Department of Intensive Care Medicine, The Third Xiangya Hospital of Central South University, Changsha, 410013, Hunan Province, PR China.
Department of Emergency Medicine, The Third Xiangya Hospital of Central South University, Changsha, 410013, Hunan Province, PR China.
Arch Biochem Biophys. 2020 Nov 30;695:108611. doi: 10.1016/j.abb.2020.108611. Epub 2020 Sep 29.
Sepsis-induced cardiac dysfunction is one of the leading complications of sepsis, contributing to the high morbidity and mortality of septic patients. Several lines of evidence have demonstrated that autophagy and pyroptosis may be involved in septic cardiac dysfunction. In this study, we examined the impact of zinc finger antisense 1 (ZFAS1) on sepsis-induced myocardial dysfunction via regulating pyroptosis and autophagy.
Mice with cecal ligation and puncture (CLP)-induced sepsis was constructed in vivo. Myocardial injury was assessed by H&E staining, immunohistochemistry (IHC) for NLRP3, caspase 1, and interleukin (IL)-1β, as well as ELISA assay for serum levels of creatine kinase (CK), CK-MB, tumor necrosis factor α (TNF-α), and IL-1β. Primary cardiomyocytes exposed to lipopolysaccharide (LPS) were established to simulate sepsis-induced cardiac dysfunction in vitro. Cell viability was examined by MTT assay and concentration of TNF-α and IL-1β was measured by ELISA. Flow cytometry, immunofluorescent staining and western blotting were performed to assess pyroptosis and autophagy. The transcriptional regulation of SP1 on ZFAS1 was determined using ChIP assay. Luciferase reporter assay was performed to verify the ZFAS1/miR-590-3p interaction. Besides, activation of AMPK/mTOR signaling was detected using western blotting.
Highly expressed ZFAS1 was observed in sepsis-induced cardiac dysfunction in the in vivo and in vitro model. Knockdown of ZFAS1 robustly abolished LPS-induced pyroptosis and attenuated the inhibition of autophagy. SP1 was identified to be an essential transcription factor to positively regulate ZFAS1 expression. Moreover, miR-590-3p functioned as a downstream effector to reverse ZFAS1-mediated sepsis-induced cardiac dysfunction. AMPK/mTOR signaling was involved in miR-590-3p-regulated autophagy and pyroptosis of cardiomyocytes. Furthermore, the regulatory network of ZFAS1/miR-590-3p on AMPK/mTOR signaling was verified in vivo.
ZFAS1, activated by SP1, aggravates the progression of sepsis-induced cardiac dysfunction via targeting miR-590-3p/AMPK/mTOR signaling-mediated autophagy and pyroptosis of cardiomyocytes.
脓毒症诱导的心脏功能障碍是脓毒症的主要并发症之一，导致脓毒症患者的发病率和死亡率居高不下。有几条证据表明自噬和焦亡可能与脓毒症性心脏功能障碍有关。在这项研究中，我们通过调节焦亡和自噬来研究锌指反义 1 (ZFAS1) 对脓毒症诱导的心肌功能障碍的影响。
在体内构建盲肠结扎和穿刺 (CLP) 诱导的脓毒症小鼠模型。通过苏木精和伊红 (H&E) 染色、NLRP3、半胱天冬酶 1 和白细胞介素 (IL)-1β 的免疫组织化学 (IHC) 以及血清肌酸激酶 (CK)、CK-MB、肿瘤坏死因子 α (TNF-α) 和 IL-1β 的 ELISA 测定评估心肌损伤。体外建立脂多糖 (LPS) 暴露的原代心肌细胞，模拟脓毒症诱导的心脏功能障碍。通过 MTT 测定评估细胞活力，通过 ELISA 测定 TNF-α 和 IL-1β 的浓度。通过流式细胞术、免疫荧光染色和 Western blot 评估焦亡和自噬。通过 ChIP 测定确定 SP1 对 ZFAS1 的转录调控。通过荧光素酶报告基因测定验证 ZFAS1/miR-590-3p 相互作用。此外，通过 Western blot 检测 AMPK/mTOR 信号的激活。
在体内和体外模型中，脓毒症诱导的心脏功能障碍中观察到高表达的 ZFAS1。ZFAS1 的敲低可显著消除 LPS 诱导的焦亡并减弱自噬的抑制。确定 SP1 是正向调节 ZFAS1 表达的必需转录因子。此外，miR-590-3p 作为下游效应物发挥作用，逆转 ZFAS1 介导的脓毒症诱导的心脏功能障碍。AMPK/mTOR 信号参与了 miR-590-3p 调节的心肌细胞自噬和焦亡。此外，在体内验证了 ZFAS1/miR-590-3p 对 AMPK/mTOR 信号的调控网络。
SP1 激活的 ZFAS1 通过靶向 miR-590-3p/AMPK/mTOR 信号介导的心肌细胞自噬和焦亡，加重脓毒症诱导的心脏功能障碍的进展。
