The Citrus Flavonoid Hesperetin Encounters Diabetes-Mediated Alzheimer-Type Neuropathologic Changes through Relieving Advanced Glycation End-Products Inducing Endoplasmic Reticulum Stress

The present study investigates whether hesperetin, a citrus flavonoid, can encounter advanced glycation end-product (AGE)-induced Alzheimer’s disease-like pathophysiological changes with the underlying mechanisms. SH-SY5Y cells pretreated with hesperetin before stimulation with AGEs (200 μg/mL) were assessed in the following experiments. Hesperetin (40 μmol/L) elevated the reduced cell viability induced by AGEs. Hesperetin ameliorated reactive oxygen species overproduction and the downregulation of superoxide dismutase, glutathione peroxidase, and catalase, triggered by AGEs. Amyloid precursor protein upregulation, accompanied by the increased production of Aβ, caused by AGEs, was reversed by hesperetin. However, hesperetin lowered β-site APP-cleaving enzyme 1 expression, inducing insulin-degrading and neprilysin expression. In addition, hesperetin downregulated the expressions of the AGEs-induced endoplasmic reticulum (ER) stress proteins, including 78-kDa glucose-regulated protein and C/EBP homologous protein, and lowered the phosphorylation of protein kinase R-like ER kinase and activating transcription factor 4. Hesperetin-pretreated cells had a minor apoptotic DNA fragmentation. Hesperetin is able to upregulate Bcl-2 protein expression, downregulate Bax expression, and decrease caspase-12/-9/-3 activity as well, indicating that it inhibits ER stress-mediated neuronal apoptosis. There is a similar effect between hesperetin and positive rosiglitazone control against Aβ aggravation of SH-SY5Y cell injury induced by AGEs. Thus, hesperetin might be a potential agent for treating glycation-induced Aβ neurotoxicity.


Introduction
Alzheimer's disease (AD) is one of the neurodegenerative diseases characterized by progressive memory deficits and cognitive decline [1]. The neuropathological symptoms of AD feature amyloid plaques, neurofibrillary tangles with neurons, and synapses loss in the brain areas linked to cognition, learning, and memory [2]. Based on the amyloid cascade hypothesis, the accumulation of amyloid β (Aβ) is a significant cause of AD [3]. Although available therapeutic methods deliver moderate relief of cognitive symptoms that cannot significantly alter these disease's progression due to AD's multifactorial and complex nature, successful therapeutic and preventive interventions are strongly needed [4].
An increasing epidemiological display shows a higher risk of developing AD in diabetic patients [5]. The pathology associated with diabetes-mediated AD involves chronic hyperglycemia accelerating the reaction between glucose and proteins that eventually cause 100 IU/mL penicillin, and 100 mg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 37 • C in a humidified 5% CO 2 incubator. SH-SY5Y cells were seeded at 1 × 10 6 cells/cm 2 in 100 mm diameter culture dishes in DMEM/F12 medium containing 10% FBS to induce neuronal differentiation. Once the cells were 40-50% confluent, differentiation was initiated by adding 10 mmol/L of retinoic acid (Sigma-Aldrich, St. Louis, MO, USA). Treatments for all the experiments were carried out after the five-day differentiation period.

Cell Viability Assay
The 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO, USA) assay used to measure the cellular metabolic activity could be an indicator of cell viability [27]. Cells were exposed to the different concentrations of hesperetin (10-40 µmol/L) or rosiglitazone (10 µmol/L) in the medium for 24 h at 37 • C in 5% CO 2 in the air. MTT (5 mg/mL of stock in PBS) was added (20 µL/well in 200 µL of cell suspension), then the medium was aspirated and the cells were incubated with MTT dye for another 4 h. At the end of incubation, the dye was taken out and 100 µL of DMSO was added to each well to solubilize the MTT formazan crystals. The absorbance of the solution in each well was measured on a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA) at 540 nm, and background optical densities were subtracted from that of each well. The net absorbance from the wells of the cells cultured with the vehicle (not treated) was taken as the 100% viability. The data are expressed as percentages compared to the vehicle-treated control.

Determination of Intracellular ROS Levels and Antioxidant Enzyme Activities
Cells were reacted with 10 µmol/L dichloro-dihydro-fluorescein diacetate (Sigma-Aldrich, St. Louis, MO, USA) at 37 • C in the dark for 10 min. The change in dichlorofluorescein fluorescence intensity was measured at an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA).
The determination of antioxidant enzyme activity, including superoxide dismutase (SOD; EC 1.15.1.1), glutathione peroxidase (GSH-Px; EC 1.11.1.9), and catalase (CAT; EC 1.11.1.6), were performed using an SOD Activity Colorimetric Assay Kit (Cat. # K335), GPx Colorimetric Assay Kit (Cat. # K762), and CAT Activity Colorimetric Assay Kit (Cat. # K773), respectively, following the procedures provided by Bio Vision, Inc. (San Francisco, CA, USA). The assay for SOD activity was determined by inhibiting the nitroblue tetrazolium reduction, with a xanthine-xanthine oxidase used as a superoxide generator [28]. The production of formazan was determined spectrophotometrically at 450 nm. The activity of GSH-Px was estimated based on the assay after the reduction in absorbance at 340 nm due to reduced nicotinamide adenine dinucleotide phosphate being consumed by glutathione reductase, with the incubation of the sample in the presence of hydrogen peroxide (H 2 O 2 ) to starting the reaction [29]. The activity of CAT was determined from the decomposition rate of H 2 O 2 to water and oxygen [30], and the absorbance was measured at 570 nm in a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA). Enzyme activities were expressed as units per milligram protein. The protein concentration was measured by using a Bio-Rad protein assay.
The activities of caspase-9 (Abcam plc., Cambridge, U.K., Cat. # ab65608) and -3 (Abcam plc., Cambridge, U.K., Cat. # ab39401) were measured by the colorimetric assay kits. In brief, aliquots of cell extracts containing 50 or 100 µg of protein were incubated for 2 h at 37 • C in a reaction buffer, with 100 µmol/L chromogenic substrates for caspase-9 (Ac-LEHD-pNA), or caspase-3 (Ac-DEVD-pNA). The caspase-9/-3-like activity was determined by measuring the cleavage substrate at 405 nm using a microplate reader. The results were expressed as relative to those obtained with vehicle-treated control.

Measurement of DNA Fragmentation
The determination of the cytoplasmic histone-associated DNA fragments (mono-and oligo nucleosomes) after induced cell death was quantitated by a cellular DNA fragmentation ELISA kit (Roche Molecular Biochemicals, Mannheim, Germany) with a primary anti-histone mouse monoclonal antibody coated to the microtiter plate, and a second anti-DNA mouse monoclonal antibody coupled to peroxidase [31]. The retained peroxidase in the immunocomplex was determined by incubating 2,2 -azino-di-[3-ethylbenzthiazoline sulfonate] as a substrate for 10 min at 20 • C. The color change was measured at a wavelength of 405 nm using a microplate reader (Spectramax M5, Molecular Devices, Sunnyvale, CA, USA). The optical density values at 405 nm were corrected for the total protein present in each sample. The data were normalized to the vehicle-treated control.

Western Blot Analysis
Cells were lysed with a lysis buffer containing a mixture of 30 mmol/L Tris-HCl, pH 7.4, 250 mmol/L Na 3 VO 4 , 5 mmol/L EDTA, 250 mmol/L sucrose, 1% Triton X-100 with protease inhibitor, and a phosphatase inhibitor cocktail. The Bio-Rad protein assay kit was used to determine the protein concentrations. The proteins (50 µg/lane) were loaded on a 12.5% polyacrylamide gel, submitted to electrophoresis, and then electroblotted onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). For the renaturation of proteins and the blocking of the unoccupied spaces, the membrane was incubated overnight at 4 • C in 0.1% Tween 20-PBS containing 5% nonfat dry milk.

Statistical Aanalysis
The results are presented as the mean ± standard deviation (SD) of five independent experiments performed triplicate (n = 5). The evaluation of statistically significant differences, one-way analysis of variance, and Dunnett range post hoc comparisons using Systat SigmaPlot version 14.0 (Systat Software Inc., San Jose, CA, USA) was used. It was considered statistically significant when the differences were at p < 0.05.

Hesperetin Improves Cell Viability in AGE-Challenged Cells
The cell viability was reduced from 88.4 to 57.1%, respectively, under the condition when SH-SY5Y cells were incubated with AGEs at 50 to 250 µg/mL concentrations for 24 h ( Figure 1A). The AGE (200 µg/mL)-induced cell viability reduction presented a timedependent tendency in SH-SY5Y cells ( Figure 1B). In this study, SH-SY5Y cells were exposed for 24 h to 200 µg/mL to start neuronal insults in the subsequent experiments. The 1 h pretreatment of hesperetin before the exposure of AGEs (200 µg/mL) improved the cell viability associated with an increasing concentration of hesperetin (10-40 µmol/L; Figure 1C). The protective potency of hesperetin was most potent at the 40 µmol/L concentration. The survival rate sustained to 83.9% in SH-SY5Y cells treated with hesperetin (40 µmol/L) before the AGE's (200 µg/mL) exposure ( Figure 1C). Pretreatment at 10 µmol/L of rosiglitazone with AGE (200 µg/mL)-cultured cells obtained a similar result in cell viability (84.3%; Figure 1C). Neither hesperetin nor rosiglitazone alone could induce cell death ( Figure 1D).

Hesperetin Alleviates Oxidative Stress in Cells under AGE Stimulation
The AGEs elevated the intracellular ROS production to 2.5-fold in untreated controls, which was reduced by hesperetin in a concentration-dependent manner ( Figure 2A). Hesperetin (40 µmol/L) pretreatment reduced the AGE-induced ROS production in SH-SY5Y cells by 35.9%, relative to the values from vehicle-treated counterparts; the results were similar to the effects produced by 10 µmol/L rosiglitazone ( Figure 2A).

Hesperetin Alleviates Oxidative Stress in Cells under AGE Stimulation
The AGEs elevated the intracellular ROS production to 2.5-fold in untreated cont which was reduced by hesperetin in a concentration-dependent manner (Figure 2A). H peretin (40 μmol/L) pretreatment reduced the AGE-induced ROS production in SH-S cells by 35.9%, relative to the values from vehicle-treated counterparts; the results w similar to the effects produced by 10 μmol/L rosiglitazone (Figure 2A).
The activities in SOD and GSH-Px were reduced markedly when SH-SY5Y cells w exposed to 24 h AGEs (200 μg/mL), but were abrogated by hesperetin pretreatment concentration-dependent manner ( Figure 2B,C). The SOD and GSH-Px activity reduct in AGE-exposed SH-SY5Y cells were reversed by rosiglitazone (10 μmol/L), with res similar to those produced by hesperetin (40 μmol/L; Figure 2B,C).
The CAT activity in SH-SY5Y cells under AGE stimulation was lower (25.9%) t the untreated control group ( Figure 2D). The pretreatment of SH-SY5Y cells with peretin (40 μmol/L) or rosiglitazone (10 μmol/L) increased the CAT activity to 3.1-3.3-fold, respectively, in AGEs incubated relative to those observed in the vehicle-tre An MTT assay used cell viability determination; the value was expressed as a percentage of the control group from untreated cells. The results are presented as the mean ± SD of five independent experiments performed triplicate (n = 5). a p < 0.05 and b p < 0.01 when compared to the data from the untreated control group (control). c p < 0.05 and d p < 0.01 when compared to the data from the vehicle (Veh)-treated cells.
The activities in SOD and GSH-Px were reduced markedly when SH-SY5Y cells were exposed to 24 h AGEs (200 µg/mL), but were abrogated by hesperetin pretreatment in a concentration-dependent manner ( Figure 2B,C). The SOD and GSH-Px activity reductions in AGE-exposed SH-SY5Y cells were reversed by rosiglitazone (10 µmol/L), with results similar to those produced by hesperetin (40 µmol/L; Figure 2B,C).
The CAT activity in SH-SY5Y cells under AGE stimulation was lower (25.9%) than the untreated control group ( Figure 2D). The pretreatment of SH-SY5Y cells with hesperetin (40 µmol/L) or rosiglitazone (10 µmol/L) increased the CAT activity to 3.1-and 3.3-fold, respectively, in AGEs incubated relative to those observed in the vehicle-treated counterparts ( Figure 2D). The results are presented a mean ± SD of five independent experiments performed triplicate (n = 5). a p < 0.05 and b p < 0.01 w compared to the data from the untreated control group (control). c p < 0.05 and d p < 0.01 when pared to the data from the vehicle (Veh)-treated cells.

Hesperetin Lessens APP Cleavage and Decreases Aβ Secretion in Cells under AGE Stimulation
AGEs elevated the APP protein levels in SH-SY5Y cells to 4.8-fold of that in the v cle-treated group ( Figure 3A). The AGE-induced upregulation of APP expression wa duced in SH-SY5Y cells pretreated with hesperetin (40 μmol/L) or rosiglitazone μmol/L) by 55.3 and 58.8%, respectively, relative to those in the vehicle-treated coun part group ( Figure 3A).
The BACE1 level in SH-SY5Y cells receiving AGE induction was 4.4-fold of the v cle-treated group. Hesperetin (40 μmol/L) pretreatment reduced the higher BACE1 le by 51.1% ( Figure 3A). In contrast, rosiglitazone did not influence BACE1 levels in SY5Y cells that only received AGE induction ( Figure 3A).
The protein levels of IDE and NEP in AGE-cultured SH-SY5Y cells were lowere 43.5 and 45.6% of those in the untreated controls, respectively. At the same time, peretin (40 μmol/L) or rosiglitazone (10 μmol/L) pretreatment resulted in the upregula of both protein levels of IDE and NEP ( Figure 3A).
The results show that AGEs caused 3.3-fold and 2.6-fold increases in the amoun Aβ1-40 and Aβ1-42 in SH-SY5Y cells, respectively, whereas rosiglitazone (10 μmol/L) treatment attenuated these enhancements ( Figure 3B). It decreased to 50.7 and 54.1 Aβ1-40 and Aβ1-42 levels, respectively, when SH-SY5Y cells received hesperetin The results are presented as the mean ± SD of five independent experiments performed triplicate (n = 5). a p < 0.05 and b p < 0.01 when compared to the data from the untreated control group (control). c p < 0.05 and d p < 0.01 when compared to the data from the vehicle (Veh)-treated cells.

Hesperetin Lessens APP Cleavage and Decreases Aβ Secretion in Cells under AGE Stimulation
AGEs elevated the APP protein levels in SH-SY5Y cells to 4.8-fold of that in the vehicletreated group ( Figure 3A). The AGE-induced upregulation of APP expression was reduced in SH-SY5Y cells pretreated with hesperetin (40 µmol/L) or rosiglitazone (10 µmol/L) by 55.3 and 58.8%, respectively, relative to those in the vehicle-treated counterpart group ( Figure 3A).
The BACE1 level in SH-SY5Y cells receiving AGE induction was 4.4-fold of the vehicletreated group. Hesperetin (40 µmol/L) pretreatment reduced the higher BACE1 levels by 51.1% ( Figure 3A). In contrast, rosiglitazone did not influence BACE1 levels in SH-SY5Y cells that only received AGE induction ( Figure 3A).
The protein levels of IDE and NEP in AGE-cultured SH-SY5Y cells were lowered to 43.5 and 45.6% of those in the untreated controls, respectively. At the same time, hesperetin (40 µmol/L) or rosiglitazone (10 µmol/L) pretreatment resulted in the upregulation of both protein levels of IDE and NEP ( Figure 3A).

Hesperetin Attenuated ER Stress in Cells under AGE Stimulation
AGEs increased the GRP78 levels in SH-SY5Y cells to 4.5-fold of the vehicle-treated control ( Figure 4A). Hesperetin (40 µmol/L) or rosiglitazone (10 µmol/L) lowered the AGE's induced higher GRP78 levels in SH-SY5Y cells by 44.9 and 52.6%, respectively ( Figure 4A).   . The band densities for protein were normalized to the β-actin band intensity. The ratio of phosphoprotein to total protein in PERK (p-PERK/PERK) and eIF2α (p-eIF2α/eIF2α) is also shown. The results are presented as the mean ± SD of five independent experiments performed triplicate (n = 5). a p < 0.05 and b p < 0.01 when compared to the data from the untreated control group (control). c p < 0.05 and d p < 0.01 when compared to the data from the vehicle (Veh)-treated cells.
AGEs caused the protein levels of ATF4 and CHOP to increase by 4.8-and 4.0-fold in SH-SY5Y cells, respectively ( Figure 4D,E). AGE-induced upregulation on ATF4 and CHOP protein expression in SH-SY5Y cells remarkably reversed in hesperetin (40 µmol/L) pretreatment (57.3% decreases in ATF4, 48.7% decreases in CHOP; Figure 4D,E). The AGEelevated higher levels of ATF4 and CHOP decreased to 49.2 and 45.7%, respectively, when the cells were pretreated with 10 µmol/L rosiglitazone ( Figure 4D,E).
AGEs caused the protein levels of ATF4 and CHOP to increase by 4.8-and 4.0-fold in SH-SY5Y cells, respectively ( Figure 4D,E). AGE-induced upregulation on ATF4 and CHOP protein expression in SH-SY5Y cells remarkably reversed in hesperetin (40 μmol/L) pretreatment (57.3% decreases in ATF4, 48.7% decreases in CHOP; Figure 4D,E). The AGE-elevated higher levels of ATF4 and CHOP decreased to 49.2 and 45.7%, respectively, when the cells were pretreated with 10 μmol/L rosiglitazone ( Figure 4D,E).
AGEs markedly decreased the Bcl-2/Bax ratio in SH-SY5Y cells, which was increased (4.1 and 4.6-fold elevation, respectively) by pretreatment with hesperetin (40 μmol/L) or rosiglitazone (10 μmol/L) ( Figure 5A). AGEs induced a 3.7-fold increase in caspase-12-like activity, which suffered a 2.1-fold and 1.9-fold reduction in SH-SY5Y cells pretreated with hesperetin (40 μmol/L) or rosiglitazone (10 μmol/L) ( Figure 5B). The caspase-like activities in caspase-9 and caspase-3 increased by 4.1-and 4.0-fold in cells under AGE stimulation ( Figure 5B). Hesperetin (40 The commercial kit quantified cytosolic histone-associated DNA fragmentation. The results are presented as the mean ± SD of five independent experiments performed triplicate (n = 5). a p < 0.05 and b p < 0.01 when compared to the data from the untreated control group (control). c p < 0.05 and d p < 0.01 when compared to the data from the vehicle (Veh)-treated cells.
An elevation of apoptotic DNA fragmentation to 4.3-fold higher was induced by AGEs, which was decreased by 50.3% when pretreated with hesperetin (40 µmol/L) ( Figure 5C). The pretreatment of rosiglitazone (10 µmol/L) alleviated the greater degree of apoptotic DNA fragmentation induced by AGEs ( Figure 5C).

Discussion
AGE-induced oxidative stress significantly influences developing neurodegenerative disorders; thus, enhancing the antioxidant system to reduce oxidative stress could be a logical therapeutic approach [7]. Flavonoid compounds are clarified to antioxidative stress by their antioxidant activity, thus representing beneficial candidates for the protection against oxidative diseases, including neurodegenerative disorders, such as AD [32]. As an in vitro model for AD studies, the retinoic acid-induced cholinergic differentiation in SH-SY5Y cells was well used [24]. Recently, it has been demonstrated that G protein-coupled estrogen receptors have a direct anti-inflammatory effect in human cholinergic neurons [33]. Although flavonoids with estrogen-like activities could reduce inflammation in cholinergic neurons to consider as prospective neuroprotectants [34], the protective mechanisms related to alleviating oxidative stress and Aβ-mediated neurotoxicity induced by AGEs is essential to be elucidated. The obtained results show that hesperetin upregulates the antioxidative capacity to contribute to SH-SY5Y cells against ROS induced by AGEs. Further research on the ability to counter oxidative stress or examine the mechanisms by which hesperetin protects against AGE-induced neuronal cellular damage is valuable.
One characteristic hallmark of AD is an Aβ accumulation caused by the formation of plaque and Aβ complex toxicity [2,3]. Evidence has shown that AGE-induced ROS increases the synthesis of Aβ through the proteolytic processing of a transmembrane protein, APP, by upregulating APP processing proteins, such as BACE1, eventually leading to the aggregation and deposition of Aβ to cause neuronal damage [7]. The Aβ levels are highly dependent on Aβ degradation, although much attention has focused on the cellular production of Aβ by amyloidogenic processing. Among the enzymes that contributed to the cleaving of Aβ, NEP and IDE are the most important, which belong to two metalloprotease family members, M16 (IDE) and M13 (NEP), respectively [35]. Aβ composed a length of 39-43 amino acid residues; 40 amino acid residues (Aβ1-40), as well as 42 amino acid residues (Aβ1-42), are the predominant isoforms [36]. Remarkably, there is the potent aggregation and associated toxicity in Aβ1-42 [37]. In AGE-stimulated SH-SY5Y cells, hesperetin pretreatment resulted in the translational downregulation of APP and BACE1 accompanied by a decrease in Aβ secretion. It revealed that hesperetin might directly ameliorate Aβ overproduction via inhibiting Aβ generation. As mentioned before, the extent of Aβ expressed in the brain is controlled by Aβ production and its enzymatic degradation clearance pathways, thus investigating the influence of hesperetin on Aβ degradation. The pretreatment of AGE-cultured SH-SY5Y cells with hesperetin shown in the upregulation of protein levels in IDE and NEP, highlights that the attenuation of Aβ accumulation by this compound is mediated by modulating Aβ-degrading enzymes to increase Aβ degradation. Therefore, these findings support the notion that the downregulation of Aβ generation and promotion of Aβ degradation are responsible mechanisms by which hesperetin protects against AGE-induced neuronal cellular damage.
Evidence indicates that the accumulation of AGEs is associated with ER stress induction in cells contributing to the pathogenesis of AD and other multiple diseases [38]. During ER stress, GRP78 cooperated with the misfolded proteins to maintain the proteins in foldable states and control the release of three UPR mediators, including inositol-requiring enzyme 1 alpha (IRE1α), PERK, and ATF6 as well [39]. The CHOP protein is the transcription of genes controlled in cell apoptosis, activated by the PERK/eIF2α/ATF4 axis under prolonged ER stress [10]. Accordingly, several evidence lines suggest that AD's pathogenesis and progression are closely linked with the UPR signaling pathway controlled byPERK [40]. Therefore, therapeutic targeting on the PERK-associated UPR signaling branches may help to develop a therapeutic approach to treat AD [40]. We observed that hesperetin pretreatment counters the AGE-induced upregulation of proteins under ER stress involving GRP78, p-PERK, p-eIF2α, and ATF4. Moreover, the increasing CHOP expression level in SH-SY5Y cells after the induction with AGEs was attenuated by hesperetin pretreatment. These data suggest the survivability of AGE-treated SH-SY5Y cells enhanced by hesperetin through suppressing ER stress-induced GRP78 to downregulate PERK/eIF2α/ATF4/CHOP, finally contributing to neuron protection.
The Bcl2-family protein expression is controlled by several transcriptional factors involving CHOP [41]. Although Bcl-2 family proteins are characterized on the mitochondrion, the proteins are localized at ER and nuclear membranes [42]. Hesperetin prevented protein downregulation in Bcl-2 and, in parallel, reduced Bax expression in SH-SY5Y cells under AGE stimulation. Therefore, the action of hesperetin on the reductions in AGE-induced ER stress may be associated with recovering the balance between the pro-and anti-apoptotic Bcl-2 family, which finally leads to inhibiting neuronal apoptosis. Prolonged ER stress can activate caspase-12 that may feed back in a caspase-9-dependent loop involving caspase-3 activation and certainly catalyze apoptosis [11]. We observed that AGEs significantly induced SH-SY5Y cell apoptosis and increased active caspase-12 that directly activated caspase-9 and the downstream effector caspase-3, supporting the ER stress-mediated activation of the caspase pathway involved in AGE-induced apoptosis [43]. Compared with AGE-treated SH-SY5Y cells, hesperetin-pretreated cells had a minor apoptotic DNA fragmentation and decreased along with the cleavage of caspase-12; in parallel, protein expression and the activity of cleaved caspase-9 and -3 expression were downregulated by hesperetin pretreatment. Our data suggest that hesperetin prevents the AGE-induced activation of apoptosis in SH-SY5Y cells by reducing the caspase-12-dependent ER stress.
There is a similar effect between hesperetin and positive rosiglitazone control against the Aβ aggravation of SH-SY5Y cell injury induced by AGE-mediated ER stress. In contrast to rosiglitazone playing a minor role in the regulation of Aβ generation [44], hesperetin lowered BACE1 protein expression to downregulate Aβ generation in SH-SY5Y cells induced by AGEs. Although PPARs have recently been identified as ahead of the times targets in researching new active drugs for AD [45], whether the activation of PPARs was involved in the neuron protection of hesperetin remains to be investigated further.
In conclusion, hesperetin protects SH-SY5Y cells against AGE-induced ROS, resulting in a decrease in the Aβ accumulation via the downregulation of Aβ generation and enhancing Aβ degradation. Additionally, hesperetin resolved PERK/eIF2α/ATF4/CHOP activation together with anti-apoptotic effects via the suppression of the caspase-12-dependent pathway in AGE-treated SH-SY5Y cells. Therefore, we conclude that hesperetin protects neuroblastoma cells against AGE-induced damage via multiple mechanisms, and thus may be an excellent option for potential pre-symptom treatment for neurodegenerative disorders augmented under glycation conditions. Data Availability Statement: All the data needed to evaluate the conclusions in the paper are present in the paper. Additional data related to this paper may be requested from the authors.