Anti-inflammatory and anti-osteoporotic effects of 2,4'-dihydroxybenzophenone

Abstract

2,4′-Dihydroxybenzophenone (DHP) is derived from the herbs of Garcinia xanthochymus; no biochemical properties have been elucidated. In this study, we investigated whether DHP alleviates LPS-induced inflammatory responses and endotoxin shock in RAW 264.7 macrophages and zebrafish larvae. In LPS-microinjected zebrafish larvae as an endotoxin shock model, DHP dramatically attenuates mortality and morphological abnormality and restored reduced heart rate. Moreover, the expression of proinflammatory mediators, including nitric oxide, tumor necrosis factor-alpha (TNF-α), and interleukin-12 (IL-12), was dramatically reduced in the presence of DHP accompanied by mitigation of macrophage and neutrophil recruitment to the inflammatory sites. We revealed that DHP inhibits LPS-induced inflammatory response by downregulating pro-inflammatory mediators in RAW 264.7 macrophages. Molecular docking data predicted that DHP possibly binds to hydrophobic pocket of myeloid differentiation factor 2 (MD2) and occlude dimerization of Toll-like receptor 4 (TLR4) and MD2. Hence, DHP downregulated the TLR4-mediated intracellular signaling pathway, decreasing the expressions of myeloid differentiation primary response 88 (MyD88), phosphorylation of IL-1 receptor-associated protein kinase-4 (p-IRAK4), and nuclear factor-κB (NF-κB) in LPS-induced RAW 264.7 macrophages. Furthermore, we revealed that DHP inhibits mitochondrial reactive oxygen species (mtROS) during LPS-induced inflammatory conditions in RAW 264.7 macrophages and zebrafish larvae accompanied by the stabilization of mitochondrial membrane potential. Altogether, our study confirmed that DHP alleviates LPS-induced inflammation and endotoxin shock in vitro and in vivo by binding to TLR4/MD2 receptor complex and subsequently reducing mtROS production.|2,4′-Dihydroxybenzophenone (DHP) is an organic compound derived from Garcinia xanthochymus, but there are no reports on its biochemical functions and bioavailability. In this study, we evaluated whether DHP affects osteoblast differentiation and activation, as well as anti-osteoporotic activity in MC3T3-E1 preosteoblasts and zebrafish larvae. In the non-toxic concentrations, DHP-treated MC3T3-E1 preosteoblasts increased alkaline phosphatase (ALP) activation and mineralization in a concentration-dependent manner, accompanied by high expression of osteoblast-specific markers, including RUNX family transcription factor 2 (RUNX2), osterix (OSX), and ALP. Consistent with data in MC3T3-E1 preosteoblasts, DHP upregulated osteoblast-specific marker genes in zebrafish larvae and simultaneously stimulated vertebral formation. we also revealed that DHP enhanced phosphorylation of glycogen synthase kinase-3β (GSK-3β) at SER9 and total expression of β-catenin in the cytosol, and markedly hastened localization of β-catenin into the nucleus. Furthermore, DHP restored prednisolone (PDS)-induced marked decrease in ALP activity and mineralization, as well as osteoblast-specific marker expression. In PDS-treated zebrafish, DHP alleviates PDS-induced osteoporosis in zebrafish larvae by restoring vertebral formation and osteoblast-specific gene expression. Taken together, these results suggest that DHP is a potential osteoanabolic candidate to treat osteoporosis by stimulating osteoblast differentiation.