Liu Xiaobin, Wang Dong, Zhang Xiaoning, Lv Meng, Liu Ge, Gu Changping, Yang Fan, Wang Yuelan
Department of Anesthesiology and Perioperative Medicine, Shandong Qianfoshan Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China.
Ann Transl Med. 2021 Jan;9(2):159. doi: 10.21037/atm-20-7983.
Previous experiments revealed phospholipid scramblase 4 (PLSCR4) mRNA to be significantly increased in a lipopolysaccharide (LPS)-induced acute respiratory distress syndrome (ARDS) model of human pulmonary microvascular endothelial cells (HPMECs); however, the effect of PLSCR4 and its mechanism have not been reported to date. The PLSCR family is thought to mediate the transmembrane movement of phospholipids (PS), and has been found to be involved in pyroptosis through combing with gasdermin D (GSDMD). We therefore speculated that PLSCR4 may contribute to cell death via pyroptosis.
To investigate the effect and mechanism of PLSCR4 in ARDS, we constructed an in vitro model of LPS-induced ARDS in HPMECs transfected with PLSCR4 small interfering RNA (siRNA) or scramble siRNA (sc siRNA). After 4 h of LPS stimulation, western blotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), tracer flux assays, and fluorescence assays were used to study the relationship between PLSCR4 and pyroptosis with regards to their impact on ARDS. We also established an ARDS mouse model which was pretreated with a liquid complex of PLSCR4 siRNA/sc siRNA-lipofectamine 2000 through the fundus venous plexus. Finally, we used DNA pull-down and protein profiling to study the potential transcription factor of PLSCR4.
It was found that when the expression of PLSCR4 was elevated, the concentration of interleukin 1 beta (IL-1β) and IL-18 decreased, along with barrier damage (P<0.05). Furthermore, HPMEC injury was reduced with more distribution of PS and N-terminal cleavage product (GSDMD-NT) of GSDMD on the external side of cell membrane. However, the pyroptosis-relevant proteins of GSDMD and caspase-1 were not obviously changed (P<0.05); we further found that when PLSCR4 was depressed, the lung injury was aggravated in the mice. In the DNA pull-down assay, P62280 remarkably increased, which suggested that P62280 might be the transcription factor for PLSCR4.
PLSCR4 alleviated pyroptosis by transporting PS to the outside of the membrane, blocking the formation of pyroptosis pores composed of GSDMD. Moreover, P62280 might be the transcription factor of PLSCR4. These insults may provide useful insights into the clinical treatment of ARDS.
先前的实验显示，在脂多糖（LPS）诱导的人肺微血管内皮细胞（HPMECs）急性呼吸窘迫综合征（ARDS）模型中，磷脂翻转酶4（PLSCR4）mRNA显著增加；然而，PLSCR4的作用及其机制迄今尚未见报道。PLSCR家族被认为介导磷脂（PS）的跨膜运动，并且已发现其通过与gasdermin D（GSDMD）结合参与细胞焦亡。因此，我们推测PLSCR4可能通过细胞焦亡导致细胞死亡。
为了研究PLSCR4在ARDS中的作用及机制，我们构建了用PLSCR4小干扰RNA（siRNA）或乱序siRNA（sc siRNA）转染的HPMECs的LPS诱导ARDS体外模型。LPS刺激4小时后，采用蛋白质免疫印迹法、免疫沉淀法、酶联免疫吸附测定（ELISA）、示踪通量测定法和荧光测定法，研究PLSCR4与细胞焦亡之间的关系及其对ARDS的影响。我们还建立了一个ARDS小鼠模型，通过眼底静脉丛用PLSCR4 siRNA/sc siRNA - 脂质体2000的液体复合物进行预处理。最后，我们使用DNA下拉和蛋白质谱分析来研究PLSCR4的潜在转录因子。
发现当PLSCR4表达升高时，白细胞介素1β（IL - 1β）和IL - 18浓度降低，同时屏障损伤减轻（P<0.05）。此外，随着PS和GSDMD的N端裂解产物（GSDMD - NT）在细胞膜外侧的分布增加，HPMEC损伤减轻。然而，GSDMD和caspase - 1的细胞焦亡相关蛋白没有明显变化（P<0.05）；我们进一步发现，当PLSCR4表达降低时，小鼠的肺损伤加重。在DNA下拉试验中，P62280显著增加，这表明P62280可能是PLSCR4的转录因子。
PLSCR4通过将PS转运到细胞膜外，阻止由GSDMD组成的细胞焦亡孔的形成，从而减轻细胞焦亡。此外，P62280可能是PLSCR4的转录因子。这些发现可能为ARDS的临床治疗提供有用的见解。
