Anti-TLR4 (aa 161-192) polyclonal antibody

Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern. Neutrophils present in the mononuclear cell fraction of Ficoll gradient separation are called low-density neutrophils (LDNs). Here we report the novel role of eCIRP on LDNs' heterogeneity in sepsis. Sepsis was induced in male C57BL/6 wild-type (WT) and CIRP(-/-)mice by cecal ligation and puncture (CLP). At 20 h after CLP, LDNs in the blood were isolated by Ficoll gradient separation, followed by staining the cells with anti-Ly6G and anti-CD11b Abs and detection by flow cytometry. Sepsis or recombinant murine CIRP (rmCIRP) injection in mice resulted in significant increase in the frequency (%) and number of Ly6G(+)CD11b(hi)and Ly6G(+)CD11b(lo)LDNs in the blood compared to sham- or vehicle-treated mice. At 20 h of CLP, CIRP(-/-)mice had significantly lower frequency and number of Ly6G(+)CD11b(hi)and Ly6G(+)CD11b(lo)LDNs in the blood compared to WT mice. In sepsis mice or rmCIRP-injected mice, compared to Ly6G(+)CD11b(lo)LDNs, the expression of CXCR4, ICAM-1, and iNOS and formation of reactive oxygen species, and neutrophil extracellular traps in Ly6G(+)CD11b(hi)LDNs in the blood were significantly increased. Treatment of WT bone marrow-derived neutrophils (BMDNs) with rmCIRP increased Ly6G(+)CD11b(hi)LDN frequency, whereas treatment of TLR4(-/-)BMDNs with rmCIRP significantly decreased the frequency of Ly6G(+)CD11b(hi)LDNs. BMDNs' stimulation with rmCIRP increased the expression of transcription factors in LDNs. eCIRP induces the formation of a proinflammatory phenotype Ly6G(+)CD11b(hi)of LDNs through TLR4. Targeting eCIRP may provide beneficial outcomes in sepsis by decreasing proinflammatory Ly6G(+)CD11b(hi)LDNs.

Magnesium is being investigated in various clinical conditions and has shown to be effective in some chronic pain models. However, it is not clear if oral magnesium use affects pain perception in acute pain. TLR4's (toll-like receptor) role in pain perception has emerged through its role in immune pathways and ion channels. The aim of this study is to investigate the effect of a single oral dose of magnesium citrate on pain conduction and whether with magnesium, the expression of TLR4 changes in the acute phase. Following a single dose of 66-mg/kg magnesium citrate administration to male Balb-c mice, pain perception (via hot-plate test), motor conduction (via electrophysiological recording, forelimb grip strength, rotarod and open-field tests), and emotional state (via elevated plus maze and forced swim test) were evaluated. In behavioral experiments, the control group was compared with applied magnesium for three different time groups (4, 8, 24 h). TLR4 expression was measured in four groups: control, magnesium (Mg), hot plate (HP), and Mg + HP. Hot plate latency was prolonged in the magnesium group (p< 0.0001) and electrophysiological recordings (p< 0.001) and forelimb grip strength measurement (p< 0.001) determined motor latency. Compared with the untreated hot plate group, TLR4 levels was lower in the brain (p= 0.023) and higher in the sciatic nerve (p= 0.001) in the magnesium-treated hot plate group. Consequently, the study indicated a single dose of magnesium citrate appeared to cause weakening in the transmission and perception of nociceptive pain. TLR4 may act as a regulator in magnesium's effects on pain perception.