Chen Kechun, Xu Bingtian, Long Lu, Wen Huizhen, Zhao Qian, Tu Xingxing, Wang Jiakang, Xu Jiangping, Wang Haitao
NMPA Key Laboratory for Research and Evaluation of Drug Metabolism & Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515, China.
Key Laboratory of Mental Health of the Ministry of Education, Southern Medical University, Guangzhou, 510515, China.
Mol Neurobiol. 2025 Mar;62(3):3376-3395. doi: 10.1007/s12035-024-04495-9. Epub 2024 Sep 17.
We have previously shown that inhibition of phosphodiesterase 4 (PDE4) protects against cerebral ischemia/reperfusion injury. However, it remains unclear whether and how PDE4 affects ferroptosis under cerebral ischemia/reperfusion conditions. In this study, we found that overexpression of PDE4B in HT-22 cells exacerbated the detrimental effects of oxygen-glucose deprivation/reoxygenation (OGD/R), including a decrease in cell viability and glutathione (GSH) levels and an increase in Fe content. PDE4B knockdown mitigated the effects of OGD/R, as evidenced by decreased oxidative stress, lactate dehydrogenase (LDH) release, Fe content, and nuclear receptor coactivator 4 (NCOA4) expression. PDE4B knockdown also enhanced the levels of GSH, ferroportin (FPN), and ferritin heavy chain 1 (FTH1). Consistently, inhibition of PDE4 by roflumilast (Roflu) produced similar effects as PDE4B knockdown. Roflu also ameliorated the morphology and membrane potential of the mitochondria. Glutathione peroxidase 4 (GPX4) knockdown blocked the effects of Roflu on cell viability and lipid peroxidation. Moreover, we found that nuclear factor erythroid 2-related factor 2 (Nrf-2) knockdown decreased GPX4 expression. In addition, Nrf-2 knockdown led to enhanced lipid peroxidation, LDH release, and iron levels, while the GSH and FPN levels decreased. More crucially, PDE4 inhibition decreased infarct volume, alleviated oxidative stress, and restored the expression levels of ferroptosis-associated proteins in middle cerebral artery occlusion/reperfusion (MCAO/R) rats. Interestingly, the GPX4 inhibitor RSL3 blocked the neuroprotective effects of Roflu in rats subjected to MCAO/R. Thus, PDE4 inhibition significantly inhibits neuronal ferroptosis by activating the Nrf-2/GPX4 pathway. These data indicate the existence of a novel mechanism underlying the neuroprotective effects of PDE4 inhibition.
我们之前已经表明，抑制磷酸二酯酶4（PDE4）可预防脑缺血/再灌注损伤。然而，PDE4在脑缺血/再灌注条件下是否以及如何影响铁死亡仍不清楚。在本研究中，我们发现HT-22细胞中PDE4B的过表达加剧了氧糖剥夺/复氧（OGD/R）的有害影响，包括细胞活力和谷胱甘肽（GSH）水平降低以及铁含量增加。PDE4B敲低减轻了OGD/R的影响，氧化应激、乳酸脱氢酶（LDH）释放、铁含量和核受体辅激活因子4（NCOA4）表达降低证明了这一点。PDE4B敲低还提高了GSH、铁转运蛋白（FPN）和铁蛋白重链1（FTH1）的水平。同样，罗氟司特（Roflu）抑制PDE4产生了与PDE4B敲低类似的效果。Roflu还改善了线粒体的形态和膜电位。谷胱甘肽过氧化物酶4（GPX4）敲低阻断了Roflu对细胞活力和脂质过氧化的影响。此外，我们发现核因子红细胞2相关因子2（Nrf-2）敲低降低了GPX4表达。此外，Nrf-2敲低导致脂质过氧化、LDH释放和铁水平升高，而GSH和FPN水平降低。更关键的是，抑制PDE4可减少大脑中动脉闭塞/再灌注（MCAO/R）大鼠的梗死体积，减轻氧化应激，并恢复铁死亡相关蛋白的表达水平。有趣的是，GPX4抑制剂RSL3阻断了Roflu对MCAO/R大鼠的神经保护作用。因此，抑制PDE4可通过激活Nrf-2/GPX4途径显著抑制神经元铁死亡。这些数据表明存在一种新的机制，是PDE4抑制发挥神经保护作用的基础。
