*2.8. Statistical Analysis*

Statistical analysis was performed using SAS 9.3 software (SAS Institute Inc., Cary, NC, USA). All results are expressed as mean ± SD and are representative of the data obtained from three independent experiments. Statistical comparisons of di fferences between groups were performed through the Student's *t* test, considering \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001 as being statistically significant.

#### **3. Results and Discussion**

#### *3.1. Nobiletin Inhibits Cytotoxicity Evoked by A*β*25-35*

To verify the neuroprotective e ffect of nobiletin, cell viability was assessed using the MTT assay and FACS. As shown in Figure 1a, nobiletin did not adversely a ffect PC12 cell viability at concentrations of 1 to 25 μM, which were used for further study. Treatment with 50 μM Aβ25-35 for 24 h induced approximately 40% cell death in comparison with the control group (*p* < 0.001; Figure 1b). However, pretreatment with 1, 10, and 25 μM nobiletin significantly increased cell viability up to 78.1% ± 7.4%, 81.3% ± 4.5%, and 82.4% ± 4.7%, respectively. Notably, 10 μM nobiletin exhibited a similar neuroprotective e ffect to that of resveratrol, a well-known positive control. Consistent with the results of the MTT assay, nobiletin significantly prevented Aβ25-35-induced cell death in FACS analysis (Figure 1c), and this e ffect was dependent on the dose of nobiletin. These data provided correlative evidence indicating that nobiletin contributes to cell survival in PC12 cells damaged by Aβ25-35.

**Figure 1.** Protective properties of nobiletin against Aβ25-35-mediated cell damage. (**a**) Evaluation of cytotoxicity nobiletin alone in PC12 cells. Cells were pretreated with nobiletin for 1 h followed by exposure to 50 μM of Aβ25–35 for 24 h, and cell viability was assessed by (**b**) MTT reduction assay and (**c**) fluorescence-activated cell sorting (FACS) analysis. (**d**) Cell cycle progression was measured by FACS. The percentage of cells in the G0/G0, S, and G0/M phases of the cell cycle was determined using the Muse 1.5 Analysis software. (**e**) Intracellular ROS production was observed by CM-H2DCFDA fluorescent dye. ### *p*<0.001, ## *p* < 0.01, and # *p* < 0.05 vs. control. \*\*\* *p* < 0.001, \*\* *p* < 0.01, and \* *p* < 0.05 vs. Aβ25–35.

Cell cycle regulation is a crucial process of cell growth and proliferation in neurons [20]. As shown in Figure 1d, Aβ25-35 significantly induced an increase in cells in the G0/G1 phase (*p* < 0.01) and a corresponding decrease in cells in the S phase and G2/M phase (*p* < 0.05), suggesting that cells had lower rates of growth and tended to be arrested at the G0/G1 transition. However, nobiletin restored Aβ25-35-mediated cell cycle dysregulation in a concentration-dependent manner, which may contribute to the enhanced cell viability effect of the compound.

Nobiletin was further evaluated for its antioxidant property in <sup>A</sup>β25-35-injured cells by ROS regulation. As indicated in Figure 1e, fluorescence intensity and large numbers of bright particles in cells were visibly increased by Aβ25-35 exposure, suggesting the presence of intracellular oxidative stress. Aβ25-35 stimulated significant increase in ROS to 100% ± 4.86% (*p* < 0.001). However, pretreatment of nobiletin decreased ROS generation in a dose-dependent manner (*p* < 0.05 and *p* < 0.001). These data were consistent with previous descriptions of the antioxidant property of nobiletin. The compound protected PC12 cells against H2O2-triggered damage by scavenging ROS, decreasing malonaldehyde (MDA), and enhancing glutathione (GSH) and superoxide dismutase (SOD) contents [21]. In addition, excessive intracellular ROS stimulates the activation of signal transduction cascades, which disturbs calcium homeostasis and leads to the initiation of apoptosis. Nobiletin prevents mitochondrial calcium overload as well as ROS generation in glutamate-induced cortical neurons [22]. In vivo, the intraperitoneal administration of nobiletin reportedly reduced tau phosphorylation, the index of protein oxidation, and protein carbonyl levels in SAMP8 mice, which were related to the recovery of GSH/glutathione disulfide (GSSG) ratio and increased glutathione peroxidase (GPx) activity [23]. These results demonstrated that the neuroprotective role of nobiletin results, at least in part, from the reduction of oxidative stress.

#### *3.2. Nobiletin Reduces <sup>A</sup>*β*25–35-Mediated Apoptosis and Caspase-3 Activation*

Intracellular oxidative stress plays a central role in the induction of neuronal apoptosis stimulated by Aβ [24]. As shown in Figure 2a, cells treated with Aβ25–35 exhibited uneven morphology of their nuclei as a result of membrane blebbing, chromatin aggregation, and nuclear condensation, whereas the nobiletin pretreated group showed dispersed and weak fluorescence that is typical of live cells. Particularly, nobiletin at 25 μM decreased apoptosis similar to the level of the positive control group. Analysis of morphology alone is not sufficient to distinguish between early and late apoptotic cells. Thus, flow cytometric analysis was employed to quantitatively analyze apoptotic cell death. When exposed to Aβ25-35, early and late apoptosis was significantly increased to 26.39% ± 2.48% and 29.06% ± 2.33%, respectively, compared with the control group (*p* < 0.001, Figure 2b). Nobiletin—at all concentrations—markedly reduced both early and late apoptosis. These results were consistent with the anti-apoptotic activity of nobiletin in endoplasmic reticulum stress-induced PC12 and I/R-exposed Kupffer cells [25].

Caspase-3 is a biomarker of oxidative-stress-stimulated cell death that has also been implicated in the final stage of apoptosis. The caspase-3 was obviously activated in the Aβ25-35-treated group (*p* < 0.001, Figure 2c). However, enhanced caspase-3 activity was dose-dependently decreased by nobiletin. Aβ-triggered apoptotic cell death is related to the reduced anti-apoptotic Bcl-2 protein and the increased pro-apoptotic molecule Bax expression. In a previous study, nobiletin displayed an anti-apoptosis effect by decreasing the ratio of Bcl-2/Bax expression in H2O2-stimulated HT22 cells [26].

**Figure 2.** Activity of nobiletin in preventing Aβ25-35-evoked apoptosis and caspase-3. (**a**) Morphological features of apoptotic cells were observed by fluorescence microscopy using Hoechst 33342 staining (magnification ×400). (**b**) Flow cytometric analysis was used to investigate the properties of nobiletin on Aβ25-35-stimulated apoptosis. The cell populations discriminated in each quadrant are live cells in the lower-left quadrant, early apoptotic cells in the lower-right quadrant, late apoptotic cells in the upper-right quadrant, and dead cells in the upper-left quadrant. (**c**) Caspase-3 activity was assessed using the caspase-3 assay kit. ### *p* < 0.001 vs. control. \*\*\* *p* < 0.001, \*\* *p* < 0.01, and \* *p* < 0.05 vs. Aβ25–35.

#### *3.3. Nobiletin Suppresses <sup>A</sup>*β*25-35-Induced Release of Inflammatory Markers*

Aβ25-35 exposure increases the release of NO and PGE2 by approximately 5-fold compared with the control (Figure 3a,b). Notably, treatment with 10 and 25 μM nobiletin exhibited similar activity as that of 50 μM resveratrol. Aβ25-35 treatment increased the expression level of pro-inflammatory cytokines, such as TNF-α and interleukin (IL)-1β, by 3-fold (Figure 3c), but the expressions were suppressed by 10 and 25 μM nobiletin.

As shown in Figure 3d, the level of iNOS stimulated by Aβ25-35 increased up to 202.5% ± 18.3% compared with the control (*p* < 0.001). However, nobiletin promptly inhibited the expression of iNOS

protein. Notably, the highest concentration of nobiletin resulted in almost complete suppression of the enzyme production (118.3% ± 15.7%; *p* < 0.001). In parallel, Aβ25-35-mediated COX-2 expression was also markedly blocked by nobiletin.

Several studies have demonstrated that nobiletin possesses strong anti-inflammatory ability in lipopolysaccharide (LPS)-induced expression of pro-inflammatory cytokines in BV2 microglial cells [27–29]. A recent in vivo study suggested that the oral administration of nobiletin (100 mg/kg/day) for 6 weeks attenuated microglial activation and secretion of pro-inflammatory mediators, leading to the restoration of memory deficits in mice [30].

**Figure 3.** Inhibitory properties of nobiletin on Aβ25-35-mediated expression of (**a**) NO, (**b**) PGE2, (**c**) TNF-α and IL-β, and (**d**) iNOS and COX-2 in PC12 cells. The cells were pretreated with nobiletin for 1 h and then exposed to Aβ25-35 for 24 h. The culture supernatant was collected to evaluate the NO and PGE2 formation. Protein expression of TNF- α, IL-β, iNOS, and COX-2 was measured by Western blot analysis. The band intensities were quantified using Image J software and normalized to β-actin. ### *p* < 0.001 vs. control. \*\*\* *p* < 0.001, \*\* *p* < 0.01, and \* *p* < 0.05 vs. Aβ25–35.

#### *3.4. Nobiletin Regulates <sup>A</sup>*β*25-35-Induced NF-*κ*B and MAPK Signaling Pathways*

Aβ25-35 obviously elevated the phosphorylation of p65 and IκB-α by 227.4% ± 19.6% and 194.4% ± 10.1%, respectively. In contrast, pretreatment with nobiletin at 10 and 25 μM remarkably repressed p65 expression (Figure 4a). Moreover, in the case of IκB-α, all doses of nobiletin indicated a significant inhibitory effect in respect to Aβ25-35 treatment.

**Figure 4.** Inhibitory properties of nobiletin in the NF-κB/MAPKs pathway. Phosphorylation of (**a**) p65 and IκB-α, and (**b**) p38, ERK1/2, and JNK was examined by Western blot analysis. The cells were pretreated with nobiletin for 1 h and then exposed to Aβ25-35 for 4 h (p65 and IκB-α) or 1 h (p38, ERK1/2, and JNK). Quantification of band intensities was conducted using Image J software and normalized to β-actin. ### *p* < 0.001 vs. control. \*\*\* *p* < 0.001, \*\* *p* < 0.01, and \* *p* < 0.05 vs. Aβ25–35.

As presented in Figure 4b, nobiletin suppressed Aβ25-35-evoked phosphorylation of p38 and JNK. In particular, the level of phosphorylated JNK was markedly suppressed when treated with 25 μM nobiletin (94.4% ± 9.8%, *p* < 0.001), indicating that the phosphorylation of MAPKs was closely related with the inhibitory property of nobiletin on Aβ25-35-evoked activation of p65 and IκBα.

Previous research has reported that BACE1 promoter transactivation is modulated by the NF-κB signaling pathway, indicating that the suppression of NF-κB leads to inhibition of BACE1 activity [9]. In our previous study, we observed that nobiletin blocked BACE1 activity, suggesting that the compound might modulate BACE1 activation by suppressing the NF-κB signaling pathway [19]. Another study reported that nobiletin inhibited H2O2-evoked cell death in HT22 murine hippocampal cells, accompanied by decreased JNK and p38 phosphorylation [26]. In rat primary astrocytes, nobiletin suppressed the overexpression of iNOS and NO by the inhibition of the NF-κB/p38 MAPK pathways.

Several animal studies clearly demonstrated that nobiletin (10–50 mg/kg) improves memory in rats exposed to chronic intracerebroventricular infusion of Aβ1-40 [31]. In addition, daily supplementation of nobiletin (10 mg/kg) for 4 months significantly lowers both <sup>A</sup>β1-40/42 and amyloid plaques in 9-month-old APP-SL 7–8 Tg mice [17]. Recently, Nakajima et al. demonstrated that nobiletin (10 mg/kg) reduces tau phosphorylation in SAMP8 mice [23].

Bioavailability is an essential factor for the development of potential therapeutic agents. In addition, the anti-AD agents must penetrate the blood–brain barrier to attain su fficient concentration for the therapeutic application within the central nervous system. When nobiletin (50 mg/kg) was administered, the content of intact nobiletin was detected in rat brains within 1 h after dosing, suggesting that this compound can rapidly cross the blood–brain barrier and reach the brain. Furthermore, the concentration of nobiletin was 2.4-fold higher in the brain than in plasma. Interestingly, nobiletin was detected for up to 24 h in the brain, whereas in the plasma, it was observed up to 9 h, suggesting that elimination of this compound from the brain was slower in comparison to plasma [32]. Of note, nobiletin has no discernible toxicity in chronic treatments in mice and humans [33].
