DHPA Protects SH-SY5Y Cells from Oxidative Stress-Induced Apoptosis via Mitochondria Apoptosis and the Keap1/Nrf2/HO-1 Signaling Pathway

Oxidative stress in the brain is highly related to the pathogenesis of Alzheimer’s disease (AD). It could be induced by the overproduction of reactive oxygen species (ROS), produced by the amyloid beta (Aβ) peptide and excess copper (Cu) in senile plaques and cellular species, such as ascorbic acid (AA) and O2. In this study, the protective effect of 5-hydroxy-7-(4′-hydroxy-3′-methoxyphenyl)-1-phenyl-3-heptanone (DHPA) on Aβ(1–42)/Cu2+/AA mixture-treated SH-SY5Y cells was investigated via in vitro and in silico studies. The results showed that DHPA could inhibit Aβ/Cu2+/AA-induced SH-SY5Y apoptosis, OH· production, intracellular ROS accumulation, and malondialdehyde (MDA) production. Further research demonstrated that DHPA could decrease the ratio of Bax/Bcl-2 and repress the increase of mitochondrial membrane potential (MMP) of SH-SY5Y cells, to further suppress the activation of caspase-3, and inhibit cell apoptosis. Meanwhile, DHPA could inhibit the Aβ/Cu2+/AA-induced phosphorylation of Erk1/2 and P38 in SH-SY5Y cells, and increase the expression of P-AKT. Furthermore, DHPA could bind to Keap1 to promote the separation of Nrf2 to Keap1 and activate the Keap1/Nrf2/HO-1 signaling pathway to increase the expression of heme oxygenase-1 (HO-1), quinone oxidoreductase-1 (NQO1), glutathione (GSH), and superoxide dismutase (SOD). Thus, our results demonstrated that DHPA could inhibit Aβ/Cu2+/AA-induced SH-SY5Y apoptosis via scavenging OH·, inhibit mitochondria apoptosis, and activate the Keap1/Nrf2/HO-1 signaling pathway.


Introduction
Alzheimer's disease (AD) is a neurodegenerative disease that causes memory loss, cognitive impairment, and behavioral dysfunction [1]. In AD brains, high concentrations of Cu 2+ are detected (about 0.4 mM) in amyloid beta (Aβ)-accumulated senile plaques [2,3]. Moreover, the Aβ-Cu 2+ complex could react with ascorbic acid (AA) (500 µM-10 mM) in the brain to continuously generate excessive hydroxyl radicals (OH·) and reactive oxygen species (ROS) via Fenton and Haber-Weiss reactions [4,5]. These ROS radicals will further induce oxidative damage to neurons and aggravate the progression of AD [6].
Mitochondria are important sources of ROS [7,8]. Excess ROS could influence the expression of B-cell lymphoma 2 (Bcl-2) and Bcl2-associated X (Bax), changing the mitochondrial membrane potential (MMP), inducing the expression of caspase-3 (cleaved), and inducing neuronal apoptosis [9]. Aβ  stock was freshly prepared by dissolving Aβ  (0.5 mg) in 20 mM NaOH (220 µL) solution. Copper stock was prepared by dissolving CuSO 4 (0.25 g) in 100 mM H 2 SO 4 (10 mL), diluted with 1 mM H 2 SO 4 to 1 mM CuSO 4 , and diluted with deionized (DI) water (100 µM). Solutions of AA (10 mM) were freshly prepared with DI water (for the CCA assay) or complete media (cell assays). The DHPA (2 mM) stock was prepared with dimethyl sulfoxide (DMSO). To prepare the Aβ   (10 µM)/Cu 2+ (5 µM)/AA (1 mM)/DHPA mixture, different stock volumes were added into the DI water (for the CCA assay) or complete media (cell assays) to obtain the desired concentrations. The final concentration of NaOH was 0.4 mmol/L, and the final concentration of DMSO was less than 0.1%. The control group solution was prepared with the same formula as the experimental group, except Aβ  , Cu 2+ , AA, or DHPA.

Cell Culture
Human neuroblastoma SH-SY5Y cells were purchased from the American Type Culture Collection (Washington, DC, USA). SH-SY5Y cells were routinely grown in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 1% streptomycin/penicillin-streptomycin at 37 • C in a humidified incubator (S111, Thermo Fisher Scientific, Waltham, MA, USA) with 5% CO 2 .

Cell Viability Assay (MTT Assay)
When SH-SY5Y cells grew to the log phase, they were digested with trypsin for counting, the cell densities were adjusted to 1 × 10 4 cells/mL (100 µL), and added to a 96well plate, 100 µL per well. Then, the plated 96-well plate was placed in an incubator for 6 h. Afterward, the cells were stably adhered to the wall, cultured with Aβ (1-42) (10 µM)/Cu 2+ (5 µM)/AA (1 mM) mixtures in the absence or presence of different concentrations of DHPA (0, 0.5, 2, 4, 6, 8, 10 µM) for 24 h; 100 µL of 10% MTT was added to each well, incubated for 4 h, were carefully removed, and 100 µL of DMSO was added. The absorbance value was detected at 490 nm with an automated microplate spectrophotometer (Spectra Max i3X, Sunnyvale, CA, USA). Cell viability is expressed as a percentage of the control.

Flow Cytometry Assay
SH-SY5Y cells (1 × 10 4 cells/mL, 1 mL) were placed on a 6-well plate for 6 h, and treated with Aβ   of different concentrations of DHPA (0, 0.5, 4, 10 µM) for 24 h at 37 • C. Cell apoptosis was detected with the Annexin V-FITC(PI) Apoptosis Analysis kit. The cells were washed and centrifuged 3 times, resuspended in a flow tube, and incubated with fluorescein isothiocyanate-(FITC-)-coupled annexin V (AV) and propidium iodide (PI) solution for 15 min. The fluorescence signal was detected with a NovoCyte flow cytometer (Agilent Technologies Inc., Palo Alto, CA, USA) and analyzed by NovoExpress software.

Detection of Hydroxyl Radical
CCA was used as a fluorescence probe for OH· determination [31] with a Hitachi F-4600 spectrofluorometer from Hitachi High-Tech Corporation (Tokyo, Japan). CCA fluorescence was recorded at 540 nm, with an excitation wavelength of 390 nm. The widths of the entrance and exit slits were both 5 nm. The OH· amounts in the solutions of the Aβ (1-42) (10 µM)/Cu 2+ (5 µM)/AA (1 mM) mixtures in different concentrations of DHPA (0.5, 4, 10 µM) were detected with a CCA probe. The fluorescence ratio was calculated as follows: where F is the CCA fluorescence intensity in each solution and F 0 is the CCA fluorescence intensity of the Aβ (1-42) (10 µM)/Cu 2+ (5 µM)/AA (1 mM) mixture.

Mitochondrial Membrane Potential Determination
A fluorescent probe 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethyl-imidacarbocyanine (JC-1) was used to detect mitochondrial membrane potential (MMP) of SH-SY5Y cells. The cell treatment method is the same as the description in Section 2.7. The cells were treated with JC-1 dye following the JC-1 kit instructions. The fluorescence intensity was recorded with a microplate reader and the MMP was obtained as the ratio of the JC-1 monomer (FI 530)/JC-1 aggregate (FI 590) fluorescence intensity.

Western Blot Analysis
Cytosolic protein and nuclear protein samples were collected from DHPA (0, 0.5, 4, 10 µM) and Aβ    (Hefei, China). Proteins were separated by polyacrylamide/sodium dodecyl sulfate gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% bovine albumin in Tris-buffered saline containing 0.05% Tween 20 (TBST), followed by incubation with the respective primary antibodies diluted in 5% (M/V) bovine albumin overnight at 4 • C. After washing with Tris-buffered saline with 0.05% Tween 20 (TBST), the membranes were incubated with an HRP-secondary antibody for 1 h. Immunoreactive bands were visualized with a chemiluminescent substrate kit and imaged with Gel Imager 721-BR10883 from BIO-RAD Co., Ltd. (Hercules, CA, USA). Bands were detected using Image-J software.

Molecular Docking
The interaction between the DHPA and Keap1 protein (PDB:1X2J, origin: Mus musculus) was investigated with a similar method reported by Liu and co-workers [23]. The molecular structure of the Keap1 protein was downloaded from the Protein Data Bank (http://www.rcsb.org (accessed on 23 May 2022)). The molecular structures of DHPA and curcumin were downloaded from http://www.chemicalbook.com (accessed on 23 May 2022). The molecular docking experiments were performed with AutoDock Vina. Moreover, the binding energies and interaction sites were analyzed by Discover Studio 2019.

Statistical Analysis
The results were calculated by using the SPSS 22.0 statistical analysis, and Origin 2018 was used for drawing. Adobe Illustrator software was used to combine graphics and draw a schematic diagram of the mechanisms. Each experiment and each group of data were repeated three times, and the results are expressed by mean ± standard deviation (mean ± SD).  /Cu 2+ /AA-Treated SH-SY5Y Cells Viability Amyloid beta (Aβ) peptides aggregated with redox-active metal ions (such as Cu 2+ and Fe 3+ ) have been found to exist in senile plaques in the brains of AD patients [32]. These metal ions can easily bind Aβ peptides to form complexes [32,33] and react with substances such as ascorbic acid (AA) and O 2 in cells to promote the production of OH [33]. Thus, in this study, the Aβ (1-42) /Cu 2+ /AA system was used as the OH production model to simulate the OH-producing process in the senile plaques of AD patients.

Effects of DHPA on Aβ
To investigate the cytotoxicity of the 5-hydroxy-7-(4 -hydroxy-3 -methoxyphenyl)-1-phenyl-3-heptanone (DHPA) to SH-SY5Y neuroblastoma cells, the cell viabilities were detected with the MTT assay. When the concentration of DHPA was at 10 µM, the cell viability was 89.9%. When the concentration of DHPA increased, the cell viability decreased. Moreover, when the concentration of DHPA was at 40 µM, the cell viability decreased to 65.7% ( Figure 1B). As shown in Figure 1C, concentrations of Cu 2+ (5 µM) and AA (1 mM) at lower levels showed almost no cell toxicity to SH-SY5Y cells. Aβ (1-42) (10 µM) reduced the cell viability to 92.3% ( Figure 1C). However, when the same concentrations of Aβ  , Cu 2+ , and AA were mixed, the cell viability decreased to 52.2%. This may have been due to the oxidative damage from OH·, which was produced by the Aβ  /Cu 2+ /AA system, and the cell toxicities from Aβ  toxic aggregates.

Effects of DHPA on Aβ(1-42)/Cu 2+ /AA-induced SH-SY5Y Cell Apoptosis
As shown in Figure 2A, cells in the control group (untreated cells) showed normal cell morphology, were well-adhered, and had clear edges. The cells in the Aβ(1 -42)/Cu 2+ /AA-treated group showed 'shrunken atrophy' and blurred ruptured boundaries; most cells were apoptotic. When the cells were treated with DHPA, the morphological damage and density reduction caused by Aβ(1-42)/Cu 2+ /AA were restored in a DHPA dosedependent manner. The shapes of the cells gradually became normal, and the edges were gradually clear, but a small number of suspended cells could still be observed. Annexin V/PI staining was also used to verify the effect of DHPA on Aβ(1-42)/Cu 2+ /AA-induced cell apoptosis. As shown in Figure 2B, with the addition of DHPA, the apoptosis rate of Aβ(1-42)/Cu 2+ /AA-treated SH-SY5Y cells decreased from 47.23% to 22.41%, which indicated that DHPA could inhibit Aβ(1-42)/Cu 2+ /AA-induced cell apoptosis in a dose-dependent manner.

Effects of DHPA on Aβ (1-42) /Cu 2+ /AA-Induced SH-SY5Y Cell Apoptosis
As shown in Figure 2A, cells in the control group (untreated cells) showed normal cell morphology, were well-adhered, and had clear edges. The cells in the Aβ  /Cu 2+ /AAtreated group showed 'shrunken atrophy' and blurred ruptured boundaries; most cells were apoptotic. When the cells were treated with DHPA, the morphological damage and density reduction caused by Aβ  /Cu 2+ /AA were restored in a DHPA dose-dependent manner. The shapes of the cells gradually became normal, and the edges were gradually clear, but a small number of suspended cells could still be observed. Annexin V/PI staining was also used to verify the effect of DHPA on Aβ  /Cu 2+ /AA-induced cell apoptosis. As shown in Figure 2B, with the addition of DHPA, the apoptosis rate of Aβ  /Cu 2+ /AA-treated SH-SY5Y cells decreased from 47.23% to 22.41%, which indicated that DHPA could inhibit Aβ  /Cu 2+ /AA-induced cell apoptosis in a dose-dependent manner.

Scavenging Effect of DHPA on Aβ (1-42) /Cu 2+ /AA-Produced OH
To investigate the reason behind the inhibition effect of DHPA on Aβ  /Cu 2+ /AAinduced cell apoptosis, the scavenging effect of DHPA on OH·, produced by the Aβ  / Cu 2+ /AA mixture, was detected with the CCA method. With the increase in the DHPA concentration (0, 0.5, 4, 10 µM), the amount of OH· decreased from 100% to 54.82% ( Figure 3). The results indicate that DHPA could inhibit the production of OH· by the Aβ  /Cu 2+ /AA mixture in a dose-dependent manner.
Antioxidants 2022, 11, x FOR PEER REVIEW 8 of 18 OH· could be produced by the Aβ(1-42)/Cu 2+ /AA system and induce oxidative damage to SH-SY5Y cells. Oxidative damage could influence the accumulation of intracellular ROS and malondialdehyde (MDA), as well as the activities of SOD and glutathione (GSH) in cells [34]. Thus, the amount of intracellular ROS and MDA, and the activities of SOD and GSH, were detected. As shown in Figure 4A,B, the level of intracellular ROS increased with the incubation of the Aβ(1-42)/Cu 2+ /AA mixture, while it reduced with the addition of DHPA (0.5, 4, 10 μM). To investigate whether the repression effect of DHPA on intracellular ROS production was only caused by a chemical reaction to the oxidants outside the cell, further experiments were performed. As shown in Figure 4C, DHPA solutions were added to cells after removing the Aβ(1-42)/Cu 2+ /AA mixture, the level of intracellular ROS was also decreased by DHPA. These results indicate that DHPA can reduce ROS production outside the cell, reduce intracellular ROS levels by its antioxidant abilities, or activate the endogenous antioxidant system in the cells. Meanwhile, the amount of MDA in SH-SY5Y was also decreased by DHPA ( Figure 4D). Furthermore, the activities of SOD and GSH in SH-SY5Y cells were improved by DHPA ( Figure 4E,F). These results indicated that DHPA could reduce intracellular ROS, lipid peroxidation, and increase the activities of SOD and GSH. 3.4. Effect of DHPA on the Accumulation of ROS and Malondialdehyde, and the Activities of Antioxidant Enzymes in Aβ  /Cu 2+ /AA-Treated SH-SY5Y Cells OH· could be produced by the Aβ  /Cu 2+ /AA system and induce oxidative damage to SH-SY5Y cells. Oxidative damage could influence the accumulation of intracellular ROS and malondialdehyde (MDA), as well as the activities of SOD and glutathione (GSH) in cells [34]. Thus, the amount of intracellular ROS and MDA, and the activities of SOD and GSH, were detected. As shown in Figure 4A,B, the level of intracellular ROS increased with the incubation of the Aβ (1-42) /Cu 2+ /AA mixture, while it reduced with the addition of DHPA (0.5, 4, 10 µM). To investigate whether the repression effect of DHPA on intracellular ROS production was only caused by a chemical reaction to the oxidants outside the cell, further experiments were performed. As shown in Figure 4C, DHPA solutions were added to cells after removing the Aβ  /Cu 2+ /AA mixture, the level of intracellular ROS was also decreased by DHPA. These results indicate that DHPA can reduce ROS production outside the cell, reduce intracellular ROS levels by its antioxidant abilities, or activate the endogenous antioxidant system in the cells. Meanwhile, the amount of MDA in SH-SY5Y was also decreased by DHPA ( Figure 4D). Furthermore, the activities of SOD and GSH in SH-SY5Y cells were improved by DHPA ( Figure 4E,F). These results indicated that DHPA could reduce intracellular ROS, lipid peroxidation, and increase the activities of SOD and GSH.

Effects of DHPA on Mitochondrial Apoptosis in Aβ (1-42) /Cu 2+ /AA-Induced SH-SY5Y Cells
The mitochondrion is the sensitive organelle to oxidative damage, which could induce pro-apoptotic factor expression and cell apoptosis [7]. As shown in Figure 5, with the incubation of Aβ  /Cu 2+ /AA, the ratio of the pro-apoptotic protein Bax to the anti-apoptotic protein Bcl-2 (Bax/Bcl-2) increased, and the mitochondrial membrane potential (MMP) significantly increased, resulting in an increase of caspase-3 (cleaved) expression. When DHPA was added to the Aβ (1-42) /Cu 2+ /AA-treated cells, the Bax/Bcl-2 ratio decreased, the MMP reduced, and the caspase-3 (cleaved) expression decreased. These results indicate that DHPA could inhibit Aβ  /Cu 2+ /AA-induced cell apoptosis via the mitochondrial apoptosis pathway.

Effects of DHPA on the Phosphorylation of MAPKs and AKT in Aβ(1-42)/Cu 2+ /AA-induced SH-SY5Y Cells
The MAPK signaling pathway plays an important role in oxidative stress-induced neuronal cell death [35]. To investigate the effects of DHPA on the MAPK pathway, the expression of Erk and P38-related proteins were studied. As shown in Figure 6, phosphorylation of Erk1/2 and P38 was significantly enhanced by Aβ(1-42)/Cu 2+ /AA stimulation, while DHPA markedly decreased the expressions of P-Erk and P-P38. Moreover, the expressions of P-AKT decreased with the incubation of Aβ(1-42)/Cu 2+ /AA but increased significantly after DHPA treatment.  The MAPK signaling pathway plays an important role in oxidative stress-induced neuronal cell death [35]. To investigate the effects of DHPA on the MAPK pathway, the expression of Erk and P38-related proteins were studied. As shown in Figure 6, phosphorylation of Erk1/2 and P38 was significantly enhanced by Aβ  /Cu 2+ /AA stimulation, while DHPA markedly decreased the expressions of P-Erk and P-P38. Moreover, the expressions of P-AKT decreased with the incubation of Aβ  /Cu 2+ /AA but increased significantly after DHPA treatment.

Effects of DHPA on the Keap1/Nrf2/HO-1 Signaling Pathway in Aβ (1-42) /Cu 2+ /AA-Treated SH-SY5Y Cells
The Keap1/Nrf2/HO-1 signaling pathway is considered an important pathway in the endogenous antioxidant system, and it is highly sensitive to oxidative stress [10]. To verify whether DHPA activated the Keap1/Nrf2/HO-1 signaling pathway in SH-SY5Y cells, some protein expressions were detected with the WB experiment. As shown in Figure 7, the amount of detectable Keap1 increased in Aβ  /Cu 2+ /AA-treated SH-SY5Y cells but decreased with the DHPA addition. The expression of Nrf2 in the cytoplasm (cyto-Nrf2) slightly increased by the Aβ  /Cu 2+ /AA treatment, and DHPA has a minor effect on cyto-Nrf2 expression. However, Aβ  /Cu 2+ /AA treatment could decrease the amount of Nrf2 in the nucleus (nucl-Nrf2), while DHPA could increase the nucl-Nrf2 amount, and result in an increase of HO-1 and NQO-1 expressions.  The Keap1/Nrf2/HO-1 signaling pathway is considered an important pathway in the endogenous antioxidant system, and it is highly sensitive to oxidative stress [10]. To verify whether DHPA activated the Keap1/Nrf2/HO-1 signaling pathway in SH-SY5Y cells some protein expressions were detected with the WB experiment. As shown in Figure

Results from Molecular Docking
Molecular docking (MD) techniques are commonly used to investigate the interactions between small molecules and the protein target [36]. To investigate the interaction between DHPA and Keap1, 1X2J (PDB ID) was selected as the crystal structure of Keap1 for the MD simulation. Compared with well-studied curcumin [21], the binding energy for the DHPA-Keap1 complex (−9.1 kcal/mol) is even lower than the one of the curcumin-Keap1 complex (−8.8 kcal/mol) (Table 1), which indicated higher binding stability of the DHPA-Keap1 complex than the curcumin-Keap1 complex [36]. At the same time, we found that the residues, namely VAL418 (bond length: 2.  Figure 8C). In addition, both DHPA and curcumin can form a hydrophobic Pi-Alkyl interaction between amino acid residues ALA366 and ALA556 and Keap1, but DHPA can also form a Pi-Pi stacked with Keap1 and TYR334 ( Figure 8C,D), which may also be one of the reasons why the DHPA-Keap1 complex is more stable.

Results from Molecular Docking
Molecular docking (MD) techniques are commonly used to investigate the interac tions between small molecules and the protein target [36]. To investigate the interaction between DHPA and Keap1, 1X2J (PDB ID) was selected as the crystal structure of Keap for the MD simulation. Compared with well-studied curcumin [21], the binding energy for the DHPA-Keap1 complex (−9.1 kcal/mol) is even lower than the one of the curcumin Keap1 complex (−8.8 kcal/mol) (Table 1), which indicated higher binding stability of th DHPA-Keap1 complex than the curcumin-Keap1 complex [36]. At the same time, w found that the residues, namely VAL418 (bond length: 2.2 Å), VAL465 (bond length: 2. Å), VAL512 (bond length: 2.0 Å), ARG415 (bond length: 2.2 Å), and GLY364 (bond length

Discussion
According to the experimental results, we believe that DHPA has an inhibition effect on Aβ/Cu 2+ /AA-induced SH-SY5Y cell apoptosis, which is mainly caused by anti-oxidation activity ( Figure 9). In the brains of AD patients, aggregated Aβ and an excess amount of Cu (~0.4 mM) were found in the senile plaque [37,38]. Moreover, research shows that toxic Aβ aggregates and oxidative stress, produced by the Aβ/Cu mixture/complex, play important roles in AD pathogenesis [33]. The Aβ-Cu 2+ complex can promote the production of H 2 O 2 by reacting with substances (e.g., ascorbic acid, AA, and O 2 ) in cells [39][40][41]. In the cellular milieu, any unbounded Cu 2+ will also react with H 2 O 2 to produce hydroxyl radicals via the Haber-Weiss reaction [33].
The imbalance of redox homeostasis could increase ROS levels and result in cell lipid peroxidation and cell apoptosis. [42]. In the present study, the Aβ  /Cu 2+ /AA system produced hydroxyl radicals ( Figure 3). Moreover, DHPA could inhibit hydroxyl radical production and protect the SH-SY5Y cells from oxidation damage induced by hydroxyl radicals (Figure 1). The amounts of intracellular ROS and MDA in SH-SY5Y cells, incubated with the Aβ  /Cu 2+ /AA system, were reduced by the DHPA treatment ( Figure 4A,B). The activities of SOD and GSH increased with the DHPA treatment ( Figure 4C,D). Moreover, the cell apoptosis of SH-SY5Y induced by the Aβ  /Cu 2+ /AA system was also inhibited by the DHPA treatment ( Figure 2).  According to the experimental results, we believe that DHPA has an inhibition effect on Aβ/Cu 2+ /AA-induced SH-SY5Y cell apoptosis, which is mainly caused by anti-oxidation activity (Figure 9). In the brains of AD patients, aggregated Aβ and an excess amount of Cu (~0.4 mM) were found in the senile plaque [37,38]. Moreover, research shows that toxic Aβ aggregates and oxidative stress, produced by the Aβ/Cu mixture/complex, play important roles in AD pathogenesis [33]. The Aβ-Cu 2+ complex can promote the production of H2O2 by reacting with substances (e.g., ascorbic acid, AA, and O2) in cells [39][40][41]. In the cellular milieu, any unbounded Cu 2+ will also react with H2O2 to produce hydroxyl radicals via the Haber-Weiss reaction [33].
The imbalance of redox homeostasis could increase ROS levels and result in cell lipid peroxidation and cell apoptosis. [42]. In the present study, the Aβ(1-42)/Cu 2+ /AA system produced hydroxyl radicals ( Figure 3). Moreover, DHPA could inhibit hydroxyl radical production and protect the SH-SY5Y cells from oxidation damage induced by hydroxyl radicals (Figure 1). The amounts of intracellular ROS and MDA in SH-SY5Y cells, incubated with the Aβ(1-42)/Cu 2+ /AA system, were reduced by the DHPA treatment ( Figure  4A,B). The activities of SOD and GSH increased with the DHPA treatment ( Figure 4C, D). Moreover, the cell apoptosis of SH-SY5Y induced by the Aβ(1-42)/Cu 2+ /AA system was also inhibited by the DHPA treatment ( Figure 2).
Apoptosis is a complex, precise, and protease-dependent molecular cascade process [43]. The Bcl-2 protein family is mainly located in the outer membrane of mitochondria, including pro-apoptotic members (Bax, etc.) and anti-apoptotic proteins (Bcl-2, etc.) [44]. Upon cell stimuli by oxidative stress, the ratio of Bax/Bcl-2 increased, which changed the MMP, activated caspase-3, and resulted in cell apoptosis [45]. Thus, the ratio of Bax/Bcl-2 and the expression level of cleaved caspase-3 play major roles in cell apoptosis [46]. Our experiments showed that Aβ(1-42)/Cu 2+ /AA stimulation increased the ratio of Bax/Bcl-2 proteins in SH-SY5Y cells, which reduced the MMP and increased the expression of cleaved caspase-3. However, the addition of DHPA provided significant protection against Aβ(1-42)/Cu 2+ /AA-induced damage by downregulating the ratio of Bax/Bcl-2 and cleaved caspase-3, alleviating MMP loss ( Figure 5). These results indicate that DHPA Apoptosis is a complex, precise, and protease-dependent molecular cascade process [43]. The Bcl-2 protein family is mainly located in the outer membrane of mitochondria, including pro-apoptotic members (Bax, etc.) and anti-apoptotic proteins (Bcl-2, etc.) [44]. Upon cell stimuli by oxidative stress, the ratio of Bax/Bcl-2 increased, which changed the MMP, activated caspase-3, and resulted in cell apoptosis [45]. Thus, the ratio of Bax/Bcl-2 and the expression level of cleaved caspase-3 play major roles in cell apoptosis [46]. Our experiments showed that Aβ  /Cu 2+ /AA stimulation increased the ratio of Bax/Bcl-2 proteins in SH-SY5Y cells, which reduced the MMP and increased the expression of cleaved caspase-3. However, the addition of DHPA provided significant protection against Aβ  /Cu 2+ /AA-induced damage by downregulating the ratio of Bax/Bcl-2 and cleaved caspase-3, alleviating MMP loss ( Figure 5). These results indicate that DHPA could protect the SH-SY5Y cells from Aβ  /Cu 2+ /AA-induced cell apoptosis via the mitochondrial apoptotic pathway.
PI3K/Akt is an important regulatory pathway of cells; it is involved in regulating cell growth, metabolism, apoptosis, etc. [47]. Akt can be activated by phosphorylation and subsequently activate multiple downstream targets to enhance cell survival. In this study, DHPA can significantly increase the expression of the P-Akt protein ( Figure 6C), suggesting that DHPA protects SH-SY5Y cells from Aβ  /Cu 2+ /AA-induced oxidative damage by inhibiting ROS accumulation, activating the PI3K/Akt pathway, and regulating the expressions of apoptosis-related proteins.
Mitogen-activated protein kinases (MAPKs) include JNK, P38, and ERK, among which, P38 can be stimulated by a variety of cytokines to produce corresponding effects and participate in regulating cell proliferation, differentiation, migration, invasion, and death [35]. In AD brains, phosphorylated (active) P38 (P-P38) levels are increased [48]. Moreover, many studies have shown a relationship between the activation of the P38/Erk pathway and programmed cell death [49,50]. In our study, DHPA significantly enhanced the expressions of P-P38 and P-Erk proteins ( Figure 6B,D). Moreover, activated P38 induces Bax translocation and mediates the activation of caspases family proteins, which induce mitochondrial apoptosis. At the same time, PI3K/Akt and MAPKs regulate Keap1/Nrf2/HO-1 signaling pathway activation and promote nuclear translocation of Nrf2 [51].
The Keap1/Nrf2/HO-1 signaling pathway is considered the "switch" for the endogenous antioxidant system. Under normal physiological conditions, the Nrf2 signaling pathway is negatively regulated by the binding of Keap1 to the Nrf2 protein [52,53]. When the cells are stimulated with oxidative stress, Nrf2 is activated, separates from Keap1, is translocated from the cytoplasm to the nucleus, and induces the expressions of NQO1 and HO-1 [19,54]. In our study, DHPA could significantly activate Nrf2 in a dose-dependent manner and increase the expressions of NQO1 and HO-1 (Figure 7), which resulted in protective activity against oxidative stress induced by Aβ  /Cu 2+ /AA in SH-SY5Y cells. The molecular mechanism of the activation effect of DHPA on the Keap1/Nrf2/HO-1 signaling pathway was investigated with MD. MD could provide the binding modes and affinities between the compounds and proteins [55]. As reported in another study, curcumin could bind to the Keap1 protein to separate the binding between Keap1 and Nrf2, promote the entrance of Nrf2 to the nucleus, and activate the antioxidant system [21]. In the present study, the binding of Keap1 to curcumin and DHPA was detected. Moreover, the results show that DHPA, similar to curcumin, could bind to Keap1 via hydrogen bonds (Figure 8) (Table 1); the binding could promote the separation of Nrf2 to Keap1, which could result in the activation of the Keap1/Nrf2/HO-1 signaling pathway.

Conclusions
To summarize, DHPA can be considered an inhibitor for Aβ  /Cu 2+ /AA-induced oxidative damage by scavenging OH, regulating mitochondria apoptosis, and activating the Keap1/Nrf2/HO-1 signaling pathway. Our results indicate that DHPA could decrease Aβ  /Cu 2+ /AA-induced OH production, intracellular ROS accumulation, and MDA production. It could also repress the ratio of Bax/Bcl-2, MMP increase, activated caspase-3 expression, and the phosphorylation of Erk1/2 and P38. Furthermore, DHPA could increase the expression of P-AKT and bind with Keap1 to promote the separation of Nrf2 to Keap1 and activate the Keap1/Nrf2/HO-1 signaling pathway to increase the expressions of HO-1, NQO1, GSH, and SOD. Therefore, the results suggest that DHPA has high antioxidative abilities and might be considered a potential drug for AD.