Ceftiofur impairs pro-inflammatory cytokine secretion through the inhibition of the activation of NF-κB and MAPK

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Abstract

Ceftiofur is a new broad-spectrum, third-generation cephalosporin antibiotic for veterinary use. Immunopharmacological studies can provide new information on the immunomodulatory activities of some drugs, including their effect on cytokine productions. For this reason, we investigated the effect of ceftiofur on cytokine productions in vitro. We found that ceftiofur can downregulate tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), but did not affect interleukin-10 (IL-10) production. We further investigated signal transduction mechanisms to determine how ceftiofur affects. RAW 264.7 cells were pretreated with 1, 5, or 10 mg/L of ceftiofur 1 h prior to treatment with 1 mg/L of LPS. Thirty minutes later, cells were harvested and mitogen activated protein kinases (MAPKs) activation was measured by Western blot. Alternatively, cells were fixed and nuclear factor-κB (NF-κB) activation was measured using immunocytochemical analysis. Signal transduction studies showed that ceftiofur significantly inhibited extracellular signal-regulated kinase (ERK), p38, and c-jun NH2-terminal kinase (JNK) phosphorylation protein expression. Ceftiofur also inhibited p65-NF-κB translocation into the nucleus. Therefore, ceftiofur may inhibit LPS-induced production of inflammatory cytokines by blocking NF-κB and MAPKs signaling in RAW264.7 cells.

Section snippets

Materials and methods

Reagents. Ceftiofur, dimethyl sulfoxide (DMSO), LPS (Escherichia coli 055:B5), and 3-(4,5-dimethylthiazol-2-y1)-2,5-dipheny-ltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Invitrogen-Gibco (Grand Island, NY). Rabbit anti-NF-κB/p65 polyclonal antibody, rabbit anti-JNK polyclonal antibody, rabbit anti-ERK polyclonal antibody, rabbit anti-p38 polyclonal antibody, mouse

The effect of ceftiofur on macrophage toxicity

Ceftiofur did not display any cellular toxicity against RAW264.7 cells over 24 h, as determined by the MTT assay (Fig. 1).

The effect of ceftiofur on LPS-induced cytokine production in vitro

TNF-α, IL-1β, IL-6, and IL-10 concentrations in the culture supernatants of RAW 264.7 cells were measured by sandwich ELISA (Fig. 2). RAW 264.7 cells treated with LPS produced significant amounts of all cytokines examined. The concentration of TNF-α was up to 18 ng/mL after LPS stimulation. However, the concentration of TNF-α in the supernatant of cells pretreated with 1, 5,

Discussion

When mononuclear phagocytes recognize LPS via cell surface receptors, they release numerous pro-inflammatory cytokines [17]. Excessive production of pro-inflammatory cytokines will result in a systemic inflammatory response syndrome typified by septic shock and the complications that are normally associated with it. It would be desirable to be able to selectively regulate cytokine production in the treatment of inflammatory diseases [18], [19], [20], [21]. Therefore, any intervention that

Acknowledgment

This work was supported by a grant from the National Natural Science Foundation of China (No. 30671586).

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    These authors contributed equally to this work.

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