Biosafety Research Institute and Laboratory of Pathology (BK21 Plus Program), College of Veterinary Medicine, Jeonbuk National University, Iksan, 54596, Republic of Korea.
Biosafety Research Institute and Laboratory of Pathology (BK21 Plus Program), College of Veterinary Medicine, Jeonbuk National University, Iksan, 54596, Republic of Korea.
Toxicology. 2020 Nov;444:152579. doi: 10.1016/j.tox.2020.152579. Epub 2020 Sep 6.
The common causes of Non-alcoholic fatty liver disease (NAFLD) are obesity, dyslipidemia, and insulin resistance. Metabolic disorders and lipotoxic hepatocyte damage are hallmarks of NAFLD. Even though amlexanox, a dual inhibitor of TRAF associated nuclear factor κB (NF-κB) activator-binding kinase 1 (TBK1) and IκB kinase epsilon (IKKε), has been reported to effectively improve obesity-related metabolic dysfunctions in mice models, its molecular mechanism has not been fully investigated. This study was designed to investigate the effects of amlexanox on in vitro nonalcoholic steatohepatitis (NASH) model induced by treatment of palmitic acid (PA, 0.4 mM), using a trans-well co-culture system of hepatocytes and Kupffer cells (KCs). Stimulation with PA significantly increased the phosphorylation levels of TBK1 and IKKε in both hepatocytes and KCs, suggesting a potential role of TBK1/IKKε in PA-induced NASH progression. Treatment of amlexanox (50 μM) showed significantly reduced phosphorylation of TBK1 and IKKε and hepatotoxicity as confirmed by decreased levels of lactate dehydrogenase released from hepatocytes. Furthermore, PA-induced inflammation and lipotoxic cell death in hepatocytes were significantly reversed by amlexanox treatment. Intriguingly, amlexanox inhibited the activation of KCs and induced polarization of KCs towards M2 phenotype. Mechanistically, amlexanox treatment decreased the phosphorylation of interferon regulator factor 3 (IRF3) and NF-κB in PA-treated hepatocytes. However, decreased phosphorylation of NF-κB, not IRF3, was found in PA-treated KCs upon amlexanox treatment. Taken together, our findings show that treatment of amlexanox attenuated the severity of PA-induced hepatotoxicity in vitro and lipoapoptosis by the inhibition of TBK1/IKKε-NF-κB and/or IRF3 pathway in hepatocytes and KCs.
非酒精性脂肪性肝病（NAFLD）的常见病因包括肥胖、血脂异常和胰岛素抵抗。代谢紊乱和脂毒性肝细胞损伤是非酒精性脂肪性肝病的特征。尽管 TRAF 相关核因子κB（NF-κB）激活激酶 1（TBK1）和 IκB 激酶ε（IKKε）双重抑制剂氨来占诺（amlexanox）已被报道可有效改善肥胖相关代谢功能障碍的小鼠模型，但它的分子机制尚未被充分研究。本研究旨在采用肝细胞和枯否细胞（KCs）的共培养转染系统，研究氨来占诺（amlexanox）对棕榈酸（PA，0.4 mM）诱导的体外非酒精性脂肪性肝炎（NASH）模型的影响。PA 刺激显著增加了肝细胞和 KCs 中 TBK1 和 IKKε 的磷酸化水平，提示 TBK1/IKKε 在 PA 诱导的 NASH 进展中可能起作用。用 50 μM 的氨来占诺（amlexanox）治疗，可显著降低 TBK1 和 IKKε 的磷酸化水平，并降低从肝细胞释放的乳酸脱氢酶水平，从而减轻肝毒性。此外，氨来占诺（amlexanox）治疗还显著逆转了 PA 诱导的肝细胞炎症和脂毒性细胞死亡。有趣的是，氨来占诺（amlexanox）抑制了 KCs 的激活，并诱导 KCs 向 M2 表型极化。在机制上，氨来占诺（amlexanox）处理降低了 PA 处理的肝细胞中干扰素调节因子 3（IRF3）和 NF-κB 的磷酸化。然而，在氨来占诺（amlexanox）处理的 PA 处理的 KCs 中，发现 NF-κB 的磷酸化减少，而不是 IRF3。总之，我们的研究结果表明，氨来占诺（amlexanox）治疗通过抑制 TBK1/IKKε-NF-κB 和/或 IRF3 通路减轻了 PA 诱导的体外肝细胞毒性和脂肪细胞凋亡的严重程度。
