Andrographolide ameliorates depressive-like behavior by targeting the CyPA–IκBα/NF-κB axis: an APOE-isoform–dependent mechanism in chronic social defeat stress

Hypothesis: We hypothesized that chronic social defeat stress (CSDS) activates the CyPA–IκBα/NF-κB–MMP9 pro-inflammatory axis in the medial prefrontal cortex (mPFC) in an APOE‑isoform‑dependent manner, and that the natural compound andrographolide ameliorates depressive‑like behavior by suppressing this axis and restoring neurotrophic support. Data and methods: CSDS was induced in male C57BL/6J mice. Depressive‑like behavior was quantified by forced swim test (FST) and tail suspension test (TST). mPFC tissue was analyzed by Western blot, MALDI‑TOF mass spectrometry, co‑immunoprecipitation (Co‑IP), and transmission electron microscopy (TEM). In vivo electrophysiology recorded pyramidal neuron firing. Network pharmacology, two‑sample Mendelian randomization (MR), molecular docking, molecular dynamics (MD) simulations (100 ns), and in silico APOE4 knockout (scMAGeCK) were used to predict and validate molecular interactions and pathway enrichment. Key findings: CSDS mice showed increased immobility in FST/TST, mPFC mitochondrial damage, reduced pyramidal neuron firing, elevated CyPA, IL‑6, TNF‑α, MMP9, PD‑1/PD‑L1, and decreased IκBα and BDNF. Co‑IP and MD simulations confirmed CyPA–IκBα interaction. MR indicated causal links of TNF‑α and IL‑6 with MDD risk. Andrographolide (5 mg/kg/day, i.p.) reversed all behavioral, ultrastructural, molecular, and electrophysiological abnormalities. Docking showed andrographolide binds CyPA (−8.2 kcal/mol) and IκBα (−5.8 kcal/mol); MD suggested it may stabilize IκBα via a methylation‑associated conformation. Bortezomib (proteasome inhibitor) produced similar effects; combined andrographolide+bortezomib yielded enhanced improvements. CSDS increased APOE4 and decreased APOE3 protein levels; andrographolide normalized this balance. In silico APOE4 knockout identified NF‑κB as the top pathway, and MD indicated APOE4 binds CyPA less efficiently than APOE3, potentially impairing CyPA clearance. Interpretation: The CyPA–IκBα/NF‑κB–MMP9 axis is a critical driver of CSDS‑induced neuroinflammation and depressive‑like behavior. Andrographolide acts on this axis at multiple nodes—CyPA and IκBα—and modulates APOE isoform expression. The convergence of effects with bortezomib and the enhanced combination outcome support a causal, targetable role for this axis. These data nominate andrographolide as a candidate for neuroinflammation‑associated MDD and provide a mechanistic framework linking APOE4 to exacerbated inflammatory signaling in depression.假说： 我们提出如下假说：慢性社交挫败应激（chronic social defeat stress, CSDS）可通过载脂蛋白E（APOE）亚型依赖的方式，激活内侧前额叶皮层（medial prefrontal cortex, mPFC）内的促炎轴CyPA–IκBα/NF-κB–MMP9；而天然化合物穿心莲内酯（andrographolide）可通过抑制该轴并恢复神经营养支持，改善抑郁样行为。 数据与方法： 本研究对雄性C57BL/6J小鼠构建慢性社交挫败应激模型。采用强迫游泳实验（forced swim test, FST）与悬尾实验（tail suspension test, TST）量化抑郁样行为。通过蛋白质免疫印迹、基质辅助激光解吸电离飞行时间质谱（MALDI-TOF mass spectrometry）、免疫共沉淀（co-immunoprecipitation, Co-IP）及透射电子显微镜（transmission electron microscopy, TEM）对内侧前额叶皮层组织进行分析。通过在体电生理学记录锥体神经元放电活动。采用网络药理学、双样本孟德尔随机化（two-sample Mendelian randomization, MR）、分子对接、分子动力学（molecular dynamics, MD）模拟（100纳秒）及计算机模拟APOE4基因敲除（scMAGeCK）技术，预测并验证分子相互作用与通路富集情况。 主要发现： 慢性社交挫败应激模型小鼠在强迫游泳实验与悬尾实验中不动时间显著延长，内侧前额叶皮层出现线粒体损伤，锥体神经元放电频率降低，CyPA、IL-6、TNF-α、MMP9、PD-1/PD-L1表达水平升高，而IκBα与BDNF表达水平下降。 免疫共沉淀与分子动力学模拟证实了CyPA与IκBα之间存在相互作用。孟德尔随机化分析显示，TNF-α与IL-6水平与重度抑郁症（Major Depressive Disorder, MDD）风险存在因果关联。 穿心莲内酯（5 mg/kg/天，腹腔注射）可逆转所有行为学、超微结构、分子层面及电生理学异常。分子对接结果显示，穿心莲内酯可与CyPA（结合能-8.2 kcal/mol）及IκBα（结合能-5.8 kcal/mol）结合；分子动力学模拟提示，其可通过与甲基化相关的构象稳定IκBα。 硼替佐米（proteasome inhibitor）可产生相似效应；穿心莲内酯与硼替佐米联合使用可进一步增强改善效果。 慢性社交挫败应激可升高APOE4蛋白水平并降低APOE3蛋白水平，而穿心莲内酯可使该平衡恢复至正常水平。计算机模拟APOE4基因敲除分析显示，NF-κB为富集程度最高的通路；分子动力学模拟表明，APOE4与CyPA的结合效率低于APOE3，可能会损害CyPA的清除过程。 解读： CyPA–IκBα/NF-κB–MMP9轴是慢性社交挫败应激诱导的神经炎症与抑郁样行为的关键驱动因素。穿心莲内酯可通过作用于该轴的多个靶点（CyPA与IκBα），并调节APOE亚型的表达。硼替佐米与穿心莲内酯的效应趋同性，以及联合使用的增强效应，均证实了该轴具有可靶向的因果性作用。本研究数据将穿心莲内酯列为神经炎症相关重度抑郁症（Major Depressive Disorder, MDD）的潜在治疗候选药物，并构建了将APOE4与抑郁症中加剧的炎症信号通路相关联的机制框架。