Anti-Neuroinflammatory Potential of a Nectandra angustifolia (Laurel Amarillo) Ethanolic Extract.

Microglia, the resident macrophage-like population in the CNS, plays an important role in the pathogenesis of many neurodegenerative disorders. Nectandra genus is known to produce different metabolites with anti-inflammatory, anti-oxidant and analgesic properties. Although the species Nectandra angustifolia is popularly used for the treatment of different types of inflammatory processes, its biological effects on neuroinflammation have not yet been addressed. In this study, we have investigated the role of a Nectandra angustifolia ethanolic extract (NaE) in lipopolysaccharide (LPS)-induced neuroinflammation in vitro and in vivo. In LPS-activated BV2 microglial cells, NaE significantly reduced the induced proinflammatory mediators TNF-α, IL-1ß, IL-6, COX-2 and iNOS, as well as NO accumulation, while it promoted IL-10 secretion and YM-1 expression. Likewise, reduced CD14 expression levels were detected in microglial cells in the NaE+LPS group. NaE also attenuated LPS-induced ROS and lipid peroxidation build-up in BV2 cells. Mechanistically, NaE prevented NF-κB and MAPKs phosphorylation, as well as NLRP3 upregulation when added before LPS stimulation, although it did not affect the level of some proteins related to antioxidant defense such as Keap-1 and HO-1. Additionally, we observed that NaE modulated some activated microglia functions, decreasing cell migration, without affecting their phagocytic capabilities. In LPS-injected mice, NaE pre-treatment markedly suppressed the up-regulated TNF-α, IL-6 and IL-1ß mRNA expression induced by LPS in brain. Our findings indicate that NaE is beneficial in preventing the neuroinflammatory response both in vivo and in vitro. NaE may regulate microglia homeostasis, not only restraining activation of LPS towards the M1 phenotype but promoting an M2 phenotype.

Beneficial role of the phytoestrogen genistein on vascular calcification.

Although soy phytoestrogen are proposed to prevent or improve postmenopausal vascular and bone diseases, the currently available data are controversial and unclear. In this study we evaluated the molecular and biochemical action of genistein on the cellular events involved in vascular calcification. Rat monocytes, aortic vascular cell and osteoblasts cultures in vitro exposed to Gen were employed. Gen down regulated the expression of cell adhesion molecules involved in stable leukocyte attachment. Using flow cytometry we found that the PE significantly diminished monocyte integrins CD11b, CD11c and CD18 expression either under basal and pro-inflammatory environment. At endothelial level, Gen also reduced Intercellular Adhesion Molecule 1 mRNA expression. On vascular muscle cells, the PE markedly reduced cell proliferation and migration. When vascular calcification was studied, muscle cells transdifferentiation into osteoblasts like cells was evaluated. Cells were cultured in osteogenic medium for 21 days. The expression of alkaline phosphatase and the presence of calcified nodules in the extracellular matrix were selected as features of muscle transdifferentiation. Calcified muscle cells exhibited higher levels of alkaline phosphatase activity and enhanced deposition of calcium nodules respect to native cells. Both osteoblastic markers were significantly reduced after Gen treatment. In contrast to this anti-osteogenic action, on bone cells Gen promoted osteoblasts growth, enhanced alkaline phosphatase activity and increased matrix mineralization. Its mitogenic action on osteoblasts directly depends on nitric oxide endothelial production stimulated by the PE. The data presented suppose a beneficial role of Gen on bone and vascular cells, with a cross link between both systems.

Vascular action of bisphosphonates: In vitro effect of alendronate on the regulation of cellular events involved in vessel pathogenesis.

In this work we investigate whether, despite the procalcific action of alendronate on bone, the drug would be able to regulate in vitro the main cellular events that take part in atherosclerotic lesion generation. Using endothelial cell cultures we showed that Alendronate (1-50µM) acutely enhances nitric oxide production (10-30min). This stimulatory action of the bisphosphonate involves the participation of MAPK signaling transduction pathway. Under inflammatory stress, the drug reduces monocytes and platelets interactions with endothelial cells induced by lipopolysaccharide. Indeed the bisphophonate exhibits a significant inhibition of endothelial dependent platelet aggregation. The molecular mechanism of alendronate (ALN) on leukocyte adhesion depends on the regulation of the expression of cell adhesion related genes (VCAM-1; ICAM-1); meanwhile the antiplatelet activity is associated with the effect of the drug on nitric oxide production. On vascular smooth muscle cells, the drug exhibits ability to decrease osteogenic transdifferentiation and extracellular matrix mineralization. When vascular smooth muscle cells were cultured in osteogenic medium for 21days, they exhibited an upregulation of calcification markers (RUNX2 and TNAP), high alkaline phosphatase activity and a great amount of mineralization nodules. ALN treatment significantly down-regulates mRNA levels of osteoblasts markers; diminishes alkaline phosphatase activity and reduces the extracellular calcium deposition. The effect of ALN on vascular cells differs from its own bone action. On calvarial osteoblasts ALN induces cell proliferation, enhances alkaline phosphatase activity, and increases mineralization, but does not affect nitric oxide synthesis. Our results support the hypothesis that ALN is an active drug at vascular level that regulates key processes involved in vascular pathogenesis through a direct action on vessel cells.