As the resident innate immune cells of the central nervous system (CNS), microglia regulate inflammatory response and restore CNS homeostasis by removing pathogens and exogenous agents, such as lipopolysaccharide (LPS) (Park
In neurotoxicological and immunological research, BV2 microglial cells have been widely used as a substitute for primary microglia (Li
Soybean, which is commonly consumed in traditional Asian diets, has been widely studied for its beneficial effects on the prevention of breast cancer, heart disease, and dementia (Lammersfeld
This study aimed to identify the potential antineuroinflammatory effects of 7,3’,4’-THIF on LPS-induced microglial activation and to explore the possible underlying mechanisms by measuring iNOS, COX-2, IL-6, and ROS production. Furthermore, the antineuroinflammatory properties of 7,3’,4’-THIF were evaluated by assessing MAPK and GSK-3β signaling activation.
7,3’,4’-THIF was obtained from Indofine Chemical Company, Inc (San Mateo, CA, USA). The purity of 7,3’,4’-THIF was >98.1%. Dimethyl sulfoxide (DMSO), 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and lipopolysaccharide (LPS) (Escherichia coli, 026:B6) were purchased from Sigma Chemical Co (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Hyclone (Logan, UT, USA). Fetal bovine serum (FBS), 0.25% trypsin–EDTA, and penicillin/streptomycin were obtained from GIBCO-BRL (Grand Island, NY, USA). Dulbecco’s phosphate-buffered saline (D-PBS) was obtained from Welgene (Gyeongsan, Korea). Anti-β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc (Dallas, TX, USA). Other primary antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). Secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc (West Grove, PA, USA). All other chemicals were of analytical grade and were purchased from Sigma Chemical Co.
BV-2 microglial cells (catalog number: CRL-2469) were obtained from ATCC (Manassas, VA, USA). BV-2 microglial cells were maintained in DMEM supplemented with 10% heat-inactivated FBS (v/v) and 0.1% penicillin/streptomycin (v/v) in a humidified atmosphere of 5% CO2 and 95% air at 37°C. When the cells reached 80-90% confluency in 100-mm2 cell culture dishes, they were dissociated with trypsin-EDTA and sub-cultured in culture dishes. LPS was prepared immediately before use as a 100 μg/ml stock and diluted in PBS to the indicated final concentration. 7,3’,4’-THIF was dissolved in DMSO and the stock solutions were added directly to the culture medium. In all experiments, cells were treated with the indicated concentrations of 7,3’,4’-THIF in serum-free DMEM with or without LPS (100 ng/mL) and control cells were treated with DMSO alone. The final concentration of solvent was always <0.1% (v/v).
BV2 microglial cells (2.5×105 cells/well in 24-well plates) were treated with vehicle (0.1% v/v DMSO) or 7,3’,4’-THIF (10, 25, or 50 μM) with or without LPS (100 ng/mL) treatment and incubated at 37°C in a 5% CO2 incubator. Cell viability was determined 24 h later by treating with the MTT solution (5 mg/mL) for 2 h. Blue formazan crystals are formed due to the action of mitochondrial dehydrogenases in viable cells on MTT. The samples were dissolved in DMSO, and absorbance was measured at 540 nm using a microplate reader (SpectraMax 250, Molecular Device, Sunnyvale, CA, USA). Results are presented as the percentage of metabolized MTT relative to that of controls, as determined by absorbance measurements.
BV2 microglial cells (2.5×105 cells/well in 24-well plates) were stimulated with vehicle (0.1% v/v DMSO) or 7,3’,4’-THIF (10, 25, or 50 μM) for 30 min before (pretreatment) or after (posttreatment) treatment with LPS (100 ng/mL). Culture supernatant was collected 24 h later, and nitrite concentration was determined by mixing 100 μL of culture medium with an equal volume of Griess reagent [0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride and 1% sulfanilamide in 5% phosphoric acid] in a 96-well plate. Nitrite concentration was calculated using the standard solution of sodium nitrite diluted in cell culture medium. Absorbance of each well was measured at 540 nm using a microplate reader (SpectraMax M2, San Jose, CA, USA).
BV2 microglial cells were seeded at a density of 5×105 cells/well in 6-well plates and incubated overnight. After pretreatment with 7,3’,4’- THIF (10, 25, or 50 μM) or vehicle (0.1% v/v DMSO) for 30 min, the cells were incubated with LPS (100 ng/mL) for 6 h. IL-6 concentration in the culture medium was determined using an IL-6 ELISA kit (Elabscience Biotechnology Co., Ltd, Wuhan, China) according to the manufacturer’s instructions.
Intracellular ROS production was assessed using the DCFH-DA kit (Abcam plc, Cambridge, UK) following the manufacturer’s protocol. In brief, BV2 microglial cells (2.5×104 cells/well in 96-well black plates) were stained with the DCFH-DA solution (20 μM) at 37°C for 45 min in the dark. The cells were rinsed with wash buffer. Vehicle (0.1% v/v DMSO) or 7,3’,4’-THIF (10, 25, or 50 μM) was added 30 min before LPS activation. After a 6-h incubation at 37°C, absorbance of each well was determined at Ex/Em=485/535 nm in the end-point mode using a microplate reader (SpectraMax M2). DCFH-DA fluorescent images were captured with a fluorescence microscope (20× magnification).
Western blotting was performed as previously described (Ko
All results were analyzed using Prism 6.0 (GraphPad Software, Inc., San Diego, CA, USA) and are expressed as mean ± SEM. Statistical analyses were performed using one-way analysis of variance, followed by either the Newman–Keuls
To examine whether 7,3’,4’-THIF is neurotoxic, the MTT assay was conducted in BV2 microglial cells with or without LPS stimulation. 7,3’,4’-THIF demonstrated no neurotoxicity at any concentration (10, 25, or 50 μM) used in this study (Fig. 2A, 2B). Therefore, these concentrations of 7,3’,4’-THIF were used in subsequent experiments.
To assess the antineuroinflammatory effects of 7,3’,4’-THIF in LPS-stimulated BV2 microglial cells, NO concentration was measured using the Griess reagent. LPS challenge significantly increased NO concentration by almost 25 μM compared with the control values (
LPS treatment activates iNOS in microglia, resulting in increased production of NO. LPS also induces COX-2 overexpression, which mediates prostaglandin and inflammatory cytokine synthesis (Kim
To examine the effects of 7,3’,4’-THIF on proinflammatory cytokine production, we measured IL-6 release using ELISA. LPS-stimulated BV2 cells showed significantly increased IL-6 production to 671.3% of the control value (
LPS-induced ROS accumulation promotes neuronal cell death and promotes excessive inflammatory responses (Cui
To further understand mechanisms underlying the action of 7,3’,4’-THIF, we examined the expression levels of ERK and JNK using western blotting. In the presence of LPS, the phosphorylation levels of ERK and JNK increased to 170.9% (
Finally, we investigated the expression levels of NF-κB, a crucial transcription factor that modulates neuroinflammatory responses in microglia. LPS significantly increased NF-κB expression to 189.4% of the control values (
The daidzein metabolite 7,3’,4’-THIF possesses potential pharmacological activities. Owing to its antioxidant, antipollutant, and antiamnesic effects, 7,3’,4’-THIF may be a novel candidate for pharmaceutical applications (Huang
Microglial activation is an important feature of neurodegenerative disorders including AD. Therefore, we assessed the effect of 7,3’,4’-THIF on neuroinflammation using LPS-stimulated BV2 microglial cells in the present study. Activated microglia increases the levels of proinflammatory cytokines IL-6, IL-1β, and TNF-α (Lull and Block, 2010). These mediators contribute to neuronal cell injury and eventually trigger inflammatory cascades (Hemmer
Evidence indicates that elevated ROS levels are associated with neurodegenerative disorders such as AD (Block
Several results suggest that the MAPK, PI3K/Akt, GSK-3β, and NF-κB signaling cascades mediate inflammatory processes. Previous studies on microglia have shown that phosphorylated MAPK, PI3K/Akt, and GSK-3β activate NF-κB, leading to elevated expression of inflammatory mediators such as iNOS, COX-2, and proinflammatory cytokines (Surh
In conclusion, as depicted in Fig. 6, 7,3’,4’-THIF, a major daidzein metabolite, effectively protected BV2 microglial cells against LPS-induced neurotoxicity, oxidative stress, and inflammatory responses. These beneficial effects may result from the inhibition of NO release and reduction of iNOS, COX-2, and proinflammatory cytokine levels. In addition, 7,3’,4’-THIF significantly suppressed LPS-induced ROS production. The antineuroinflammatory effects of 7,3’,4’-THIF may result from the inhibition of MAPK and NF-κB signaling pathways. Further studies are warranted to investigate whether 7,3’,4’-THIF protects against inflammation-induced neuronal injury in animal models. 7,3’,4’-THIF may be a promising approach for the treatment of inflammation-related neurodegenerative disorders.
This work was supported by a grant (NRF-2017R1A2B2002428) from the Basic Science Research Program through the National Research Foundation of South Korea.
The authors have no conflicts of interest to declare.