Antineuroinflammatory Effect of and Its Molecules Coumarin and Amburoside A by Inhibiting the MAPK Signaling Pathway in LPS-Activated BV-2 Microglial Cells.

Microglia plays an important role in the neuroinflammatory response, identified as one of the major factors in the development and progression of neurodegenerative diseases. and its bioactive compounds, including coumarin (CM), vanillic acid (VA), and amburoside A (AMB), exert antioxidant, anti-inflammatory, and neuroprotective activities, on 6-OHDA-induced neurotoxicity in rat mesencephalic cells determined by our group. The present study investigated the anti-inflammatory effect of the dry extract from (DEAC), CM, AMB, and VA on lipopolysaccharide- (LPS-) stimulated microglial cells and elucidated the possible molecular mechanism of action. The DEAC was characterized by HPLC-PDA (chemical markers: CM, AMB, and VA). The BV-2 microglial cell line was pretreated with increasing concentrations of DEAC, CM, AMB, or VA in the presence or absence of LPS to evaluate the toxicity and anti-inflammatory activity. The cytotoxicity of DEAC, CM, AMB, or VA on BV-2 cells was evaluated by the MTT test, the free radical scavenging activity of test drugs was investigated, and the nitric oxide (NO) production was determined using the Griess reagent, while cytokine levels were measured by ELISA. The expressions of toll-like receptor 4 (TLR-4), nuclear factor kappa B (NF-B), MAPK members (JNK and ERK1/2), and iNOS were determined through Western blot analysis. DEAC, CM, AMB, or VA (5-100 g/mL) did not induce any detectable cytotoxicity in BV-2 cells. All test drugs (100 g/mL) showed free radical scavenging activity (hydroxyl and superoxide radicals); however, only DEAC, CM, and AMB (5-100 g/mL) significantly reduced NO production. DEAC (100 g/mL), as well as CM (50 and 100 g/mL) and AMB (25 g/mL), reduced at least 50% of NO produced and markedly decrease the production of TNF- and IL-6 but they did not significantly affect IL-10 levels. Only DEAC (100 g/mL) and AMB (25 g/mL) reduced the expression of iNOS, and they did not affect arginase activity. DEAC (100 g/mL) suppressed the activation of the MAPKs JNK and ERK1/2 in LPS-activated BV-2 cells but it did not suppress the expression of TLR-4 nor the phosphorylation of NF-B. In conclusion, DEAC, CM, and AMB exerted anti-inflammatory activity in LPS-activated microglial cells as observed by the reduction in the production of inflammatory mediators and the expression of iNOS. We identified the MAPK signaling pathway as a probable mechanism of action to the anti-inflammatory effects observed.