Pharmacological mechanism underlying anti-inflammatory, anti-nociceptive and neuroprotective activities of anomalin and capillarisin

Abstract

In our continuous search for novel anti-inflammatory and analgesic agents from traditional medicinal plants, Saposhnikovia divaricata, Sesli resinosum, Leonurus japonicus and Artemisia capillaris have been focused of our investigations. Anomalin, calipteryxin, (3´S,4´S)-3´, 4´-disenecioyloxy-3´,4´-dihydroseselin), 15, 16-epoxy-3α-hydroxylabda-8,13(16),14-trien-7-one and capillarisin are considered to exhibit anti-inflammatory activity. Based on significant anti-inflammatory effects, anomalin and capillarisin were studied in detail. To clarify the cellular signaling mechanisms underlying the anti-inflammatory action of anomalin and capillarisin, the effect of both the compounds were investigated on the production of inflammatory molecules in LPS-stimulated murin macrophages. Anomalin and capillarisin dose-dependently inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA and protein expression levels in LPS-stimulated RAW 264.7 macrophages. Molecular analysis using quantitative real time polymerase chain reaction (qRT-PCR) revealed that several pro-inflammatory cytokines were reduced by anomalin and capillarisin, and this reduction correlated with the down-regulation of the NF-κB signaling pathway. Additionally, MAPKs and Akt pathways were also focused under investigation. The time course experimental results showed that both the compounds exhibited remarkable inhibitory effects on the activation of Akt and MAPKs proteins. Due to the considerable anti-inflammatory effects of anomalin and capillarisin, CFA- and carrageenan-induced acute and chronic pain models were next focused. The animal models for anti-hyperalgesic and anti-allodynic effects revealed that anomalin and capillarisin significantly inhibited CFA- and carrageenan-induced pain by using Randall Selitto and Von Frey filaments. Another set of experiments observed that anomalin and capillarisin inhibited CFA- and carrageenan-induced paw edema in acute and chronic models, and it did not cause any apparent toxicity. To elucidate the molecular mechanism underlying the anti-nociceptive effect of anomalin and capillarisin, various pain signaling pathways (NF-κB, CREB, and MAPKs/AP-1) that are involved were examined. Intraperitoneal pre-treatment of anomalin and capillarisin exhibited potent inhibitory effects on direct mediators of hyperalgesia (iNOS, COX-2 and MAPKs proteins). The release of CFA-induced plasma nitrite and paw tissue hyperalgesic cytokine (TNF-α) was reduced remarkably. Another signaling molecule, adenosine 5'-triphosphate (ATP) in plasma and substance P (SP) in paw tissue were markedly suppressed by anomalin and capillarisin. Moreover, in vitro experiment demonstrated that anomalin and capillarisin exhibited promising neuroprotective and anti-neuroinflammatory effects against sodium nitroprussid (SNP)-induced N2a neuroblastoma cells. Anomalin and capillarisin were observed remarkable inhibitory activity against STZ-induced type I diabetic animal neuropathic pain model. Hence, these results clearly demonstrated that anomalin and capillarisin showed substantial anti-inflammatory and analgesic effects in a consistent manner and their mechanisms involve the inhibition of the NF-κB, CREB, and MAPKs/AP-1 signaling pathways. Thus, the present study showed that anomalin and capillarisin could be developed for the management of acute and chronic inflammatory pain.