Korean J Physiol Pharmacol.  2015 May;19(3):219-228. 10.4196/kjpp.2015.19.3.219.

Dieckol Attenuates Microglia-mediated Neuronal Cell Death via ERK, Akt and NADPH Oxidase-mediated Pathways

• Cui Y
• Park JY
• Wu J
• Lee JH
• Yang YS
• Kang MS
• Jung SC
• Park JM
• Yoo ES
• Kim SH
• Ahn Jo S
• Suk K
• Eun SY

• KMID: 1791422
• DOI: http://doi.org/10.4196/kjpp.2015.19.3.219

Abstract

Excessive microglial activation and subsequent neuroinflammation lead to synaptic loss and dysfunction as well as neuronal cell death, which are involved in the pathogenesis and progression of several neurodegenerative diseases. Thus, the regulation of microglial activation has been evaluated as effective therapeutic strategies. Although dieckol (DEK), one of the phlorotannins isolated from marine brown alga Ecklonia cava, has been previously reported to inhibit microglial activation, the molecular mechanism is still unclear. Therefore, we investigated here molecular mechanism of DEK via extracellular signal-regulated kinase (ERK), Akt and nicotinamide adenine dinuclelotide phosphate (NADPH) oxidase-mediated pathways. In addition, the neuroprotective mechanism of DEK was investigated in microglia-mediated neurotoxicity models such as neuron-microglia co-culture and microglial conditioned media system. Our results demonstrated that treatment of anti-oxidant DEK potently suppressed phosphorylation of ERK in lipopolysaccharide (LPS, 1 microg/ml)-stimulated BV-2 microglia. In addition, DEK markedly attenuated Akt phosphorylation and increased expression of gp91(phox), which is the catalytic component of NADPH oxidase complex responsible for microglial reactive oxygen species (ROS) generation. Finally, DEK significantly attenuated neuronal cell death that is induced by treatment of microglial conditioned media containing neurotoxic secretary molecules. These neuroprotective effects of DEK were also confirmed in a neuron-microglia co-culture system using enhanced green fluorescent protein (EGFP)-transfected B35 neuroblastoma cell line. Taken together, these results suggest that DEK suppresses excessive microglial activation and microglia-mediated neuronal cell death via downregulation of ERK, Akt and NADPH oxidase-mediated pathways.

Keyword

Akt; Dieckol; gp91(phox); Microglia; Neuron-microglia co-culture

MeSH Terms

Adenine
Cell Death*
Cell Line
Coculture Techniques
Culture Media, Conditioned
Down-Regulation
Microglia
NADP*
NADPH Oxidase
Neuroblastoma
Neurodegenerative Diseases
Neurons*
Neuroprotective Agents
Niacinamide
Phosphorylation
Phosphotransferases
Reactive Oxygen Species
Adenine
Culture Media, Conditioned
NADP
NADPH Oxidase
Neuroprotective Agents
Niacinamide
Phosphotransferases
Reactive Oxygen Species

Figure

• 

  Fig. 1 Dose determination for DEK treatment. (A) Cell viabilities were examined in BV-2 microglia using MTT assay to determine the safe and appropriate doses of DEK treatment. BV-2 cells were treated for 25 h with the indicated concentration of DEK. (B, C) Microglial cells were pretreated with DEK for 1 h and then stimulated with LPS (1 µg/ml) in the presence of DEK for 24 h. Effects of DEK on NO release were examined in culture media of LPS-stimulated primary microglial cells (B) and BV-2 microglial cell line (C) using Griess reagent. Values are the mean±S.E.M. of four samples in one independent experiment. The data were replicated in three repeated independent experiments. ###p<0.001 as compared to untreated control group and **p<0.01; ***p<0.001 as compared to LPS alone-treated group.

• 

  Fig. 2 Neuroprotective effects of DEK against microglial-mediated neuronal cell death. (A) BV-2 microglial cells were pretreated with DEK (50 µM) for 6 h. After wash-out, microglial cells were further incubated with LPS (1 µg/ml) for 24 h in the absence of DEK. MTT assay-based cell viabilities of HT-22 neurons were measured after different conditioned media treatment for 24 h. Groups are summarized as follows: the conditioned media from control BV-2 cells (Control-CM); LPS was added to the conditioned media from control BV-2 cells (Control CM+LPS); the conditioned media from LPS alone-treated BV-2 cells (LPS-CM); the conditioned media from LPS-treated BV-2 cells after DEK pretreatment (LPS/DEK-CM). (B) BV-2 microglial cells were pretreated with DEK (50 µM) for 6 h and then washed. B35-EGFP neurons were added to these BV-2 microglial cells to construct neuron-microglia co-culture system. Then, LPS (1 µg/ml) was stimulated for 24 h in the absence of DEK. The numbers of B35-EGFP neuronal cells were assessed by counting EGFP-positive cells under a fluorescent microscope. Fluorescent images of five random fields per well were captured and counted. Values are the mean±S.E.M. of four samples in one independent experiment. The data were replicated in three repeated independent experiments. ###p<0.001 as compared to the untreated control group and ***p<0.001 as compared to LPS alone-treated group.

• 

  Fig. 3 Inhibitory effects of DEK on ERK phosphorylation in LPS-stimulated microglia. BV-2 microglial cells were pretreated with DEK (50 µM) for 1 h and then stimulated with LPS (1 µg/ml) in the presence of DEK for 30 min. Total proteins were subjected to SDS-PAGE, and probed with antibodies against phospho-ERK (A) and phospho-p38 (B) using Western blot analysis. Optical densities of individual protein bands were normalized to the corresponding levels of ERK and p38. Values are the mean±S.E.M. of four samples in one independent experiment. The data were replicated in three repeated independent experiments. #p<0.05, ###p<0.001 as compared to untreated control group and *p<0.05; ***p<0.001 as compared to LPS alone-treated group.

• 

  Fig. 4 Inhibitory effects of DEK on Akt-mediated pathways in LPS-stimulated microglia. BV-2 microglial cells were pretreated with DEK (50 µM) for 1 h and then stimulated with LPS (1 µg/ml) in the presence of DEK for 30 min. Total proteins were subjected to SDS-PAGE, and probed with antibodies against phospho-Akt. (A) Optical densities of individual protein bands were normalized to the corresponding levels of Akt. Levels of NF-κB p65 subunit in nuclear fraction (B) and IκBα in cytosol fraction (C) were determined using Western blot analysis. Optical densities of individual protein bands were normalized to the corresponding levels of β-actin or housekeeping nuclear protein TATA-binding protein (TBP). Values are the mean±S.E.M. of four samples in one independent experiment. The data were replicated in three repeated independent experiments. #p<0.05, ##p<0.01; ###p<0.001 as compared to untreated control group and *p<0.05; **p<0.01 as compared to LPS alone-treated group.

• 

  Fig. 5 Inhibitory effects of DEK on gp91phox expression in LPS-stimulated microglia. (A) The time course of LPS-induced expression of gp91phox, a catalytic component of NADPH oxidase complex, was investigated. BV-2 microglial cells were stimulated with LPS (1 µg/ml) for different time periods. Thereafter, protein fractions were extracted and subjected to Western blot analysis with anti-gp91phox antibody. (B) BV-2 cells were pretreated with DEK (50 µM) for 1 h and then stimulated with LPS (1 µg/ml) in the presence of DEK for 6 h, based on the time course data. Thereafter, protein fractions were extracted and subjected to Western blot analysis to investigate the effect of DEK on LPS-induced gp91phox expression. (C) BV-2 microglial cells were pretreated with apocynin and DPI, as NADPH oxidase inhibitors, for 1 h and then stimulated with LPS (1 µg/ml) in the presence of apocynin and DPI for 24 h. The amount of NO released in culture media was examined using Griess reagent to investigate effects of NADPH oxidase inhibitors on LPS-induced NO release. (D) Free radical scavenging activity of DEK was performed in cell-free system using DPPH assay. (E) The time course of LPS-induced intracellular ROS levels was investigated by spectrofluorometer using ROS-sensitive fluorescent dye DCF-DA. (F) BV-2 cells were pretreated with DEK (1, 10 and 50 µM) for 1 h and then stimulated with LPS (1 µg/ml) in the presence of DEK for 30 min. The intracellular ROS levels were determined by spectrofluorometer using ROS-sensitive fluorescent dye DCF-DA. Optical densities of individual protein bands were normalized to the corresponding levels of β-actin. Values were expressed as mean±S.E.M. #p<0.05, ##p<0.01 ###p<0.001 as compared to untreated control group and *p<0.05 **p<0.01; ***p<0.001 as compared to LPS alone-treated group.

• 

  Fig. 6 Neuroprotective effects of the blockade of PI3K/Akt and NADPH oxidase against microglia-mediated neuronal cell death. Neuroprotective effects of the blockade of PI3K/Akt and NADPH oxidase were investigated against microglia-mediated neuronal cell death in microglial conditioned media system (A) and in neuronmicroglia co-culture system (B). Wortmannin (100 nM), LY294002 (25 µM) and apocynin (500 µM) were pretreated in both experimental systems and then, neuronal cell deaths were evaluated as shown in Fig. 2 (See 'Materials and methods' for the detailed description). Values are the mean±S.E.M. of four samples in one independent experiment. The data were replicated in three repeated independent experiments. ###p<0.001 as compared to untreated control group and **p<0.01 ***p<0.001 as compared to LPS alone-treated group.

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