Differences in Proinflammatory Property of Six Subtypes of Peroxiredoxins and Anti-Inflammatory Effect of Ligustilide in Macrophages.

Abstract

BACKGROUND:Peroxiredoxins (Prxs) are proposed to function as damage-associated molecular patterns (DAMPs) and contribute to post-ischemic neuroinflammation and brain injury by activating Toll-like receptor (TLR) 4 at the acute and subacute phases after ischemic stroke. However, there are few studies concerning the inflammatory profiles of six distinct subtypes of Prxs (Prx1-Prx6). Our previous study demonstrated that the protective effect of ligustilide (LIG) against cerebral ischemia was associated with inhibition of neuroinflammatory response and Prx/TLR4 signaling in rats. Herein, the present study explored the inflammatory members of Prxs and the effect of LIG on their inflammatory responses in macrophages. METHODOLOGY/PRINCIPAL FINDINGS:The murine RAW264.7 macrophages were treated with each of exogenous recombinant Prxs at a range of 1 to 50 nM for 24 h. The WST-1 test showed that Prx3 exhibited a significant cytotoxicity, whereas the rest five Prxs did not affect cellular viability. The quantitative measurements with spectrometry or ELISA indicated that three subtypes, Prx1, Prx2 and Prx4, increased production of proinflammatory mediators, including nitric oxide (NO) metabolites, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in a concentration-dependent manner. Immunostaining demonstrated that 20 nM Prx1, Prx2 or Prx4 significantly increased expression of TLR4 and iNOS and nuclear translocation of NF-κB p65. However, Prx5 and Prx6 showed no poinflammatory effect in macrophages. Remarkably, LIG treatment effectively inhibited the inflammatory response induced by Prx1, Prx2 and Prx4. CONCLUSION:Three members of Prxs, Prx1, Prx2 and Prx4, are inflammatory DAMPs that induce TLR4 activation and inflammatory response in macrophages, which is effectively inhibited by LIG. These results suggest that inflammatory Prxs-activated macrophages may provide a novel cellular model for screening the potential inhibitors of DAMPs-associated inflammatory diseases such as stroke. Moreover, selective blocking strategies targeting the inflammatory subtypes of Prxs probably provide promising therapeutic approaches with a prolonged time window for stroke.