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

Abstract

Background Endothelial 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. Methods The 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. Results The 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. Conclusions Our 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

The role and therapeutic potential of SIRTs in sepsis

“…Inhibitors of these molecules can reverse the LPS-induced downregulation of SIRT4 expression, attenuate the inflammatory cascade triggered in endothelial cells by LPS, and mitigate their damage and death. This suggests that the functional effects of miR-15b-5p and PCSK9 can, at least in part, be attributed to their ability to modulate SIRT4 expression ( 72 ). Earlier research also found that SIRT4 could mitigate the LPS-induced expression of pro-inflammatory cytokines and adhesion molecules in HUVECs by blocking the nuclear translocation of NF-κB p65 ( 73 ).…”

PCSK9 inhibitors ameliorate arterial stiffness in ACS patients: evidences from Mendelian randomization, a retrospective study and basic experiments

“…According to published methods, 10 mmol/L β-gp, 20ug/m dexamethasone and 50ug/ mL L-ascorbic acid were added to the routine medium to induce osteogenesis of VSMCs for 7 days, the medium were changed every 2/3 days (24-29). To mimic the effects of PCSK9i in vivo, 100 μg/mL of reagent-grade purified evolocumab (Selleck, America) was used to co-incubate with VSMCs for 7 days during osteogenesis induction (30)(31)(32). To mimic the stimulation of VSMCs by PCSK9 protein in vivo, refer to previous research (33), 0 μg/mL, 0.55 μg/mL, 1.1 μg/mL, 2.2 μg/mL, and 4.4 μg/mL of recombinant human PCSK9 (MedChemExpress, USA) were co-incubated with VSMCs for 7 days, and the concentration exhibiting the highest intracellular calcium content was chosen for subsequent experimental procedures.…”

Novel insights into the regulatory role of N6-methyladenosine methylation modified autophagy in sepsis

“…Conversely, inhibition of the autophagy pathway may likewise ameliorate sepsis-induced organic depression. In the pathogenesis of sepsis, for instance, it has become clear that suppressing autophagy mechanisms by targeting SIRT4, MAPKs, and Nrf2 pathways may be a useful strategy for protecting organ function [177][178][179]. Indeed, it has also been demonstrated that autophagy is activated early in the onset of sepsis, but that as the condition progresses, autophagic activity declines [180,181].…”