Department of Gastrointestinal Surgery, The Affiliated Huai'an No. 1 People's Hospital of Nanjing Medical University, Huaian, People's Republic China.
Department of Critical Care Medicine, The First Affiliated Hospital of Xinjiang Medical University, Urumqi, People's Republic China.
Shock. 2024 Mar 1;61(3):477-489. doi: 10.1097/SHK.0000000000002249. Epub 2023 Nov 16.
Objective: Autophagy elevation in endotoxemia plays a protective role by negatively regulating the pyroptosis of vascular endothelial cells, but the molecular mechanisms are still poorly understood. The present study aimed to identify the mechanism underlying autophagy and pyroptosis in endotoxemia. Methods: Bioinformatics analysis and whole-gene transcriptome sequencing prediction were used to identify the endotoxemia-related lncRNA-miRNA-mRNA axis of interest. Human umbilical vein endothelial cells (HUVECs) were activated by lipopolysaccharide (LPS) to mimic the inflammatory environment encountered in endotoxemia. Autophagy and pyroptosis of LPS-treated HUVECs were assessed in response to the knockdown of MALAT1 (metastasis-associated lung adenocarcinoma transcript 1)/miR-433-3p (miRNA-433-3p)/RPTOR (regulatory-associated protein of mTOR). The binding affinity of MALAT1, miR-433-3p, and RPTOR was detected by RNA pull-down and luciferase activity assays. The endothelial cell-specific RPTOR knockout mice were developed and rendered septic using LPS induction to verify the role of RPTOR in autophagy, pyroptosis, and inflammatory response in vivo . Results: The in vitro experiments indicated that LPS could stimulate HUVECs to highly express RPTOR, and its knockdown enhanced cellular autophagy and restricted pyroptosis to curb inflammatory responses. Mechanically, MALAT1 is competitively bound to miR-433-3p to release RPTOR expression, thereby promoting pyroptosis and aggravating endotoxemia. In vivo experiments further confirmed that the knockdown of RPTOR activated autophagy and curtailed pyroptosis in septic mice. Conclusion: MALAT1 is highly expressed in endotoxemia. MALAT1 promotes RPTOR expression by competitively absorbing miR-433-3p, inhibits LPS-activated HUVEC cell autophagy, promotes cell death, enhances LPS-induced inflammatory activation of vascular endothelial cells, and ultimately promotes the progression of endotoxemia.
内毒素血症中自噬的升高通过负向调节血管内皮细胞的细胞焦亡发挥保护作用，但分子机制仍知之甚少。本研究旨在确定内毒素血症中自噬和细胞焦亡的机制。
采用生物信息学分析和全基因转录组测序预测，确定内毒素血症相关 lncRNA-miRNA-mRNA 轴。用脂多糖（LPS）激活人脐静脉内皮细胞（HUVEC）模拟内毒素血症中遇到的炎症环境。根据 MALAT1（转移相关肺腺癌转录物 1）/miR-433-3p（miRNA-433-3p）/RPTOR（mTOR 调节相关蛋白）的敲低，评估 LPS 处理的 HUVEC 中的自噬和细胞焦亡。通过 RNA 下拉和荧光素酶活性测定检测 MALAT1、miR-433-3p 和 RPTOR 的结合亲和力。利用 LPS 诱导建立内皮细胞特异性 RPTOR 敲除小鼠，验证 RPTOR 在体内自噬、细胞焦亡和炎症反应中的作用。
体外实验表明，LPS 可刺激 HUVEC 高度表达 RPTOR，其敲低增强细胞自噬并限制细胞焦亡以抑制炎症反应。机制上，MALAT1 与 miR-433-3p 竞争性结合以释放 RPTOR 表达，从而促进细胞焦亡并加重内毒素血症。体内实验进一步证实，RPTOR 的敲低可激活脓毒症小鼠的自噬并抑制细胞焦亡。
MALAT1 在内毒素血症中高表达。MALAT1 通过竞争性吸收 miR-433-3p 促进 RPTOR 表达，抑制 LPS 激活的 HUVEC 细胞自噬，促进细胞死亡，增强 LPS 诱导的血管内皮细胞炎症激活，最终促进内毒素血症的进展。
