Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
School of Life and Pharmaceutical Sciences, Dalian University of Technology, Panjin, 124221, China.
Cell Death Dis. 2021 Feb 11;12(2):174. doi: 10.1038/s41419-020-03243-w.
Fulminant hepatitis (FH) is an incurable clinical syndrome where novel therapeutics are warranted. Withaferin A (WA), isolated from herb Withania Somnifera, is a hepatoprotective agent. Whether and how WA improves D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced FH is unknown. This study was to evaluate the hepatoprotective role and mechanism of WA in GalN/LPS-induced FH. To determine the preventive and therapeutic effects of WA, wild-type mice were dosed with WA 0.5 h before or 2 h after GalN treatment, followed by LPS 30 min later, and then killed 6 h after LPS treatment. To explore the mechanism of the protective effect, the macrophage scavenger clodronate, autophagy inhibitor 3-methyladenine, or gene knockout mouse lines NLR family pyrin domain containing 3 (Nlrp3)-null, nuclear factor-erythroid 2-related factor 2 (Nrf2)-null, liver-specific AMP-activated protein kinase (Ampk)a1 knockout (Ampka1) and liver-specific inhibitor of KB kinase β (Ikkb) knockout (Ikkb) mice were subjected to GalN/LPS-induced FH. In wild-type mice, WA potently prevented GalN/LPS-induced FH and inhibited hepatic NLRP3 inflammasome activation, and upregulated NRF2 and autophagy signaling. Studies with Nrf2-null, Ampka1, and Ikkb mice demonstrated that the hepatoprotective effect was independent of NRF2, hepatic AMPKα1, and IκκB. Similarly, 3-methyladenine cotreatment failed to abolish the hepatoprotective effect of WA. The hepatoprotective effect of WA against GalN/LPS-induced FH was abolished after macrophage depletion, and partially reduced in Nlrp3-null mice. Consistently, WA alleviated LPS-induced inflammation partially dependent on the presence of NLRP3 in primary macrophage in vitro. Notably, WA potently and therapeutically attenuated GalN/LPS-induced hepatotoxicity. In conclusion, WA improves GalN/LPS-induced hepatotoxicity by targeting macrophage partially dependent on NLRP3 antagonism, while largely independent of NRF2 signaling, autophagy induction, and hepatic AMPKα1 and IκκB. These results support the concept of treating FH by pharmacologically targeting macrophage and suggest that WA has the potential to be repurposed for clinically treating FH as an immunoregulator.
暴发性肝炎（FH）是一种无法治愈的临床综合征，需要新的治疗方法。从草药印度萝芙木中分离出的 Withaferin A（WA）是一种肝保护剂。WA 是否以及如何改善 D-半乳糖胺（GalN）/脂多糖（LPS）诱导的 FH 尚不清楚。本研究旨在评估 WA 在 GalN/LPS 诱导的 FH 中的肝保护作用和机制。为了确定 WA 的预防和治疗作用，野生型小鼠在 GalN 处理前 0.5 小时或处理后 2 小时给予 WA，然后 30 分钟后给予 LPS，然后在 LPS 处理后 6 小时处死。为了探讨保护作用的机制，用巨噬细胞清除剂 clodronate、自噬抑制剂 3-甲基腺嘌呤、NLR 家族包含吡啶结构域的 3（Nlrp3）-null、核因子-红细胞 2 相关因子 2（Nrf2）-null、肝特异性 AMP 激活蛋白激酶（Ampk）a1 敲除（Ampka1）和肝特异性 KB 激酶β（Ikkb）敲除（Ikkb）小鼠进行 GalN/LPS 诱导的 FH。在野生型小鼠中，WA 强烈预防 GalN/LPS 诱导的 FH，并抑制肝 NLRP3 炎性体激活，并上调 NRF2 和自噬信号。用 Nrf2-null、Ampka1 和 Ikkb 小鼠进行的研究表明，这种肝保护作用与 NRF2、肝 AMPKα1 和 IκκB 无关。同样，3-甲基腺嘌呤共处理不能消除 WA 的肝保护作用。巨噬细胞耗竭后，WA 对 GalN/LPS 诱导的 FH 的保护作用被消除，而在 Nlrp3-null 小鼠中则部分降低。同样，WA 减轻 LPS 诱导的炎症反应部分依赖于体外原代巨噬细胞中 NLRP3 的存在。值得注意的是，WA 强力且治疗性地减轻 GalN/LPS 诱导的肝毒性。总之，WA 通过靶向巨噬细胞部分依赖于 NLRP3 拮抗作用来改善 GalN/LPS 诱导的肝毒性，而与 NRF2 信号、自噬诱导以及肝 AMPKα1 和 IκκB 无关。这些结果支持通过药理学靶向巨噬细胞治疗 FH 的概念，并表明 WA 具有作为免疫调节剂重新用于临床治疗 FH 的潜力。
