Cell viability and LDH release were measured by MTS and LDH assay kit. Pro-inflammatory mediators and cytokines production including NO, PGE2, IL-1β, IL-6 and TNF-α were detected by Elisa assay. Additionally, western blot was used to detect the expression of inflammatory proteins associated with the MAPKs, JAK/STAT, NF-κB and Nrf2/HO-1 signaling pathways. Moreover, immunofluorescence assay and EMSA were performed to determine the Nrf2 translocation and the binding-DNA activity of NF-κB, respectively.
LTC at 0.5-2 μg/ml significantly increased cell viability and decreased LDH, NO, PGE2, IL-1β, IL-6 and TNF-α production in Oxygen-Glucose Deprivation/Reoxygenation (OGD/R) and LPS-induced BV2 microglia cells. Meanwhile, LTC not only decreased the protein expressions of iNOS, COX-2, but also down-regulated phosphorylation of ERK1/2, p38, and up-regulated HO-1 expression via nuclear translocation of Nrf2. LTC can significantly inhibit the phosphorylation of JAK1/STAT3 and reduce the translocation of NF-κB from cytosol to nucleus as well as the binding-DNA activity. PC12 cell pretreated with LTC- condition medium (CM) significantly alleviated lipopolysaccharide (LPS)-induced neurotoxicity and increased PC12 cell viability in a dose-dependent manner.
The present study showed that LTC exhibited a strong anti-neuroinflammatory activity and neuroprotective effects on LPS-stimulated BV2 microglia cells and PC12 cells.