GDF15 regulated by HDAC2 exerts suppressive effects on oxygen and glucose deprivation-induced pyroptosis via NLRP3/Caspase-1/ GSDMD pathway

Purpose: Pyroptosis, inflammation-related programed cell death mediated by NLRP3 inflammasome, plays a critical role 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 (OGD)-induced pyroptosis. Methods: HT22 neurons were subjected to OGD to simulate cerebral hypoxic-ischemic injury. HT22 neurons were transfected with plasmids to overexpress GDF15, or shRNAs constructs to silence GDF15. ELISA assay was used to detect GDF15, interleukin-1β (IL-1β), IL-18, and neuron specific enolase (NSE) in culture medium. Cells pyroptosis was measured by flow cytometery. Chromatin immuno-precipitation assay was used to detect interaction of H3K27ac with GDF15 promoter. Results: OGD exposure caused significant increases in medium levels of IL-1, IL-18 and NSE, percentages of pyroptotic cells, and NLRP3, caspase-1, and gasdermin D (GSDMD) protein, which were markedly reversed by GDF15 overexpression (p-value < 0.001). GDF15 knockdown resulted in significant increases in medium levels of IL-1, IL-18 and NSE, percentages of pyroptotic cells, and caspase-1 and GSDMD protein (p-value < 0.001). The GDF15 knockdown-induced effects were counteracted by treatment with NLRP3 inhibitor. OGD decreased H3K27ac enrichment with promoter of GDF15 to down-regulate GDF15, but was compromised by co-treatment with HDAC2 inhibitor. Conclusion: Our data demonstrates that GDF15 attenuates OGD/R-induced pyroptosis through NLRP3/Caspase-1/GSDMD pathway. HDAC2 is involves in mediating OGD-induced GDF15 down-regulation via H3K27ac enrichment. GDF15 overexpression and HDAC2 inhibition hold potential as useful therapeutic strategies for neuroprotection.

目的：焦亡（Pyroptosis）是由NLRP3炎性小体（NLRP3 inflammasome）介导的炎症相关程序性细胞死亡，在脑缺血缺氧损伤的发病机制中发挥关键作用。本研究旨在探讨生长分化因子15（growth differentiation factor 15，GDF15）在氧糖剥夺（oxygen-glucose deprivation，OGD）诱导的焦亡中的生物学功能。方法：采用氧糖剥夺（OGD）处理HT22神经元以模拟脑缺血缺氧损伤。将HT22神经元通过质粒转染以过表达GDF15，或通过短发夹RNA（shRNAs）构建体沉默GDF15。采用酶联免疫吸附试验（enzyme-linked immunosorbent assay，ELISA）检测细胞培养基中GDF15、白细胞介素-1β（interleukin-1β，IL-1β）、IL-18以及神经元特异性烯醇化酶（neuron specific enolase，NSE）的水平。采用流式细胞术检测细胞焦亡情况。采用染色质免疫沉淀（chromatin immuno-precipitation，ChIP）实验检测H3K27乙酰化修饰（H3K27ac）与GDF15启动子的相互作用。结果：OGD处理可显著升高细胞培养基中IL-1β、IL-18及NSE的水平、细胞焦亡率，以及NLRP3、半胱氨酸天冬氨酸蛋白酶-1（caspase-1）和焦孔素D（gasdermin D，GSDMD）的蛋白表达水平，而过表达GDF15可显著逆转上述变化（p值<0.001）。沉默GDF15可显著升高细胞培养基中IL-1β、IL-18及NSE的水平、细胞焦亡率，以及caspase-1和GSDMD的蛋白表达水平（p值<0.001）。GDF15沉默诱导的上述效应可被NLRP3抑制剂处理所抵消。OGD处理可降低GDF15启动子区域的H3K27ac富集水平，从而下调GDF15的表达，而联合组蛋白去乙酰化酶2（HDAC2）抑制剂处理可逆转这一现象。结论：本研究数据表明，GDF15可通过NLRP3/Caspase-1/GSDMD通路减轻氧糖剥夺复氧（OGD/R）诱导的细胞焦亡，发挥神经保护作用。HDAC2可通过调控H3K27ac富集介导OGD诱导的GDF15表达下调。过表达GDF15及抑制HDAC2有望成为潜在的神经保护治疗策略。