Stabilization of Nrf2 by tBHQ prevents LPS-induced apoptosis in differentiated PC12 cells
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- Khodagholi, F. & Tusi, S.K. Mol Cell Biochem (2011) 354: 97. doi:10.1007/s11010-011-0809-2
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The inflammatory reaction plays an important role in the pathogenesis of the neurodegenerative disorders. tert-butylhydroquinone (tBHQ) exhibits a wide range of pharmacological activities including anti-oxidative and anti-inflammatory action. In this study, we tried to elucidate possible effects of tBHQ on lipopolysaccharide (LPS)-induced inflammatory reaction and its underlying mechanism in neuron-like PC12 cells. tBHQ inhibited LPS-induced generation of reactive oxygen species (ROS) and elevation of intracellular calcium level. It also inhibited LPS-induced cyclooxygenase 2 (COX-2), TNF-α, nuclear factor KappaB (NF-kB), and caspase-3 expression in a dose-dependent manner while stabilizing nuclear factor-erythroid 2 p45-related factor 2. Moreover, the phosphorylations of p38, ERK1/2, and JNK were suppressed by tBHQ. These results suggest that the anti-inflammatory properties of tBHQ might result from inhibition of COX-2 and TNF-α expression, inhibition of NF-kB nuclear translocation along with suppression of MAP kinases (p38, ERK1/2, and JNK) phosphorylation in PC12 cells, so may be a useful agent for prevention of inflammatory diseases.
KeywordsLipopolysaccharideMitogen-activated protein kinaseNF-κBPC12 cellstBHQ
Antioxidant responsive elements
Central nervous system
Kelch-like ECH-associated protein 1
3-[4,5-Dimethylthiazol-2-yl]-2, 5-dephenyl tetrazolium bromide
Nerve growth factor
Nuclear factor-erythroid 2 p45-related factor 2
Reactive oxygen species
Much evidence supports the hypothesis that apoptosis, an evolutionarily conserved form of cell death, is one of the predominant signaling pathways involved in pathophysiology of neurodegenerative diseases [1, 2]. This pathway can be triggered by a variety of cytotoxic stressors, such as lipopolysaccharide (LPS), which induce activation of executioner caspases and other signaling cascades that ultimately lead to apoptotic destruction of the cells [3, 4]. LPS is an endotoxin and a constituent of the outer membrane of gram-negative bacteria. In response to LPS, neurons are readily activated and undergo dramatic morphological and physiological transformations [5, 6]. Neurons were traditionally believed to be passive bystanders in neuroinflammation but more recent evidence suggests that neurons themselves can generate inflammatory molecules.
Pro-inflammatory biomarkers such as TNF-α and COX-2 which are both effector genes regulated by the NF-κB pathway, have been noted  to exhibit greater induction in Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2)-deficient mice as compared with wild-type mice, indicating that ablation of Nrf2 seems to accelerate inflammatory reactions. Besides, as constitutively active NF-κB occurs in inflammation  and as Nrf2 is implicated in it  we selected these two important transcriptional regulators to explore the potential for their putative cross-talk in inflammation.
Among a wide range of natural and synthetic small molecules with diverse chemical backgrounds that are potent inducers of Nrf2 activity [9–11]; tert-butylhydroquinone (tBHQ) which is approved for human use is of particular interest. Moreover, augmentation of Nrf2 activity, via pre-treatment with chemical inducer tBHQ, potently inhibits LPS-induced microglial activation . Therefore in this study, we investigated the neuroprotective and antiapoptotic roles of Nrf2 activation in neuron-like PC12 cells via suppression of LPS-induced inflammation and apoptosis. In addition, we provided some insights into its mechanism of action.
Materials and methods
Antibodies directed against caspase-3, NF-κB, phospho-p38 MAP kinase (Thr180/Tyr182), phospho-SAPK/JNK (Thr183/Tyr185), p38 MAP kinase, SAPK/JNK, and TNF-α and β-actin were obtained from Cell Signaling Technology. Phospho-ERK1/2, ERK1/2, and COX-2 antibodies were obtained from ABCAM and ABR-Affinity BioReagents, respectively. Antibodies directed against Nrf2 (C-20) and Lamin B2 were purchased from Santa Cruz Biotechnology. Fura-2/AM was obtained from Invitrogen. All the other reagents, unless otherwise stated, were from Sigma-Aldrich (St. Louis, MO).
Cell culture and PC12 differentiation
Rat pheochromocytoma (PC12) cells obtained from Pasteur Institute (Tehran, Iran) were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma, Aldrich), supplemented with 5% fetal bovine serum, 10% horse serum, and 1% antibiotic mixture comprising penicillin–streptomycin, in a humidified atmosphere at 37°C with 5% CO2. Growth medium was changed three times a week. The cells were differentiated by treating with nerve growth factor (NGF) (50 ng/ml) every other day for 6 days.
Differentiated PC12 cells, plated in 75 cm2 culture flasks, were incubated with different concentrations (10, 20, and 40 μM) of tBHQ for 24 h prior to our experiments, then the cells were treated with lipopolysaccharide (1 mg/ml) for 18 h.
Measurement of cell viability
Cell viability was determined by the conventional MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) reduction assay. The dark blue formazan crystals formed in intact cells were solubilized in DMSO and the absorbance was measured at 550 nm. Results were expressed as the percentages of reduced MTT, assuming the absorbance of control cells as 100%.
AO/EB double staining
Apoptosis was determined morphologically after staining the cells with AO/EB followed by fluorescence microscopy inspection. Briefly, differentiated PC12 cells were seeded in a 96-well plate and were treated with different concentrations (10, 20, and 40 μM) of tBHQ followed by adding lipopolysaccharide (1 mg/ml). After 18 h, the cells were harvested and washed three times with PBS and were adjusted to a density of 106 cells/ml of PBS. AO/EB solution (1:1 v/v) was added to the cell suspension in a final concentration of 100 μg/ml. The cellular morphology was evaluated by fluorescence microscope (Zeiss, Germany).
Western blot analysis
For Western blot analysis, the cells were lysed in buffer containing complete protease inhibitor cocktail. The total proteins were electrophoresed in 12% SDS-PAGE gels, transferred to polyvinylidene fluoride membranes and probed with specific antibodies. Immunoreactive polypeptides were detected by chemiluminescence using enhanced ElectroChemiLuminescence (ECL) reagents (Amersham Bioscience, USA) and subsequent autoradiography. Quantification of the results was performed by densitometric scan of films. Data analysis was done by ImageJ, measuring integrated density of bands after background subtraction. Protein concentrations were determined according to Bradford’s method . A standard plot was generated using bovine serum albumin. Nuclear and cytoplasmic proteins were isolated as described by Kutuk and Basaga .
Measurement of glutathione levels
The concentration of glutathione (GSH) was determined in whole cell lysate using dithionitrobenzoic acid (DTNB) method at 412 nm .
Measurement of lipid peroxidation
Malondialdehyde (MDA) levels were measured by the double heating method . The method is based on spectrophotometric measurement of the purple color generated by the reaction of thiobarbituric acid (TBA) with MDA. Briefly, 0.5 ml of cell lysate was mixed with 2.5 ml of trichloroacetic acid (TCA, 10% w/v) solution followed by boiling in a water bath for 15 min. After cooling to room temperature, the samples were centrifuged at 3,000 rpm for 10 min and 2 ml of each sample supernatant was transferred to a test tube containing 1 ml of TBA solution (0.67% w/v). Each tube was then placed in boiling water for 15 min. After cooling to room temperature, the absorbance was measured at 532 nm with respect to the blank solution.
Superoxide dismutase activity assay
Superoxide dismutase (SOD) activity was measured based on the extent inhibition of amino blue tetrazolium formazan formation in the mixture of nicotinamide adenine dinucleotide, phenazine methosulphate, and nitroblue tetrazolium (NADH–PMS–NBT), according to the method of Kakkar et al. . Assay mixture contained 0.1 ml of cell lysate, 1.2 ml of sodium pyrophosphate buffer (pH 8.3, 0.052 M), 0.1 ml of phenazine methosulphate (186 μM), 0.3 ml of NBT (300 μM), and 0.2 ml of NADH (750 μM). Reaction was started by addition of NADH. After incubation at 30°C for 90 s, the reaction was stopped by addition of 0.1 ml of glacial acetic acid. Reaction mixture was stirred vigorously with 4.0 ml of n-butanol. Color intensity of the chromogen in butanol was measured spectrophotometrically at 560 nm. One unit of enzyme activity was defined as that amount of enzyme which caused 50% inhibition of NBT reduction/mg protein.
Catalase activity assay
Catalase (CAT) activity was measured by the method of Aebi . Briefly, 200 μl of cell lysate was added to a cuvette containing 1.995 ml of 50 mM phosphate buffer (pH 7.0). Reaction was started by addition of 1.0 ml of freshly prepared 30 mM H2O2. The rate of decomposition of H2O2 was measured spectrophotometrically at 240 nm.
Measurement of intracellular ROS
The fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCF-DA) was used to monitor intracellular accumulation of ROS. For this purpose, the DCFH-DA solution (10 μM) was added to the suspension of the cells (1 × 106/ml), the mixture was incubated at 37°C for 1 h. Cells were then washed twice with PBS and finally, the fluorescence intensity was measured by Varian-spectrofluorometer, model Cary Eclipse with excitation and emission wavelengths of 485 and 530 nm, respectively.
Measurement of intracellular calcium level
After the treatments, PC12 cells were collected and prepared to generate a 0.5 ml cell suspension for every sample. Fura-2/AM (final concentration 5 μM) was added to the cell suspension. The suspension was shaken at 37°C for 1 h, and then centrifuged twice at 1,000 rpm for 5 min. The cells were re-suspended in HEPES buffer solution, containing NaCl 132, KCl 3, glucose10 HEPES 10, and CaCl2 2 mM, pH 7.4, and finally the fluorescence intensity was measured by Varian-spectrofluorometer, model Cary Eclipse with excitation and emission wavelengths of 340 and 500 nm, respectively.
All data were represented as the mean ± S.D. Comparison between groups was made by one-way analysis of variance (ANOVA) followed by a specific post-hoc test to analyze the difference. The statistical significances were achieved when P < 0.05 (* or # or ^ P < 0.05, ** or ## P < 0.01, and *** or ### P < 0.001).
Effect of different concentrations of LPS on cell viability in PC12 cells
tBHQ suppresses LPS-induced apoptosis in PC12 neurons
To evaluate neuroprotective effect of tBHQ, differentiated PC12 cells were plated in 96-well plate, treated with different doses of tBHQ, and exposed to LPS 24 h later. After 18 h, the cells were assayed for cell viability. As the results shown in Fig. 1b, pretreatment with tBHQ dose dependently protected neurons against LPS-induced apoptosis. Treatment of the cells with different doses of tBHQ (10, 20, and 40 μM), increased cell viability to 70, 79, and 86%, respectively. At these concentrations, tBHQ alone did not have any cytotoxic effect in PC12 cells.
The neuroprotective effect of tBHQ was further confirmed by morphological observation (Fig. 1c). After incubation with 50 ng/ml NGF for 6 days, PC12 cells displayed a neuronal phenotype that resembles that of the sympathetic neurons. Morphological observation indicated that a majority of NGF-differentiated PC12 cells ceased division and extended neuritic processes. Exposure to LPS for 18 h caused heterogeneity in their shape, loss of connections between neural cells, and mostly detachment from the cultured plate surface. As shown in Fig. 1c, pretreatment of cells with tBHQ, alleviated the cell damage.
Morphological evaluation of apoptosis
Acridine orange/ethidium bromide (AO/EB) staining discriminates live cells from dead ones on the basis of membrane integrity. AO is a cell-permeable nucleic acid selective cationic dye which is taken up by both the viable and nonviable cells and emits green fluorescence if intercalates into double stranded nucleic acid (DNA). EB intercalates and stains DNA, providing a red-orange fluorescence. Although it does not stain healthy cells, it can be used to identify apoptotic cells which have much more permeable membranes. The result obtained from AO/EB double staining is represented in Fig. 1d. In this method, viable cells show uniform bright green nuclei with organized structure, while apoptotic cells have orange to red nuclei with condensed or fragmented chromatin. As shown in Fig. 1e, an analysis of the stained cells indicated that pretreatment of the cells with different concentrations of tBHQ (10, 20, and 40 μM) has significantly reduced the extent of cell apoptosis compared to those observed among the cells exposed solely to LPS.
tBHQ inhibits caspase-3 activation in neuron-like PC12 cells
tBHQ results in induction of Nrf2 in PC12 neurons
tBHQ increases the glutathione level in differentiated-PC12 cells
Effects of tBHQ on lipid peroxidation, glutathione level, and antioxidant enzyme activities in LPS-treated PC12 cells
MDA (nmol/mg protein)
SOD (U/mg protein)
CAT (μmol/mg protein)
GSH (μmol/mg protein)
0.61 ± 0.04
41.70 ± 1.45
2.50 ± 0.08
6.50 ± 0.12
0.91 ± 0.02**
20.82 ± 2.16**
1.39 ± 0.06**
3.72 ± 0.15**
tBHQ (10 μM)
0.66 ± 0.03
41.29 ± 2.29
2.42 ± 0.13
7.10 ± 0.19*
tBHQ (10 μM) + LPS
0.84 ± 0.02#
25.54 ± 1.41#
1.80 ± 0.08#
4.55 ± 0.22#
tBHQ (20 μM)
0.62 ± 0.02
39.90 ± 1.71
2.35 ± 0.10
7.22 ± 0.19*
tBHQ (20 μM) + LPS
0.80 ± 0.01#
30.17 ± 0.87#
1.94 ± 0.05#
5.30 ± 0.33#
tBHQ (40 μM)
0.67 ± 0.03
43.53 ± 1.16
2.44 ± 0.07
7.76 ± 0.15*
tBHQ (40 μM) + LPS
0.70 ± 0.02##
39.32 ± 0.66##
2.30 ± 0.04##
6.44 ± 0.20##
tBHQ reduces lipid peroxidation and rescues loss of the activity of antioxidant enzymes LPS-treated PC12 cells
Treatment of PC12 cells with 1 mg/ml LPS for 18 h, increased the intracellular MDA level, while preincubation of cells for 24 h, with different concentrations of tBHQ (10, 20, and 40 μM) markedly attenuated the changes in the MDA level (Table 1). In addition, PC12 cells treated with LPS caused the decrease in the activities of SOD and CAT. However, 18 h pretreatment with different concentrations of tBHQ (10, 20, and 40 μM) significantly increased their activity (Table 1).
tBHQ inhibits LPS-induced intracellular ROS generation and intracellular calcium elevation
tBHQ results in inhibition of nuclear translocation of NF-κB in PC12 cells
tBHQ inhibits MAP kinase phosphorylation in PC12 neurons
tBHQ results in inhibition of COX-2 and TNF-α expressions in PC12 neurons
Previous studies have revealed that LPS-induced inflammatory mediator production can be attenuated by antioxidants like curcumin and vitamin E [30, 31]. tBHQ is one of the most common antioxidants with a variety of pharmacological activities including anti-inflammatory potential . Nonetheless, little information is available with respect to the molecular mechanisms underlying the anti-inflammatory effect of tBHQ. Here, we focused on the mechanisms underlying its protective effects against LPS in PC12 cells. PC12 cell is an established cell line that is derived from rat pheochromocytoma . It has been used extensively as an in vitro model system to study neuronal cell fate, including survival, proliferation, differentiation, and apoptosis [32–34]. On the other hand, LPS is one of the most potent microbial inducers of inflammation, and of the cascade of intracellular events which may lead to cell death . Our data showed that treatment of PC12 cells with LPS resulted in cell death, which was significantly decreased in the presence of tBHQ, in a dose-dependent manner.
To further examine the involvement of important cellular components in cell fate, we focused first on Nrf2, as tBHQ activates it [24, 25]. The ARE is present in the promoters of genes encoding antioxidant enzyme and phase ΙΙ detoxification enzyme such as glutathione synthetase, CAT, and SOD [36, 37]. Previous studies have shown that Nrf2 induction is a common strategy for cells to combat chemical toxicity [38–40]. In the present study, we observed that LPS caused ROS formation representing a serious hazard for the cell, as they can oxidize macromolecules, thus damaging proteins. Here, we could cope with the harmful effects of ROS through induction of Nrf2. tBHQ treatment prevented accumulation of these species, along with increase of SOD, CAT, and GSH. This safeguard system enabled PC12 neurons to control and later exploit the reactivity of ROS for signal transduction. ROS are, in fact, implicated as second messenger in an array of biological responses.
In an attempt to explore detailed mechanism of cell defense, we determined the effect of tBHQ treatment on activation of nuclear factor-kappa B (NF-kB) family transcription factors [41–45]. These transcription factors are master coordinators of immunity, inflammation, differentiation, and cell survival [46–48]. Two main pathways for NF-kB activation have been reported so far [47, 49]. The canonical pathway—triggered in response to proinflammatory cytokines and stress such as LPS. The non-canonical pathway—engaged by various members of the TNF-R family such as lymphotoxin (LT)β and BAFF receptors. In the present study, we showed that tBHQ inhibits the canonical NF-κB pathway. In addition, caspase-3 can be activated by ROS. Therefore, the suppressive effect of tBHQ on translocation of NF-κB and also on the expression of active forms of caspase-3 shows that tBHQ suppresses NF-κB activation along with suppression of ROS production.
Increased defense against reactive oxygen metabolites was further confirmed by a decrease in activation of mitogen-activated protein kinases (MAPKs), which controls various cellular activities including gene expression, mitosis, differentiation, and cell survival/apoptosis  and is induced following cellular stress or cytokine signaling [51, 52]. In mammals, the MAPK cascades are composed of three distinct signaling molecules, the c-Jun N-terminal (JNK) cascade, the p38 MAPK cascade, and the extracellular signal-regulated kinase (ERK) cascade. Based on our results, tBHQ shows neuroprotective effect against LPS-induced cell death through inhibition of MAPKs.
Indeed, it has been shown that ROS act as second messengers activating MAPKs and NF-κB and result in further expression of proinflammatory cytokines [51, 52], that collectively lead to inflammation and tumorigenesis in different cell types [53, 54]. Among the inflammatory cytokines, TNF-α plays a key role in regulating inflammation, mostly through the induction of other inflammatory cytokines [55, 56]. This study demonstrates that tBHQ inhibits production of TNF-α in LPS-stimulated PC12 cells in a dose-dependent manner. In addition, in this condition the transcription of pro-inflammatory mediator COX-2 decreased. Decrease of intracellular Ca2+ was another important factor that was shown in the presence of tBHQ. LPS exposure increased Ca2+ concentration in the cytoplasm . Marked elevation in Ca2+ triggers different processes of degradation, such as ROS formation  and activation of several hydrolytic enzymes . Therefore, the interactions between Ca2+ and ROS are complex, and Ca2+ plays an important role in the development of oxidative and inflammative injuries.
In summary, Nrf2-mediated up-regulation of antioxidative and other cytoprotective enzymes confers cellular protection against inflammation by reducing deleterious production of pro-inflammatory mediators, and also NF-κB, showing the possible crosstalk between Nrf2 and NF-κB. Moreover, tBHQ was found to suppress the phosphorylations of ERK1/2, JNK, and p38. These findings provide a partial molecular explanation for the anti-inflammatory properties of tBHQ.
This work was supported by Shahid Beheshti University of Medical Sciences Research Funds. The authors thank MS. Joodi for her help in preparing the manuscript.