MiR-15b-5p and PCSK9 inhibition reduces lipopolysaccharide-induced endothelial dysfunction by targeting SIRT4

Cellular & Molecular Biology Letters

Research

Raffaele Marfella¹  Celestino Sardu¹  Luigi Mele²  Elisa Martino³  Nunzia D’Onofrio³  Maria Luisa Balestrieri³  Giuseppe Campanile⁴  Anna Balestrieri⁵ 

[1] Department of Advanced Clinical and Surgical Sciences, University of Campania Luigi Vanvitelli, Piazza Miraglia, 80138, Naples, Italy;Department of Experimental Medicine, University of Campania Luigi Vanvitelli, Via Luciano Armanni 5, 80138, Naples, Italy;Department of Precision Medicine, University of Campania Luigi Vanvitelli, Via L. De Crecchio 7, 80138, Naples, Italy;Department of Veterinary Medicine and Animal Production, University of Naples Federico II, Via F. Delpino 1, 80137, Naples, Italy;Food Safety Department, Istituto Zooprofilattico Sperimentale del Mezzogiorno, Via Salute 2, 80055, Portici, Italy;

关键词: microRNA-15b-5p;    PCSK9;    SIRT4;    Sepsis;    Endothelial inflammation;    Pyroptosis;    Autophagy;   

DOI  :  10.1186/s11658-023-00482-5

 received in 2023-04-21, accepted in 2023-08-01,  发布年份 2023

来源: Springer

【 摘 要 】

BackgroundEndothelial dysfunction and deregulated microRNAs (miRNAs) participate in the development of sepsis and are associated with septic organ failure and death. Here, we explored the role of miR-15b-5p on inflammatory pathways in lipopolysaccharide (LPS)-treated human endothelial cells, HUVEC and TeloHAEC.MethodsThe miR-15b-5p levels were evaluated in LPS-stimulated HUVEC and TeloHAEC cells by quantitative real-time PCR (qRT–PCR). Functional experiments using cell counting kit-8 (CCK-8), transfection with antagomir, and enzyme-linked immunosorbent assays (ELISA) were conducted, along with investigation of pyroptosis, apoptosis, autophagy, and mitochondrial reactive oxygen species (ROS) by cytofluorometric analysis and verified by fluorescence microscopy. Sirtuin 4 (SIRT4) levels were detected by ELISA and immunoblotting, while proprotein convertase subtilisin-kexin type 9 (PCSK9) expression was determined by flow cytometry (FACS) and immunofluorescence analyses. Dual-luciferase reporter evaluation was performed to confirm the miR-15b-5p–SIRT4 interaction.ResultsThe results showed a correlation among miR-15b-5p, PCSK9, and SIRT4 levels in septic HUVEC and TeloHAEC. Inhibition of miR-15b-5p upregulated SIRT4 content, alleviated sepsis-related inflammatory pathways, attenuated mitochondrial stress, and prevented apoptosis, pyroptosis, and autophagic mechanisms. Finally, a PCSK9 inhibitor (i-PCSK9) was used to analyze the involvement of PCSK9 in septic endothelial injury. i-PCSK9 treatment increased SIRT4 protein levels, opposed the septic inflammatory cascade leading to pyroptosis and autophagy, and strengthened the protective role of miR-15b-5p inhibition. Increased luciferase signal validated the miR-15b-5p–SIRT4 binding.ConclusionsOur in vitro findings suggested the miR-15b-5p–SIRT4 axis as a suitable target for LPS-induced inflammatory pathways occurring in sepsis, and provide additional knowledge on the beneficial effect of i-PCSK9 in preventing vascular damage by targeting SIRT4.Graphical Abstract

【 授权许可】

CC BY   
© University of Wroclav 2023

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