Osteoblasts and osteoclasts are the two most important cells in bone remodeling. When the number of osteoclasts or bone resorption function is increased, it can cause many kinds of common diseases because of the excessive loss of bone mass.Osteoclasts are the only cells in the human body to absorb bone, and in the physiological conditions, to maintain normal bone tissue metabolic balance.The increased number of osteoclasts or the enhanced absorption of osteoclasts could result in many diseases including osteoporosis, osteoarthritis, Paget’s disease and bone metastasis. Although the traditional drugs and new drugs are effective in the treatment of osteoclast related diseases, but there are still some complications such as bone necrosis and tumor. Recently, natural compounds as a drug in the regulation of osteoclast formation, prevention and treatment of osteoclast associated osteolysis disease began to gain increasing attentions. In the previous stage of screening drugs which may inhibit osteoclast differentiation, we found that traditional Chinese medicine Gambogic acid could effectively inhibit osteoclast differentiation and its bone resorption function. Therefore, this study will further explore the molecular biology mechanism of gambogic acid in the inhibition of osteoclast differentiation and maturation process, and to study the role of gambogic acid in the prevention and treatment of osteoclast related diseases, thus to provide the basis of cell biology and animal science for clinical application.Objective(1) To explore the molecular biological mechanism of gambogic acid in inhibiting osteoclast differentiation and maturation. (2) To determine the safety and efficacy of gambogic acid in OVX model. (3) To provide the basis for the clinical application of Chinese Medicine.MethodsSix-week-old female C57BL/6 mice were used in the present study. BMMs (bone marrow macrophages) were isolated from whole bone marrow. After the formation of osteoclasts, cells were stained for TRAP, and TRAP-positive cells with more than three nuclei were counted as osteoclasts. The cytotoxic effect of GBA was assessed using CCK-8 assays, and the IC50 was gained from the results. Bone absorption assay was used to evaluate the impact of different concentration of GBA on the bone resorption function of osetoclasts. Resorption pits were imaged using a scanning electron microscope and the bone resorption area was quantified using ImageJ software. Western blotting was used to examine the target signalling pathway of GBA in inhibiting the differentiation and bone resorption function of osteoclasts. We next established an OVX-induced osteoporosis model to determine the effect of GBA on osteoporosis in vivo. After 1 week, mice in the GBA-low and GBA-high groups were injected intraperitoneally with GBA at a dose of 0.5 or 2 mg/kg respectively twice per week for 4 weeks. Mice in the sham and vehicle groups received PBS. At the end of the experiment, the mice were killed and both the femurs and tibiae were excised and fixed in 4%(w/v) paraformaldehyde for histological and μ CT (micro-computed tomography) analysis respectively.ResultsWe demonstrated that cell viability was not affected by GBA at concentrations lower than 120 nM. However, GBA at concentrations higher than 240 nM was cytotoxic to BMMs. The calculated IC 50 for GBA in BMMs was 320 nM. Together, these results demonstrate that GBA suppresses osteoclast differentiation in a dose-dependent manner. BMMs were cultured on bone slices with or without various concentrations of GBA. Extensive bone resorption pits were observed on the surface of bone slices in the control group. In contrast, the size of the resorption area was decreased to approximately 60% of that of the control group by treatment with 60 nM GBA. Moreover, osteoclasts treated with 80 nM GBA showed only approximately 20% resorptive area. Very few resorption pits were observed on the bone slices treated with 120 nM GBA. These data demonstrated a potential inhibitory effect of GBA on osteoclastic bone resorption.We applied GBA in a murine model of OVX-induced osteoporosis, and the morphology of OVX mice treated with various concentration of GBA or vehicle was analyzed using micro-CT, H&E and TRAP staining. BV/TV after OVX was significantly reduced in comparison with that in the sham group. An overall protective effect of GBA against OVX-induced bone loss was observed. The administration of GBA produced a higher Tb.N and reduced Tb.Sp.We explored further the underlying mechanisms by which GBA regulates osteoclast formation and function. Thus the expression of osteoclast-specific genes was examined. In response to stimulation by RANKL, osteoclast-related genes were up-regulated in the control group. GBA suppressed osteoclastic gene expression in bone in a dose-dependent manner and time-dependent manner. Together, these data demonstrate further that GBA suppresses osteoclast formation in vitro.Critical signalling pathways involved in osteoclast differentiation including NF-κB (nuclear factor κB), PI3K (phosphoinositide 3-kinase)/Akt and MAPK (mitogen-activated protein kinase) signalling pathways were investigated in order to elucidate the possible underlying mechanisms. Phosphorylation of p38 was observed after stimulation with RANKL for 5 min. In contrast, the reduced activation of p38 was observed in the GBA-treated group exposed to RANKL. Similarly, JNK phosphorylation was also increased after 5 min of RANKL exposure, but GBA attenuated this JNK phosphorylation. The phosphorylation of Akt was increased after stimulation with RANKL for 10 min, but GBA reduced the phosphorylation of Akt. In contrast, GBA had no obvious effect on ERK and NF-κB activation. Moreover, expression of c-Fos and c-Jun, which are downstream of p38 and JNK, was reduced dramatically by GBA treatment. These data suggest that GBA may exert an inhibitory effect on p38, JNK and Akt activation.GBA was administered further in the absence or presence of anisomycin, a compound capable of activating JNK and p38 signalling cascades. Osteoclast formation was inhibited in cells treated with GBA alone. However, in cells treated with both GBA and anisomycin, impaired osteoclastogenesis was rescued and mature osteoclasts were observed. In addition, we detected that the phosphorylation of the JNK and p38 pathways was higher in the GBA with anisomycin group than the GBA-only group. We also performed computational molecular docking using mouse JNK1, JNK2 and p38 kinase domain models to determine whether GBA can bind directly to JNK and p38 proteins. GBA formed some favourable connections and docked comfortably within the JNK1, JNK2 and p38 ATP-binding sites. Our data demonstrate that GBA could suppress osteoclast formation in vitro by inhibiting JNK and p38 phosphorylation partially, thus preventing OVX-induced osteolysis in vivo.ConclusionGBA inhibits osteoclast formation and function both in vitro and in vivo, suggesting that GBA has potential value in the treatment of osteoclast-related diseases.