Department of Dentistry, Chung Shan Medical University Hospital, Taichung 40201, Taiwan.
School of Dentistry, Chung Shan Medical University, Taichung 40201, Taiwan.
Int J Mol Sci. 2022 Oct 4;23(19):11773. doi: 10.3390/ijms231911773.
Rutin, also called quercetin-3-rhamnosyl glucoside, is a natural flavonol glycoside present in many plants. Rutin is used to treat various diseases, such as inflammation, diabetes, and cancer. For polymeric biomaterials, triethylene glycol dimethacrylate (TEGDMA) is the most commonly used monomer and serves as a restorative resin, a dentin bonding agent and sealant, and a bone cement component. Overall, TEGDMA induces various toxic effects in macrophages, including cytotoxicity, apoptosis, and genotoxicity. The aim of this study was to investigate the protective mechanism of rutin in alleviating TEGDMA-induced toxicity in RAW264.7 macrophages. After treatment with rutin, we assessed the cell viability and apoptosis of TEGDMA-induced RAW264.7 macrophages using an methylthiazol tetrazolium (MTT) assay and Annexin V-FITC/propidium iodide assay, respectively. Subsequently, we assessed the level of genotoxicity using comet and micronucleus assays, assessed the cysteinyla aspartate specific proteinases (caspases) and antioxidant enzyme (AOE) activity using commercial kits, and evaluated the generation of reactive oxygen species (ROS) using a dichlorodihydrofluorescein diacetate (DCFH-DA) assay. We evaluated the expression of heme oxygenase (HO)-1, the expression of nuclear factor erythroid 2 related factor (Nrf-2), and phosphorylation of AMP activated protein kinase (AMPK) using the Western blot assay. The results indicated that rutin substantially reduced the level of cytotoxicity, apoptosis, and genotoxicity of TEGDMA-induced RAW264.7 macrophages. Rutin also blocked the activity of caspase-3, caspase-8, and caspase-9 in TEGDMA-stimulated RAW264.7 macrophages. In addition, it decreased TEGDMA-induced ROS generation and AOE deactivation in macrophages. Finally, we found that TEGDMA-inhibited slightly the HO-1 expression, Nrf-2 expression, and AMPK phosphorylation would be revered by rutin. In addition, the HO-1 expression, Nrf-2 expression, and AMPK phosphorylation was enhanced by rutin. These findings indicate that rutin suppresses TEGDMA-induced caspase-mediated toxic effects through ROS generation and antioxidative system deactivation through the Nrf-2/AMPK pathway. Therefore, rutin has the potential to serve as a novel antitoxicity agent for TEGDMA in RAW264.7 macrophages.
芦丁,也称为槲皮素-3-鼠李糖苷,是一种天然类黄酮糖苷,存在于许多植物中。芦丁用于治疗各种疾病,如炎症、糖尿病和癌症。对于聚合生物材料,三甘醇二甲基丙烯酸酯(TEGDMA)是最常用的单体,用作修复树脂、牙本质粘结剂和密封剂以及骨水泥成分。总的来说,TEGDMA 会在巨噬细胞中引起各种毒性作用,包括细胞毒性、细胞凋亡和遗传毒性。本研究旨在探讨芦丁缓解 RAW264.7 巨噬细胞中 TEGDMA 诱导的毒性的保护机制。用芦丁处理后,我们分别使用甲基噻唑四唑(MTT)测定法和 Annexin V-FITC/碘化丙啶测定法评估 TEGDMA 诱导的 RAW264.7 巨噬细胞的细胞活力和细胞凋亡。随后,我们使用彗星和微核测定法评估遗传毒性,使用商业试剂盒评估半胱天冬氨酸特异性蛋白酶(caspases)和抗氧化酶(AOE)的活性,并使用二氯二氢荧光素二乙酸酯(DCFH-DA)测定法评估活性氧物种(ROS)的生成。使用 Western blot 测定法评估血红素加氧酶(HO)-1 的表达、核因子红细胞 2 相关因子(Nrf-2)的表达和 AMP 激活蛋白激酶(AMPK)的磷酸化。结果表明,芦丁可显著降低 TEGDMA 诱导的 RAW264.7 巨噬细胞的细胞毒性、细胞凋亡和遗传毒性水平。芦丁还阻断了 TEGDMA 刺激的 RAW264.7 巨噬细胞中 caspase-3、caspase-8 和 caspase-9 的活性。此外,它减少了 TEGDMA 诱导的巨噬细胞中 ROS 的产生和 AOE 的失活。最后,我们发现 TEGDMA 轻微抑制了 HO-1 表达、Nrf-2 表达和 AMPK 磷酸化,而芦丁可逆转这一现象。此外,芦丁还增强了 HO-1 表达、Nrf-2 表达和 AMPK 磷酸化。这些发现表明,芦丁通过 ROS 生成和抗氧化系统失活来抑制 TEGDMA 诱导的 caspase 介导的毒性作用,这是通过 Nrf-2/AMPK 通路实现的。因此,芦丁有可能成为 RAW264.7 巨噬细胞中 TEGDMA 的新型解毒剂。