Circ-Phkb promote LPS-induced acute lung injury via the TLR4/MyD88/NF-κB /CCL2 axis. Circ-Phkb promote LPS-induced acute lung injury via the TLR4/MyD88/NF-κB /CCL2 axis

Our results showed that both circRNA and mRNA profiles are different from those of the sham group. A significant upregulation of circ-Phkb was observed in NR8383 cells and rats exposed to LPS. When circ-Phkb expression was reduced in NR8383 cells, apoptosis and inflammation were greatly reduced, and cell proliferation was increased, while overexpression of circ-Phkb have the opposite effect. In terms of mechanism, circ-Phkb suppression inhibits CCL2 expression via TLR4/MyD88/NF-κB pathway. Overall design: In this study, we obtained differential circRNA and mRNA expression profiles and possible ceRNA networks through circRNA sequencing, mRNA sequencing and bioinformatics analysis of lung tissues from an LPS-induced ALI rat model. Furthermore, qRT-PCR assays were performed to screen for circ-Phkb in ALI rat lung tissues, alveolar macrophages and LPS-induced NR8383 cells. Induction with or without LPS was then conducted with circ-Phkb siRNA and overexpression lentivirus in NR8383. Proliferation was detected by CCK8 and Edu staining, and apoptosis was detected by TUNEL assay and cytometry. Inflammatory factors were detected using ELISA. Finally, WB was used to detect the apoptosis-related proteins and the activation of the TLR4/MyD88/NF-κB pathway.

本研究结果显示，环状RNA（circRNA）与信使RNA（mRNA）的表达谱均与假手术组存在显著差异。在经脂多糖（LPS）处理的NR8383细胞与大鼠体内，均观测到circ-Phkb的显著上调。当NR8383细胞中circ-Phkb的表达被抑制时，细胞凋亡与炎症反应均显著减轻，细胞增殖能力增强；而circ-Phkb过表达则会产生相反的效应。在机制层面，circ-Phkb的抑制可通过Toll样受体4（TLR4）/髓系分化因子88（MyD88）/核因子κB（NF-κB）通路抑制趋化因子配体2（CCL2）的表达。研究整体设计：本研究通过对脂多糖（LPS）诱导的急性肺损伤（ALI）大鼠模型的肺组织进行环状RNA测序、信使RNA测序及生物信息学分析，获取了差异表达的环状RNA与信使RNA谱，以及潜在的内源竞争RNA（ceRNA）调控网络。此外，本研究通过实时荧光定量聚合酶链反应（qRT-PCR）实验，在急性肺损伤大鼠肺组织、肺泡巨噬细胞以及脂多糖诱导的NR8383细胞中筛选circ-Phkb的表达情况。随后，我们使用circ-Phkb小干扰RNA（siRNA）与过表达慢病毒处理NR8383细胞，并分别施加脂多糖诱导与不施加脂多糖诱导。采用细胞计数试剂盒-8（CCK8）与5-乙炔基-2'-脱氧尿苷（Edu）染色检测细胞增殖能力，通过末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）测定与流式细胞术检测细胞凋亡情况。采用酶联免疫吸附测定（ELISA）检测炎症因子水平。最后，通过蛋白质印迹法（WB）检测凋亡相关蛋白的表达以及Toll样受体4（TLR4）/髓系分化因子88（MyD88）/核因子κB（NF-κB）通路的激活状态。