白藜芦醇通过增加miR-181a-5p的表达,抑制脂多糖介导的破骨细胞前体RAW 264.7细胞的活化。

Hai-Yan Xue, Ming-Wei Liu, Guang Yang
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引用次数: 2

摘要

白藜芦醇(Resveratrol, Res)具有抗炎症和抗骨质疏松功能。我们通过脂多糖(LPS)刺激破骨细胞前体RAW 264.7细胞释放炎症因子来评估Res对破骨细胞分化的影响。本研究采用LPS (1 ng/L)体外诱导Raw 264.7炎症损伤模型。实验采用25 ng/mL M-CSF + 30 ng/mL RANKL或1 μg/L LPS诱导破骨细胞生成。我们使用细胞计数试剂盒-8测定RAW 264.7细胞的相对细胞存活率。然后,采用酶联免疫吸附法测定炎症标志物的丰度,如白细胞介素-1β (IL-1β)、肿瘤坏死因子-α (TNF-α)和IL-6。随后,应用Western blot分析评估磷酸化转化生长因子β活化激酶1 (P-TAK1)蛋白、TNF受体相关因子6 (TRAF6)、核因子κ b抑制蛋白(i -κB)、磷酸化i -κB -α (p - i -κB -α)和核因子κB65 (NF-κB65)的丰度。实时聚合酶链反应检测miR-181a-5p、TRAF6、特异基因降钙素受体(CTR)、活化T核因子1 (NFATC1)、组织蛋白酶K (CTSK)、基质金属蛋白酶(MMP)-9 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,与模型组比较,Res治疗组大鼠上清炎症因子TNF-α、IL-1β、IL-6的表达程度明显减弱(p < 0.05)。此外,在RAW 264.7细胞中,miR-181a-5p的表达程度显著升高,而与模型组相比,Res治疗组p - i -κ b -α、p - tak1、NF-κB65和TRAF6的表达显著降低(p < 0.05)。Res治疗组在破骨细胞分化和骨吸收过程中,CTR、NFATC1、MMP-9、CTSK和TRAP mRNA表达水平显著降低。结果提示,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,其潜在机制可能与Res通过增加miR-181a-5p表达抑制TRAF6/TAK1通路活性有关。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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

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.

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International Journal of Immunopathology and Pharmacology
International Journal of Immunopathology and Pharmacology Immunology and Microbiology-Immunology
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期刊介绍: International Journal of Immunopathology and Pharmacology is an Open Access peer-reviewed journal publishing original papers describing research in the fields of immunology, pathology and pharmacology. The intention is that the journal should reflect both the experimental and clinical aspects of immunology as well as advances in the understanding of the pathology and pharmacology of the immune system.
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