Nutr Res Pract.  2023 Oct;17(5):899-916. 10.4162/nrp.2023.17.5.899.

Neuroprotective effects of hesperetin on H ₂ O ₂ -induced damage in neuroblastoma SH-SY5Y cells

• Moon HR
• Yun JM

• KMID: 2546947
• DOI: http://doi.org/10.4162/nrp.2023.17.5.899

Abstract

BACKGROUND/OBJECTIVES
Oxidative stress is a fundamental neurodegenerative disease trigger that damages and decimates nerve cells. Neurodegenerative diseases are chronic central nervous system disorders that progress and result from neuronal degradation and loss. Recent studies have extensively focused on neurodegenerative disease treatment and prevention using dietary compounds. Heseperetin is an aglycone hesperidin form with various physiological activities, such as anti-inflammation, antioxidant, and antitumor. However, few studies have considered hesperetin’s neuroprotective effects and mechanisms; thus, our study investigated this in hydrogen peroxide ( H ₂ O ₂ )-treated SH-SY5Y cells.
MATERIALS/METHODS
SH-SY5Y cells were treated with H ₂ O ₂ (400 µM) in hesperetin absence or presence (10–40 µM) for 24 h. Three-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assays detected cell viability, and 4′,6-diamidino-2-phenylindole staining allowed us to observe nuclear morphology changes such as chromatin condensation and apoptotic nuclei. Reactive oxygen species (ROS) detection assays measured intracellular ROS production; Griess reaction assays assessed nitric oxide (NO) production. Western blotting and quantitative polymerase chain reactions quantified corresponding mRNA and proteins.
RESULTS
Subsequent experiments utilized various non-toxic hesperetin concentrations, establishing that hesperetin notably decreased intracellular ROS and NO production in H ₂ O ₂ -treated SH-SY5Y cells (P < 0.05). Furthermore, hesperetin inhibited H ₂ O ₂ -induced inflammation-related gene expression, including interluekin-6, tumor necrosis factor-α, and nuclear factor kappa B (NF-κB) p65 activation. In addition, hesperetin inhibited NFκB translocation into H ₂ O ₂ -treated SH-SY5Y cell nuclei and suppressed mitogen-activated protein kinase protein expression, an essential apoptotic cell death regulator. Various apoptosis hallmarks, including shrinkage and nuclear condensation in H ₂ O ₂ -treated cells, were suppressed dose-dependently. Additionally, hesperetin treatment down-regulated Bax/ Bcl-2 expression ratios and activated AMP-activated protein kinase-mammalian target of rapamycin autophagy pathways.
CONCLUSION
These results substantiate that hesperetin activates autophagy and inhibits apoptosis and inflammation. Hesperetin is a potentially potent dietary agent that reduces neurodegenerative disease onset, progression, and prevention

Keyword

Neurodegenerative disease; hydrogen peroxide; inflammation; apoptosis; hesperetin

Figure

• 

  Fig. 1 Hesperetin increased cell viability in H2O2-treated SH-SY5Y cells.(A) Cells were exposed to various H2O2 concentrations (100-600 µM) for 24 h, and the MTT assay was used to measure cell viability. (B) Cells were pretreated with hesperetin at several concentrations and then induced with or without 400 µM H2O2 for 24 h. Experiments were performed in triplicate, and results are presented as the mean ± SD. Different letters indicate significant differences (P < 0.05) as determined by Duncan’s multiple range test.H2O2, hydrogen peroxide; MTT, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide.

• 

  Fig. 2 Hesperetin suppressed ROS and NO production levels in H2O2-induced SH-SY5Y cells.(A) The DCFDA assay detected intracellular ROS accumulation. A fluorescence plate reader estimated intracellular ROS levels at Ex/Em = 485/535 nm. (B) SH-SY5Y cells were treated with hesperetin (10–40 µM) for 2 h and then exposed to H2O2 for 24 h before harvest. The culture supernatant was assayed using the Griess reagent to estimate the NO concentration. Experiments were performed in triplicate, and results are presented as the mean ± SD. Different letters indicate significant differences (P < 0.05) as determined by Duncan’s multiple range test.H2O2, hydrogen peroxide; ROS, reactive oxygen species; NO, nitric oxide.

• 

  Fig. 3 Hesperetin inhibited inflammatory cytokine secretion factor in H2O2-treated SH-SY5Y cells.SH-SY5Y cells were treated with hesperetin (10–40 µM) for 2 h and then exposed to H2O2 for 24 h before harvest. (A) An ELISA kit measured IL-6 and TNF-α secretion. TNF-α and COX-2 expression levels were measured using (A) immunoblotting, and (D–E) densities were normalized to β-actin using ImageJ software. (D) TNF-α and (E) COX-2 levels. Experiments were performed in triplicate, and results are presented as the mean ± SD. Different letters indicate significant differences (P < 0.05) as determined by Duncan’s multiple range test.H2O2, hydrogen peroxide; TNF, tumor necrosis factor; IL, interleukin; COX-2, cyclooxygenase-2; ELISA, enzyme-linked immunosorbent assay.

• 

  Fig. 4 Hesperetin’s effects on NF-κB activation in H2O2-treated SH-SY5Y cells.NF-κB and p-IκBα proteins levels were measured using (A) immunoblotting, and (B-E) densities were normalized to β-actin using ImageJ software. (B) p-IκBα (cytosol), (C) NF-κB (nuclear). Cells were harvested, and (D) NF-κB mRNA expression in H2O2-induced SH-SY5Y cells was evaluated. Data are presented as the mean ± SD. Different letters indicate significant differences (P < 0.05) as determined by Duncan’s multiple range test. (E) SH-SY5Y cells were treated with hesperetin and fixed with 4% paraformaldehyde. After blocking with an appropriate buffer, cells were incubated with antibodies. Next, DAPI staining confirmed cell nuclei. Signals were quantified using fluorescence microscopy at 400× magnification.H2O2, hydrogen peroxide; NF, nuclear factor; p-, phosphorylated; DAPI, 4′,6-diamidino-2-phenylindole.

• 

  Fig. 5 Hesperetin inhibited H2O2-induced apoptosis in H2O2-treated SH-SY5Y cells.Procaspase-3 protein expression levels were determined using (A) immunoblotting, and (B) densities were normalized to β-actin using ImageJ software. Bax and Bcl-2 protein expression levels were measured using (C) immunoblotting, and (D) densities were normalized to β-actin using ImageJ software. (D) Bax/Bcl-2 levels. (E, F) The relative mRNA expression levels are depicted after normalization against β-actin mRNA expression. The data are expressed relative to untreated cells’ mRNA levels, which was arbitrarily defined as 1. Experiments were performed in triplicate, and the results are presented as the mean ± SD. Data were analyzed by applying the 2−ΔΔCT method. Different letters indicate significant differences (P < 0.05) as determined by Duncan’s multiple range test. (G) SH-SY5Y cells were treated with hesperetin (10–40 µM) for 24 h and fixed with 4% paraformaldehyde. A fluorescence microscope assessed signal quantification at 400× magnification.H2O2, hydrogen peroxide; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2.

• 

  Fig. 6 Hesperetin’s effect on the MAPK signaling pathway in H2O2-treated SH-SY5Y cells.p-ERK, p-JNK, and p-p38 expression levels were measured using (A) immunoblotting, and (B-D) densities were normalized to β-actin using ImageJ software. (B) p-ERK, (C) p-JNK, and (D) p-p38 levels. Experiments were performed in triplicate, and results are presented as the mean ± SD. Different letters indicate significant differences (P < 0.05) as determined by Duncan’s multiple range test.MAPK, mitogen-activated protein kinase; H2O2, hydrogen peroxide; p-, phosphorylated; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; p38, p38 mitogen-activated protein kinases.

• 

  Fig. 7 Hesperetin induces autophagy through the SIRT1-AMPK-mTOR pathway.SIRT1-AMPK-mTOR pathway protein expression levels were measured using (A) immunoblotting, and (B-D) densities were normalized to β-actin using ImageJ software. (B) p-AMPK/AMPK, (C) p-mTOR/mTOR, and (D) SIRT1 (nuclear) levels. Experiments were performed in triplicate, and results are presented as the mean ± SD. Different letters indicate significant differences (P < 0.05) as determined by Duncan’s multiple range test.H2O2, hydrogen peroxide; AMPK, AMP-activated protein kinase; mTOR, mammalian target of rapamycin; Sirt1, NAD-dependent deacetylase sirtuin-1; p-, phosphorylated.

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