Bupivacaine inhibits the TLR4‐ and TLR2‐Myd88/NF‐κB pathways in human leukocytes

Abstract

Local anesthetics have anti-inflammatory effects. Because most previous experiments were performed with supra-therapeutic concentrations, we measured the effects of clinically relevant concentrations of bupivacaine on the Toll like receptor 4 (TLR4)-and TLR2-myeloid differentiation primary response 88 (MyD88)-nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) pathways. We measured tumor necrosis factor alpha (TNF-α) and prostaglandin E2 (PGE2) release, p38 mitogen-activated protein kinase (MAP-kinase) phosphorylation and translocation of NF-κB in human peripheral blood mononuclear cells (hPBMCs) and human monocytes challenged with lipopolysaccharide (LPS) or tripalmitoylated lipopeptide Pam3CysSerLys4 (Pam3CSK4) in the presence or absence of bupivacaine. Similarly, we measured the effect of bupivacaine on HEK293 cells expressing the hTLR4 and the hTLR2 genes and challenged with LPS or Pam3CSK4. Finally, molecular docking simulations of R(+)-and S (À)-bupivacaine binding to the TLR4-myeloid differentiation protein 2 (MD-2) complex and to the TLR2/TLR1 heterodimer were performed. In PBMCs, bupivacaine from 0.1 to 100 μM inhibited LPS-induced TNF-α and PGE2 secretion, phosphorylation of p38 and nuclear translocation of NF-κB in monocytes. Bupivacaine similarly inhibited the effects of Pam3CSK4 on TNF-α secretion. Bupivacaine inhibited the effect of LPS on HEK293 cells expressing the human TLR4 receptor and the effect of Pam3CSK4 on HEK293 cells expressing the human TLR2 receptor. Molecular docking showed that bupivacaine binds to the MD-2 co-receptor of TLR4 and to the TLR2 receptor. Contrary to numerous experiments performed with supratherapeutic doses, our results were obtained with concentrations of bupivacaine as low as 0.1 μM. We conclude that bupivacaine modulates the inflammatory reactions such as those observed after surgery or trauma, at least partly by inhibiting the TLR4-and TLR2-NF-κB pathways.Abbreviations: AP-1, activating protein-1; CD14, cluster of differentiation protein 14; COX-2, cyclooxygenase 2; ELISA, enzyme-linked immunosorbent assay; IL1-β, interleukin 1 beta; MAP-kinase, mitogen-activated protein kinase; MD-2, myeloid differentiation protein 2; MyD88, myeloid differentiation primary response 88; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; Pam3CSK4, tripalmitoylated lipopeptide Pam3CysSerLys4; PBMC, peripheral blood mononuclear cell; PGE2, prostaglandin E2; qPCR, real-time polymerase chain reaction; RNA, ribonucleic acid; TLR, Toll like receptor; TNF-α, tumor necrosis factor alpha; TRIF, TIR-domain-containing adapter-inducing interferon-β; XlogP3, predicted logarithm of the n-octanol/water partition coefficient.Marie Binczak and Emilien Purenne equally participated in this study.

Pro‐inflammatory cytokines mediating senescence of vascular endothelial cells in atherosclerosis

“…The NF-κB pathway plays an important regulatory role in inflammatory responses, cell proliferation, differentiation, apoptosis, and senescence [73][74][75][76][77][78]. And various stimulus factors, including pro-inflammatory factors, can induce phosphorylation of inhibitors of NF-κB (IκBs), promote nuclear translocation of NF-κB, and regulate the expression of target genes [79][80][81].…”