Introduction

Mitochondria are the major organelles responsible for the synthesis of ATP in mammalian cells [1,2,3]. Furthermore, mitochondria are an important source of reactive oxygen species (ROS), such as radical anion superoxide (O2 −·) and hydrogen peroxide (H2O2) [4]. Mitochondrial damage may lead to bioenergetics impairment and cell death due to the release of cytochrome c to the cytosol [5]. Indeed, mitochondrial dysfunction has been viewed in several human disorders, such as neurodegeneration and cardiovascular diseases [6,7,8]. Moreover, mitochondria are a target of several toxicants that increase the production of reactive species by the organelles [9,10,11,12,13]. Therefore, mitochondrial protection is of pharmacological interest. In this context, the number of publications involving the mitochondrial medicine field has increased, as recently reported [14].

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is the master regulator of the redox environment in mammalian cells [15,16,17]. Under physiological conditions, Nrf2 is maintained in the cytosol through the binding with the Kelch-like ECH-associated protein 1 (Keap1) [18]. Increased levels of pro-oxidant agents and/or xenobiotics cause the release of Nrf2 from Keap1 and its translocation to the cell nucleus, where this transcription factor upregulates the expression of several enzymes by binding to the antioxidant responsive element (ARE) present in the genes of these proteins [19]. Nrf2 controls the expression of the catalytic (GCLC) and regulatory (GCLM) subunits of γ-glutamate-cysteine ligase (γ-GCL), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S-transferase (GST), among others [20]. In this context, Nrf2 is an important regulator of both synthesis and metabolism of reduced glutathione (GSH) in mammalian cells [15].

Naringenin (NGN; 5,7-dihydroxy-2-(4-hydroxyphenyl)-2,3-dihydrochromen-4-one; MW 272.25 g/mol) is a flavanone found in citrus fruits and has been viewed as an potent inducer of Nrf2 presenting cytoprotective and antioxidant capacities in several cell types [21, 22]. Furthermore, NGN induces anti-inflammatory effects in both in vitro and in vivo experimental models [23, 24]. Recently, Lou et al. [25] have published that NGN suppressed the 6-hydroxydopamine-induced neurotoxicity by an Nrf2-dependent mechanism in SH-SY5Y cells. Nonetheless, the role of NGN as a possible protective agent regarding mitochondrial function has not been studied yet.

Therefore, we investigated in the present work whether and how NGN would protect mitochondria in an experimental model using human neuroblastoma SH-SY5Y cells exposed to H2O2, a pro-oxidant agent widely utilized to induce redox impairment experimentally.

Materials and Methods

Materials

Plastic materials used to perform cell culture were acquired from Corning, Inc (NY, USA) and Beckton Dickson (NJ, USA). Reagents necessary to culture cell have been obtained from Sigma (MO, USA). Other chemicals and assay kits utilized here were obtained from different manufacturers, as described below.

Cell Culture and Treatments

The human dopaminergic neuroblastoma SH-SY5Y cells have been acquired from the American Type Culture Collection (Manassas, VA, USA) and were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 HAM nutrient medium (1:1 mixture) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1000 units/mL penicillin, 1000 µg/mL streptomycin, and 2.5 µg/mL amphotericin B in a 5% CO2 humidified incubator at 37 °C.

In order to induce mitochondrial dysfunction, redox impairment, and cell death in SH-SY5Y cells, we utilized H2O2 at 300 µM for different periods of incubation according to each specific assay, as previously reported by us [26,27,28]. NGN at 20–80 µM was administrated to the cells 2 h before exposure to H2O2 for additional 3 or 24 h, according to each assay.

Evaluation of Cell Viability

Cell viability was studied by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as previously reported [29].

Quantification of Mitochondria-Related Apoptotic Factors

We quantified the levels of Bcl-2, Bax, cytochrome c (mitochondrial and cytosolic), and cleaved PARP by using commercial ELISA assay kits based on the instructions of the manufacturer (Abcam, MA, USA). We evaluated the activities of the apoptotic enzymes caspase-9 and caspase-3 through the utilization of fluorimetric assay kits according to the instructions of the manufacturer (Abcam, MA, USA). DNA fragmentation determination in cell lysates was performed by using a commercial ELISA kit based on the instructions of the manufacturer (Roche, Germany).

Quantification of the Production of Intracellular Reactive Oxygen Species (ROS)

In order to quantify the production of intracellular ROS, we utilized the nonpolar compound 2′-7′-dichlorodihydrofluorescein diacetate (DCFH-DA) assay, as previously described [30].

Evaluation of NO· Production

The production of NO· was quantified by using a commercial kit according to the instructions of the manufacturer (Abcam, MA, USA).

Quantification of Malondialdehyde (MDA), Protein Carbonyl, and 8-Oxo-dG Levels

In order to evaluate the levels of both total and mitochondrial MDA and protein carbonyl, and nuclear 8-oxo-dG content, we used commercial kits (Abcam, MA, USA) following the instructions of the manufacturer.

Examination of Mitochondrial 3-Nitrotyrosine Levels

The levels of 3-nitrotyrosine in mitochondrial membranes were evaluated by using a polyclonal antibody to 3-nitrotyrosine (Calbiochem, Germany) in an indirect ELISA assay, as previously reported [28, 31].

Determination of GSH Levels

The levels of total and mitochondrial GSH were determined according to the protocol of a commercial kit based on the instructions of the manufacturer (Abcam, MA, USA).

Isolation of Mitochondria

Mitochondria were isolated from SH-SY5Y cells by washing and re-suspending the cells in a buffer (250 mM sucrose, 10 mM KCl, 1 mM EGTA, 1 mM EDTA, 1 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulphonyl floride, 1 mM benzamidine, 1 mM pepstatin A, 10 mg/mL leupeptin, 2 mg/mL aprotonin, and 20 mM HEPES, pH 7.4). After several differential centrifugations, samples containing purified mitochondria were collected [32].

Extraction of Submitochondrial Particles (SMP)

After mitochondrial isolation, the organelles were frozen and thawed (three times) rendering superoxide dismutase-free SMP. This solution presenting SMP was washed (twice) by using a buffer (140 mM KCl, 20 mM Tris-HCl, pH 7.4), causing Mn-superoxide dismutase leakage from the organelles. We applied this protocol to verify the quantification of O2 −· production and to study the effects of H2O2 and/or NGN on the levels of markers of lipid peroxidation, protein carbonylation, and protein nitration in mitochondrial membranes [33].

Evaluation of Enzyme Activities

We analyzed the enzyme activities of aconitase, α-ketoglutarate dehydrogenase (α-KGDH), succinate dehydrogenase (SDH), complex I, and complex V by using commercial kits according to the instructions of the manufacturer (Abcam, MA, USA).

Quantification of the ATP Levels

We examined the levels of ATP by the utilization of commercial kit following the instruction of the manufacturer (Abcam, MA, USA).

Determination of MMP

We analyzed MMP by using a commercial kit applying the tetraethylbenzimidazolylcarbocyanide iodine (JC-1) following the instructions of the manufacturer (Abcam, MA, USA).

siRNA Transfection

In order to knockdown the transcription factor Nrf2, we performed transient transfection of SH-SY5Y cells by using Nrf2 siRNA based on the recommendations of the manufacturer (Santa Cruz, CA, USA).

Statistical Analyses

Statistical analyses were performed in the present work by utilizing the GraphPad 5.0 software. Data are shown as the mean ± standard error of the mean (S.E.M) of three or five independent experiments each done in triplicate; p values were considered significant when p < 0.05. Differences between the experimental groups were checked by one-way ANOVA followed by the post hoc Tukey’s test.

Results

NGN Prevented Cell Death in SH-SY5Y Cells Exposed to H2O2

According to Fig. S1, NGN at different concentrations (20–80 µM) decreased the effect of H2O2 on the viability of SH-SY5Y cells. NGN at 80 µM efficiently prevented the decrease in the levels of Bcl2 (Fig. S2A), as well as suppressed the increase in the levels of Bax in cells exposed to H2O2 (Fig. S2B). NGN blocked cytochrome c release to the cytosol (Fig. S2C), consequently maintaining the levels of this protein in the mitochondria of SH-SY5Y cells treated with H2O2 (Fig. S2D). In this regard, NGN blocked the activation of caspase-9 (Fig. S3A) and caspase-3 (Fig. S3B), leading to decreased cleavage of PARP (Fig. S3C) and fragmentation of DNA (Fig. S3D).

NGN Induced Antioxidant Effects in SH-SY5Y Cells Treated with H2O2

As depicted in Fig. 1a, pretreatment with NGN at 80 µM decreased the production of reactive species in SH-SY5Y cells administrated with H2O2. NGN reduced lipid peroxidation (Fig. 1b) and protein carbonylation (Fig. 1c) in H2O2-treated SH-SY5Y cells. Additionally, NGN enhanced the levels of GSH, as well as prevented the H2O2-induced decrease in the levels of this antioxidant in SH-SY5Y cells (Fig. 1d). NGN also decreased the effect of H2O2 regarding oxidative damage in DNA, as shown in Fig. 2. NGN pretreatment also reduced the production of O2 −· (Fig. 3a) and NO• (Fig. 3b) in stressed SH-SY5Y cells.

Fig. 1
figure 1

The effects of NGN on the intracellular production of ROS (a) and on the levels of lipid peroxidation (b), protein carbonylation (c), and cellular GSH (d) in SH-SY5Y cells exposed to H2O2. The cells were treated with NGN at 80 µM for 2 h prior administration of H2O2 at 300 µM for additional 24 h. ROS production was examined 3 h after exposure to H2O2 due to the high reactivity of these chemical species. Data are shown as the mean ± SEM of three or five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey’s test, *p < 0.05 different from the control group; # different from H2O2-treated group; a different from the control group

Full size image
Fig. 2
figure 2

The effects of NGN on the levels of 8-oxo-dG in SH-SY5Y cells exposed to H2O2. The cells were treated with NGN at 80 µM for 2 h prior administration of H2O2 at 300 µM for additional 24 h. Data are shown as the mean ± SEM of three or five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey’s test, *p < 0.05 different from the control group; # different from H2O2-treated group

Full size image
Fig. 3
figure 3

The effects of NGN on the production of O2 −· (a) and NO· (b) in SH-SY5Y cells exposed to H2O2. The cells were treated with NGN at 80 µM for 2 h prior administration of H2O2 at 300 µM for additional 3 h due to the high reactivity of these chemical species. Data are shown as the mean ± SEM of three or five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey’s test, *p < 0.05 different from the control group; # different from H2O2-treated group

Full size image

NGN Induced Mitochondrial Protection in SH-SY5Y Cells Administrated with H2O2

As shown in Fig. 4a, NGN prevented the H2O2-induced loss of MMP in SH-SY5Y cells. Moreover, NGN decreased lipid peroxidation (Fig. 4b), protein carbonylation (Fig. 4c), and protein nitration (Fig. 4d) in mitochondrial membranes obtained from SH-SY5Y cells treated with H2O2. NGN also upregulated the levels of GSH in the mitochondria of SH-SY5Y cells, as well as efficiently prevented the H2O2-induced decrease in mitochondrial GSH in this experimental model (Fig. 5).

Fig. 4
figure 4

The effects of NGN on the MMP (a) and on the levels of lipid peroxidation (b), protein carbonylation (c), and protein nitration (d) in mitochondrial membranes obtained from SH-SY5Y cells exposed to H2O2. The cells were treated with NGN at 80 µM for 2 h prior administration of H2O2 at 300 µM for additional 24 h. Data are shown as the mean ± SEM of three or five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey’s test, *p < 0.05 different from the control group; # different from H2O2-treated group

Full size image
Fig. 5
figure 5

The effects of NGN on the levels of GSH in mitochondria obtained from SH-SY5Y cells exposed to H2O2. The cells were treated with NGN at 80 µM for 2 h prior administration of H2O2 at 300 µM for additional 24 h. Data are shown as the mean ± SEM of three or five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey’s test, *p < 0.05 different from the control group; # different from H2O2-treated group; a different from the control group

Full size image

On the other hand, NGN prevented bioenergetics deficits induced by H2O2 in SH-SY5Y, as demonstrated in Fig. 9. NGN prevented the H2O2-mediated inhibition of the TCA cycle enzymes aconitase (Fig. 6a), α-ketoglutarate dehydrogenase (Fig. 6b), and succinate dehydrogenase (Fig. 6c). Furthermore, NGN reduced the impact of the treatment with H2O2 on the activity of complex I (Fig. 7a) and complex V (Fig. 7b), as well as on the levels of ATP (Fig. 7c) in SH-SY5Y cells.

Fig. 6
figure 6

The effects of NGN on the activities of aconitase (a), α-ketoglutarate dehydrogenase (b), and succinate dehydrogenase (c) in SH-SY5Y cells exposed to H2O2. The cells were treated with NGN at 80 µM for 2 h prior administration of H2O2 at 300 µM for additional 24 h. Data are shown as the mean ± SEM of three or five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey’s test, *p < 0.05 different from the control group; # different from H2O2-treated group

Full size image
Fig. 7
figure 7

The effects of NGN on the activities of the complexes I (a) and V (b) and on the levels of ATP in SH-SY5Y cells exposed to H2O2. The cells were treated with NGN at 80 µM for 2 h prior administration of H2O2 at 300 µM for additional 24 h. Data are shown as the mean ± SEM of three or five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey’s test, *p < 0.05 different from the control group; # different from H2O2-treated group

Full size image

NGN Induced Mitochondrial Protection by a Mechanism Dependent on Nrf2 in SH-SY5Y cells

As depicted in Fig. 8, silencing of Nrf2 by using siRNA strategy suppressed the effect of NGN on the levels of GSH in the mitochondria of SH-SY5Y cells exposed to H2O2. In a similar way, Nrf2 knockdown abrogated the effects of NGN on the activity of aconitase (Fig. 9a), complex I (Fig. 9b), and complex V (Fig. 9c). NGN also failed to prevent the H2O2-induced loss of MMP in SH-SY5Y cells treated with siRNA against Nrf2 (Fig. 10).

Fig. 8
figure 8

The effects of Nrf2 silencing (48 h) on the levels of GSH in mitochondria obtained from SH-SY5Y cells exposed to NGN and/or H2O2. Data are shown as the mean ± SEM of three or five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey’s test, *p < 0.05 different from the cells transfected with scrambled control (NC) siRNA and treated with NGN and H2O2

Full size image
Fig. 9
figure 9

The effects of Nrf2 silencing (48 h) on the activities of aconitase (a), complex I (b), and complex V (c) in SH-SY5Y cells exposed to NGN and/or H2O2. Data are shown as the mean ± SEM of three or five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey’s test, * p < 0.05 different from the cells transfected with scrambled control (NC) siRNA and treated with NGN and H2O2

Full size image
Fig. 10
figure 10

The effects of Nrf2 silencing (48 h) on MMP in SH-SY5Y cells exposed to NGN and/or H2O2. Data are shown as the mean ± SEM of three or five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey’s test, *p < 0.05 different from the cells transfected with scrambled control (NC) siRNA and treated with NGN and H2O2

Full size image

NGN Induced Cytoprotection by an Nrf2-Dependent Mechanism in SH-SY5Y cells

Silencing of Nrf2 suppressed the NGN-induced cytoprotection in SH-SY5Y cells exposed to H2O2 (Fig. 11).

Fig. 11
figure 11

The effects of Nrf2 silencing (48 h) on the viability of SH-SY5Y cells exposed to NGN and/or H2O2. Data are shown as the mean ± SEM of three or five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey’s test, *p < 0.05 different from the cells transfected with scrambled control (NC) siRNA and treated with NGN and H2O2

Full size image

Discussion

In the herein presented work, we found that NGN pretreatment prevented mitochondrial dysfunction in SH-SY5Y cells exposed to H2O2. NGN suppressed the H2O2-induced redox impairment in mitochondrial membranes and attenuated the effects of this pro-oxidant regarding mitochondrial function, as assessed through the quantification of the activities of enzymes involved in the TCA cycle and in the oxidative phosphorylation system. Furthermore, NGN abrogated the pro-apoptotic signaling promoted by H2O2 in this experimental model. The mitochondria-related effects, as well as the cytoprotective action of NGN, were abolished by siRNA targeting Nrf2, demonstrating that this transcription factor may take a central role in mediating the beneficial actions elicited by NGN.

In fact, Nrf2, in addition to its role in the maintenance of the redox environment, has been linked to mitochondrial function and dynamics in different cell types, as reported by other research groups [34, 35]. Nrf2 is involved in the regulation of the expression of mitochondria-located antioxidant enzymes, such as Mn-superoxide dismutase (Mn-SOD) and GPx [36,37,38,39,40], and also takes a role in the control of mitochondria-related bioenergetics functions [41,42,43]. Recently, Nrf2 has been associated with the modulation of mitochondrial biogenesis in mammalian cells [44, 45].

In this regard, there is increasing interest in natural compounds that may regulate mitochondrial function and redox biology due to the central role these organelles present in the control of cellular homeostasis [46,47,48,49,50,51,52,53,54,55,56,57]. We have demonstrated that other bioactive molecules exhibit the ability to upregulate Nrf2, causing mitochondrial protection in SH-SY5Y cells exposed to different chemical stressors [26,27,28, 58,59,60,61,62]. These bioactive molecules share a common mechanism involving upregulation of the production of GSH in both total and mitochondrial samples. GSH is the major non-enzymatic antioxidant in mammalian cells, and is also utilized in the phase II detoxification reactions by the enzyme GST [63, 64]. GSH is utilized by GPx in the conversion of H2O2 into water, decreasing the risk this pro-oxidant agent generates hydroxyl radical (•OH) through either Fenton or Haber–Weiss reactions [15, 65]. Thus, GSH may take an important role in the NGN-induced cytoprotection in H2O2-treated SH-SY5Y cells. This effect of NGN would be particularly interesting in the case of both prevention and treatment of neurodegenerative diseases, such as Parkinson’s disease and Alzheimer’s disease, in which it has been observed decreased levels of both enzymatic and non-enzymatic antioxidant defenses [66,67,68,69]. Moreover, mitochondrial dysfunction and redox disturbances have been described as taking a pivotal role in the progression of those conditions [70]. Attenuation of mitochondrial dysfunction may lead to decreased production of reactive species and accumulation of markers of redox imbalance, which also present toxic effects, such as MDA, acrolein, and 4-hydroxynonenal [71]. Actually, we found that NGN pretreatment reduced the levels of MDA in both total and mitochondrial samples obtained from the cells exposed to H2O2. Thus, NGN is very likely to block the vicious cycle involving the production of reactive species and the accumulation of toxic agents in cells undergoing redox impairment. However, it remains to be fully understood exactly how this polyphenol interferes in cellular redox biology causing cytoprotection.

Overall, NGN induced mitochondrial protection in SH-SY5Y cells exposed to H2O2 by a mechanism dependent on the Nrf2 transcription factor. Future research should be done in in vivo experimental model focusing on different mammalian brain areas.