Xiao Hua, Chen Wei, Lu Darong, Shi Guixin, Xia Xiangping, Yao Shengtao
Medical College of Soochow University, No. 1, Shizi Street, Gusu District, Suzhou 215000, China.
Department of Neurosurgery, Affiliated Hospital of Zunyi Medical University, 149 Dalian Road, Huichuan District, Zunyi 563000, China.
Toxicol Res (Camb). 2024 Jul 25;13(4):tfae112. doi: 10.1093/toxres/tfae112. eCollection 2024 Aug.
Pyroptosis, inflammation-related programed cell death mediated by NLRP3 inflammasome, is involved in the pathogenesis of cerebral hypoxic-ischemic injury. Our study aims to explore the biological role of growth differentiation factor (GDF)15 in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal pyroptosis.
HT22 neurons were subjected to OGD/R to simulate cerebral hypoxic-ischemic injury. Cells were transfected with plasmids to overexpress GDF15, or lentiviral-based shRNAs constructs to silence GDF15. ELISA assay was used to detect GDF15, IL-1β, IL-18, and neuron specific enolase (NSE) levels. Cell pyroptosis was measured by flow cytometery. Chromatin immunoprecipitation assay was used to detect interaction of H3K27ac with GDF15 promoter. GDF15, NLRP3, Caspase-1 p20 and GSDMD-N expressions were measured by Western blotting.
Patients with malignant middle cerebral artery infarction showed decreased GDF15, but increased IL-1β, IL-18, and NSE levels in serum compared to healthy controls. OGD/R treatment caused significant increases in the levels of IL-1β, IL-18 and NSE, percentages of pyroptotic cells, and expressions of NLRP3, Caspase-1 p20, and GSDMD in HT22 cells, which were markedly reversed by GDF15 overexpression. However, GDF15 knockdown resulted in neuronal injury similar to those observed in OGD/R treatment. The GDF15 knockdown-induced effects were counteracted by treatment with NLRP3 inhibitor. OGD/R decreased the enrichment of H3K27ac in the promoter of GDF15 to down-regulate GDF15, but was compromised by co-treatment with HDAC2 inhibitor.
Our data demonstrates that GDF15 attenuates OGD/R-induced pyroptosis through NLRP3 inflammasome. HDAC2 is involved in mediating OGD-induced GDF15 down-regulation via H3K27ac modification. GDF15 overexpression and HDAC2 inhibition hold potential as useful therapeutic strategies for neuroprotection.
焦亡是由NLRP3炎性小体介导的炎症相关程序性细胞死亡，参与脑缺氧缺血性损伤的发病机制。本研究旨在探讨生长分化因子（GDF）15在氧糖剥夺/复氧（OGD/R）诱导的神经元焦亡中的生物学作用。
对HT22神经元进行OGD/R处理以模拟脑缺氧缺血性损伤。用质粒转染细胞以过表达GDF15，或用基于慢病毒的短发夹RNA构建体转染以沉默GDF15。采用酶联免疫吸附测定（ELISA）检测GDF15、白细胞介素（IL）-1β、IL-18和神经元特异性烯醇化酶（NSE）水平。通过流式细胞术检测细胞焦亡。采用染色质免疫沉淀测定法检测组蛋白H3赖氨酸27乙酰化（H3K27ac）与GDF15启动子的相互作用。通过蛋白质免疫印迹法检测GDF15、NLRP3、半胱天冬酶-1 p20和Gasdermin D-N（GSDMD-N）的表达。
与健康对照相比，恶性大脑中动脉梗死患者血清中GDF15水平降低，但IL-1β、IL-18和NSE水平升高。OGD/R处理导致HT22细胞中IL-1β、IL-18和NSE水平显著升高，焦亡细胞百分比以及NLRP3、半胱天冬酶-1 p20和GSDMD的表达显著增加，而GDF15过表达可显著逆转这些变化。然而，GDF15基因敲低导致的神经元损伤与OGD/R处理所观察到的损伤相似。用NLRP3抑制剂处理可抵消GDF15基因敲低诱导效应。OGD/R降低了GDF15启动子中H3K27ac的富集，从而下调GDF15，但HDAC2抑制剂共同处理可减弱这种下调作用。
我们的数据表明，GDF15通过NLRP3炎性小体减轻OGD/R诱导的焦亡。HDAC2通过H3K27ac修饰参与介导OGD诱导的GDF15下调。过表达GDF15和抑制HDAC2有望成为神经保护的有效治疗策略。
