1. Introduction

Wheat is one of the major cereals consumed worldwide. Wheat consists of the endosperm

(80–85%), germ (2.5–3%), and bran (10–14%) [1]. Compared to the endosperm and bran fractions,

wheat germ contains higher proportions of sulfur compounds, dietary fibers (lignins, oligosaccharides,

and phytic acids), B vitamins, vitamin E, phytosterols (mainly sitosterol and campesterol), flavonoids,

total choline, vitamin E, flavonoids, and choline [1]. Wheat germ is specifically rich in linoleic acid,

which is susceptible to oxidative reactions and thus affects the long-term storage of wheat flour

quality [2]. Also, wheat germ has a high protein content and is considered as a source of good vegetable

proteins [2]. Despite its nutritional value, the major use of wheat germ generated during milling is

limited to animal feed. It is estimated that approximately 25,000,000 tons of wheat germ are produced annually [3]. Therefore, it is important to determine the heath-enhancing potential of wheat germ after

applying various processing technologies.

2.3. Animals

Male Balb/c mice aged 7 weeks were purchased from Koatech (Pyungtek, South Korea) and

underwent 1 week of adjustment prior to experiments. The animal protocol (KHUASP(GC)-19-005)

was approved by the Institutional Animal Care and Use Committee of Kyung Hee University, and mice

were cared for according to the specifications of the US National Research Council for the Care and

Use of Laboratory Animals (1996).

2.4. Macrophage Isolation

Mice were injected intraperitoneally with 2 mL of 3.5% sterile thioglycollate (BD, Sparks, MD,

USA). Four days later, the mice were sacrificed via CO2 inhalation and peritoneal exudate cells were

harvested by injecting 7 mL of cold DMEM (HyClone, Logan, UT, USA) plus 1% fetal bovine serum

(FBS; HyClone) and 1% penicillin-streptomycin. After centrifugation, the cells were resuspended in

DMEM plus 10% FBS and 1% penicillin-streptomycin and counted using a Countess II Automated Cell

Counter (Thermo Scientific, Bothell, WA, USA). The cells were then plated and incubated overnight at

37 °C, and the non-adherent cells were removed.

2.5. Cell Viability Assay

Cell viability was tested using an MTT assay. Cells in 96-well plates were incubated for 24 h

with increasing concentrations of UWG, EWG and DMBQ and then the culture medium was removed.

MTT (final concentration 0.5 mg/mL) (Sigma) was added to each well for 1 h, and then the media was

removed. DMSO was added and incubated for 15 min to solubilize the MTT. Optical density was

measured at 570 nm with an iMark microplate reader (Bio-Rad, Hercules, CA, USA). Cell viability was

expressed as a percentage of control cells.

2.6. Western Blotting

To detect iNOS, COX2, and HO-1, cells were stimulated with or without 100 ng/mL LPS (Sigma)

and simultaneously incubated with UWG, EWG, or DMBQ for 24 h. For IκBα and phosphate MAPK,

cells were pretreated with UWG, EWG, or DMBQ for 1 h and then stimulated with LPS for 15 min.

For nuclear Nrf2, cells were incubated with UWG or EWG for the indicated time period. Whole cell

lysates were prepared by resuspending the cells in RIPA buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1

mM EDTA; 20mM NaF; 0.5% NP-40; 1% Triton X-100) plus a phosphatase inhibitor cocktail (Sigma) and

a protease inhibitor cocktail (Quartett, Berlin, Germany). Nuclear protein lysates were prepared using the Nuclear Extraction Kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s protocol.

Protein concentration was determined using the Bradford assay. Protein lysates were separated on an

8% or 10% sodium dodecyl sulfate-polyacrylamide gel and were then transferred to a polyvinylidene

fluoride membrane. The membranes were blocked with 5% skim milk in Tris-buffered saline with

0.1% Tween 20 (TBST) for 1 h then incubated overnight at 4°C with primary antibodies against

iNOS, COX2 (Cayman Chemical, Ann Arbor, MI, USA), HO-1, IκBα, glyceraldehyde 3-phosphate

dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-JNK, JNK,

phospho-p38, p38, phospho-ERK1/2, ERK1/2, Nrf2, or lamin B1 (Cell Signaling Technology, CA, USA)

diluted 1:1000 in 5% skim milk in TBST. The blots were washed with TBST and incubated for 1 h with

anti-rabbit horseradish peroxidase-conjugated antibody (diluted 1:5000 in 5% skim milk in TBST).

The protein bands were detected with EzWestLumi plus (ATTO, Tokyo, Japan) and analyzed using an

EZ-Capture MG (ATTO).

2.7. Cytokine Analysis

The levels of TNF-α, IL-6, IL-12p70, and IL-10 in the supernatants and sera were determined

using the appropriate DuoSet ELISA Development Systems (R&D Systems, Minneapolis, MN, USA)

according to the manufacturer’s protocol.

2.8. Luciferase Assay

RAW264.7 cells were transfected with the pGL4.32 containing five copies of an NF-κB response

element or pGL4.44 containing six copies of an AP-1 responsive element and the firefly luciferase

reporter gene (luc2P) (Promega, Madison, WI, USA). The transfected RAW264.7 cells were plated into

96 well plates and incubated at 37 °C overnight. After changing the media, cells were pretreated with

UWG, EWG, or DMBQ for 2 h and then stimulated with LPS for 6 h. Luciferase activity was measured

using the Dual-Glo®luciferase assay system (Promega, Madison, WI, USA).

2.9. In vivo Experiments

Mice were randomly divided into normal, control, 0.3 g/kg UWG, 3 g/kg UWG, 0.3 g/kg EWG, and 3 g/kg EWG groups (n = 5 for the normal group and n = 13 for the control and treatment groups).

UWG and EWG suspended in water were orally given to mice once daily for 4 weeks. Mice in the normal and control groups were given an equal amount of water. The control, UWG, and EWG groups were intraperitoneally injected with 1.3 mg/kg of LPS at the end of the experiment. After 1 h, the mice

were anesthetized with ether, and blood was collected via cardiac puncture. Serum was obtained and stored at −20 °C for further analysis.

2.10. Real-Time RT PCR

RNA from the liver was isolated using Tri-RNA Reagent (Favorgen, Kaohsiung, Taiwan) and

reverse-transcribed into cDNA using the High Capacity cDNA Reverse Transcription kit (Applied

Biosystems, Foster, CA, USA). Real-time PCR was performed using SYBR Green Mix (Applied

Biosystems, Foster, CA, USA) on a StepPlus One real-time PCR system (Applied Biosystems, Foster, CA,

USA). Quantification of gene expression was determined by the standard curve calculation method.

Target gene was normalized to GAPDH.

2.11. Statistical Analysis

The data are presented as the mean ± SD. The two-tailed Student’s t-test or one-way analysis of

variance was applied to compare the differences between groups. If the statistical analysis showed that

the differences between multiple groups were significant, Tukey’s post-hoc test was used for further

comparison. All statistical analyses were performed using IBM SPSS software, version 22.0 (Chicago,

IL, USA). P-values less than 0.05 were considered statistically significant.