A Nitroalkene Benzoic Acid Derivative Targets Reactive Microglia and Prolongs Survival in an Inherited...
(20 mL, 369.2 mmol), ammonium acetate (2.55 g, 33.1 mmol),
and acetic acid (30 mL) were sequentially introduced into a
round bottom flask; then it was placed with stirring in an oil
bath preheated at 90 °C for 4 h. The reaction mixture was then
allowed to cool, the precipitate was isolated via filtration and
wash with cold water. After drying the desired product was
obtained as a yellow solid (4.20 g, 82%). mp 270–272 °C
(decomposition is observed). 1H-NMR (acetone-d6): δ = 8.30
(d, J = 13.6 Hz, 1H, CH=CHNO2); 8.18 (d, J = 13.6 Hz, 1H,
CH=CHNO2); 8.00 (d, J = 8.4 Hz, 2H, H-Ar); 7.97 (d, J =
8.4 Hz, 2H, H-Ar).
intraperitoneally injected with LPS (1 mg/kg). Differences in
luciferase activity were compared between groups after 3 h
following LPS injection. To accomplish that, 150 mg/kg of
the substrate D-luciferin (#K9918PE, XenoLight) dissolved in
PBS, pH 7.4 was injected intraperitoneally to each mouse.
Mice were anesthetized with isoflurane (3.0% induction,
2.5% maintenance), placed in a ventral position in the light-
tight imaging chamber, and imaged 10 min after luciferin
injection using bioluminescence and X-ray modes (5 s acqui-
sition, performed in Preclinical In-Vivo Xtreme II Optical/X-
ray imaging system, Bruker, USA). Changes in NF-κB activ-
ity were calculated after comparing the differences of lumi-
nescence between groups, using ImageJ software.
13C-NMR: δ = 167.1 (C=O); 140.1 (CH=CHNO2); 138.3
(CH=CHNO2); 134.9 (C4-Ar); 133.8 (C1-Ar); 130.3
(C2,C3,C5,C6-Ar). MS (IE, 70 eV): m/z(%) = 193 (M+, 86),
148 (51, M+–CO2H), 121 (10, M+–CH=CHNO2), 102 (43:
148–NO2), 91 (85: 121–H2C=O), 77 (100, Ar).
LPS Challenge in Mice C57BL/6 mice were randomized divid-
ed into 3 groups (n = 3 mice, per group) and intraperitoneally
injected with BANA (50 mg/kg), benzoic acid (50 mg/kg), or
vehicle (DMSO 10% v/v in phosphate buffer). After 1 h ani-
mals were intraperitoneally injected with LPS (10 mg/kg) or
PBS. Two hours following injection mice were sacrificed, and
after peritoneal wash, blood was extracted and were both
stored to measure IL-1β with a commercially available
ELISA kit (BD OptEIA).
BANA Electrophilic Reactivity The electrophilic reactivity of
the BANA compound was determined by analyzing the UV-
Visible spectra of the reaction between BANA and β-
mercaptoethanol. BANA (30 μM) was incubated with β-
mercaptoethanol (300 μM) in phosphate buffer (20 mM)
pH = 7.4, and scans were taken each minute up to 10 min.
Sulforhodamine B Assay Cell viability was performed by the
sulforhodamine B assay. Briefly, cells were placed in a 96-
multi-well plate and incubated with different concentrations of
BANA (5–110 μM) for 24 h. After media removal, cells were
washed twice with PBS pH = 7.4. Cells were fixed with tri-
chloroacetic acid for 1 h at 4 °C and then washed five times
with distilled water. A total of 50 μL of sulforhodamine B
0.4% m/v in acetic acid 1% v/v was added to each well and
incubated 30 min at room temperature. After staining, the
plate was washed at least five times with acetic acid 1% v/v.
Once the plate was dry the protein-bound dye was dissolved in
10 mM Tris and the absorbance at 570 nm was read using a
microplate spectrophotometer. The concentration which killed
half of the tested cells in culture (LC50) was determined using
GraphPad Prism 7.03.
Study of Nuclear Translocation of NF-κB in HT-29 Reporter
Cell Line The effect of BANA on nuclear translocation of
NF-κB was studied by using the HT-29 NF-κB reporter cell
line as previously described [31]. Briefly, cells were treated
with increasing doses of BANA (10, 20, and 30 μM), benzoic
acid (30 μM), dimethyl fumarate (30 μM), or vehicle
(DMSO) for 3 h. After treatment, cells were stimulated and
coincubated with TNFα (1 ng/ml) for 24 h. Finally, cells were
trypsinized and resuspended for flow cytometry analysis.
Cells were analyzed using an Attune NxT Flow Cytometer
(Thermo Fisher Scientific) equipped with 488 and 405 nm
lasers. Attune NxT Software was used for data acquisition
and FlowJo v.10 for data analysis. Green Fluorescent
Protein and propidium iodide fluorescence emissions were
detected using band-pass filters 530/30 and 695/40, respec-
tively. For each sample, 10,000 counts gated on a forward
scatter versus side scatter dot plot, excluding doublets, were
recorded. Only single living cells (cells that excluded
propidium iodide) were considered for results comparison.
Analysis of NF-κB Activation In Vivo For NF-κB in vivo imag-
ing studies, we used the NF-κB-RE-Luc random transgenic
mouse model (Taconic, BALB/c-Tg(Rela-luc)31Xen) aged
6–8 weeks. These animals carry a transgene containing 6
NF-κB-responsive elements (RE) from the CMVα (immedi-
ate early) promoter placed upstream of a basal SV40 promot-
er, and a modified firefly luciferase cDNA (Promega pGL3).
The reporter is inducible by LPS and TNFα intraperitoneal
injection [30]. Animals were randomized divided into four
groups and intraperitoneally injected with increasing doses
of BANA (10, 20, and 30 mg/kg), benzoic acid (30 mg/kg),
dimethyl fumarate (30 mg/kg), or vehicle (DMSO 10% (v/v)/
PEG 20% (v/v) in phosphate buffer). After 2 h, animals were
Effect of BANA on Nuclear Translocation of NF-κB in BV2
Murine Microglial Cell Line The effect of BANA on nuclear
translocation of NF-κB was studied by immunocytochemistry
using murine microglia cell line BV2. Cells were treated with
BANA (10, 20, and 30 μM), benzoic acid (30 μM), or dimeth-
yl fumarate (30 μM) for 3 h. After treatment, cells were stim-
ulated with LPS (100 ng/ml) for 30 min. Then, cells were
washed and fixed with PFA 4% for 20 min at 4 °C and washed
with PBS. Immunocytochemistry was performed as follows: