Nurr1: A vital participant in the TLR4-NF-κB signal pathway stimulated by α-synuclein in BV-2 cells.

Parkinson's disease (PD) is a multi-factorial neurodegenerative disease. Abnormal α-synuclein protein aggregate and sustained microglia activation contribute to the pathogenic processes of PD. However, the relationship between α-synuclein and microglia-mediated neuroinflammation remains unclear. We purified α-synuclein after overexpression in Escherichia coli and then used it to stimulate BV-2 cells or primary microglia cells from wild type or toll-like receptor 4 (TLR4)-defective mice. Enzyme linked immunosorbent assay (ELISA) and real-time PCR results confirmed that α-synuclein could enhance the production of tumor necrosis factor α (TNF-α) through TLR4 activation. Western blotting results confirmed the involvement of the TLR4/PI3K/AKT/GSK3ß signal pathway in the inflammatory response. Nuclear factor kappa B (NF-κB) could translocate to the nucleus, promoting the expression of TNF-α when stimulated by α-synuclein in BV-2 cells. Nurr1 suppressed the production of TNF-α via interaction with NF-κB/p65 and inhibiting its nuclear translocation. In addition, both NF-κB and Nurr1 appeared to be regulated by the TLR4-mediated signal pathway. Our work demonstrated that TLR4 recognized α-synuclein and activated downstream signaling mechanisms leading to the release of pro-inflammatory mediators that are contra-balanced by Nurr1 expression. In conclusion, Nurr1 is a novel participant in the neuroinflammation stimulated by α-synuclein, thus the regulation of Nurr1 may be a novel neuroprotective target for PD treatment.

The molecular mechanism of polygalasaponin F-mediated decreases in TNFα: emphasizing the role of the TLR4-PI3K/AKT-NF-κB pathway.

Polygalasaponin F (PS-F), an oleanane-type triterpenoid saponin extracted from Polygala japonica, decreases the release of the inflammatory cytokine tumor necrosis factor α (TNFα), but the precise molecular mechanisms by which this event occurs are not fully understood. To study the anti-neuroinflammatory mechanisms of PS-F, enzyme-linked immunosorbent assay was used to detect the secretion of TNFα from BV-2 microglial cells. Nuclear proteins extracted from BV-2 microglial cells stimulated by lipopolysaccharide (LPS) and pretreated with/without inhibitors were measured by Western blotting, and cell viability was evaluated by MTT analysis. The results indicated that inhibition of toll-like receptor (TLR) 4 (CLI-095 1 µg/ml), phosphatidylinositol 3-kinase (PI3K) (Ly294002 10 µM) or IκBα phosphorylation (Bay11-7082 10 µM) completely prevents the release of TNFα induced by LPS without affecting cell viability and attenuated the nuclear translocation of p65 stimulated by LPS. In addition, PS-F exhibited a similar trend regarding TNFα release, AKT phosphorylation and NF-κB translocation. These results suggest that PS-F reduces neuroinflammatory cytokine secretion through the regulation of the TLR4-PI3K/AKT-NF-κB signaling pathway.

The molecular mechanism of rotenone-induced α-synuclein aggregation: emphasizing the role of the calcium/GSK3ß pathway.

Environmental toxin exposure is associated with the development of Parkinson's disease (PD), and environmental factors can influence the onset of the majority of sporadic PD cases via genetically mediated pathways. Rotenone, a widespread pesticide, induces Parkinsonism and the formation of Lewy bodies in animals; however, the molecular mechanism that underlies α-synuclein aggregation remains unclear. Here, we assessed the aggregation of α-synuclein in PC12 cells with or without cross-linking following rotenone exposure via a variety of methods, including western blotting, immunofluorescence and electron microscopy. We demonstrated that rotenone increased the intracellular calcium levels and induced the aggregation and phosphorylation of α-synuclein in a calcium-dependent manner. Aggregated α-synuclein is typically degraded by autophagy, and rotenone impaired this process. The attenuation of autophagy and α-synuclein alterations were reversed by scavenging calcium. Calcium regulates the activity of AKT-glycogen synthase kinase 3 (GSK3)ß. We demonstrated that rotenone attenuated the phosphorylation of AKT and GSK3ß, and the elimination of calcium reversed these phenomena. As a GSK3ß inhibitor, lithium promoted autophagy and decreased the aggregation and phosphorylation of α-synuclein. GSK3ß activation through overexpression depressed autophagy and increased the total protein level and phosphorylation of α-synuclein. These results suggest that rotenone-induced α-synuclein aggregation is mediated by the calcium/GSK3ß signaling pathway.

Polygalasaponin F inhibits secretion of inflammatory cytokines via NF-κB pathway regulation.

To study the anti-neuroinflammatory mechanisms of polygalasaponin F (PS-F), ELISA method was used to detect the secretion of inflammatory cytokines. Western blot was used to detect the protein expression and phosphorylation levels. Immunofluorescence assay was used to observe the NF-κB nuclear translocation. PS-F could inhibit the release of inflammatory cytokines TNF-α and NO induced by lipopolysaccharides (LPS) and reduce the expression of inducible nitric oxide synthases (iNOS). As for MAPK-signaling pathway, PS-F could only inhibit the phosphorylation levels of p38 MAPK, but did not significantly affect the phosphorylation levels of JNK and ERK1/2 protein kinases. PS-F could inhibit NF-κB nuclear translocation in a dose-dependent manner. The results of Western blot assay were consistent with immunofluorescence assays. Meanwhile, p38-specific inhibitor SB203580 (20 µM) and p65-specific inhibitor PDTC (100 µM) were, respectively, administered as a positive control. In addition, PS-F could significantly inhibit the cytotoxicity of conditioned medium prepared by LPS-stimulated BV-2 microglia (LPS conditioned media) to neuronal PC12 cells and improve cell viability. PS-F inhibits the secretions of neuroinflammatory cytokines by the regulation of NF-κB-signaling pathway.