Glia maturation factor deficiency suppresses 1-methyl-4-phenylpyridinium-induced oxidative stress in astrocytes - PubMed (original) (raw)

Glia maturation factor deficiency suppresses 1-methyl-4-phenylpyridinium-induced oxidative stress in astrocytes

Mohammad Moshahid Khan et al. J Mol Neurosci. 2014 Aug.

Abstract

Inflammation is closely intertwined with pathogenesis of Parkinson's disease (PD). Increasing evidence suggests that inhibition of glia-mediated inflammation might represent a promising therapeutic target for PD. Glia maturation factor (GMF), an inflammatory protein, predominantly localized in astrocytes is previously isolated, sequenced and cloned in our laboratory. In the present investigation, we demonstrate that GMF-deficiency in astrocytes upregulates the antioxidant status and limit the extent of lipid peroxidation and production of reactive oxygen species (ROS) along with diminished nuclear factor-κB-mediated inflammatory responses in 1-methyl-4-phenylpyridinium (MPP(+))-induced toxicity. Primary astrocytes obtained from wild-type (Wt) and GMF-deficient (GMF-KO) mice were treated with 5, 10, and 20 μM MPP(+) for 24, 48, and 72 h in vitro. Our results show decreased release of ROS and increased level of glutathione in astrocytes obtained from GMF-KO mice when compared to astrocytes derived from Wt mice following MPP(+) treatment. Additionally, we found decreased activity of NF-κB, and reduced levels of proinflammatory tumor necrosis factor- α, interleukin-1β (IL-1β), IL-17, IL-33, and chemokine (C-C motif) ligand 2 (CCL2) in GMF-KO astrocytes when compared to Wt astrocytes. Our overall results suggest that GMF-KO astrocytes are significantly resistant to MPP(+) toxicity when compared to Wt astrocytes.

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Figures

Fig. 1

Fig. 1

Primary cultures of astrocytes derived from Wt and GMF-KO mice were incubated with various doses of MPP+ (0–100 μM) for 24 and 48 h. MPP+-induced cytotoxicity was measured by MTT reduction and LDH release assays. a MTT assay show higher cell viability in GMF-KO astrocytes when compared to Wt astrocytes, and b Wt astrocytes released more LDH when compared to GMF-KO astrocytes following MPP+ treatments. *p<0.05 (_n_=4), compared with the Wt astrocytes and GMF-KO astrocytes treated with MPP+ by student t test analysis

Fig. 2

Fig. 2

Primary cultures of astrocytes derived from Wt and GMF-KO mice were incubated with MPP+ (5, 10, 20 μM) for 24, 48, and 72 h. Levels TBARS (a), GSH (b), and ROS (c) were assayed in the cell lysate. TBARS and GSH (_n_=4); ROS (_n_=5); Values are means±SEM. *p<0.05, compared with the Wt astrocytes and GMF-KO astrocytes treated with MPP+

Fig. 3

Fig. 3

Reduction in NF-κB activity in GMF-KO astrocytes compared to Wt primary mouse astrocytes. Astrocytes were seeded and treated with 20 μM MPP+ for 24, 48, and 72 h and levels of NF-κB activity were measured by ELISA according to manufacturer's protocol. A significant time-dependent suppression of NF-κB activation following MPP+ treatment was seen in GMF-KO astrocytes as compared with Wt astrocytes. Values are means±SEM (_n_=6). *p<0.05, compared with the Wt astrocytes and GMF-KO astrocytes treated with MPP+

Fig. 4

Fig. 4

Downregulation of MPP+-dependent proinflammatory iNOS expression in GMF-KO astrocytes. Astrocytes derived from Wt and GMF-KO mice were incubated with MPP+ (20 μM) for 72 h. Cell lysates (35 μg protein per lane) were subjected to SDS-polyacrylamide gel electrophoresis followed by electroblotting. The blots were probed with anti-iNOS antibody and anti-β-actin antibody. Actin served as internal marker showing equal sample loading. Bands intensity was measured by densitometry and quantified using NIH-Image J software. Expression of iNOS was significantly decreased in GMF-KO astrocytes when compared to Wt astrocytes at 20 μM concentration of MPP+ after 72 h. Values are means±SEM (_n_=3). *p<0.05, compared with the Wt astrocytes and GMF-KO astrocytes treated with MPP+, AU arbitrary units

Fig. 5

Fig. 5

Nitric oxide (NO) levels were significantly decreased in GMF-KO astrocytes following MPP+ treatment in a dose- (5, 10, and 20 μM) and time-dependent (24, 48, and 72 h) manner. Astrocytes were incubated with MPP+ for 24, 48, and 72 h and then the culture media were collected for NO assay using commercial kit. Values are expressed as means±SEM (_n_=5). *p<0.05, compared with the Wt astrocytes and GMF-KO astrocytes treated with MPP

Fig. 6

Fig. 6

Reduced expressions of inflammatory cytokines/chemokine in primary cultures of GMF-KO astrocytes compared to Wt astrocytes following MPP+ treatment. Astrocytes were incubated with MPP+ for 24, 48, and 72 h at 5, 10, and 20 μM MPP+. After the incubation period was over the culture media were collected for the assay of proinflammatory cytokines and chemokine by ELISA. Levels of TNF-α, IL-1β, IL-17, IL-33, and CCL2 were significantly decreased in the GMF-KO astrocytes when compared to the Wt astrocytes in dose- (5, 10, and 20 μM) and time-dependent manner (24, 48, and 72 h) following MPP+ treatment. Values are means±SEM (_n_=5). *p<0.05, compared with the Wt astrocytes and GMF-KO astrocytes treated with MPP+

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