* Environmental Science Institute, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China;
Toxicol Sci. 2013 Nov;136(1):86-96. doi: 10.1093/toxsci/kft180. Epub 2013 Aug 22.
Microcystin-leucine-arginine (MCLR) is an environmental toxin from harmful algae, which has been linked to hepatotoxicity with high risks associated with liver disease. In this study, we explored the role of MCLR in NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways, which are important in regulating inflammatory and immune responses, in human hepatoma cell line HepG2 and primary mouse hepatocytes (PMHs). By in vitro cell-free and luciferase reporter systems, Western blotting with antiphospho-inhibitory protein of NF-κB (IκBα)/c-Jun N-terminal kinase (JNK)/extracellular signal-regulated kinase 1/2 (ERK1/2) antibody, it was found that at noncytotoxic concentrations (≤ 20 nM MCLR in PMHs, 1-1000 nM in HepG2), MCLR treatment alone promoted activation of NF-κB and MAPK pathways and modulated TNF-α-induced activation of the 2 pathways in both cell models. By ELISA assay, MCLR was found to induce production of proinflammatory cytokine IL-6 in PMHs. At cytotoxic concentrations (≥ 50 nM MCLR in PMHs), MCLR dramatically reduced cell viability and damaged cell morphology in PMHs, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and transmission electron microscopy analysis. These results suggest that MCLR below 20 nM has significant immunomodulatory activities through activation of NF-κB and MAPK signaling pathways, and PMHs are more sensitive to MCLR-induced cytotoxicity than HepG2. To our knowledge, this is the first report showing the immunomodulatory role of MCLR in hepatocytes. Our results provide a better understanding of the molecular mechanisms underlying MCLR-induced hepatotoxicity.
微囊藻氨酸-亮氨酸-精氨酸（MCLR）是一种来自有害藻类的环境毒素，与肝病相关的肝毒性高风险有关。在这项研究中，我们探讨了 MCLR 在核因子-κB（NF-κB）和丝裂原活化蛋白激酶（MAPK）信号通路中的作用，该通路在调节炎症和免疫反应中起着重要作用，我们在人肝癌细胞系 HepG2 和原代小鼠肝细胞（PMHs）中进行了研究。通过体外无细胞和荧光素酶报告系统，用抗磷酸化 NF-κB（IκBα）/c-Jun N-末端激酶（JNK）/细胞外信号调节激酶 1/2（ERK1/2）抗体进行 Western blot，结果发现，在非细胞毒性浓度（≤20 nM MCLR 在 PMHs 中，1-1000 nM 在 HepG2 中）下，MCLR 处理本身就可促进 NF-κB 和 MAPK 通路的激活，并调节这两种细胞模型中 TNF-α诱导的这两条通路的激活。通过 ELISA 测定，发现 MCLR 可诱导 PMHs 中促炎细胞因子 IL-6 的产生。在细胞毒性浓度（≥50 nM MCLR 在 PMHs 中）下，MCLR 显著降低了 PMHs 的细胞活力并破坏了细胞形态，这通过 3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴盐测定法和透射电子显微镜分析得到证实。这些结果表明，在低于 20 nM 的浓度下，MCLR 通过激活 NF-κB 和 MAPK 信号通路具有显著的免疫调节活性，并且 PMHs 比 HepG2 对 MCLR 诱导的细胞毒性更敏感。据我们所知，这是首次报道 MCLR 在肝细胞中的免疫调节作用。我们的结果为理解 MCLR 诱导的肝毒性的分子机制提供了更好的认识。
