Kuriya Kenji, Itoh Shimon, Isoda Akihiro, Tanaka Shoki, Nishio Masahiro, Umekawa Hayato
Department of Life Sciences, Graduate School of Bioresources, Mie University, 1577 Kurimamachiya, Tsu, Mie, 514-8507, Japan.
Mie Study Center, The Open University of Japan, 1234 Ishinden, Tsu, Mie, 514-0061, Japan.
Biochem Biophys Rep. 2023 Aug 30;35:101536. doi: 10.1016/j.bbrep.2023.101536. eCollection 2023 Sep.
Osteoclasts are multinucleated cells formed by macrophage cell fusion that are responsible for bone resorption. Previously, we found that treating osteoclastic progenitor cells with (-)-epigallocatechin gallate (EGCg) increased cell fusion. In this study, we aimed to identify factors involved in the cell fusion induced by EGCg.
We hypothesized that EGCg-induced oxidative stress might be involved in cell fusion, and used macrophage cell line RAW264.7 cells. We evaluated cell fusion activity after adding the antioxidants N-acetyl-l-cysteine (NAC) or catalase in addition to EGCg. The mRNA expressions of genes related to cell fusion and bone resorption were quantified by real-time PCR. Finally, we added hydrogen peroxide and examined its effects on cell fusion and TRAP activity.
EGCg-induced cell fusion was strongly inhibited by the addition of NAC in a dose-dependent manner (EGCg with 5 mM NAC; decreased to 1.5%; < 0.05), while the inhibitory effect of catalase was limited (EGCg with 500 U/mL catalase; decreased to 27.7%; < 0.05). DC-STAMP expression was significantly upregulated by EGCg compared with the untreated group, and the upregulation was significantly suppressed by 5 mM NAC. Conversely, Nfatc1 and TRAP expression were not upregulated by EGCg. These results suggest that EGCg induces DC-STAMP expression reactive oxygen species production, which regulates cell fusion but does not affect the osteoclastic pathway. Although treatment with hydrogen peroxide promoted the formation of multinucleated cells, no increase in TRAP activity was observed, which was similar to EGCg treatment.
This study suggests that the increased cell fusion by EGCg may be induced by oxidative stress due to reactive oxygen species production.
破骨细胞是由巨噬细胞融合形成的多核细胞，负责骨吸收。此前，我们发现用（-）-表没食子儿茶素没食子酸酯（EGCg）处理破骨细胞祖细胞可增加细胞融合。在本研究中，我们旨在确定参与EGCg诱导的细胞融合的因素。
我们假设EGCg诱导的氧化应激可能参与细胞融合，并使用巨噬细胞系RAW264.7细胞。除EGCg外，我们添加抗氧化剂N-乙酰-L-半胱氨酸（NAC）或过氧化氢酶后评估细胞融合活性。通过实时PCR定量与细胞融合和骨吸收相关的基因的mRNA表达。最后，我们添加过氧化氢并检查其对细胞融合和抗酒石酸酸性磷酸酶（TRAP）活性的影响。
添加NAC以剂量依赖性方式强烈抑制EGCg诱导的细胞融合（5 mM NAC与EGCg共同处理；降至1.5%；<0.05），而过氧化氢酶的抑制作用有限（500 U/mL过氧化氢酶与EGCg共同处理；降至27.7%；<0.05）。与未处理组相比，EGCg显著上调了树突状细胞特异性跨膜蛋白（DC-STAMP）的表达，而5 mM NAC显著抑制了这种上调。相反，EGCg未上调活化T细胞核因子c1（Nfatc1）和TRAP的表达。这些结果表明，EGCg通过诱导DC-STAMP表达和活性氧产生来调节细胞融合，但不影响破骨细胞途径。虽然过氧化氢处理促进了多核细胞的形成，但未观察到TRAP活性增加，这与EGCg处理相似。
本研究表明，EGCg导致的细胞融合增加可能是由活性氧产生引起的氧化应激诱导的。
