AWRK6, A Synthetic Cationic Peptide Derived from Antimicrobial Peptide Dybowskin-2CDYa, Inhibits Lipopolysaccharide-Induced Inflammatory Response

Abstract

Lipopolysaccharides (LPS) are major outer membrane components of Gram-negative bacteria and produce strong inflammatory responses in animals. Most antibiotics have shown little clinical anti-endotoxin activity while some antimicrobial peptides have proved to be effective in blocking LPS. Here, the anti-LPS activity of the synthetic peptide AWRK6, which is derived from antimicrobial peptide dybowskin-2CDYa, has been investigated in vitro and in vivo. The positively charged α-helical AWRK6 was found to be effective in blocking the binding of LBP (LPS binding protein) with LPS in vitro using ELISA. In a murine endotoxemia model, AWRK6 offered satisfactory protection efficiency against endotoxemia death, and the serum levels of LPS, IL-1β, IL-6, and TNF-α were found to be attenuated using ELISA. Further, histopathological analysis suggested that AWRK6 could improve the healing of liver and lung injury in endotoxemia mice. The results of real-time PCR and Western blotting showed that AWRK6 significantly reversed LPS-induced TLR4 overexpression and IκB depression, as well as the enhanced IκB phosphorylation. Additionally, AWRK6 did not produce any significant toxicity in vivo and in vitro. In summary, AWRK6 showed efficacious protection from LPS challenges in vivo and in vitro, by blocking LPS binding to LBP, without obvious toxicity, providing a promising strategy against LPS-induced inflammatory responses.

Keywords: AWRK6; antimicrobial peptide; inflammatory response; lipopolysaccharides (LPS).

Figure 1

AWRK6 inhibited the binding of lipopolysaccharides (LPS) with LPS binding protein (LBP) in vitro. (A) The helical wheel projection of AWRK6. (B) The inhibition effects of AWRK6 on the binding of LBP with LPS when AWRK6 was added 1 h before LBP. (C) The inhibition effects of AWRK6 on the binding of LBP with LPS when AWRK6 and LBP were added at the same time. (D) The inhibition effects of AWRK6 on the binding of LBP with LPS when AWRK6 was added after incubation with LBP for 15, 30, 60, and 120 min. (E) The neutralization effects of AWRK6 on LPS, detected by LAL assay. * p < 0.05 compared with the 0 (B, C, and E) or con (D) groups.

Figure 1

AWRK6 inhibited the binding of lipopolysaccharides (LPS) with LPS binding protein (LBP) in vitro. (A) The helical wheel projection of AWRK6. (B) The inhibition effects of AWRK6 on the binding of LBP with LPS when AWRK6 was added 1 h before LBP. (C) The inhibition effects of AWRK6 on the binding of LBP with LPS when AWRK6 and LBP were added at the same time. (D) The inhibition effects of AWRK6 on the binding of LBP with LPS when AWRK6 was added after incubation with LBP for 15, 30, 60, and 120 min. (E) The neutralization effects of AWRK6 on LPS, detected by LAL assay. * p < 0.05 compared with the 0 (B, C, and E) or con (D) groups.

Figure 2

AWRK6 prevented LPS-induced lethal endotoxemia in mice. (A) The survival curves of LPS-induced endotoxemic mice treated with a single dose of AWRK6 (2.5, 5, or 10 mg/kg, n = 30). (B) The survival curves of endotoxemic mice treated with 10 mg/kg AWRK6 at different time points (n = 30). (C–F) The effects of AWRK6 treatment (10 mg/kg) on the serum levels of LPS, IL-1β, IL-6, and TNF-α in the endotoxemic mice. PMB was used as a positive control. * p < 0.05 compared with the LPS group.

Figure 3

AWRK6 protected liver and lung injury in endotoxemia mice. (A) Histopathological analysis of HE-stained sections from liver in endotoxemia mice. (B) Histopathological analysis of HE-stained sections from lung in endotoxemia mice. PMB was used as a positive control. Bar indicates 100 nm.

Figure 4

AWRK6 relieved LPS-induced systemic immune response by blocking TLR4/NF-κB activation. (A,B) The mRNA levels of two genes involved in immune response, TLR4 and IκB in primary macrophages were analyzed by real-time PCR. (C,D) The mRNA levels of TLR4 and IκB in RAW 264.7 cells were analyzed by real-time PCR. (E) The expression of TLR4, IκB, and p-IκB was detected by Western blotting. (F) The quantification of Western blotting was applied with ImageJ. (G) AWRK6 inhibited LPS-induced immune response by blocking TLR4/NF-κB activation (the arrows indicate induction and T bar indicates inhibition). * p < 0.05 compared with LPS group, # p < 0.05 compared with the LPS + PMB group.

Figure 5

AWRK6 did not produce any significant toxicity in vivo and in vitro. (A) The survival curves of mice treated with a single dose of AWRK6 (n = 10). (B–E) The cell viabilities of primary murine hepatocytes, splenocytes, macrophages, and renal tubular epithelial cells incubated with AWRK6 and PMB. * p < 0.05 compared with the 0 group.