Pinocembrin attenuates lipopolysaccharide-induced inflammatory responses in Labeo rohita macrophages via the suppression of the NF-κB signalling pathway

Abstract

Pinocembrin is a flavonoid that has been reported to exhibit various pharmacological and biological activities including antimicrobial, antioxidant, and anti-inflammatory. To explore the anti-inflammatory activity of pinocembrin in a fish cell line, we investigated its ability to regulate the inflammatory mediators elevated by lipopolysaccharide (LPS) in Labeo rohita head-kidney (HK) macrophages. HK macrophages of L. rohita were treated with LPS (1 (mu g mL(-1)) in the presence or absence of pinocembrin. We examined the inhibitory effect of pinocembrin on LPS-induced nitric oxide (NO) and prostaglandin E-2 (PGE(2)) production. The inhibitory effect of pinocembrin on nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was investigated by RT-PCR and western blot. The effect of pinocembrin on pro-inflammatory cytokines (tumour necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta)) and anti-inflammatory cytokine IL-10 was investigated by ELISA and RT-PCR. The phosphorylation of three mitogen activated protein kinases (MAPKs) ERK, JNK, and p38 was analysed by western blot. Pinocembrin inhibited LPS-induced productions of NO and PGE2, and also markedly inhibited TNF-alpha, IL-1 beta, iNOS, and COX-2 production in a concentration-dependent manner. In addition, TNF-alpha and IL-1 beta mRNA expression levels decreased significantly, while IL-10 mRNA expression increased (P < 0.05) with pinocembrin pre-treatment. RT-PCR and western blot analysis showed that pinocembrin decreased both the mRNA and protein expression levels of LPS-induced iNOS and COX-2 in HK macrophages. Pinocembrin suppressed the phosphorylation of MAPK in LPS-stimulated HK macrophages. Further, pinocembrin significantly inhibited LPS-induced NF-kappa B transcriptional activity via the attenuation of I kappa B alpha degradation. Taken together, pinocembrin reduced the levels of pro-inflammatory mediators, such as iNOS, COX-2, TNF-alpha, and IL-1 beta, by inhibiting NF-kappa B activation via the suppression of ERK and p38 phosphorylation, and by attenuating the degradation of I kappa B alpha. These results suggest that pinocembrin is a potential novel candidate for the treatment of inflammatory conditions in L. rohita macrophages. (C) 2016 Elsevier Ltd. All rights reserved.