Resveratrol suppresses lipopolysaccharide-mediated activation of osteoclast precursor RAW 264.7 cells by increasing miR-181a-5p expression

Abstract

Resveratrol (Res) has anti-inflammation and antiosteoporosis functions. We evaluated the effect of Res on osteoclast differentiation by releasing inflammatory cytokines from osteoclast precursor RAW 264.7 cells stimulated by lipopolysaccharide (LPS). In the study, LPS (1 ng/L) was used to induce the Raw 264.7 inflammatory injury model in vitro. A total of 25 ng/mL M-CSF + 30 ng/mL RANKL or plus 1 μg/L LPS was used to induce osteoclastogenesis in the experiments. We utilized the Cell Counting Kit-8 assay to measure the relative cell survival of RAW 264.7 cells. Then, enzyme-linked immunosorbent assays were utilized to measure the abundance of inflammatory markers, such as interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and IL-6. Subsequently, Western blot analysis was applied to assess the abundance of phosphorylated transforming growth factor beta-activated kinase 1 (P-TAK1) protein, TNF receptor-associated factor 6 (TRAF6), nuclear factor-κB inhibitor protein (IκB), phosphorylated IκB-α (P-IκB-α), and nuclear factor κB65 (NF-κB65). mRNA expression levels of miR-181a-5p, TRAF6, specific gene calcitonin receptor (CTR), activated T nuclear factor 1 (NFATC1), cathepsin K (CTSK), and matrix metalloproteinase (MMP)-9 were determined via a real-time polymerase chain reaction. Osteoclast bone resorption function was determined. Finally, tartrate-resistant acid phosphatase (TRAP) staining was performed.The results found that Compared with the model group, the degrees of expressions of supernatant inflammatory factors TNF-α, IL-1β, and IL-6 were substantially attenuated in the Res treatment group ( p < 0.05). Furthermore, the extent of miR-181a-5p expression in the RAW 264.7 cells significantly increased, whereas P-IκB-α, P-TAK1, NF-κB65, and TRAF6 expressions significantly decreased in the Res treatment group as opposed to the model group ( p < 0.05). The CTR, NFATC1, MMP-9, CTSK, and TRAP mRNA expression levels were substantially reduced during osteoclast differentiation and bone resorption in the Res treatment group.The results suggest that Res can reduce the RAW 264.7 cell differentiation into osteoclasts and relieve LPS-stimulated osteoporosis, and the underlying mechanism may be associated with the Res-inhibited activity of the TRAF6/TAK1 pathway through the increased miR-181a-5p expression.

CX3CL1 promotes M1 macrophage polarization and osteoclast differentiation through NF-κB signaling pathway in ankylosing spondylitis in vitro

“…Besides, the NF-κB signaling pathway regulates osteoclast differentiation [ 43 ]. Under external environmental stimulation, the NF-κB signaling pathway is activated, inducing the expression of multiple osteoclast-specific genes in the nucleus, including MMP-9, TRAP, CTSK [ 44 ]. In addition, the NF-κB signaling pathway can also promote osteoclast differentiation by regulating the Wnt signaling pathway [ 45 ].…”

Therapeutic effects of resveratrol on epigenetic mechanisms in age‐related diseases: A comprehensive review

“…It is possible that these effects are related to the fact that miR‐181a‐5p disapproval can increase the levels of the Sirt1 and PI3K proteins (Zhu et al, 2021). RSV has been found to decrease the expression of NF‐κB65, P‐IκB‐α, and P‐TAK1 in RAW 264.7 cells (Xue et al, 2023). This suggests that RSV may reduce the differentiation of RAW 264.7 cells into osteoclasts and alleviate osteoporosis caused by LPS.…”

“…This suggests that RSV may reduce the differentiation of RAW 264.7 cells into osteoclasts and alleviate osteoporosis caused by LPS. These effects may be related to RSV‐induced suppression of TRAF6/TAK1 axis (Xue et al, 2023).…”