Vanillin Protects Dopaminergic Neurons against Inflammation-Mediated Cell Death by Inhibiting ERK1/2, P38 and the NF-κB Signaling Pathway

Abstract

Neuroinflammation plays a very important role in the pathogenesis of Parkinson's disease (PD). After activation, microglia produce pro-inflammatory mediators that damage surrounding neurons. Consequently, the inhibition of microglial activation might represent a new therapeutic approach of PD. Vanillin has been shown to protect dopaminergic neurons, but the mechanism is still unclear. Herein, we further study the underlying mechanisms in lipopolysaccharide (LPS)-induced PD models. In vivo, we firstly established rat models of PD by unilateral injection of LPS into substantia nigra (SN), and then examined the role of vanillin in motor dysfunction, microglial activation and degeneration of dopaminergic neurons. In vitro, murine microglial BV-2 cells were treated with vanillin prior to the incubation of LPS, and then the inflammatory responses and the related signaling pathways were analyzed. The in vivo results showed that vanillin markedly improved the motor dysfunction, suppressed degeneration of dopaminergic neurons and inhibited microglial over-activation induced by LPS intranigral injection. The in vitro studies demonstrated that vanillin reduces LPS-induced expression of inducible nitric oxide (iNOS), cyclooxygenase-2 (COX-2), IL-1β, and IL-6 through regulating ERK1/2, p38 and NF-κB signaling. Collectively, these data indicated that vanillin has a role in protecting dopaminergic neurons via inhibiting inflammatory activation.

Keywords: MAPK; NF-κB; Parkinson’s disease; inflammation; microglia; vanillin.

Figure 1

Vanillin administration improves the motor dysfunction by lipopolysaccharide (LPS) intranigral injection. Rats were treated with the vehicle or vanillin (5, 10, or 20 mg/kg/day) for three days prior to LPS intranigral injection and this lasted for 24 days. The number of turns after two (A); and four weeks (B) was induced by apomorphine in Parkinson’s disease (PD) model rats. # p < 0.01 vs. the sham-operated control rats; and * p < 0.05 and ** p < 0.01 vs. the vehicle treated LPS-injected rats.

Figure 2

Vanillin administration increases the survival rate of dopaminergic neurons in the substantia nigra (SN). The PD model rats were anaesthetized and decapitated to obtain the SN after 24 days of vanillin administration. (A) Immunohistochemical results of tyrosine hydroxylase (TH)-positive cells, scale bar represents 1.0 mm; (B) The survival ratio of the dopaminergic neurons; and (C) TH expression. # p < 0.01 vs. the sham-operated control rats; and. * p < 0.05 and ** p < 0.01 vs. the vehicle treated LPS-injected rats.

Figure 3

Vanillin inhibits LPS-induced activation of microglia in the SN. The PD model rats were anaesthetized and decapitated to obtain the SN after 24 days of vanillin administration. (A) immunohistochemical results of ionized calcium-binding adaptor molecule-1 (IBA-1)-positive cells, scale bar represents 100 μm; (B) the number of IBA-1-positive cells; (C) O-X42 expression. # p < 0.01 vs. the sham-operated control rats; and. ** p < 0.01 vs. the vehicle treated LPS-injected rats.

Figure 4

Vanillin attenuates the production of inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX-2) induced by LPS in BV-2 cells. Cells were pretreated with vanillin (100, 200, 300, 400 and 500 nM) for 1 h and were then incubated with LPS (1 µg/mL) for 4 h. The mRNA levels of iNOS (A); and COX-2 (B) in BV-2 cells were examined by quantitative real-time PCR. The protein levels of iNOS (C,D) and COX-2 (C, E) were determined by Western blot. The data were normalized to β-actin. # p < 0.01 vs. no treated group. * p < 0.05 and ** p < 0.01 vs. the vanillin-untreated LPS-stimulated group.

Figure 5

Effects of vanillin on LPS-induced proteins production and mRNA expression of pro-inflammatory cytokines in BV-2 cells were examined. The mRNA levels of IL-1β (A), IL-6 (C) and TNF-α (E) in BV-2 cells were examined by quantitative real-time PCR. Cells were pretreated with vanillin (100, 200, 300, 400 and 500 nM) for 1 h, and then incubated with LPS (1 µg/mL) for 4 h; the expression level of IL-1β (B), IL-6 (D) and TNF-α (F) was determined via ELISA. Cells were pretreated with vanillin (500 nM) for 1 h, and then incubated with LPS (1 µg/mL) for 12 h. # p < 0.01 vs. no treated group. ** p < 0.01 vs. the vanillin-untreated LPS-stimulated group.

Figure 5

Effects of vanillin on LPS-induced proteins production and mRNA expression of pro-inflammatory cytokines in BV-2 cells were examined. The mRNA levels of IL-1β (A), IL-6 (C) and TNF-α (E) in BV-2 cells were examined by quantitative real-time PCR. Cells were pretreated with vanillin (100, 200, 300, 400 and 500 nM) for 1 h, and then incubated with LPS (1 µg/mL) for 4 h; the expression level of IL-1β (B), IL-6 (D) and TNF-α (F) was determined via ELISA. Cells were pretreated with vanillin (500 nM) for 1 h, and then incubated with LPS (1 µg/mL) for 12 h. # p < 0.01 vs. no treated group. ** p < 0.01 vs. the vanillin-untreated LPS-stimulated group.

Figure 6

Vanillin regulates MAPKs and NF-κB activation in LPS-stimulated BV-2 cells. (A) the expression of MAPKs and NF-κB p65 was examined via Western blot. The phosphorylation ratio of ERK1/2 (B); p38 (C); JNK1/2 (D); and NF-κB p65 (E) was quantified. Each value was then expressed relative to the control (LPS no treatment), which was set as 1.00. # p < 0.01 vs. no control. ** p < 0.01 vs. treated with LPS alone within the same time point.