Efeito anti-inflamatório do extrato seco padronizado e ativos (cumarina e amburosídio A) de Amburana cearensis em modelos experimentais de neuroinflamação e doença de Parkinson: Papel da via TLR-4/NF- ĸB/MAPK

Abstract

Studies have demonstrated the role of neuroinflammation in Parkinson's disease (PD) and other neurodegenerative diseases, which have an important role of microglial cells among the cellular tissues involved. Given the restrictions of current pharmacotherapy for PD, the search for new drugs is essential. Amburana cearensis extract and/or flag have demonstrated several pharmacological activities, such as anti-inflammatory, antioxidant and neuroprotective, as previously determined by studies in our laboratory. Given the above, the study aimed to investigate the anti-inflammatory and antioxidant effects of the dry extract of cultivated A. cearensis(ESAC) and chemical constituents (coumarin - CM, amburoside A -AMB and vanillic acid - VA) in models of neuroinflammation in cells microglia (BV-2 lineage), neuronal cells (PC-12 lineage) and in vivo PD model. Therefore, the effect of ESAC and actives (CM, AMB and AV) on the activation of microglial cells induced by LPS was investigated, measured by the experience of the concentration of pro-inflammatory mediators and/or oxidants, such as nitrite

(Griees), cytokines (ELISA) and iNOS protein expression. We also evaluated the role of pro- inflammatory gene transcription factors (NF-κB p65 and MAPK), belonging to the two main

inflammatory signaling pathways (IkB/NF-κB p65 and MAPKs), using the Western blotting technique. The free radical scavenging activity of the tested drugs was also investigated. To assess whether the extract's anti-inflammatory action confers neuroprotection, its effect on neuronal cell culture (PC-12) was investigated, as well as its effect on a PD model in rats induced by unilateral injection of 6-OHDA (12 μg/animal) in the right striatum. All tested drugs (100 μg/mL) experienced free radical (HOand O2-

) scavenging activity. Addition of ESAC (1- 100 μg/mL), CM (1-100 μg/mL), AMB (1-100 μg/mL), or AV (1-100 μg/mL) to BV-2 cells in culture did not alter cell viability (MTT test) and significantly increased nitric oxide (NO) production induced by LPS (0.5 μg/mL) in BV-2 cells, showing the following relationship AMB>ESAC>CM>AV. The results appreciated that only ESAC and AMB significantly reduced iNOS (Western blotting) protein expression. ESAC, AMB and CM reduced tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) concentrations but did not significantly affect IL-10 levels. It should also be noted that AMB had an immunomodulatory effect, as evidenced by the increase in urea concentration. The reduction in inflammatory mediators emitted by ESAC was due to the decrease in phosphorylation of the MAPK pathway (JNK and ERK1/2). Furthermore, ESAC (100 μg/mL), present in the conditioned medium, prevented the loss of viability, and produced NO release from PC-12 neuronal cells exposed to 6-OHDA (10 μg/mL) and to the environment (originating from LPS-stimulated BV-2 cells). In the in vivo

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study, it was shown that a dose of 200 mg/Kg of ESAC reversed the motor deficits induced by 6-OHDA, in addition to raising superoxide dismutase (SOD) levels and reducing nitrite levels and lipid peroxidation. The study determined in an unprecedented way the anti-inflammatory and antioxidant effect of ESAC and actives (CM and AMB) that seem to be associated with the inhibition of the MAPKs pathway, in addition to the neuroprotective effect of ESAC on the motor deficit induced by 6-OHDA.