Trauma center, 36657The First Hospital Affiliated of Kunming Medical University, Kunming, China.
Department of Emergency, 36657The First Hospital Affiliated of Kunming Medical University, Kunming, China.
Int J Immunopathol Pharmacol. 2023 Jan-Dec;37:3946320231154995. doi: 10.1177/03946320231154995.
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 ( < 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 ( < 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.
白藜芦醇（Res）具有抗炎和抗骨质疏松作用。我们通过释放脂多糖（LPS）刺激的破骨细胞前体 RAW 264.7 细胞中的炎性细胞因子来评估 Res 对破骨细胞分化的影响。在该研究中，使用 LPS（1ng/L）在体外诱导 Raw 264.7 炎症损伤模型。采用 25ng/ml M-CSF+30ng/ml RANKL 或加 1μg/L LPS 诱导破骨细胞生成实验。我们利用细胞计数试剂盒-8 测定 RAW 264.7 细胞的相对细胞存活率。然后，酶联免疫吸附测定法用于测量炎性标志物（如白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）和 IL-6）的丰度。随后，采用 Western blot 分析评估磷酸化转化生长因子β激活激酶 1（P-TAK1）蛋白、肿瘤坏死因子受体相关因子 6（TRAF6）、核因子-κB 抑制剂蛋白（IκB）、磷酸化 IκB-α（P-IκB-α）和核因子κB65（NF-κB65）的丰度。通过实时聚合酶链反应测定 miR-181a-5p、TRAF6、特异性基因降钙素受体（CTR）、活化 T 核因子 1（NFATC1）、组织蛋白酶 K（CTSK）和基质金属蛋白酶（MMP）-9 的 mRNA 表达水平。测定破骨细胞骨吸收功能。最后进行抗酒石酸酸性磷酸酶（TRAP）染色。结果发现，与模型组相比，Res 治疗组上清液炎性因子 TNF-α、IL-1β和 IL-6 的表达程度明显减弱（<0.05）。此外，Res 治疗组 RAW 264.7 细胞中 miR-181a-5p 的表达明显增加，而 P-IκB-α、P-TAK1、NF-κB65 和 TRAF6 的表达明显降低（<0.05）。在 Res 治疗组中，破骨细胞分化和骨吸收过程中 CTR、NFATC1、MMP-9、CTSK 和 TRAP 的 mRNA 表达水平明显降低。结果表明，Res 可减少 RAW 264.7 细胞向破骨细胞的分化，缓解 LPS 刺激的骨质疏松症，其作用机制可能与 Res 通过增加 miR-181a-5p 的表达抑制 TRAF6/TAK1 通路的活性有关。
