Yang Kang, Han Qing-Tong, Xing Rong-Xue, Li Zhi-Ying, Xu Lin-Tao, Chen Lu-Zhou, Xiang Lan, Ren Dong-Mei, Hu Qing-Wen, Wang Xiao-Ning, Shen Tao
State Key Laboratory of Discovery and Utilization of Functional Components in Traditional Chinese Medicine, Shandong University, Jinan, 250012, China; Key Lab of Chemical Biology (MOE), School of Pharmaceutical Sciences, Shandong University, Jinan, 250012, China; Shandong Engineering Research Center for Traditional Chinese Medicine Standard, School of Pharmaceutical Sciences, Shandong University, Jinan, 250012, China; Shandong Key Laboratory of Bioactive Components and Translational Research of Traditional Chinese Medicine, Jinan, 250012, China.
Shandong Key Laboratory of Bioactive Components and Translational Research of Traditional Chinese Medicine, Jinan, 250012, China.
Redox Biol. 2025 May;82:103621. doi: 10.1016/j.redox.2025.103621. Epub 2025 Mar 27.
Kelch ECH-associating protein 1 (Keap1)-Nuclear factor erythroid 2-related factor 2 (Nrf2) axis is crucial for regulating oxidative stress and inflammatory responses in acute pneumonia. Sphaeropsidin A (SA) is a antioxidant diterpenoid isolated from Sphaeropsis sapinea f. sp. cupressi, discovered as a novel Nrf2 agonist by our research group previously. However, the accurate function and mechanism of SA in treating acute pneumonia are still unknown.
The therapeutic effect of SA was evaluated in LPS-induced acute pneumonia in mice. The underlying mechanism of action was then analyzed by transcriptomics. The direct target of SA was identified through the synthesis of SA-biotin probe, and the binding amino acid residues were found and verified by LC-MS/MS analysis and site-specific mutation. Finally, knockout mice were employed to verify the mechanism of SA.
Our data indicated that SA significantly inhibited LPS-induced acute pneumonia in mice via up-regulating Nrf2, inhibiting NLRP3 inflammasome and NF-κB activation, and identified Keap1 as the direct target of SA. Specifically, the effective dose of SA in mice was only 2 mg/kg. SA selectively covalent bound to Keap1 in cysteine 151 residue (Cys151). SA mediated the activation of Nrf2 and reduced the level of ROS, thereby inhibiting the NF-κB and NLRP3 inflammasome. Besides, SA formed hydrogen bond with ASP48 of ASC, blocking its oligomerization and inhibiting the activation of NLRP3 inflammasome.
This study indicates that SA might be a new covalent molecule of Keap1 to activate Nrf2, and is a promising drug candidate or lead molecule for the therapy of acute pneumonia through regulating Nrf2/NF-κB/NLRP3 inflammasome axis.
Kelch ECH 相关蛋白 1（Keap1）-核因子红细胞 2 相关因子 2（Nrf2）轴对于调节急性肺炎中的氧化应激和炎症反应至关重要。球壳菌素 A（SA）是一种从松材球壳孢中分离出的抗氧化二萜类化合物，此前被本研究小组发现是一种新型的 Nrf2 激动剂。然而，SA 在治疗急性肺炎中的准确功能和机制仍不清楚。
在 LPS 诱导的小鼠急性肺炎模型中评估 SA 的治疗效果。然后通过转录组学分析其潜在作用机制。通过合成 SA-生物素探针鉴定 SA 的直接靶点，并通过液相色谱-串联质谱分析和位点特异性突变找到并验证结合氨基酸残基。最后，利用基因敲除小鼠验证 SA 的作用机制。
我们的数据表明，SA 通过上调 Nrf2、抑制 NLRP3 炎性小体和 NF-κB 激活，显著抑制 LPS 诱导的小鼠急性肺炎，并确定 Keap1 为 SA 的直接靶点。具体而言，SA 在小鼠中的有效剂量仅为 2mg/kg。SA 选择性地与 Keap1 的半胱氨酸 151 残基（Cys151）共价结合。SA 介导 Nrf2 的激活并降低 ROS 水平，从而抑制 NF-κB 和 NLRP3 炎性小体。此外，SA 与 ASC 的 ASP48 形成氢键，阻止其寡聚化并抑制 NLRP3 炎性小体的激活。
本研究表明，SA 可能是一种激活 Nrf2 的新型 Keap1 共价分子，是通过调节 Nrf2/NF-κB/NLRP3 炎性小体轴治疗急性肺炎的有前景的候选药物或先导分子。
