Caffeic Acid Inhibits RANKL and TNF-α-induced Phosphorylation of p38 Mitogen-activated Protein Kinase in RAW-D Cells

Abstract

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.

Effects of Caffeic Acid and Its Derivatives on Bone: A Systematic Review

“… 40 Four in vitro studies used CA doses between 0.1–5 µM. 35 , 37 , 38 , 40 Ang et al 36 used doses between 0–0.3 µM and Sandra et al 41 and Sandra and Ketherin 39 used a dose of 10 µg/mL (55.5 µM). The treatment period was 5–7 days for the differentiation of osteoclasts.…”

“… ↓ RANKL and TNFα-induced osteoclastogenesis in RAW-D cells. ↓ RANKL and TNFα-induced NF-κB activity in RAW-D cells Sandra and Ketherin 39 Cell line: RAW-D cells Mode of disease induction: RANKL and TNFα-induced osteoclastogenesis Treatment: 10 µg/mL of caffeic acid for 2 hours Negative control: untreated cells Positive control: n.a. ↓ RANKL and TNFα-induced osteoclastogenesis and phosphorylation of p38 MAPK compared with negative control Kwon et al 37 Cell line: RAW264.7 cells Mode of disease induction: RANKL- induced osteoclastogenesis Treatment: 0.1, 1 and 5 µM of CAPE for 5 days Negative control: untreated cells Positive control: n.a.…”

“…CADPE was shown to suppress RANKL-induced activation of TRAF6 activation and the subsequent signalling pathways in multiple osteoclast progenitors, such as BMMs, 38 RAW264.7 38 and RAW D cells. 39 Sandra and Ketherin suggested that the downregulation of p38 is the key step of CA-mediated osteoclastogenesis. 39 Upon activation, p38 initiates osteoclastogenesis by inducing NF-κB and NFATc1 expression.…”

“… 39 Sandra and Ketherin suggested that the downregulation of p38 is the key step of CA-mediated osteoclastogenesis. 39 Upon activation, p38 initiates osteoclastogenesis by inducing NF-κB and NFATc1 expression. 67 , 68 Inhibition of p38 MAPK reduces RANKL (canonical) and TNFα-induced (non-canonical) osteoclast formation.…”

Zinc Administration Affects Bronchial Mucosal NF-κB p105/p50, p-NF-κB p65, IL-8, and IL-1β of Zinc-deficient Rats

“…(3) Zinc is an essential component of several enzymes and cofactors in signaling pathways. Controlling inflammatory signaling pathway through correlated underlying factors such as interleukin (IL)-1b (4-6) and IL-8 (7), and also transcription factors, such as NF-κB (6)(7)(8)(9), is required for normal development (10,11), apoptotic induction of tumor/ cancer cell (12)(13)(14), and cellular function. (15) Zinc plays a role in the inflammatory system by a variety of mechanisms such as protecting the mucociliary cytoplasmic apparatus (tubulin and basal bodies), and inhibiting Nuclear Factor (NF)-κB translocation into the nucleus, which prevents the subsequent expression of proinflammatory cytokines.…”