The Effect Of Andrographolide On Ovariectomy Mice And Osteoclasts

BACKGROUND:Osteoporosis is one of the common diseases in elderly people and postmenopausal women, which often lead to the increase in bone fragility and raise the risk of fracture.Bone is a hard but dynamic organ. Which remodeling itself by two processes-namely bone resorption (by osteoclasts) and bone formation (by osteoblasts). The balance within these two contrary processes maintains bone homeostasis. However, when bone resorption by osteoclasts exceeds bone formation by osteoblasts, osteolytic disorders such as osteoporosis, rheumatoid arthritis, Paget’s disease will happened.Osteoclasts are terminally differentiated cells which differentiate from monocyte/macrophage lineage. With the stimulation of many cytokines such as M-CSF and Receptor activator of NF-κB ligand (RANKL), the osteoclast precursors fused and form the multinucleated osteoclasts. RANKL play a key role in osteoclastogenesis, survival and function. RANKL could band to its receptor RNAK on osteoclasts or their precursors, then regulate the expression of target genes through canonical and non-canonical NF-κB pathway.Andrographolide (AP), a bicyclic diterpenoid lactone, is a product isolated from the whole plant or leaves of Andrographis paniculata. Which is one of the effective ingredients in Andrographis paniculata and possesses the activities of anti-inflammation, antibacterial, antipyretic antiviral, antitumor and immune regulation. There is a report suggests that andrographolide could alveolar bone resorption induced by inflammation. However this is not direct research reveal the effect of andrographolide on postmenopausal osteoporosis. Therefore, in this study, we conduct experiments to test the effects of andrographolide on osteoporosis and explore the underlying potential mechanisms.METHODS:1. We established the C57BL/6J OVX (ovariectomized) mice model, and divided them into 4 groups randomly (Sham group, OVX group, AP 1 mg/kg group, AP 5mg/kg group, n=6). The AP 1 mg/kg group and AP 5 mg/kg group were intraperitoneal injected with AP in physiological saline, while the sham and OVX groups received physiological saline as vehicles. All injections were administered every two days. After treatment of six weeks, the tibias of mice were isolated and scanned by Micro-CT, to reconstructed the 3D model of tibias and analyzed the bone resorption parameters, including BV/TV (bone volume per tissue volume), Tb.N (trabecular number), Tb.Th (trabecular thickness), Tb.Sp (trabecular separation), and cortical bone parameter Cor.Th (Cortex thickness).2. We used the safe dosage of andrographolide to interrupt the RANKL-induced osteoclastogenesis. The cells were dyed with TRACP and nucleus≥3 cells were counted as osteoclasts. The number of osteoclasts in different group were compared. We tested the cytotoxicity of andrographolide on BMMs through MTS. The expression of osteoclast-related genes were tested by Real-time PCR.3. To explore the regulation effect of andrographolide on RANKL signaling pathway, Luciferase Reporter Gene Assay were performed using RAW264.7 which stabled transferred 3κB-Luc-SV40 or pNFATcl-TA-Luc luciferase construct. Cells were pre-treated with andrographolide for 1 hour followed by stimulation with RANKL. Luciferase activity were detected and compared within groups. The regulation effect of andrographolide on RANKL-induced protein level of NF-κB、NFATc1、ERK were detected by Western blot.RESULTS:1. The Micro-CT analysis suggested that intraperitoneal injection of 1 mg/kg andrographolide could not change the trabecular and cortical bone parameters. Treatment with 5 mg/kg andrographolide could increase BV/TV (P<0.01), Tb.N (P<0.01), decrease Tb.Sp (P<0.01) significantly, while have not significant effect on Cor.Th.2. Andrographolide inhibited osteoclastogenesis in a dose-depended manner. The MTS test result showed that andrographolide exerts no cytotoxic effect on BMMs within the dosage from 0 μM to 20 μM. Real-time PCR suggested that 1 μM andrographolide could inhibit the gene expression of TRACP (P<0.01),5 μM andrographolide could inhibit the gene expression of TRACP% Ctsk and Atp6v0d2 (P<0.01), while 10 μM andrographolide could inhibit the gene expression of TRACP、Ctsk、Atp6v0d2 and NFATc1(P<0.001).3. From experiments about the mechanism, we found that 1 μM andrographolide could impede the transcriptional activity of NF-κB (P<0.05) and NFATc1 (P<0.001).10μM andrographolide impeded the transcriptional activity more powerfully (NF-κB P<0.001, NFATc1 P<0.001) and inhibited the protein level of NF-κB, NFATcl and ERK.CONCLUSION:Andrographolide could inhibited the bone loss induced by ovariectomy, increase tibia BV/TV, Tb.N, and decrease Tb.Sp in OVX mice, while have not significant effect on Cor.Th. This anti-osteoporosis effect were derived from the negative regulation of andrographolide on NF-κB, NFATc1 and ERK signaling pathways.Thus, AP have the prospect to be developed as a potential therapeutic agent for osteoporosis and other osteoclast-related diseases.