B ACKGROUND:Caffeic acid inhibits osteoclastogenesis by downregulating expression of Cathepsin K and Nuclear Factor of Activated T cells (NFATc)1, as well as inhibiting activity of Nuclear Factor kB (NFkB). Meanwhile TNF Receptor-associated Factor (TRAF)6 was not influenced by caffeic acid. In order to investigate further caffeic acid's mechanism in inhibiting osteoclastogenesis, regulation of caffeic acid on p38 Mitogen-activated Protein Kinase (MAPK) was investigated.METHODS: RAW-D cells were pretreated with/without caffeic acid and treated with/without 20 ng/mL RANKL and 1 ng/mL TNFα for 0.2, 1, 6, and 12 hour. Tartrate Resistant Acid Phosphatase (TRAP) staining was performed. Then, western blot analysis was performed to detect p38 MAPK and phosphorylated-p38 MAPK. Resulted protein bands were quantified and statistically analyzed. RESULTS:Under induction of 20 ng/mL RANKL and 1 ng/ mL TNF-α, RAW-D cells were successfully differentiated into TRAP + osteoclast-like polynuclear cells. Under treatment of 20 ng/mL of RANKL and 1 ng/mL of TNF-α for 0.2 or 1 hour, significant (p=0,000, T test) increment of phosphorylated p38 MAPK was observed as compared with control. Pretreatment of 10 µg/mL caffeic acid significantly (p=0.000, T test) suppressed the 20 ng/mL of RANKL and 1 ng/mL of TNF-α-induced phosphorylation of p38 MAPK.

Caffeic acid, a natural substance found majorly in fruits, grains, and herbs, is known to have therapeutic benefits. One of which is to inhibit bone resorption by targeting osteoclastogenesis through inhibition of the Cathepsin K, p38 Mitogen-activated Protein Kinase (MAPK), Nuclear Factor of Activated T-cells c1 (NFATc1) and Nuclear Factor kB (NFkB). Besides p38 MAPK, the c-Jun N-terminal kinase (JNK) / stress-activated protein kinases (SAPK), another member of MAPK family, has been reported to play important roles in osteoclastogenesis. Hence, current study was undertaken in order to investigate mechanism of Caffeic Acid towards JNK/SAPK pathway. Tartrate Resistant Acid Phosphatase (TRAP) staining was performed on caffeic acid-treated and RANKL-TNFα-induced RAW-D cells. Western blot analysis was performed to detect JNK/SAPK and phosphorylated-JNK/SAPK. Protein bands were quantified and statistically analyzed. Treatment of 10 μg/mL Caffeic Acid inhibited 20 ng/mL RANKL and 1 ng/mL TNFα-induced RAW-D differentiation into TRAP+ osteoclast-like polynuclear cells. Induction of 20 ng/mL of RANKL and 1 ng/mL of TNFα for 0.2 or 1 hour, significantly increase phosphorylation of JNK/SAPK as compared with control. Treatment of 10 µg/mL Caffeic Acid significantly inhibited the 20 ng/mL of RANKL and 1 ng/mL of TNFα-induced phosphorylation of JNK/SAPK. Taken together, Caffeic Acid could inhibit the RANKL and TNFα-induced osteoclastogenesis through JNK/SAPK.

Osteoclast activities are responsible for the resorption of bone cells found in several bone diseases, one of which is periodontitis and arthritis. The downregulating signals of Receptor Activator of Nuclear Factor kB (RANK)-RANK Ligand and Tumor Necrosis Factor  (TNF)-a are the major cause of the bone destruction. Studies and experiments have been performed to overcome this matter. Various medications are now available to treat bone-related diseases, targeting the specific aspect of the signaling. Synthetic drugs such as denosumab and bisphosphonates have complex pharmacological action and have been the leading choice in treatment. Evidence in studies proved that natural resources including herbal products have potential application to therapy for bone loss, with caffeic acid and Caffeic Acid Phenethyl Ester (CAPE) showing significant inhibitory results and Chinese herbs such as Herba epimedii (Yín Yáng Huò) and Fructus psoraleae(Bǔ Gǔ Zhī) proved to contain components that give similar effects to estrogen. The purpose of this review is to discuss the therapy value of available synthetic and natural therapeutic agents. Understanding the mechanisms of both agents will not only clarify their function as therapeutic agents, but can also be the key to the treatment of diseases caused by bone resorption by targeting specific aspects of osteoclastogenesis.Keywords: osteoclastogenesis, TNF, RANKL, bone resorption, natural resource, signaling, treatment

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