MATERIALS AND METHODS: The effects of mitragynine on the mRNA and protein expression of COX-1 and COX-2 and the production of prostaglandin E(2) (PGE(2)) were investigated in LPS-treated RAW264.7 macrophage cells. Quantitative RT-PCR was used to assess the mRNA expression of COX-1 and COX-2. Protein expression of COX-1 and COX-2 were assessed using Western blot analysis and the level of PGE(2) production was quantified using Parameter™ PGE(2) Assay (R&D Systems).
RESULTS: Mitragynine produced a significant inhibition on the mRNA expression of COX-2 induced by LPS, in a dose dependent manner and this was followed by the reduction of PGE(2) production. On the other hand, the effects of mitragynine on COX-1 mRNA expression were found to be insignificant as compared to the control cells. However, the effect of mitragynine on COX-1 protein expression is dependent on concentration, with higher concentration of mitragynine producing a further reduction of COX-1 expression in LPS-treated cells.
CONCLUSIONS: These findings suggest that mitragynine suppressed PGE(2) production by inhibiting COX-2 expression in LPS-stimulated RAW264.7 macrophage cells. Mitragynine may be useful for the treatment of inflammatory conditions.
METHODS: The inhibitory effect of chrysin, kaempferol, morin, silibinin, quercetin, diosmin and hesperidin upon nitric oxide (NO), prostaglandin E(2) (PGE(2)) and tumour necrosis factor-alpha (TNF-alpha) secretion from the LPS-induced RAW 264.7 monocytic macrophage was assessed and IC(50) values obtained. Flavonoids that showed reasonable inhibitory effects in at least two out of the three assays were combined in a series of fixed IC(50) ratios and reassessed for inhibition of NO, PGE(2) and TNF-alpha. Dose-response curves were generated and interactions were analysed using isobolographic analysis.
RESULTS: The experiments showed that only chrysin, kaempferol, morin, and silibinin were potent enough to produce dose-response effects upon at least two out of the three mediators assayed. Combinations of these four flavonoids showed that several combinations afforded highly significant synergistic effects.
CONCLUSIONS: Some flavonoids are synergistic in their anti-inflammatory effects when combined. In particular chrysin and kaempferol significantly synergised in their inhibitory effect upon NO, PGE(2) and TNF-alpha secretion. These findings open further avenues of research into combinatorial therapeutics of inflammatory-related diseases and the pharmacology of flavonoid synergy.
MATERIALS AND METHODS: An in vitro monocyte recruitment model utilizing THP-1 and HUVECs was developed to evaluate TNF-α-induced monocyte adhesion and trans-endothelial migration. To study the role of Nrf2 for MA-mediated anti-inflammatory effects, Nrf2 inhibitor ML385 was used as the pharmacological inhibitor. The expression of Nrf2, monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule 1 (VCAM-1), cluster of differentiation 36 (CD36), and scavenger receptor type A (SR-A) in HUVECs and THP-1 macrophages were investigated using RT-qPCR and Western blotting. The NF-κB activity was determined using NF-κB (p65) Transcription Factor Assay Kit.
KEY FINDINGS: The results showed opposing effects of MA on Nrf2 expression in HUVECs and THP-1 macrophages. MA suppressed TNF-α-induced Nrf2 expression in HUVECs, but enhanced its expression in THP-1 macrophages. Combined effects of MA and ML385 suppressed MCP-1, VCAM-1, and SR-A expressions. Intriguingly, at the protein level, ML385 selectively inhibited SR-A but enhanced CD36 expression. Meanwhile, ML385 further enhanced MA-mediated inhibition of NF-κB activity in HUVECs. This effect, however, was not observed in THP-1 macrophages.
SIGNIFICANCE: MA attenuated foam cell formation by suppressing VCAM-1, MCP-1, and SR-A expression, as well as NF-κB activity, possibly through Nrf2 inhibition. The involvement of Nrf2 for MA-mediated anti-inflammatory effects however differs between HUVECs and macrophages. Future investigations are warranted for a detailed evaluation of the contributing roles of Nrf2 in foam cells formation.
METHODS: The release of prostaglandin E2 (PGE2) and pro-inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1β in a culture supernatant was determined by ELISA. Determination of cyclooxygenase-2 (COX-2) protein and the activation of MAPKs molecules (JNK, ERK and p38 MAPK), NF-κB and Akt in LPS-induced U937 human macrophages were investigated by immunoblot technique. The relative gene expression levels of COX-2 and pro-inflammatory cytokines were measured by using qRT-PCR. The major metabolites of P. amarus were qualitatively and quantitatively analyzed in the extract by using validated reversed-phase high performance liquid chromatography (HPLC) methods.
RESULTS: P. amarus extract significantly inhibited the production of pro-inflammatory mediators (TNF-α, IL-1β, PGE2) and COX-2 protein expression in LPS-induced U937 human macrophages. P. amarus-pretreatment also significantly downregulated the increased mRNA transcription of pro-inflammatory markers (TNF-α, IL-1β, and COX-2) in respective LPS-induced U937 macrophages. It downregulated the phosphorylation of NF-κB (p65), IκBα, and IKKα/β and restored the degradation of IκBα, and attenuated the expression of Akt, JNK, ERK, and p38 MAPKs phosphorylation in a dose-dependent manner. P. amarus extract also downregulated the expression of upstream signaling molecules, TLR4 and MyD88, which play major role in activation of NF-κB, MAPK and PI3K-Akt signaling pathways. The quantitative amounts of lignans, phyllanthin, hypophyllahtin and niranthin, and polyphenols, gallic acid, geraniin, corilagin, and ellagic acid in the extract were determined by HPLC analysis.
CONCLUSION: The study revealed that P. amarus targeted the NF-κB, MAPK and PI3K-Akt signaling pathways to exert its anti- inflammatory effects by downregulating the prospective inflammatory signaling mediators.