Lv Ling-Xia, Wen Mei, Lv Fei, Ji Tai-Bing, Fu Hua-Li, Man Ning
Respiratory and Critical Care Medicine, Wuhan Asia General Hospital, Wuhan, Hubei, China.
Kaohsiung J Med Sci. 2022 Feb;38(2):87-96. doi: 10.1002/kjm2.12452. Epub 2021 Sep 16.
Pyroptosis is a novel proinflammatory programmed cell death process. This study was designed to investigate the functional mechanisms of long noncoding RNA growth arrest-specific transcript 5 (lncRNA GAS5) on lipopolysaccharide (LPS)-induced human bronchial epithelial cell (HBEC) pyroptosis. LPS was used to induce pyroptosis in HBECs, followed by the detection of the expression of GAS5, forkhead box O3 (FOXO3), and nuclear factor E2-related factor 2/heme oxygenase 1 (Nrf2/HO-1) signaling pathway-related factors. Cell viability was evaluated using CCK-8 assay, lactate dehydrogenase (LDH) release was assessed by LDH assay kit and caspase-1 activity by flow cytometry. Furthermore, expression of NOD-like receptor family pyrin domain containing 3 and pyroptosis-related proteins was evaluated using Western blot analysis, while enzyme-linked immunosorbent assay was used to determine the levels of inflammatory factors. The interaction between GAS5 and FOXO3 was confirmed using bioinformatic prediction, RNA immunoprecipitation assay, RNA pull-down, and dual-luciferase reporter gene assay. Treatment of HBECs with LPS upregulated the expression of GAS5 and FOXO3, resulting in the inactivation of the Nrf2/HO-1 signaling pathway. On the other hand, inhibition of both GAS5 and FOXO3 promoted cell viability, reduced LDH release, pyroptosis, and inflammatory response in LPS-induced HBECs. Furthermore, FOXO3 could interact with GAS5, while FOXO3 overexpression reversed the inhibitory effect of GAS5 knockdown on cell pyroptosis. Thus, mechanistically, inhibition of FOXO3 activates the Nrf2/HO-1 pathway to suppress LPS-induced pyroptosis in HBECs. This study revealed that GAS5 knockdown attenuates FOXO3 expression thereby activating the Nrf2/HO-1 pathway to inhibit LPS-induced pyroptosis in HBECs. These findings may contribute to identifying novel targets that inhibit pyroptosis in HBECs.
细胞焦亡是一种新型的促炎性程序性细胞死亡过程。本研究旨在探讨长链非编码RNA生长停滞特异性转录本5（lncRNA GAS5）对脂多糖（LPS）诱导的人支气管上皮细胞（HBEC）焦亡的作用机制。采用LPS诱导HBECs发生焦亡，随后检测GAS5、叉头框O3（FOXO3）以及核因子E2相关因子2/血红素加氧酶1（Nrf2/HO-1）信号通路相关因子的表达。使用CCK-8法评估细胞活力，通过乳酸脱氢酶（LDH）检测试剂盒评估LDH释放，并通过流式细胞术检测半胱天冬酶-1活性。此外，采用蛋白质免疫印迹分析评估含NOD样受体家族pyrin结构域蛋白3及焦亡相关蛋白的表达，同时使用酶联免疫吸附测定法测定炎症因子水平。利用生物信息学预测、RNA免疫沉淀测定、RNA下拉及双荧光素酶报告基因测定证实GAS5与FOXO3之间的相互作用。用LPS处理HBECs可上调GAS5和FOXO3的表达，导致Nrf2/HO-1信号通路失活。另一方面，抑制GAS5和FOXO3均可促进LPS诱导的HBECs的细胞活力，减少LDH释放、细胞焦亡及炎症反应。此外，FOXO3可与GAS5相互作用，而FOXO3过表达可逆转GAS5敲低对细胞焦亡的抑制作用。因此，从机制上讲，抑制FOXO3可激活Nrf2/HO-1通路，从而抑制LPS诱导的HBECs焦亡。本研究表明，GAS5敲低可减弱FOXO3表达，从而激活Nrf2/HO-1通路，抑制LPS诱导的HBECs焦亡。这些发现可能有助于确定抑制HBECs焦亡的新靶点。
