Sulforaphane Induces Glioprotection After LPS Challenge

Abstract

Sulforaphane is a natural compound that presents anti-inflammatory and antioxidant properties, including in the central nervous system (CNS). Astroglial cells are involved in several functions to maintain brain homeostasis, actively participating in the inflammatory response and antioxidant defense systems. We, herein, investigated the potential mechanisms involved in the glioprotective effects of sulforaphane in the C6 astrocyte cell line, when challenged with the inflammogen, lipopolysaccharide (LPS). Sulforaphane prevented the LPS-induced increase in the expression and/or release of pro-inflammatory mediators, possibly due to nuclear factor κB and hypoxia-inducible factor-1α activation. Sulforaphane also modulated the expressions of the Toll-like and adenosine receptors, which often mediate inflammatory processes induced by LPS. Additionally, sulforaphane increased the mRNA levels of nuclear factor erythroid-derived 2-like 2 (Nrf2) and heme oxygenase-1 (HO1), both of which mediate several cytoprotective responses. Sulforaphane also prevented the increase in NADPH oxidase activity and the elevations of superoxide and 3-nitrotyrosine that were stimulated by LPS. In addition, sulforaphane and LPS modulated superoxide dismutase activity and glutathione metabolism. Interestingly, the anti-inflammatory and antioxidant effects of sulforaphane were blocked by HO1 pharmacological inhibition, suggesting its dependence on HO1 activity. Finally, in support of a glioprotective role, sulforaphane prevented the LPS-induced decrease in glutamate uptake, glutamine synthetase activity, and glial-derived neurotrophic factor (GDNF) levels, as well as the augmentations in S100B release and Na⁺, K⁺ ATPase activity. To our knowledge, this is the first study that has comprehensively explored the glioprotective effects of sulforaphane on astroglial cells, reinforcing the beneficial effects of sulforaphane on astroglial functionality.

Introduction

Sulforaphane (1-Isothiocyanato-4-(methylsulfinyl)-butane) is a natural isothiocyanate, found in cruciferous vegetables, such as broccoli, cauliflower, cabbage and kale (Bricker et al. 2014), that presents major anti-inflammatory and antioxidant properties. This molecule has demonstrated therapeutic potential in a wide variety of in vitro and in vivo experimental models, as well as in clinical settings (Liu et al. 2014; Tortorella et al. 2015; Fernandes et al. 2016; Axelsson et al. 2017; Ruhee et al. 2020; Tang et al. 2020). Sulforaphane, and its metabolites (sulforaphane-glutathione and sulforaphane-N-acetylcysteine), have shown high bioavailability and a wide distribution throughout the body and several pharmacokinetic studies have shown that orally administered sulforaphane can reach many target tissues at µM levels in an active form (Clarke et al. 2011; Bricker et al. 2014). Moreover, sulforaphane demonstrates a relevant ability to cross the blood–brain barrier, consequently acting in the central nervous system (CNS) and protecting neural cells in different models of brain injuries (Tarozzi et al. 2013; Bricker et al. 2014; Tortorella et al. 2015).

Of the neural cells, astrocytes make an important contribution to the physiological maintenance of the CNS. They provide energy metabolites, trophic factors, and antioxidant defenses, including glutathione (GSH) and superoxide dismutase (SOD), to other neural cells, particularly neurons (Bolaños 2016; García-Cáceres et al. 2019; Valori et al. 2019). Astrocytes also participate in the regulation and metabolism of neurotransmitters, especially glutamate, as well as ionic homeostasis, in addition to promoting neural remodeling and neurogenesis during development or tissue damage (Allen and Eroglu 2017; Vainchtein and Molofsky 2020). Importantly, astrocytes participate in immune responses during diseases or due to the presence of pathogens or potentially harmful molecules (Jensen et al. 2013; Colombo and Farina 2016; Greenhalgh et al. 2020), by expressing pattern-recognition receptors, including Toll-like receptors (TLR), which are responsible for recognizing these damaging stimuli and for evoking inflammatory responses (Anderson et al. 2014; Bobermin et al. 2019). Astroglial cells also express adenosine receptors (A₁, A_2A, A_2B, and A₃), which are able to modulate glial responses associated with neuroinflammation (Gessi et al. 2013; Bobermin et al. 2019).

The bacterial inflammogen, lipopolysaccharide (LPS), has been used experimentally to study inflammatory responses, since it binds to TLR4, leading to nuclear factor κB (NFκB) activation and the production of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) (Gorina et al. 2011; Bobermin et al. 2019). Moreover, the LPS-driven inflammatory response is associated with an increased production of reactive oxygen and nitrogen species (ROS/RNS) (Yoo et al. 2008; Bellaver et al. 2015; Wang and Zhao 2019) and, notably, the upregulation of both inflammatory and oxidative pathways contributes to cell damage and death, which are related to neurodegeneration and other brain disorders (Aguilera et al. 2018).

The beneficial effects of sulforaphane have been associated with its powerful ability to activate nuclear factor erythroid-derived 2-like 2/antioxidant responsive element (Nrf2/ARE) signaling (Danilov et al. 2009; Bergström et al. 2011; Pan et al. 2014; Dwivedi et al. 2016; Axelsson et al. 2017; Corssac et al. 2018; Uddin et al. 2020). Activation of Nrf2 is responsible for the induction of the gene expression of important metabolic, antioxidant and detoxification enzymes, including heme oxygenase-1 (HO1) (Wakabayashi et al. 2010; Dai et al. 2020). HO1 is an inducible enzyme that provides cellular resistance against stressful conditions, such as oxidative stress and inflammation (Chen 2014; Ryter and Choi 2016).

In the current study, we investigated potential mechanisms involved in the glioprotective effects of sulforaphane on LPS-challenged astroglial cells. For this, we evaluated: (i) cellular viability and morphology; (ii) inflammatory signaling—mRNA expression and/or content of TNF-α, IL-1β, NFκB, inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), high mobility group box 1 (HMGB1), hypoxia-inducible factor-1α (HIF-1α), HO1, TLR4, TLR2, A₁, A_2A, A_2B, and A₃; (iii) the effect of zinc protoporphyrin IX (ZnPP IX), a HO1 inhibitor, on the release of TNF-α, IL-1β, IL-6, interleukin-10 (IL-10) and on the transcriptional activity of NFκB; (iv) the oxidative imbalance—NADPH oxidase (NOX) activity, the levels of superoxide and 3-nitrotyrosine, the activities of SOD, catalase (CAT) and glutathione peroxidase (GPx), GSH levels and glutamate cysteine ligase (GCL) activity and expression, as well as the effect of HO1 inhibition on their activities; (v) the release of glial-derived neurotrophic factor (GDNF) and S100B, excitatory amino acid transporter 1 (EAAC1) expression and glutamate uptake, glutamine synthase (GS) mRNA expression and activity, and Na⁺, K⁺ ATPase activity; (vi) mRNA expression of Nrf2 and sirtuin 1 (SIRT1). This is the first study that comprehensively explores the glioprotective effects of sulforaphane. Our main findings indicate that sulforaphane prevented LPS-induced inflammatory and oxidative responses.

Materials and Methods

C6 Astroglial Cell Maintenance

The C6 astrocyte-like cell line was obtained from the American Type Culture Collection (Rockville, MA, USA). These cells have been widely used to study astroglial functions (oxidative/nitrosative and inflammatory responses, trophic factor release, glutamate uptake, GS activity, among others) and signaling pathways, similarly to primary astrocyte cultures (Quincozes-Santos et al. 2014, 2017). C6 astroglial cells were cultured in low glucose DMEM (pH 7.4), supplemented with 5% fetal bovine serum (FBS), 2.5 mg/ml fungizone (Gibco/Invitrogen) and 100 U/l gentamicin, at 37 °C and 5% CO₂ (dos Santos et al. 2006). During the exponential phase of growth, cells were detached from the culture flasks using 0.05% trypsin/ethylene-diamine tetraacetic acid (EDTA) and seeded at a density of 5 × 10³ cells/cm². Subsequently, C6 cells were maintained for 3 days until they reached confluence.

Sulforaphane and LPS Treatments

To evaluate the effect of sulforaphane (Sigma-Aldrich), we incubated C6 cells in the presence of three different concentrations of sulforaphane (1, 5 and 10 µM, dissolved in DMSO) during 4 h. To assess the effect of LPS and the glioprotective effect of sulforaphane, C6 cells were pre-incubated with 5 µM sulforaphane for 1 h at 37 °C in an atmosphere with 5% CO₂ in serum-free DMEM. Subsequently, 10 µg/ml LPS (Sigma-Aldrich) were added for 3 h. To investigate the putative role of HO1 in the glioprotective effect of sulforaphane on the LPS-induced glial response, we co-incubated sulforaphane with ZnPP IX (10 μM; Sigma-Aldrich), a specific HO1 inhibitor (Vargas et al. 2008; Quincozes-Santos et al. 2014). All treatments began after cells reached confluence. It should be noted that the final concentration of DMSO (used as vehicle) did not present any effect on C6 astroglial cells.

Cell Viability and Membrane Integrity Assays

The MTT (methylthiazolyldiphenyl-tetrazolium bromide; Sigma-Aldrich) reduction assay was used to verify cell viability after sulforaphane or LPS treatment. MTT was incubated at a final concentration of 50 µg/ml for 30 min at 37 °C in an atmosphere of 5% CO₂. The medium was then removed for dissolving MTT crystals in DMSO, and absorbance was measured at 560 and 650 nm (Bobermin et al. 2012). The results are expressed as percentages relative to the control values.

For membrane integrity evaluation, propidium iodide (PI) incorporation was performed. C6 astroglial cells were incubated with 7.5 µM PI for 30 min at 37 °C, in an atmosphere with 5% CO₂. Loss in membrane integrity causes fluorescent nuclei labeling with PI, for which the optical density was determined with Optiquant software (Packard Instrument Company).

Cytoskeleton Analysis—Actin Labeling

C6 astroglial cells were fixed for 20 min using 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). Cells were then permeabilized for 10 min in PBS containing 0.2% Triton X-100 and incubated with 10 µg/ml rhodamine-labeled phalloidin (Sigma-Aldrich) in PBS for 45 min. Cell nuclei were stained with 0.2 µg/ml of 4′,6′-diamidino-2-phenylindole (DAPI) for 20 min. C6 astroglial cells were analyzed using a Nikon microscope and photographed with a digital camera (DXM1200C) and a TE-FM Epi-Fluorescence accessory.

RNA Extraction and Quantitative RT-PCR

Total RNA was extracted from C6 astroglial cells using TRIzol Reagent (Invitrogen) (Bobermin et al. 2019). The relative absorbance ratio at 260/280 nm was measured to assess the concentration and purity of RNA. The synthesis of complementary DNA (cDNA) from 0.5 μg of total RNA was performed according to the manufacturer’s instructions, using the Applied Biosystems™ HighCapacity cDNA Reverse Transcription Kit (Applied Biosystems) in a 20 μl reaction. The messenger RNA (mRNA) encoding TNF-α (#Rn99999017_m1), IL-1β (#Rn00580432_m1), HMGB1 (#Rn02377062_g1), NFκB p65 (#Rn01502266_m1), COX2 (#Rn01483828_m1), TLR2 (#Rn02133647_s1), TLR4 (#Rn00569848_m1), adenosine receptors A₁ (#Rn00567668_m1), A_2a (#Rn00583935_m1), A_2b (#Rn00567697_m1), A₃ (#Rn00563680_m1), iNOS (#Rn00561646_m1), Nrf2 (#Rn00582415_m1), HO1 (#Rn01536933_m1), EAAC1 (#Rn00564705_m1), GCL (#Rn00689046_m1), GS (#Rn01483107_m1), SIRT1 (#Rn01428096_m1), and β-actin (#Rn00667869_m1) were quantified using the TaqMan inventory primers and the probes identified above (Applied Biosystems), employing the Applied Biosystems 7500 Fast system. Target mRNA levels were normalized to β-actin levels. The results were analyzed employing the 2^−ΔΔCt method (Livak and Schmittgen 2001) and expressed relative to the control levels.

NFκB Transcriptional Activity

Nuclear content, which was isolated from lysed cells with Igepal CA-630 and centrifuged, was assayed for NFκB p65 using a commercial ELISA kit with some modifications (Invitrogen, catalog #85-86081). The results are expressed as percentages relative to the control levels.

HIF-1α Transcriptional Activity

The levels of HIF-1α measured in lysed cells, using a commercial ELISA kit (Elabscience, catalog #E-EL-R0513) with some modifications. The results are expressed as pg/mg protein.

Inflammatory Response Measurement

Extracellular levels of TNF-α, IL-1β, IL-6 and IL-10 were measured in the culture medium using ELISA kits. The results are expressed in pg/ml and the average minimum sensitivities of detection for the kits were: 16.0 pg/ml for TNF-α (Invitrogen, catalog #88-7340-22); 4.0 pg/ml for IL-1β (Invitrogen, catalog #BMS630); 12.0 pg/ml for IL-6 (Invitrogen, catalog #BMS625); and 1.5 pg/ml for IL-10 (Invitrogen, catalog #BMS629).

NADPH Oxidase (NOX) Activity

NOX activity was assessed in cell lysates suspended in sodium phosphate buffer with 140 mM KCl and a protease mixture inhibitor. A modified luminescence assay was performed (Abid et al. 2007), using lucigenin as the electron acceptor generated by the NOX complex. NADPH was used as the substrate at concentrations (1 μM–1 mM) that fell well within the linear range of the assay, when using 5 μM of lucigenin. A standard curve generated with xanthine/xanthine oxidase was used to convert the data to relative light units/min/mg of protein. The results are expressed as percentages relative to the control conditions.

Cellular Superoxide Levels

A superoxide anion assay kit (Sigma-Aldrich, catalog #CS1000) was used to determine the superoxide levels in C6 astroglial cells (Quincozes-Santos et al. 2014). Briefly, this method evaluates the oxidation of luminol by superoxide anions, which results in the formation of chemiluminescence light. Thus, superoxide formation increases chemiluminescence in lysed cells. The kit includes a positive control (xanthine/xanthine oxidase superoxide anion producing system) and a negative control (superoxide dismutase enzyme repressing system). The chemiluminescence of control cells was arbitrarily set at 100% and the results are expressed as percentages relative to the control.

Measurement of 3-Nitrotyrosine Levels

The levels of 3-NT, a marker of protein damage mediated by RNS, were measured using an ELISA kit (Abcam, catalog #ab116691), according to the manufacturer’s instructions with some modifications. The results are expressed as ng/mg protein.

Superoxide Dismutase (SOD) Activity

SOD activity in lysed cells was determined using the RANSOD kit (Randox, catalog #SD125). The results are expressed as percentages relative to the control.

Catalase (CAT) Activity

CAT activity in cell lysates (50 μg protein) suspended in a reaction medium (20 mM H₂O₂, 0.1% Triton X-100, 10 mM potassium phosphate buffer, pH 7.0) was assayed by the method described previously (Aebi 1984), which consists of accompanying the decrease in absorbance, measured at 240 nm. One unit (U) of enzyme activity is defined as 1 μmol of H₂O₂ consumed per minute. The results are expressed as percentages relative to the control.

Glutathione Peroxidase (GPx) Activity

RANSEL kit (Randox, catalog #RS505) was used to evaluate GPx activity in lysed cells. The results are expressed as percentages relative to the control.

Glutathione (GSH) Levels

Intracellular levels of GSH were assessed as previously described (Browne and Armstrong 1998) in cell lysates suspended in a sodium phosphate (100 mM)/KCl buffer (140 mM), pH 8.0, containing 5 mM EDTA. Protein was precipitated with 1.7% meta-phosphoric acid, followed by centrifugation. The supernatant was then incubated with o-phthaldialdehyde (at a concentration of 1 mg/ml methanol) at 22 °C for 15 min. A calibration curve (GSH solutions—0 to 500 μM) was performed and fluorescence was measured using excitation and emission wavelengths of 350 and 420 nm, respectively. The results are expressed in nmol/mg protein.

Glutamate Cysteine Ligase (GCL) Activity

GCL was assayed according to Seelig et al., with slight modifications (Seelig and Meister 1985), in cell lysates suspended in a sodium phosphate (100 mM)/KCl buffer (140 mM), pH 8.0, containing 5 mM EDTA. The activity of GCL was determined after monitoring the NADH oxidation at 340 nm in sodium phosphate/KCl (pH 8.0) containing 5 mM Na₂-ATP, 2 mM phosphoenolpyruvate, 10 mM l-glutamate, 10 mM l-α-amino-butyrate, 20 mM MgCl₂, 2 mM Na₂-EDTA, 0.2 mM NADH, and 17 μg of pyruvate kinase/lactate dehydrogenase. The results are expressed in nmol/mg protein/min.

GDNF and S100B Measurements

Extracellular levels of GDNF and S100B were measured in the culture medium of cells using ELISA. The results are expressed in pg/ml for GDNF (Abcam; catalog #ab213901), and the average minimum sensitivity was 4.0 pg/ml. For S100B, we used a homemade ELISA protocol, as previously described (Leite et al. 2008), and the results are expressed in ng/ml.

Glutamate Uptake

The glutamate uptake assay was performed using H³-labeled glutamate (dos Santos et al. 2006). Briefly, C6 astroglial cells were rinsed once with PBS and were incubated at 37 °C in HBSS containing the following components (in mM): 137 NaCl, 5.36 KCl, 1.26 CaCl₂, 0.41 MgSO₄, 0.49 MgCl₂, 0.63 Na₂HPO₄, 0.44 KH₂PO₄, 4.17 NaHCO₃, and 5.6 glucose, adjusted to pH 7.4. Subsequently, 0.1 mM l-glutamate and 0.33 μCi/ml l-[3,4-³H] glutamate were added to initiate the assay. After 10 min, the incubation was stopped by removal of the medium and rinsing the cells twice with ice-cold HBSS. The cells were then lysed in a 0.5 M NaOH solution and incorporated radioactivity was measured using a scintillation counter. Sodium-independent uptake was determined using ice-cold N-methyl-d-glucamine instead of sodium chloride. Sodium-dependent glutamate uptake was obtained by subtracting the sodium-independent uptake from the total uptake. The results are expressed as nmol/mg protein/min.

Glutamine Synthetase (GS) Activity

The activity of GS was determined, as previously described (dos Santos et al. 2006). Briefly, the cell homogenate was added to a reaction mixture containing 10 mM MgCl₂, 50 mM L-glutamate, 100 mM imidazole–HCl buffer (pH 7.4), 10 mM 2-mercaptoethanol, 50 mM hydroxylamine–HCl. The addition of 10 mM ATP started the reaction, which was continued for 15 min at 37 °C. A solution containing 370 mM ferric chloride, 670 mM HCl and 200 mM trichloroacetic acid was then added to stop the reaction. After centrifugation, the absorbance of the supernatant was measured at 530 nm. A calibration curve was prepared using γ-glutamyl hydroxamate and treated with ferric chloride reagent. The results are expressed in μmol/mg protein/h.

Na⁺K⁺-ATPase Activity Assay

Na⁺K⁺-ATPase assay was conducted in a reaction mixture (final volume of 200 μl) contained (in mM) 5 MgCl₂, 80 NaCl, 20 KCl and 40 Tris–HCl, pH 7.4. The addition of ATP to a final concentration of 3 mM initiated the reaction. Control reactions were carried out under the same conditions with the addition of 1 mM ouabain. Na⁺K⁺-ATPase activity was calculated as the difference between the two assays, according to the method of Wyse et al. (2000). Released inorganic phosphate (Pi) was measured by the method of Chan et al. (1986). The specific activity of the enzyme was calculated as nmol Pi released/min/mg of protein.

Protein Assay

Protein content was measured using Lowry's method with bovine serum albumin as a standard (Lowry et al. 1951).

Statistical Analysis

Each set of results was obtained from three independent experiments performed in triplicate. One-way or two-way analysis of variance (ANOVA), followed by Tukey’s test, were performed to statistically analyze the differences among groups. Correlations were analyzed by Pearson correlation coefficient. All analyses were performed using the Graphpad Prism 7 software. Values of P < 0.05 were considered significant. a indicates differences from basal conditions; b indicates differences from LPS challenge.

Results

Effects of Sulforaphane and LPS on Cell Viability, Membrane Integrity and Cellular Morphology

We first evaluated the cellular viability (measured by MTT reduction) and membrane integrity (by PI incorporation) of C6 astroglial cells challenged with LPS (10 µg/ml) for 1, 3 or 6 h. We did not observe changes in cellular viability (Fig. 1a) or membrane integrity (data not shown) at any time of exposure. Additionally, we tested the effect of different concentrations of sulforaphane (1, 5 and 10 µM) for 4 h, which also had no effect on cellular viability (Fig. 1b) or membrane integrity (data not shown). Based on these data and previous studies (Fernandes et al. 2015; Bellaver et al. 2015), we chose to treat C6 cells with 5 µM of sulforaphane for 1 h (pre-treatment), followed by the addition of LPS (10 µg/ml) for a further 3 h (in the presence of sulforaphane), to investigate its glioprotective effects on the LPS-induced glial response. With regard to the actin cytoskeleton of C6 astroglial cells, no changes were observed after incubation with sulforaphane and/or LPS, compared to control conditions (Fig. 1c).

Fig. 1.

Effects of LPS and sulforaphane on cellular viability and morphology. MTT reduction was assessed in C6 astroglial cells challenged with LPS (10 µg/ml) for 1, 3 or 6 h (a), or incubated with sulforaphane (1, 5 or 10 µM) for 4 h (b). For actin cytoskeleton labeling (c), C6 cells were pre-treated with sulforaphane (5 µM), followed by the addition of LPS (10 µg/ml) for 3 h. For MTT reduction (a, b), the data are expressed as percentages relative to the control conditions and represent the mean ± S.E.M. of three independent experimental determinations performed in triplicate and statistically analyzed by one-way ANOVA, followed by Turkey’s test. Panel C shows representative images of the rhodamine-phalloidin labeling merged with the DAPI staining, from at least 3 experiments performed in duplicate. Scale bars = 100 μm. SFN sulforaphane

Sulforaphane Suppresses LPS-Induced Inflammatory Signaling in Astroglial Cells

C6 astroglial cells were challenged with 10 µg/ml LPS for 3 h. As expected, significant increases were observed in the mRNA expressions of pro-inflammatory cytokines, such as TNF-α and IL-1β (P < 0.0001; Fig. 2a, b). However, when cells were exposed to LPS in the presence of sulforaphane (5 µM), the increases in mRNA expression levels of these cytokines were abolished.

Fig. 2.

Glioprotective effect of sulforaphane against the LPS-induced inflammatory response. C6 astroglial cells were pre-treated with sulforaphane (5 µM), followed by the addition of LPS (10 µg/ml) for 3 h. The following parameters were then evaluated; mRNA expression of TNF-α (a), IL-1β (b), p65 NFκB (c), COX2 (d), iNOS (e) and HMGB1 (f); HIF1-α intracellular levels (g); mRNA expression of HO1 (h). Data are presented as means ± S.E.M. of at least five independent experimental determinations, which were analyzed by two-way ANOVA followed by Turkey’s test (P < 0.05 was considered significant). a indicates differences from control conditions; b indicates differences from LPS stimulation. SFN sulforaphane

NFκB signaling is a central controller of inflammation. Accordingly, LPS induced mRNA expression of p65 NFκB (P < 0.0001; Fig. 2c). Consistent with the anti-inflammatory activity of sulforaphane, this molecule inhibited the LPS-induced upregulation of NFκB, and downregulated the mRNA expression of p65 NFκB under control conditions (P < 0.05). Additionally, sulforaphane prevented the LPS-induced mRNA expression of the NFκB targets, COX2 (P < 0.0001; Fig. 2d) and iNOS (P < 0.0001; Fig. 2e). In the absence of LPS, sulforaphane also attenuated the expression of COX2 (P < 0.001). mRNA levels of the alarmin, HMGB1, however, were not affected (Fig. 2f). Furthermore, the content of HIF1-α, another transcription factor associated with inflammatory processes, was increased after LPS exposure (P < 0.05), and sulforaphane prevented this elevation (Fig. 2g).

In contrast, HO1 can counteract inflammatory responses, as it is a signal upstream from NFκB, while sulforaphane is reported to be a potent inducer of HO1. Accordingly, we found that sulforaphane markedly upregulated the mRNA expression of HO1 in astroglial cells, under control conditions (P < 0.0001), in addition to abolishing the significant decrease in mRNA levels of HO1, induced by LPS (P < 0.0001; Fig. 2h).

To further investigate the anti-inflammatory activity of sulforaphane, and its mechanisms, we evaluated the release of inflammatory cytokines and the transcriptional activity of NFκB after LPS challenge, in the presence of ZnPP IX, an inhibitor of HO1 activity. LPS stimulated the release of the pro-inflammatory cytokines, TNF-α (P < 0.0001; Fig. 3a), IL-1β (P < 0.0001; Fig. 3b) and IL-6 (P < 0.05; Fig. 3c), while decreasing the extracellular levels of the anti-inflammatory cytokine, IL-10 (P < 0.05; Fig. 3d). LPS also increased nuclear content of p65 NFκB (P < 0.05; Fig. 3e). Sulforaphane prevented of all these effects and augmented IL-10 release (P < 0.05) under control conditions (in the absence of LPS). However, when C6 astroglial cells were incubated in the presence of ZnPP IX, sulforaphane was no longer able to prevent the LPS-induced inflammatory response.

Fig. 3.

Anti-inflammatory effects of sulforaphane were dependent on HO1 activity. C6 astroglial cells were pre-treated with sulforaphane (5 µM) in the presence of the HO1 inhibitor, ZnPP IX (10 µM), followed by the addition of LPS (10 µg/ml) for 3 h. The extracellular levels of TNF-α (a), IL-1β (b), IL-6 (c) and IL-10 (d) were measured, as well as nuclear content of p65 NFκB (e). Data are presented as means ± S.E.M. of four independent experimental determinations performed in triplicate, which were analyzed by two-way ANOVA followed by Turkey’s test (P < 0.05 was considered significant). a indicates differences from control conditions; b indicates differences from LPS stimulation. SFN sulforaphane

The mRNA expression of TLR4, an important receptor for mediating the effects of LPS, was increased (P < 0.0001) in astroglial cells stimulated with LPS; however, this increase was prevented by sulforaphane (Fig. 4a). The expression of TLR2, which (like TLR4) is able to trigger NFκB signaling and inflammation in glial cells, did not change in response to LPS, but was decreased (P < 0.0001) by sulforaphane under control conditions (Fig. 4b).

Fig. 4.

Sulforaphane modulates the expressions of TLR and adenosine receptors. C6 astroglial cells were pre-treated with sulforaphane (5 µM), followed by the addition of LPS (10 µg/ml) for 3 h. The mRNA expression of TLR4 (a), TLR2 (b), A₁ (c), A_2a (d), A_2b (e) and A₃ (f) was evaluated. Data are presented as mean ± S.E.M. of at least five independent experimental determinations, which were analyzed by two-way ANOVA followed by Turkey’s test (P < 0.05 was considered significant). a indicates differences from control conditions; b indicates differences from LPS stimulation. SFN sulforaphane

Moreover, several studies have identified the modulatory effects of adenosine receptors on inflammatory conditions. LPS was observed to decrease the mRNA levels of the A₁ (P < 0.01) and A_2a (P < 0.0001) subtypes, while A_2b and A₃ were not affected (Fig. 4c–f). Again, sulforaphane prevented the LPS-induced effects.

The Antioxidant Effects of Sulforaphane in Astroglial Cells Challenged with LPS are Dependent on HO1

LPS can also stimulate the production of ROS/RNS, where NOX participates significantly in superoxide anion synthesis. LPS increased NOX activity (Fig. 5a; P < 0.05), as well as superoxide production in the C6 cells (P < 0.05; Fig. 5b). Sulforaphane prevented both effects; in turn the effects of sulforaphane were abolished in the presence of ZnPP IX, indicating the involvement of HO1 in this mechanism. Consistent with the increase in iNOS expression, LPS augmented 3-NT levels (P < 0.05; Fig. 5c), a marker of protein damage mediated by RNS. Sulforaphane also abrogated this effect, in a HO1-dependent manner.

Fig. 5.

Antioxidant effects of sulforaphane are also dependent on HO1. C6 astroglial cells were pre-treated with sulforaphane (5 µM), in the presence of the HO1 inhibitor, ZnPP IX (10 µM), followed by the addition of LPS (10 µg/ml) for 3 h. NOX activity (a), superoxide levels (b), 3-NT levels (c), the activities of SOD (d), CAT (e) and GPx (f), GSH levels (g), GCL activity (h) and GCL mRNA expression (i) were evaluated. Data are presented as means ± S.E.M. of at least four independent experimental determinations performed in triplicate, which were analyzed by two-way ANOVA followed by Turkey’s test (P < 0.05 was considered significant). a indicates differences from control conditions; b indicates differences from LPS stimulation. SFN sulforaphane

As an oxidative imbalance may contribute to impaired antioxidant enzymatic defenses, we evaluated the activities of SOD, CAT and GPx after LPS challenge and sulforaphane treatment. C6 astroglial cells challenged with LPS showed a slight increase in SOD activity (P < 0.05; Fig. 5d). Sulforaphane restored the activity of SOD to control values, but not in the presence of HO1 inhibitor. However, the activities of CAT and GPx (Fig. 5e, f, respectively) were not altered by LPS nor sulforaphane.

GSH is an essential antioxidant molecule for astroglial cells. Interestingly, both LPS and sulforaphane per se were able to increase the intracellular content in GSH (P < 0.05; Fig. 5g). In LPS plus sulforaphane-treated astroglial cells, the increment of GSH levels was even higher (P < 0.05). The effect of LPS on GSH levels seems to be independent of GCL, the rate-limiting enzyme for GSH biosynthesis, since LPS downregulated both GCL activity (P < 0.05; Fig. 5h) and mRNA levels (P < 0.001; Fig. 5i). In contrast, the sulforaphane-mediated increase in GSH levels was associated with an increased activity (P < 0.05) and mRNA expression (P < 0.001) of GCL. Furthermore, in the presence of the HO1 inhibitor, sulforaphane failed to modulate GSH levels and GCL activity, showing the involvement of HO1 in these effects (Fig. 5g, h, respectively).

Sulforaphane Modulates Specific Astroglial Functions

The release of trophic factors is an important astroglial function. LPS decreased GDNF levels, when compared to control conditions (P < 0.05; Fig. 6a). Sulforaphane prevented this decrease and, per se, induced an increase in GDNF release (P < 0.05). Conversely, LPS increased S100B extracellular levels (P < 0.05), and sulforaphane prevented this effect (Fig. 6b).

Fig. 6.

Effect of sulforaphane on specific astroglial functions. C6 astroglial cells were pre-treated with sulforaphane (5 µM), followed by the addition of LPS (10 µg/ml) for 3 h. The release of GDNF (a) and S100B (b), mRNA expression of EAAC1 (c), glutamate uptake (d), GS mRNA expression (e), GS activity (f), and Na⁺, K⁺ ATPase activity (g) were evaluated. Data are presented as means ± S.E.M. of at least four independent experimental determinations, performed in triplicate, which were analyzed by two-way ANOVA followed by Turkey’s test (P < 0.05 was considered significant). a indicates differences from control conditions; b indicates differences from LPS stimulation. SFN sulforaphane

Glutamate uptake is another essential function of astroglial cells for maintaining brain homeostasis. The mRNA expression of the major glutamate transporter in C6 astroglial cells, EAAC1, was decreased after LPS exposure (P < 0.05; Fig. 6c). Sulforaphane abrogated this effect, in addition to upregulating EAAC1 under control conditions (P < 0.001). When glutamate uptake was evaluated, a similar profile was observed; LPS decreased glutamate uptake (P < 0.05), and sulforaphane was able to abolish this decrease (Fig. 6d). Additionally, sulforaphane increased glutamate uptake per se (P < 0.05).

Glutamate can be converted into glutamine by GS. The mRNA levels and enzymatic activity of this specific astroglial enzyme were decreased after LPS challenge (Fig. 6e; P < 0.05; Fig. 6f; P < 0.05). Sulforaphane prevented both of these effects of LPS, restoring mRNA levels and GS activity to control levels. Furthermore, glutamate metabolism is dependent on the activity of Na⁺, K⁺ ATPase. LPS increased Na⁺, K⁺ ATPase activity in astroglial cells (P < 0.05; Fig. 6g), while sulforaphane maintained its activity at control values.

Underlying Mechanisms Involved in the Glioprotection Exerted by Sulforaphane

Nrf2 is a transcriptional factor is an upstream signal of NFκB and HO1 that is associated with cytoprotective mechanisms (Wakabayashi et al. 2010). Here, we demonstrated that sulforaphane markedly upregulated the mRNA expression of Nrf2 (P < 0.0001) in astroglial cells, compared to control conditions, in addition to totally preventing the significant decrease in Nrf2 mRNA levels, induced by LPS (P < 0.0001; Fig. 7a). Moreover, we found positive correlations between the mRNA expression of Nrf2 and genes associated with key astroglial functions, including HO1, GCL, EAAC1 and GS (Fig. 7c–f), and a negative correlation with p65 NFκB expression (Fig. 7g), reinforcing the role of Nrf2 in sulforaphane-mediated glioprotection.

Fig. 7.

Effect of sulforaphane on Nrf2 and SIRT1 expression. C6 astroglial cells were pre-treated with sulforaphane (5 µM), followed by the addition of LPS (10 µg/ml) for 3 h. The mRNA expressions of Nrf2 (a) and SIRT1 (b) were evaluated. The correlations between the mRNA expression of Nrf2 and HO1 (c), Nrf2 and GCL (d), Nrf2 and EAAC1 (e), Nrf2 and GS (f), and Nrf2 and p65 NFκB (g) were also determined by Pearson correlation coefficient, assuming P < 0.05. Data are presented as means ± S.E.M. of at least five independent experimental determinations, which were analyzed by two-way ANOVA followed by Turkey’s test (P < 0.05 was considered significant). a indicates differences from control conditions; b indicates differences from LPS stimulation. SFN sulforaphane

The SIRT1 pathway is also classically associated with cytoprotection and may counteract inflammatory responses and redox imbalances. This pathway can be diet activated by caloric restriction, polyphenols (mainly resveratrol), and other supplements (Allard et al. 2009; Li et al. 2015). Thus, we investigated whether sulforaphane could modulate the expression of SIRT1; however, no changes in the mRNA levels of SIRT1 (Fig. 7b) in response to sulforaphane were observed.

Discussion

The relationship between health and diet has been increasingly explored, including for the prevention and treatment of brain diseases. We investigated the potential glioprotective effects of sulforaphane, due to its anti-inflammatory and antioxidant properties, in C6 astroglial cells when induced by LPS. This bacterial endotoxin has been widely used for the induction of inflammation in both in vitro and in vivo studies, particularly because it binds to TLR4, which activates several intracellular pathways and modulates the expression of numerous inflammatory mediators (Guerra et al. 2011; Gorina et al. 2011; Rosciszewski et al. 2018). LPS is also an important and classical tool for studying astroglial inflammatory responses and glial activation (Guerra et al. 2011; Gorina et al. 2011; Villarreal et al. 2014; Parada et al. 2015; Bellaver et al. 2015; Bobermin et al. 2019). As a result of such an inflammatory challenge, astroglial cells can switch from having a protective role to a harmful phenotype. The identification of a glioprotective molecule, such as sulforaphane, with the ability to prevent early inflammatory responses that avoid this shift, would be promising for the development of glial-targeted protective strategies.

Although cells were incubated with LPS for a short time, this molecule triggered a significant inflammatory response in astroglial cells, at both the transcriptional and post-translational levels. It is important to note that an upregulation in the mRNA expression of pro-inflammatory cytokines can contribute to the progression or persistence of the inflammatory response. Importantly, sulforaphane prevented all of the inflammatory alterations induced by LPS. Sulforaphane also modulated the expression of the sensors of infection and tissue injury, TLR2 and TLR4 (Hanke and Kielian 2011), and this may represent another mechanism by which sulforaphane may attenuate astroglial inflammatory activation. Previously, we showed that the expressions of TLR2 and TLR4 can be modulated by adenosine receptors, which are involved in resveratrol-mediated glioprotection (Bobermin et al. 2019). The adenosine receptors (A₁, A_2a, A_2b and A₃) are involved in physiological and pathological processes in the CNS, including inflammation in the glial cells (Haselkorn et al. 2010; Gessi et al. 2013; Merighi et al. 2015; Bobermin et al. 2019). Sulforaphane prevented LPS-induced downregulation of A₁ and A_2a. Since the activation of adenosine receptors in glial cells can suppress signaling pathways that induce the production of pro-inflammatory mediators (Haselkorn et al. 2010; Newell et al. 2015), the ability of sulforaphane to maintain the basal expression of these receptors, even under inflammatory conditions, may contribute to its anti-inflammatory activity. To the best of our knowledge, this is the first study showing a modulatory effect of sulforaphane on adenosine receptor expression in glial cells.

ROS/RNS have an essential role in inflammatory responses, and the link between them may be explored for therapeutic purposes. LPS induces NOX (Yoo et al. 2008; Sharma and Nehru 2015) and iNOS (Moriyama et al. 2016; Wang and Zhao 2019) activities, in turn generating superoxide anion and NO, respectively. These molecules can react with each other, leading to the production of the powerful oxidant species, peroxynitrite. Our findings show that sulforaphane can prevent LPS-induced changes in NOX activity, superoxide levels, iNOS expression and 3-NT levels. With regard to enzymatic antioxidant defenses, while SOD activity was slightly increased by LPS, probably as a compensatory mechanism to attenuate the increase in superoxide production, sulforaphane was able to maintain SOD activity at control levels. Moreover, the activities of CAT and GPx were not affected by either LPS or by sulforaphane. Thus, enzymatic antioxidant defenses were maintained under our experimental conditions, particularly in the presence of sulforaphane.

The NFκB and Nrf2 signaling pathways are known to modulate inflammatory and oxidative responses, generally in opposite manners. Sulforaphane prevented the LPS-induced increases in both the mRNA expression and nuclear content of p65 NFκB, in astroglial cells, in addition to decreasing p65 NFκB expression under basal conditions. Similarly, HIF-1α is an important factor involved in inflammatory and redox responses (Palazon et al. 2014), and NFκB can directly modulate HIF-1α expression (van Uden et al. 2008). The mechanisms underlying the anti-inflammatory activity of sulforaphane in astroglial cells also involved the inhibition of the LPS-mediated increases in p65 NFκB and HIF-1α. In contrast, Nrf2 transcriptionally induces antioxidant and anti-inflammatory enzymes (Niture et al. 2014; Dai et al. 2020). HO1 acts as an adaptive defense mechanism to protect cells from damage, being able to modulate NFκB activation (Wakabayashi et al. 2010). Sulforaphane has been demonstrated to be a powerful inducer of Nrf2/HO1 pathway in several tissues (Pan et al. 2014; Dwivedi et al. 2016; Feng et al. 2017; Corssac et al. 2018; Dai et al. 2020; Uddin et al. 2020), thereby representing an important protective mechanism. In our study, sulforaphane also upregulated the mRNA expression of Nrf2, suggesting that it has the ability to control both the activation and expression of this transcription factor. Furthermore, HO1 was upregulated in astroglial cells, indirectly corroborating the induction of Nrf2 by sulforaphane. Importantly, when astroglial cells were incubated with a HO1 inhibitor, the anti-inflammatory and antioxidant activities of sulforaphane were abolished, reinforcing the mechanistic role of HO1 described for other glioprotective molecules (Quincozes-Santos et al. 2013, 2014; Santos et al. 2015; Bobermin et al. 2015; Arús et al. 2017).

The release of trophic factors by glial cells is known to influence the functionality of the CNS (Markiewicz and Lukomska 2006). GDNF can act as a regulator of glial activation, particularly with regard to neuroinflammation (Rocha et al. 2012; Allen et al. 2013). Sulforaphane improved the extracellular levels of GDNF in astroglial cells, reinforcing its glioprotective action. It is important to point out that a relationship exists between HO1 and GDNF, since HO1 is able to enhance the expression of GDNF (Hung et al. 2010). The S100B protein is another potential glial-derived trophic factor; however, extracellular S100B has been used as a marker of glial activation, in response to injury stimuli (Gonçalves et al. 2008; Donato et al. 2009). Importantly, the LPS-induced increase in S100B extracellular levels was prevented by sulforaphane.

Astroglial cells also participate in glutamate metabolism. Sulforaphane demonstrated a positive effect on glutamate uptake, both in the presence of LPS and in unstimulated astroglial cells. Moreover, the electrochemical Na⁺ gradient, mainly maintained by the Na⁺, K⁺-ATPase pump, is used for glutamate transporters as the driving force for glutamate uptake (Bélanger et al. 2011). In our study, Na⁺, K⁺-ATPase activity increased in response to LPS, but not in the presence of sulforaphane. It should be taken into consideration that this increase in Na⁺, K⁺-ATPase may represent a mechanism to stimulate the activity of glutamate transporters to compensate the decrease in glutamate uptake, induced by LPS.

The mRNA expression and activity of GS were decreased in LPS-exposed C6 astroglial cells. Any impairment in GS activity might limit the ability of astroglial cells to metabolize glutamate and supply neurons with metabolic substrates (Schousboe 2019). Importantly, sulforaphane was able to maintain this astroglial function, which along with the modulation of glutamate uptake, may preserve the functioning of the glutamate-glutamine cycle (Feng et al. 2017). Glial cells also produce GSH, whose first step is catalyzed by the enzyme GCL, which is transcriptionally induced by Nrf2 (Lu 2013). Although LPS decreased both the mRNA levels and the activity of GCL, the GSH content was increased, probably as an early compensatory mechanism in response to the inflammatory stimulus. However, inflammatory responses have been associated with GSH depletion (Lee et al. 2010; Arús et al. 2017), and it has been demonstrated that LPS may decrease GSH levels in astrocytes (Wang and Zhao 2019). In contrast, sulforaphane was able to reestablish both mRNA expression and the activity of GCL in LPS-stimulated astroglial cells and increased both in unstimulated cells, resulting in ameliorated GSH levels.

The physiological and pathological significance of LPS treatment, particularly considering the differences between in vivo and in vitro experimental models, should be mentioned as a possible limitation of this study. In this regard, data from astroglial cell cultures and animals subjected to LPS and sulforaphane treatments might confirm and expand the knowledge about glioprotective effects of this compound.

The heat maps presented in Fig. 8 summarize the main changes induced by LPS and the protective effects of sulforaphane in astroglial cells. Herein, we explored the mechanism by which sulforaphane is able to counteract the damaging effects of LPS in astroglial cells, focusing on the Nrf2/HO1 pathway as a central mechanism. In particular, glioprotection is closely associated with this signaling pathway, and astroglial cells have been shown to be the predominant cell type for activation of Nrf2 (Liddell 2017). Furthermore, alterations in adenosine receptor signaling and the relationship between NFκB transcriptional activity and HIF-1α seem to play an important role in sulforaphane-mediated glioprotection. In summary, this study indicates that sulforaphane may represent a promising experimental therapeutic intervention for targeting astroglial cells.

Fig. 8.

Heat maps representing the main results observed in C6 astroglial cells. The levels of cytokines, trophic factors and transcriptional factors (a), as well as the relative mRNA expression of key molecules (b) in the different experimental groups are represented. Each square represents the mean of the group and the color scale is shown at the right

Author contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by LDB, FWB, TMdS and AQS. The manuscript was written by LDB, ABK, ATSW, CAG and AQS and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

This study was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS), Universidade Federal do Rio Grande do Sul and Instituto Nacional de Ciência e Tecnologia para Excitotoxicidade e Neuroproteção (INCTEN/CNPq).

Data Availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Compliance with Ethical Standards

Conflict of interest

The authors have no conflicts of interest.