AIM OF THE STUDY: To determine the inhibitory properties of FD aqueous extract on pro-inflammatory mediators involved in lipopolysaccharide (LPS)-induced microglial cells.
METHODS: Vitexin and isovitexin in the extract were quantified via high performance liquid chromatography (HPLC). The extract was evaluated for its cytotoxicity activity via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Pre-treatment with the extract on LPS-induced microglial cells was done to determine its antioxidant and anti-neuroinflammatory properties by measuring the level of reactive oxygen species (ROS), nitric oxide (NO), tumour necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) via 2'-7'-dichlorofluorescin diacetate (DCFDA) assay, Griess assay and Western blot respectively.
RESULTS: The extract at all tested concentrations (0.1 μg/mL, 1 μg/mL, 10 μg/mL, 100 μg/mL) were not cytotoxic as the percentage viability of microglial cells were all above ~80%. At the highest concentration (100 μg/mL), the extract significantly reduced the formation of ROS, NO, TNF-α, IL-1β and IL-6 in microglial cells induced by LPS.
CONCLUSION: The extract showed neuroprotective effects by attenuating the levels of pro-inflammatory and cytotoxic factors in LPS-induced microglial cells, possibly by mediating the nuclear factor-kappa B (NF-κB) signalling pathway.
MATERIALS AND METHODS: The inhibitory effects of hexane (LHXN), dichloromethane (LDCM), ethyl acetate (LEA) and methanol (LMEOH) extracts from leaves of PS on Aβ-induced production and mRNA expression of pro-inflammatory mediators in BV-2 microglial cells were assessed using colorimetric assay with Griess reagent, ELISA kit and real-time RT-PCR respectively. Subsequently, MTT reduction assay was used to evaluate the neuroprotective effects of PS leaf extracts against Aβ-induced microglia-mediated neurotoxicity in SH-SY5Y neuroblastoma cells. The levels of tau proteins phosphorylated at threonine 231 (pT231) and total tau proteins (T-tau) were determined using ELISA kits.
RESULTS: Polar extracts of PS leaves (LEA and LMEOH) reduced the Aβ-induced secretion of pro-inflammatory cytokines (IL-1β and TNF-α) in BV-2 cells by downregulating the mRNA expressions of pro-inflammatory cytokines. The inhibition of nitric oxide (NO) production could be due to the free radical scavenging activity of the extracts. In addition, conditioned media from Aβ-induced BV-2 cells pre-treated with LEA and LMEOH protected SH-SY5Y cells against microglia-mediated neurotoxicity. Further mechanistic study suggested that the neuroprotective effects were associated with the downregulation of phosphorylated tau proteins.
CONCLUSIONS: The present study suggests that polar extracts of PS leaves confer neuroprotection against Aβ-induced microglia-mediated neurotoxicity in SH-SY5Y cells by attenuating tau hyperphosphorylation through their anti-inflammatory actions and could be a potential therapeutic agent for Alzheimer's disease.