During our efforts to find bioactive natural products with anti-inflammatory activity, we isolated gigantol from the whole plants of Cymbidium goeringii (Orchidaceae) by activity-guided chromatographic fractionation. Gigantol was found to have potent inhibitory effects on LPS-induced nitric oxide (NO) and prostaglandin E (2) (PGE (2)) production in RAW 264.7 cells. Consistent with these findings, gigantol suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein and mRNA levels in RAW 264.7 cells in a concentration-dependent manner. Our data also indicate that gigantol is a potent inhibitor of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) release and influenced the mRNA expression levels of these cytokines in a dose-dependent manner. Furthermore, a reporter gene assay for nuclear factor kappa B (NF-kappaB) and an electromobility shift assay (EMSA) demonstrated that gigantol effectively inhibited the activation of NF-kappaB, which is necessary for the expression of iNOS, COX-2, TNF-alpha, IL-1beta and IL-6. Thus, our studies suggest that gigantol inhibits LPS-induced iNOS and COX-2 expression by blocking NF- kappaB activation.

Leucine-rich repeat kinase 2 (LRRK2) is a Ser/Thr kinase having mixed lineage kinase-like and GTPase domains, controlling neurite outgrowth and neuronal cell death. Evidence suggests that LRRK2 is involved in innate immune response signaling, but the underlying mechanism is yet unknown. A novel protein inhibitor of phosphatase 3B, RCAN1, is known to positively regulate inflammatory signaling through modulation of several intracellular targets of interleukins in immune cells. In the present study, we report that LRRK2 phosphorylates RCAN1 (RCAN1-1S) and is markedly up-regulated during interleukin-1β (IL-1β) treatment. During IL-1β treatment, LRRK2-mediated phosphorylation of RCAN1 promoted the formation of protein complexes, including that between Tollip and RCAN1. LRRK2 decreased binding between Tollip and IRAK1, which was accompanied by increased formation of the IRAK1-TRAF6 complex. TAK1 activity was significantly enhanced by LRRK2. Furthermore, LRRK2 enhanced transcriptional activity of NF-κB and cytokine IL-8 production. These findings suggest that LRRK2 might be important in positively modulating IL-1β-mediated signaling through selective phosphorylation of RCAN1.

Pancreatic stellate cells (PSCs) are activated by inflammatory stimuli, such as TNF-α or viral infection. Activated PSCs play a crucial role in the development of chronic pancreatitis. Polyinosinic-polycytidylic acid (poly (I:C)) is structurally similar to double-stranded RNA and mimics viral infection. Docosahexaenoic acid (DHA) exhibits anti-inflammatory activity. It inhibited fibrotic mediators and reduced NF-κB activity in the pancreas of mice with chronic pancreatitis. The present study aimed to investigate whether DHA could suppress cytokine expression in PSCs isolated from rats. Cells were pre-treated with DHA or the antioxidant N-acetylcysteine (NAC) and stimulated with TNF-α or poly (I:C). Treatment with TNF-α or poly (I:C) increased the expression of monocyte chemoattractant protein 1 (MCP-1) and chemokine C-X3-C motif ligand 1 (CX3CL1), which are known chemoattractants, and enhanced intracellular and mitochondrial reactive oxygen species (ROS) production and NF-κB activity, but reduced mitochondrial membrane potential (MMP). Increased intracellular and mitochondrial ROS accumulation, cytokine expression, MMP disruption, and NF-κB activation were all prevented by DHA in TNF-α-or poly (I:C)-treated PSCs. NAC suppressed TNF-α-or poly (I:C)-induced expression of MCP-1 and CX3CL1. In conclusion, DHA inhibits poly (I:C)-or TNF-α-induced cytokine expression and NF-κB activation by reducing intracellular and mitochondrial ROS in PSCs. Consumption of DHA-rich foods may be beneficial in preventing chronic pancreatitis by inhibiting cytokine expression in PSCs.

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