Protective Effect of Rutin on Triethylene Glycol Dimethacrylate-Induced Toxicity through the Inhibition of Caspase Activation and Reactive Oxygen Species Generation in Macrophages

Abstract

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.

Keywords: TEGDMA; antioxidant system; apoptosis; cytotoxicity; genotoxicity; macrophage; reactive oxygen species; rutin.

Figure 1

Effect of rutin on TEGDMA-induced cytotoxicity in RAW264.7 macrophages. After RAW264.7 macrophages were incubated with rutin at 0, 10, 30, and 100 μM for 30 min, the cells were incubated with or without TEGDMA at 3 μM for 24 h. (A) The image of morphological changes was taken using the inverted microscope at the actual magnification 200×. (B) Cytotoxicity was represented as the percentage of the control group, which indicated 0% cytotoxicity rate in the treatment with TEGDMA and rutin at 0 μM. Values are expressed as mean ± S.D. of three times per group. * Represents significant difference between the indicated and control groups; # between the indicated and TEGDMA groups, p < 0.05.

Figure 2

Effect of rutin on TEGDMA-induced necrosis and apoptosis expression in RAW264.7 macrophages. Apoptosis of RAW264.7 cells was measured by flow cytometry using Annexin V-FITC/PI apoptosis detection kit. (A) The dot-plots of flowcytometry from the annexin V-FITC/PI staining assay are shown. (B) Quantitative analysis of the percentage of viable cells (Annexin V-FITC negative and PI negative), necrotic cells (Annexin V-FITC negative and PI positive), and apoptotic cells (Annexin V-FITC positive) was presented. Values are expressed as mean ± S.D. of three times per group. * Represents significant difference between the indicated and control groups, p < 0.05. # Means significant difference between the indicated and TEGDMA groups, p < 0.05.

Figure 3

Effect of rutin on TEGDMA-induced genotoxicity via COMET and MN assay in RAW264.7 macrophages. (A) was gel electrophoresis of DNA damage from COMET assay. (B,C) were quantifications of tail length and tail moment, respectively. (D) was the quantifications of MN formation. Values are expressed as mean ± S.D. of three times per group. * Represents significant difference between the indicated and control groups, p < 0.05. # Means significant difference between the indicated and TEGDMA groups, p < 0.05.

Figure 4

Effect of rutin on TEGDMA-induced caspases activities in RAW264.7 macrophages. Values are expressed as mean ± S.D. of three times per group. * Represents significant difference between the indicated and control groups, p < 0.05. # Means significant difference between the indicated and TEGDMA groups, p < 0.05.

Figure 5

Effect of rutin on TEGDMA-induced ROS generation in RAW264.7 macrophages. Values are expressed as mean ± S.D. of three times per group. * Represents significant difference between the indicated and control groups, p < 0.05. # Means significant difference between the indicated and TEGDMA groups, p < 0.05.

Figure 6

Effect of rutin on TEGDMA-reduced AOE activities in RAW264.7 macrophages. Values are expressed as mean ± S.D. of three times per group. * Represents significant difference between the indicated and control groups, p < 0.05. # Means significant difference between the indicated and TEGDMA groups, p < 0.05.

Figure 7

Effect of rutin on HO-1 and Nrf-2 expression in TEGDMA-treated RAW264.7 macrophages. Expression of HO-1 and Nrf-2 was determined using Western blotting with indicated antibodies. Representative blots and quantitative results are shown. Values are expressed as mean ± S.D. of three times per group. * Represents significant difference between the indicated and control groups, p < 0.05. # Means significant difference between the indicated and TEGDMA groups, p < 0.05.

Figure 8

Effect of rutin on AMPK phosphorylation in TEGDMA-treated RAW264.7 macrophages. Phosphorylation of AMPK was determined using Western blotting with indicated antibodies. Representative blots and quantitative results are shown. Values are expressed as mean ± S.D. of three times per group. * Represents significant difference between the indicated and control groups, p < 0.05. # Means significant difference between the indicated and TEGDMA groups, p < 0.05.