Curcumin exerts anti-inflammatory and antioxidative properties in 1-methyl-4-phenylpyridinium ion (MPP(+))-stimulated mesencephalic astrocytes by interference with TLR4 and downstream signaling pathway

Abstract

Neuroinflammation is closely associated with the pathophysiology of neurodegenerative diseases including Parkinson's disease (PD). Recent evidence indicates that astrocytes also play pro-inflammatory roles in the central nervous system (CNS) by activation with toll-like receptor (TLR) ligands. Therefore, targeting anti-inflammation may provide a promising therapeutic strategy for PD. Curcumin, a polyphenolic compound isolated from Curcuma longa root, has been commonly used for the treatment of neurodegenerative diseases. However, the details of how curcumin exerts neuroprotection remain uncertain. Here, we investigated the protective effect of curcumin on 1-methyl-4-phenylpyridinium ion-(MPP(+)-) stimulated primary astrocytes. Our results showed that MPP(+) stimulation resulted in significant production of tumor necrosis factor (TNF)-α, interleukin (IL-6), and reactive oxygen species (ROS) in primary mesencephalic astrocytes. Curcumin pretreatment decreased the levels of these pro-inflammatory cytokines while increased IL-10 expression in MPP(+)-stimulated astrocytes. In addition, curcumin increased the levels of antioxidant glutathione (GSH) and reduced ROS production. Our results further showed that curcumin decreased the levels of TLR4 and its downstream effectors including NF-κB, IRF3, MyD88, and TIRF that are induced by MPP(+) as well as inhibited the immunoreactivity of TLR4 and morphological activation in MPP(+)-stimulated astrocytes. Together, data suggest that curcumin might exert a beneficial effect on neuroinflammation in the pathophysiology of PD.

Keywords: 1-Methyl-4-phenylpyridinium ion; Astrocytes; Curcumin; Neuroinflammation; Oxidative stress; Toll-like receptor 4.

Fig. 1

Effect of curcumin on cell viability in primary astrocytes. Cells were incubated with different concentrations of curcumin (0, 1, 2, 4, 8, or 16 μM) for 48 h. Cell viability was determined by MTT assay. Data are expressed as the mean ± standard deviation. **P < 0.01, compared with control untreated astrocytes

Fig. 2

Curcumin inhibits the MPP⁺-induced activation of primary astrocytes. GFAP immunostaining in primary cultured astrocytes. Curcumin reduced GFAP expression compared with the MPP⁺-stimulated astrocytes. Magnification, ×400, scale bars = 20 μm

Fig. 3

Effect of curcumin on MPP⁺-induced cytokine production in primary astrocytes. Expression levels of a TNF-α, b IL-6, and c IL-10 in cell culture supernatants were measured. Curcumin downregulates TNF-a and IL-6 expression while it increased IL-10 production in MPP⁺-stimulated astrocytes. Data are expressed as the mean ± standard deviation. ^## P < 0.01, compared with control untreated astrocytes; *P < 0.05, **P < 0.01, compared with MPP⁺-stimulated astrocytes

Fig. 4

Effect of curcumin on MPP⁺-induced oxidative stress in primary astrocytes. a Expression levels of ROS in astrocytes were determined by monitoring a conversion of DCF H-DA to DCF. b Expression levels of GSH in the medium of primary astrocytes. Data are expressed as the mean ± standard deviation. ^## P < 0.01, compared with control untreated astrocytes; **P < 0.01, compared with MPP⁺-stimulated astrocytes

Fig. 5

Curcumin inhibits MPP⁺-induced NF-κB and IRF3 activation in primary astrocytes. Real-time PCR was performed to detect the mRNA levels of a NF-κB, b IRF3, c MyD88, and d TIRF/TRAM in primary astrocytes. e Western blot analysis was performed to determine the protein levels of NF-κB, IRF3, MyD88, and TIRF/TRAM. f Relative protein levels were quantified by a gray analysis with normalization to β-actin. Data are presented as mean ± SD; ^## P < 0.01, compared with control untreated astrocytes; *P < 0.05, **P < 0.01, compared with MPP⁺-stimulated astrocytes

Fig. 6

Curcumin attenuates the expression and immunoreactivity of TLR4 in MPP⁺-induced astrocytes. a Real-time PCR analysis was performed to determine TLR4 mRNA level of TLR4 in primary astrocytes. b The protein levels of TLR4 were determined by Western blot analysis. Relative TLR4 protein level was quantified by a gray analysis with normalization to β-actin. c The immunofluorescence of TLR4 in astrocytes was determined. TLR4 was shown in red and DAPI was shown in blue. Magnification, ×400, scale bars = 20 μm. Data are presented as mean ± SD; ^## P < 0.01, compared with control untreated astrocytes; *P < 0.05, compared with MPP⁺-stimulated astrocytes (color figure online)