**1. Introduction**

Amyloid-β peptide (Aβ) is the major component of amyloid plaque. Aβ is a characteristic marker of Alzheimer's disease (AD) and is believed to initiate the pathological cascade of the disease. Aβ is a 39 to 43 amino acid peptide formed by β- and γ-secretases that catalyze the splitting of amyloid precursor protein (APP) [1]. Aβ accumulation triggers the cascade of events, such as reactive oxygen species (ROS) production, cell cycle dysregulation, tau phosphorylation, and inflammation, which, ultimately, lead to the death of neurons [2,3].

The detrimental role of Aβ in the stimulation of oxidative stress has been reported in various studies. In a transgenic mouse model, Aβ increased the levels of protein and lipid oxidation markers, including protein carbonyls, 4-hydroxy-2-nonenal, and 3-nitrotyrosine [4,5]. The observed oxidative injury appeared to be dependent on the methionine 35 of Aβ peptide [6]. Notably, Aβ can regulate ROS generation and ROS can reciprocally promote overproduction of Aβ in a vicious cycle [7].

Nuclear factor-kappa B (NF-κB) regulates Aβ homeostasis through transcriptional upregulation of various related enzymes and proteins [8]. Under physiological conditions, NF-κB regulates the expression rates of genes for APP, β-secretase 1 (BACE1) and several components of the γ-secretase complex. However, activation of NF-κB by Aβ regulates the transcription of all these genes and

over-stimulates Aβ production [9]. Mitogen-activated protein kinases (MAPKs), such as extracellular protein regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38, can activate the NF-κB pathway to produce a series of inflammatory factors.

Nobiletin (2-(3,4-dimethoxyphenyl)-5,6,7,8-tetramethoxychromen-4-one) is one of the major polymethoxyflavones in the peel of citrus fruits, including oranges, mandarins, limes, and lemons [10]. Various pharmacological e ffects attributed to nobiletin include antioxidant, anti-inflammatory, anti-diabetic, anti-atherogenic and anti-carcinogenic activities [11–15]. The neuroprotective property of nobiletin has been demonstrated in several recent studies. The compound prevents ischemic brain injury by regulating the Akt/cAMP-response element-binding protein (CREP)/Bcl-2 pathway in Sprague–Dawley rats. Nobiletin reduces Aβ-stimulated memory impairment in several AD animal models, including olfactory bulbectomy mice, APP-SL 7–5 Tg mice and 3XTg-AD mice [16–18].

Our previous study demonstrated the ability of nobiletin to non-competitively inhibit BACE1 via hydrogen-bond-mediated interactions with allosteric residues of the enzyme [19]. However, the primary molecular mechanism underlying the neuroprotective e ffect of nobiletin on Aβ-induced oxidative stress and inflammation has not ye<sup>t</sup> been clearly demonstrated, which prompted the present investigation of the possible e ffects of nobiletin on PC12 cell models.

#### **2. Materials and Methods**

#### *2.1. Cell Culture and A*β*25-35 Stock Solution*

PC12 cells were cultured in RPMI1640 medium supplemented with 10% horse serum, 5% fetal bovine serum, and penicillin (100 U/mL) and treated with nobiletin (Sigma-Aldrich, St. Louis, MO, USA) for 24 h at 37 ◦C in a 5% CO2 incubator. Aβ25-35 (Sigma-Aldrich) was dissolved in dimethylsulfoxide (DMSO) at an initial concentration of 10 mM and diluted with phosphate bu ffered saline (PBS). Aβ25-35 solution was incubated at 37 ◦C for 48 h to permit aggregation before use.

#### *2.2. Cell Viability Analysis*

Cell viability was assessed using the established 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PC12 cells (1×10<sup>5</sup> cells/well in a 96-well plate) were incubated with MTT reagen<sup>t</sup> for 3 h at 37 ◦C. The medium was removed and the formazan crystals produced by the reduction of MTT were dissolved in DMSO. The absorbance due to the formazan crystals was measured at 570 nm using a model ELX808 spectrophotometer (BioTek, Winooski, VT, USA).

#### *2.3. Intracellular ROS Analysis*

PC12 cells were stained with CM-H2DCFDA in minimum essential medium without serum at 37 ◦C for 30 min in the dark and resuspended in Hank's balanced salt solution. Cells were placed on glass slides and cultured overnight. Imaging ROS was done by fluorescence spectrophotometry with excitation and emission wavelengths of 485 and 528 nm, respectively, using a model FLX800 spectrometer (BioTek).

#### *2.4. Apoptosis Assay by Hoechst 33342 Staining*

PC12 cells were harvested with PBS and fixed in 4% paraformaldehyde for 20 min at 25 ◦C and then washed with PBS before being exposed to Hoechst 33342 for 15 min in the dark. The apoptotic morphology was observed using fluorescence microscopy (Olympus Optical Co., Tokyo, Japan) at 400× magnification.

#### *2.5. Fluorescence-Activated Cell Sorting (FACS) Analysis*

PC12 cells were cultured in 24-well plates (5 × 10<sup>5</sup> cells/well) and incubated with 50 μM Aβ25-35 for 24 h after pretreatment with various concentrations of nobiletin for 1 h. After incubation, cells were collected and analyzed using the MuseTM Cell Analyzer (Merck Millipore, Darmstadt, Germany). Briefly, FACS analysis following staining with annexin V and 7-aminoactinomycin D (7-AAD) was performed to detect apoptosis, and cell viability was determined using a DNA-binding dye. Cells were fixed in cold 70% ethanol and stained with propidium iodide (PI), a membrane impermeant dye, to analyze the cell cycle.

#### *2.6. Assessment of Levels of Nitric Oxide (NO) and Prostaglandin E2 (PGE2)*

PC12 cells (2 × 106) were seeded on 6-cell plates containing medium and incubated with Aβ25-35 for 24 h in the presence or absence of nobiletin. The formation of NO2 −, a stable end product that has been extensively used as an indicator of NO accumulation, was assessed using Griess reagent. The media were mixed with Griess reagen<sup>t</sup> at RT for 10 min and the NO levels were analyzed using the aforementioned ELX808 microplate reader at 570 nm.

The supernatants were mixed with primary antibody solution and PGE2 conjugate for 2 h, followed by washing and addition of stop solution. The absorbance was measured at 450 nm using the ELX808 microplate reader.

#### *2.7. Western Blot Analysis*

Proteins (40 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. The blots were blocked for 2 h at RT with 5% skim milk in 0.1% Tween 20 in PBS (PBST) and then incubated overnight with specific primary antibodies at 4 ◦C. The primary antibodies used were against β-actin (1:1000), tumor necrosis factor-alpha (TNFα; 1:1000), cyclooxygenase-2 (COX-2; 1:1000), inducible NO synthase (iNOS; 1:1000), p-65 (1:1000), phospho (p)-IκBα (1:1000), p-JNK (1:1000), p-p38 (1:1000), and p-ERK (1:1000). Subsequently, horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody or anti-goat IgG was used. Protein bands were visualized using the EZ-capture device (Atto, Tokyo, Japan).
