Medical Research Center, Southwest Hospital, the Third Military Medical University, Chongqing, 400038, China.
Department of Pharmacology, College of Pharmacy, the Third Military Medical University, Chongqing 400038, China.
Sci Rep. 2016 Dec 22;6:39735. doi: 10.1038/srep39735.
Excessive activation of the TLR4 signalling pathway is critical for inflammation-associated disorders, while negative regulators play key roles in restraining TLR4 from over-activation. Naringenin is a citrus flavonoid with remarkable anti-inflammatory activity, but the mechanisms underlying its inhibition of LPS/TLR4 signalling are less clear. This study investigated the molecular targets and therapeutic effects of naringenin in vitro and in vivo. In LPS-stimulated murine macrophages, naringenin suppressed the expression of TNF-α, IL-6, TLR4, inducible NO synthase (iNOS), cyclo-oxygenase-2 (COX2) and NADPH oxidase-2 (NOX2). Naringenin also inhibited NF-κB and mitogen-activated protein kinase (MAPK) activation. However, it did not affect the IRF3 signalling pathway or interferon production, which upregulate activating transcription factor 3 (ATF3), an inducible negative regulator of TLR4 signalling. Naringenin was demonstrated to directly increase ATF3 expression. Inhibition of AMPK and its upstream calcium-dependent signalling reduced ATF3 expression and dampened the anti-inflammatory activity of naringenin. In murine endotoxaemia models, naringenin ameliorated pro-inflammatory reactions and improved survival. Furthermore, it induced AMPK activation in lung tissues, which was required for ATF3 upregulation and the enhanced anti-inflammatory activity. Overall, this study reveals a novel mechanism of naringenin through AMPK-ATF3-dependent negative regulation of the LPS/TLR4 signalling pathway, which thereby confers protection against murine endotoxaemia.
TLR4 信号通路的过度激活对炎症相关疾病至关重要，而负调控因子则在抑制 TLR4 的过度激活方面发挥关键作用。柚皮素是一种具有显著抗炎活性的柑橘类黄酮，但它抑制 LPS/TLR4 信号的机制尚不清楚。本研究在体外和体内研究了柚皮素的分子靶点和治疗效果。在 LPS 刺激的小鼠巨噬细胞中，柚皮素抑制了 TNF-α、IL-6、TLR4、诱导型一氧化氮合酶（iNOS）、环氧化酶-2（COX2）和 NADPH 氧化酶-2（NOX2）的表达。柚皮素还抑制了 NF-κB 和丝裂原活化蛋白激酶（MAPK）的激活。然而，它不影响 IRF3 信号通路或干扰素的产生，干扰素上调激活转录因子 3（ATF3），ATF3 是 TLR4 信号的一种诱导性负调控因子。柚皮素被证明可以直接增加 ATF3 的表达。抑制 AMPK 及其上游钙依赖性信号通路会降低 ATF3 的表达，并减弱柚皮素的抗炎活性。在小鼠内毒素血症模型中，柚皮素改善了促炎反应并提高了存活率。此外，它在肺组织中诱导 AMPK 激活，这是 ATF3 上调和增强抗炎活性所必需的。总的来说，这项研究揭示了柚皮素通过 AMPK-ATF3 依赖性负调控 LPS/TLR4 信号通路的新机制，从而为小鼠内毒素血症提供了保护。
