A two-herb formula inhibits osteoclastogenesis and suppresses NF-kB and MAPK pathways

Ethnopharmacological relevance: An herbal formula RL, comprising Rosae Multiflorae Fructus (the dried fruit of Rosa multiflora Thunb.) and Lonicerae Japonicae Flos (the newly bloomed flower or dried flower-bud of Lonicera japonica Thunb.), was traditionally used for treating inflammatory diseases in China.

Aim of the study: A standardized ethanolic extract of RL (RLE for short) alleviates bone erosion in collagen-induced arthritis (CIA) rats, and suppresses nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signalling in LPS-stimulated RAW264.7 macrophages. The two pathways have been implicated in osteoclastogenesis. This study aimed to determine if RLE inhibits osteoclastogenesis, and if inhibiting NF-κB and MAPK signalling is involved in the effect.

Materials and methods: RLE was prepared as previously described. Contents of gallic and chlorogenic acid in RLE are 0.0763% and 2.6399%, respectively. CIA rats and receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis were used as experimental models. Tartrate-resistant acid phosphatase (TRAP) staining and resorption pit assays were used to evaluate osteoclastogenesis and osteoclastic bone resorption, respectively. Protein levels were detected using immunoblotting. mRNA levels were detected using RT-qPCR.

Results: RLE reduced osteoclasts in joints of CIA rats. In in vitro assays, RLE inhibited RANKL-induced osteoclastogenesis and decreased bone resorption function of osteoclasts. Mechanistic studies revealed that RLE lowered protein levels of phospho-p65 (Ser536), phosphoextracellular regulated protein kinases (ERK) (Thr202/Tyr204) and phospho-p38 (Thr180/Tyr182) in joints of CIA rats. In RANKL-stimulated RAW 264.7 macrophages, RLE suppressed the phosphorylation/activation of IκB-α (Ser 32), p65 (Ser536), p38 (Thr180/Tyr182) and ERK (Thr202/Tyr204), decreased the nuclear localization of p65, nuclear factor of activated T-cells cytoplasmic 1 and c-Fos, and lowered mRNA levels of osteoclast marker/function-related genes TRAP, cathepsin k, matrix metalloproteinase-9 and osteoclast-associated receptor.

Conclusions: RLE reduces osteoclasts in joints of CIA rats and inhibits RANKL-induced osteoclastic differentiation of RAW264.7 macrophages. Inhibition of NF-κB and MAPK pathways is involved in the effects of RLE. This study provides further pharmacological justifications for the use of RL in inflammatory disease management, and provides additional pharmacological basis for developing RLE as a modern anti-arthritic drug.