Protective Effects of Curcumin on Manganese-Induced BV-2 Microglial Cell Death

Abstract

Curcumin, a bioactive component in tumeric, has been shown to exert antioxidant, anti-inflammatory, anticarcinogenic, hepatoprotective, and neuroprotective effects, but the effects of curcumin against manganese (Mn)-mediated neurotoxicity have not been studied. This study examined the protective effects of curcumin on Mn-induced cytotoxicity in BV-2 microglial cells. Curcumin (0.1–10 µM) dose-dependently prevented Mn (250 µM)-induced cell death. Mn-induced mitochondria-related apoptotic characteristics, such as caspase-3 and -9 activation, cytochrome c release, Bax increase, and Bcl-2 decrease, were significantly suppressed by curcumin. In addition, curcumin significantly increased intracellular glutathione (GSH) and moderately potentiated superoxide dismutase (SOD), both which were diminished by Mn treatment. Curcumin pretreatment effectively suppressed Mn-induced upregulation of malondialdehyde (MDA), total reactive oxygen species (ROS). Moreover, curcumin markedly inhibited the Mn-induced mitochondrial membrane potential (MMP) loss. Furthermore, curcumin was able to induce heme oxygenase (HO)-1 expression. Curcumin-mediated inhibition of ROS, down-regulation of caspases, restoration of MMP, and recovery of cell viability were partially reversed by HO-1 inhibitor (SnPP). These results suggest the first evidence that curcumin can prevent Mn-induced microglial cell death through the induction of HO-1 and regulation of oxidative stress, mitochondrial dysfunction, and apoptotic events.

Manganese (Mn) is an essential trace element required for normal metabolism in development and various physiological processes. However, excessive accumulation of Mn affects the central nervous system (CNS) and produces manganism, which is Parkinson’s disease (PD)-like symptoms.¹⁾ Several epidemiological studies have suggested that Mn overexposure is associated with specific occupational groups including miners, welders, dry-cell battery manufacturers, and farmers using Mn containing fungicide maneb.²⁾ In addition, combustion of methylcyclopentadienyl Mn tricarbonyl (MMT) as an anti-knock additive in gasoline may extensively release Mn particles in the air and generate the environmental chronic Mn exposure.³⁾ Mn can cross the blood–brain barrier (BBB) and accumulate primarily in basal ganglia and substantia nigra as high as 200–300 µM concentrations in experimental animal models.⁴⁾ Although the exact mechanisms of Mn toxicity in the CNS are not known, Mn mainly accumulates in mitochondria and induces oxidative stress in neurons and glial cells.^5–7)

Recent studies suggest that natural dietary and herbal products are harmless to human beings and are useful materials to treat neurological disorders.^8,9) Curcumin (1,7-bis[4-hydroxy-3-methoxy phenyl]-1,6-heptadiene-3,5-dione), an active compound of dietary spice tumeric (Curcuma longa), has been shown to antioxidant, anti-inflammatory, anti-mutagenic, and potential neuromodulatory properties.^10,11) Accumulating evidences have implicated that curcumin is easily crosses the BBB and may play a useful role in attenuating neurodegenerative diseases like Alzheimer’s disease (AD), PD, and stroke.^12,13) Further, curcumin effectively protected neurons and/or glial cells against various neurotoxins.^14,15) However, the effect of curcumin against Mn-induced cell death has not been studied. In the present study, we examined the possible protective effect of curcumin against Mn-induced BV-2 microglial cell damage.

MATERIALS AND METHODS

Chemicals and Reagents

Curcumin, manganese chloride and other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). Tin protoporphyrin IX dichloride (SnPP) was purchased from Tocris Bioscience (Minneapolis, MN, U.S.A.). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), other culture supplements and antibody against cytochrome c oxidase subunit 4 (COX-4) were obtained from Invitrogen (Carlsbad, CA, U.S.A.). Antibodies against actin, Bax, Bcl-2, cytochrome c and heme oxygenase (HO)-1 were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, U.S.A.). Antibody against cleaved Caspase-3 was from Cell Signaling Technology Inc. (Danvers, MA, U.S.A.).

Cell Culture and Chemical Treatments

The murine microglial cell line, BV2, was maintained in DMEM supplemented with 10% FBS and antibiotics at 37°C in 5% CO₂. BV2 cells were cultured at a seeding density of 1.5×10⁵ cells/mL and the culture medium was changed to low-serum medium (0.5% FBS) before any chemical treatment to reduce the serum effect and to prevent the direct interaction between the treated chemicals. When indicated, BV-2 cells were exposed to MnCl₂ for maximum 24 h with or without pretreatment with curcumin for 1 h.

Cell Viability Assay

Cell viability was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay. After cells were treated and the culture medium was removed, 50 µL of MTT solution (1 mg/mL in phosphate buffered saline; PBS) was added to each well in 96-well culture plate and incubated at 37°C for 4 h. Afterwards, the medium was carefully removed and the formazan crystals were dissolved in 100 µL of 100% dimethyl sulfoxide (DMSO) for 15 min on orbital shaker. The cell viability was determined by measuring the optical density (OD) at 540 nm using a microplate reader (VersaMax; Molecular Devices, Sunnyvale, CA, U.S.A.). In a given experiment, each treatment was performed in triplicate and result was expressed as a percentage of untreated control.

Immunoblot Analysis

Total cell proteins were prepared from the BV-2 cells grown under various experimental conditions for immunoblots analysis. Briefly, cells were washed twice with PBS and then lysed on ice with RIPA buffer (1% Nonidet P-40, 0.5% Na deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 0.1 mg/mL phenylmethylsulfonyl fluoride, 30 mg/mL aprotinin, and 1 mM sodium orthovanadate in PBS). Resulting lysates were centrifuged at 14000×g for 10 min at 4°C and the supernatants were collected as the total protein lysates. To detect cytochrome c in the cytosol and mitochondria, BV-2 cells were lysed with mitochondrial/cytosolic fraction kit (Biovision Inc., Mountain View, CA, U.S.A.) according to the manufacturer’s protocol. The protein samples (each 30 µg) were separated on SDS-polyacrylamide gel electrophoresis (PAGE) and electrotransferred to polyvinylidine difluoride (PVDF) membrane. The membranes were blocked in TBST blocking buffer (5% nonfat dry milk in Tris-buffered saline, pH 7.4, containing 0.1% Tween 20) for 2 h at room temperature and incubated with primary antibodies for cleaved Caspase-3 (1 : 1000 dilution), cytochrome c (1 : 1000 dilution), Bax (1 : 1000 dilution), Bcl-2 (1 : 2000 dilution), HO-1 (1 : 4000 dilution), COX-4 (1 : 4000 dilution), or actin (1 : 4000 dilution) for overnight at 4°C. The membranes were washed three times with TBST buffer (0.1% Tween 20 in Tris-buffered saline, pH 7.4) and then reacted with horse radish peroxidase (HRP)-conjugated secondary antibodies (1 : 2000 dilution) for 2 h at room temperature. After washing again with TBST buffer, membranes were reacted with enhanced chemiluminescence (ECL) reagent (GE Healthcare, Pittsburgh, PA, U.S.A.) and exposed on X-ray film. The immunoreactive bands were quantified by densitometric analysis.

Measurement of Reduced Glutathione (GSH)

The level of intracellular GSH was measured using the fluorescent dye monochlorobimane (MCB) as previously described.¹⁶⁾ Briefly, BV-2 cells cultured in black 96-well culture plates were washed with Hank’s balanced salt solution (HBSS). Cells were incubated with MCB (40 µM) over 20 min at room temperature in the dark. After twice washing with HBSS, fluorescence intensity was determined at 355 nm (excitation) and 460 nm (emission) in a fluorescence microplate reader (SpectraMax M2, Molecular Devices). GSH level was calculated from a standard curve constructed using known amounts of GSH and the values were expressed in micromoles per mg of protein.

Measurement of Cellular Superoxide Dismutase (SOD) Activity

SOD activity was measured by a previously described method with slight modifications.¹⁷⁾ BV-2 cells grown under various experimental conditions were washed twice with PBS, scraped off and collected into Eppendorf tube. Cells were lysed in ice-cold PBS by sonication and centrifuged at 15000×g for 5 min at 4°C. The supernatant was used immediately for the measurement. Each 25 µL of supernatant was mixed with 200 µL of reaction buffer (50 mM potassium phosphate buffer, pH 7.8, 1.33 mM diethylenetriaminepentaacetic acid, 1.0 U/mL catalase, 70 µM nitroblue tetrazolium, 0.2 mM xanthine, 50 µM bathocuproinedisulfonic acid, 0.13 mg/mL bovine serum albumin) at 37°C for 3 min and then reacted with 25 µL of xanthine oxidase (0.1–0.2 U/mL) at 37°C for 5 min. Formation of formazan blue from the reaction was measured using a microplate reader with 560 nm wavelength. After recording SOD activity, the values were calculated as units per mg of cellular protein.

Measurement of Lipid Peroxidation

The lipid peroxidation was estimated by measuring malondialdehyde (MDA) levels. Cell homogenate was prepared by sonication and centrifuged at 3000×g for 10 min at 4°C. The resulting 100 µL of supernatant was mixed with 900 µL of reaction buffer (150 mM Tris–HCl buffer, pH 7.1, 1 mM FeSO₄, 1.5 mM ascorbic acid) and incubated for 15 min at 37°C, and then stopped the reaction by adding 1 mL of trichloroacetic acid (10%). After add 2 mL of thiobarbituric acid (0.375%), sealed and heated for 15 min in a boiling water bath to release MDA (the product of lipid peroxidation) from proteins. Then the reactant was cooled off to 4°C and centrifuged at 4000×g for 10 min. The amount of MDA formed in supernatant was measured at 532 nm. The levels of MDA were expressed micromoles per mg of protein.

Measurement of Intracellular Reactive Oxygen Species (ROS) and Peoxynitrite

Intracellular ROS generation was measured using fluorescent dichlorofluorescein (DCF) from nonfluorescent 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) and the production of peroxynitrite (ONOO⁻) was examined by using dihydrorhodamine 123 (DHR 123). Briefly, BV-2 cells were treated with various experimental conditions and washed with HBSS. After 30 min of incubations with 20 µM H₂DCFDA or 50 µM DHR 123, cells were rinsed twice with HBSS and then the fluorescence intensity was measured at 485 nm/535 nm (excitation/emission) in a fluorescence microplate reader (SpectraMax M2, Molecular Devices).

Measurement of Caspase Activity

Caspase-3 and -9 activities were measured by colorimetric assay as previously described.¹⁸⁾ Briefly, Cells were lysed with ice-cold lysis buffer (50 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.4, 1 mM dithiothreitol (DTT), 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.1% CHAPS), incubated for 5 min on ice, and centrifuged at 10000×g for 10 min at 4°C. Subsequent protein samples (10 µg) were incubated with 200 µM of substrates (Ac-DEVD-p-nitroanilide (pNA) for Caspase-3 and Ac-LEHD-pNA for Caspase-9, respectively) at 25°C. Formation of pNA from the reaction was measured at 405 nm wavelength over 1 h.

Measurement of Mitochondrial Membrane Potential (MMP)

MMP was determined using the fluorescent dye JC-1. Mitochondria-specific lipophilic cationic fluorescent dye, JC-1 can enter the mitochondria selectively and accumulates as red aggregates in healthy cells, but it exists as green monomers in the cytosol when the mitochondrial membrane collapsed during apoptosis. Thus, the red-to-green fluorescence ratio indicates mitochondrial membrane damage. BV-2 cells were pretreated with curcumin (10 µM) and/or SnPP (3 µM) for 1 h and then treated with Mn (250 µM) for 16 h. Cells were incubated with JC-1 (5 µg/mL) for 15 min at 37°C in the dark, and then washed with PBS. The fluorescence intensity was measured at 535 nm/590 nm (excitation/emission) for red and 485 nm/535 nm (excitation/emission) for green using a fluorescence multimode microplate reader (Infinite 200; Tecan, Grodig, Austria). The result was calculated as the ratio of red/green fluorescence and the value was expressed as a relative percentage over non-treated control sample.

Statistical Analysis

Results were expressed as the mean±standard error of the mean (S.E.M.) The data were analyzed using by SPSS 12.0 software package (SPSS Inc., Chicago, IL, U.S.A.). A one-sample t-test was used for comparisons between two groups. Comparisons between multiple groups were analyzed using one-way factorial ANOVA and the Duncan’s post hoc test. Statistics was evaluated at the significance level 2α=0.05.

RESULTS

Protective Effect of Curcumin against Mn-Induced Toxicity in BV-2 Microglia

BV-2 cells are derived from C57BL/6 newborn mice immortalized with v-raf/v-myc retrovirus and widely used to study microglial activation and pathogenesis because this cell line expresses CD40 and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, some representative microglial markers.^19,20) In this study, BV-2 cells were adopted as a suitable model system to investigate the role of curcumin against Mn-induced microglial cell death.

Initial studies were performed to examine the cytotoxic response of BV-2 cells to Mn at various concentrations. As shown in Fig. 1A, incubation of BV-2 cells with 10–500 µM Mn for 24 h resulted in significant cell death in a dose-dependent manner, and 250 µM Mn induced approximately 60% cell death. Hence we did subsequent experiments using 250 µM Mn. Curcumin itself at concentrations of 0.1–10 µM showed no cytotoxicity in BV-2 cells (Fig. 1A). To investigate the effect of curcumin on Mn-induced microglial cell death, BV-2 cells were pretreated with 0.1–10 µM curcumin for 1 h, followed by 250 µM Mn treatment for 24 h. Mn-induced cytotoxicity was attenuated by curcumin dose-dependently, and especially pretreatment with 10 µM curcumin significantly increased cell survival to 90% after 24 h (Fig. 1B).

Fig. 1. Curcumin Reduces MnCl₂-Induced Cytotoxicity in BV-2 Microglia

Cell viability was determined by MTT reduction assay. (A) Various concentrations of MnCl₂ (10–500 µM) or curcumin (0.1–10 µM) were treated to BV-2 cells for 24 h. (B) BV-2 cells were pretreated with different concentrations of curcumin (0.1–10 µM) for 1 h, and exposed to 250 µM MnCl₂ for 24 h. All values represent the mean±S.E.M. from three independent experiments in triplicate. * p<0.05, compared with untreated cells. ^# p<0.05, compared with MnCl₂ treatment alone.

Curcumin Inhibited Mn-Induced Apoptotic Proteins Expression

Caspase-3 and several mitochondria-associated proteins, such as cytochrome c, Bax, and Bcl-2 play critical roles in mitochondrial oxidative damage and apoptosis.²¹⁾ We performed immunoblot analysis to reveal the expression of these proteins (Fig. 2). The representative apoptotic marker, Caspase-3 was markedly activated and expressed as cleaved forms by Mn treatment. However, curcumin pretreatment effectively reduced the cleaved Caspase-3 expression.

Fig. 2. Effects of Curcumin on MnCl₂-Induced Apoptotic Signaling Markers in BV2 Microglia

(A) Cells were pretreated with curcumin (10 µM) for 1 h, followed by treatment with MnCl₂ (250 µM) for 16 h (cytochrome c, Bax, Bcl-2) or 24 h (cleaved Caspase-3). The expression of apoptotic cell death markers, cleaved Caspase-3, cytochrome c, Bax, and Bcl-2 was determined by immunoblot analysis. Actin or COX-4 (for mitochondrial protein) expression was also detected to normalization. (B) The changes in expression level of proteins were quantified by densitometric analysis and assessed as the ratio against the value of untreated cells. Results represent mean±S.E.M. from three independent experiments. * p<0.05 and ** p<0.01, compared with untreated control group. ^# p<0.05, compared with MnCl₂ treatment alone.

It has been known that mitochondrial dysfunction is accompanied by Bcl-2 down-regulation, Bax up-regulation, and the release of cytochrome c from mitochondria to the cytosol.²²⁾ In our experiment, Mn induced a significant release of cytochrome c to the cytosol (3.01-fold of control). However, the Mn-induced cytochrome c release was attenuated by curcumin (2.01-fold of control). In addition, Mn treatment induced an increase in Bax expression (2.23-fold of control) but a decrease in Bcl-2 expression (0.77-fold of control). However, pretreatment with curcumin prior to Mn treatment significantly attenuated the changes of these proteins. Interestingly, curcumin per se 1.5-fold up-regulated the anti-apoptotic Bcl-2 compare to the control.

Effects of Mn Treatment and/or Curcumin Pretreatment on GSH, SOD, and MDA Levels

To investigate the involvement of oxidative stress damage in Mn-induced BV-2 cell cytotoxicity, we measured GSH, SOD, and MDA levels. As shown in Table 1, the levels of intracellular GSH and SOD (anti-oxidative enzymes) were decreased after treatment with Mn (250 µM) for 16 h to 68.4% of control and 88.9% of control, respectively. On the other hand, curcumin (10 µM) single treatment significantly elevated GSH (4.1-fold of control) and caused small enhancement of SOD levels (1.1-fold of control). Moreover, curcumin pretreatment resulted in significant increase in both GSH and SOD levels in a dose-dependent manner.

Table 1. Effects of Curcumin Pretreatment on GSH, SOD, and MDA Levels

Group	GSH (µmol/mg protein)	SOD (U/mg protein)	MDA (µmol/mg protein)
Control	3.23±0.55	27.15±4.12	4.96±0.22
MnCl2 (250 µM)	2.21±0.43	24.15±2.25	7.41±1.01
Curcumin (10 µM)	13.22±1.41*	30.13±5.18	4.72±0.15
MnCl2+Curcumin (0.1 µM)	2.54±0.38	24.91±5.63	7.59±1.34
MnCl2+Curcumin (1 µM)	4.58±0.59#	26.04±3.72	6.48±0.82
MnCl2+Curcumin (10 µM)	7.67±0.97##	28.84±3.89	5.57±0.71#

The values represent the mean±S.E.M., n=6 each. * p<0.01, statistical significance compared with control group. ^# p<0.05, ^## p<0.01, statistical significance compared with MnCl₂ treated cells.

Mn treatment led to a remarkable increase of MDA (oxidative damage marker) levels (1.5-fold of control). However, pretreatment with curcumin inhibited Mn-induced MDA increase in BV-2 cells dose-dependently (1 µM curcumin, 87.4%; 10 µM curcumin, 75.2% of Mn-treated cells).

Curcumin Induced HO-1 Up-Regulation and Inhibited Mn-Induced ROS and ONOO⁻ Production

HO-1 has been known to exert anti-oxidative and cytoprotective effects response to various conditions.²³⁾ As shown in Fig. 3, treatment with various concentrations of curcumin significantly increased HO-1 protein expression dose-dependently (1 µM curcumin, 1.52-fold; 10 µM curcumin, 2.78-fold of control). However, pretreatment with SnPP (3 µM, a specific HO-1 inhibitor) significantly suppressed the curcumin-induced HO-1 expression. In addition, Mn (250 µM) did not modulate HO-1 protein expression in BV-2 microglial cells.

Fig. 3. Induction of Heme Oxygenase (HO)-1 Expression by Curcumin

BV-2 cells were pretreated with curcumin (0.1–10 µM) with or without SnPP (HO-1 inhibitor, 3 µM) for 1 h and then exposed to MnCl₂ (250 µM) for 16 h. The expression of HO-1 protein was detected by immunoblot analysis and the expression level was quantified by densitometric analysis. The expression level of actin protein was used for normalization. Results are representative of three separate experiments. * p<0.01 as compared with untreated control.

To determine the changes of oxidative species during the Mn-induced cell damage and curcumin-mediated protection, we measured ROS and peroxynitrite (ONOO⁻) production using fluorescent dye H₂DCFDA and DHR 123, respectively. As shown in Fig. 4, Mn exposure increased ONOO⁻ (1.6-fold of control) and more significantly increased total ROS (4.5-fold of control), whereas pretreatment with curcumin (0.1–10 µM) dose-dependently lowered the ROS and ONOO⁻ generation. Of interest, the presence of HO-1 inhibitor (SnPP, 3 µM) significantly blocked the effect of curcumin on the production of ROS. SnPP alone did not influence ROS generation in microglial cells (data not shown).

Fig. 4. Curcumin Reduced the MnCl₂-Induced Reactive Oxygen Species (ROS) Production

BV2 cells were pretreated with various concentration of curcumin (0.1–10 µM) with or without SnPP (HO-1 inhibitor, 3 µM) for 1 h, followed by MnCl₂ (250 µM) treatment for 16 h. The intracellular ROS production and peroxynitrite (ONOO⁻) generation were respectively determined by using fluorogenic H₂DCFDA and DHR 123. All values were expressed as a percentage of fluorescence intensity to the untreated control. Data are the mean±S.E.M. of three independent experiments in triplicate. * p<0.01 as compared with control. ^# p<0.01 as compared with MnCl₂ alone. ** p<0.01 as compared with curcumin+MnCl₂-treated group.

Role of HO-1 in the Protective Effects of Curcumin

To evaluate the importance of HO-1 induction by curcumin in microglia, a pharmacological approach was used with specific HO-1 inhibitor, SnPP. As shown in Fig. 5A, SnPP augmented the Mn-induced Caspase-9 and -3 activities. Additionally, SnPP partly abolished the curcumin-mediated suppressive effect on these caspases (Fig. 5A).

Fig. 5. Role of HO-1 in the Curcumin-Mediated Cytoprotection

BV2 cells were pretreated with 3 µM SnPP (HO-1 inhibitor) for 1 h followed by 10 µM curcumin for another 1 h, and then treated with 250 µM MnCl₂. Caspase-9 activity (A) and mitochondrial membrane potential (MMP) (B) were respectively evaluated at 16 h by using colorimetric substrate Ac-LEHD-pNA and the fluorescent JC-1 dye. Caspase-3 activity (A) and cell viability (B) of BV-2 cells were detected at 24 h by colorimetric assay using a substrate Ac-DEVD-pNA and an MTT reduction assay. Data are the mean±S.E.M. from three independent experiments. * p<0.05 and ** p<0.01, compared with untreated control group. ^# p<0.05 as compared with MnCl₂ alone.

The changes of MMP were evaluated by using a specific mitochondria fluorescent dye JC-1. As shown in Fig. 5B, Mn treatment significantly decreased MMP to 62.6% of control, and SnPP augmented the loss of MMP to 33.6% of control group. Although the Mn-induced MMP loss was relieved by curcumin (91.4% of control), SnPP reduced the curcumin effect on the MMP maintenance. Furthermore, loss of cell viability was significantly increased by SnPP (45% cell viability of control) when compare with Mn alone treatment condition (60% cell viability of control). In addition, SnPP pretreatment partly reduced the effect of curcumin on cell viability (Fig. 5B). SnPP alone did not significantly affect Caspase-9 and -3 activities, MMP maintenance, or BV-2 cell viability (Fig. 5). These results indicate that HO-1 mediates the protective effects of curcumin against Mn toxicity in BV-2 microglia.

DISCUSSION

Curcumin, a naturally occurring polyphenol found in tumeric, has been reported to exhibit therapeutic potential for various diseases including cancers, psoriasis, and Alzheimer’s disease.²⁴⁾ Recent studies have also revealed that curcumin has protective effects against PD-related neurotoxicants, such as MPP⁺, rotenone, salsolinol, and 6-hydroxydopamine (6-OHDA) in neurons and/or glial cells.^14,15,25) However, the effects of curcumin against Mn toxicity in microglia have not been reported. Therefore, this study investigated that curcumin can suppress Mn-induced cytotoxicity in BV-2 microglial cells.

Several studies using Mn reported the alteration of iron homeostasis in the brain and the induction of neuroinflammation and oxidative stress.^26,27) Mn can induce neuronal damage directly and also indirectly enhance neuronal cell death through activation of microglia.²⁸⁾ Microglia easily respond to Mn and release ROS and inflammatory mediators, such as proinflammatory cytokines, nitric oxide (NO), and prostaglandins, inducing detrimental toxicity to neighboring neurons.²⁷⁾ Although there are limited reports on the effects of Mn-induced toxicity on glial cells, some studies suggested that self-produced ROS can stimulate degradation of ferritin in microglia and subsequent microglial cell death.^29,30) However, little is known about the molecular mechanisms responsible for Mn toxicity in microglia.

Previous studies demonstrated that Mn caused mitochondrial complex I and/or complex II activity in various cell types.^5,31) In agreement with previous studies, our results revealed that Mn (250 µM) induced typical mitochondrial function changes, such as pro-apoptotic cytochrome c release into the cytosol, Bax increase, anti-apoptotic Bcl-2 decrease, and Caspase-3 activation in BV-2 cells (Fig. 2). However, curcumin pretreatment significantly suppressed those changes. Previous study reported that curcumin effectively protected mitochondria from oxidative damage and attenuated apoptosis in cortical neurons.³²⁾ Moreover, curcumin prevented H₂O₂-induced apoptotic cell death in microglia.³³⁾ However, as far as we are aware, the protective effect of curcumin against Mn-induced cytotoxicity in microglia has not been studied until now. Our results clearly demonstrated for the first time that curcumin can prevent mitochondria-related apoptotic damage from Mn toxicity in microglial cells.

Because the nervous system is vulnerable to oxidative stress, there is a complex antioxidant defense system. GSH, an endogenous cysteine-containing tripeptide (L-γ-glutamyl-L-cysteinyl-glycine), is the ubiquitously existing antioxidant thiol in most cells. SOD also acts as primary antioxidative defense enzyme to prevent further generation of ROS.^34,35) Adversely, increased oxidative stress accumulates product of lipid peroxidation, such as MDA. Recent study suggested that excessive Mn caused depletion of cellular GSH and over production of MDA in the striatum of mouse.³⁶⁾ However, curcumin has been shown to decrease the elevated level of MDA and prevent decline of antioxidant enzymes in experimental animal models.^37,38) In accordance with previous studies, our results revealed that the reduction of GSH and SOD, but the elevation of MDA by Mn treatment in BV-2 cells. Of interest, these changes were significantly reversed by curcumin pretreatment (Table 1). Thus, we propose that curcumin protects microglial cells by enhancing antioxidant enzymes and by inhibiting lipid peroxidation.

Curcumin is an electrophilic compound triggering activation of transcription factor nuclear factor-E2-related factor 2 (Nrf2), which normally sequestered by cytoskeleton-associated protein Keap1. Therefore, curcumin enables Nfr2 to translocate into the nucleus, which results in bind to the antioxidant-response element (ARE) and subsequent induction of detoxifying enzymes and cytoprotective proteins, such as HO-1 and γ-glutamyl cysteine synthetase.^39,40)

Cells lacking HO-1 are vulnerable to oxidatively induced cytotoxicity, and HO-1 exerts potent antioxidant defense activity in various cell types including glial cells.^41,42) Previous studies suggested that natural antioxidants, such as baicalein and quercetin, protect glial cells via induction of HO-1.^43,44) Recent study suggests that curcumin can also protect microglial cells via up-regulation of HO-1 under an lipopolysaccharide (LPS)-mediated inflammatory environment.⁴⁵⁾ However, the role of HO-1 in curcumin against Mn-induced microglial cytotoxicity was not studied. Our results clearly demonstrated that curcumin induced HO-1 up-regulation in BV-2 cells (Fig. 3). On the other hand, attenuation of HO-1 expression by specific inhibitor SnPP significantly diminished curcumin-mediated ROS suppression (Fig. 4). In addition, pretreatment with HO-1 inhibitor SnPP aggravated the Mn-induced cytotoxicity and decreased the protective effects induced by curcumin (Fig. 5). These results supporting the essential involvement of HO-1 in the curcumin-mediated microglial cell protection against Mn-induced cytotoxicity.

Accumulating evidences suggested that various signaling pathways have been implicated in HO-1 expression and regulation.^23,43) Therefore, further studies are required to fully explain the action of curcumin and the mechanism of HO-1 induction in microglial cells.

CONCLUSION

This study demonstrated for the first time that curcumin can protect microglial cells from cytotoxic effects of Mn. Curcumin significantly suppressed Mn-induced apoptotic events, such as Caspase-9 and -3 enzyme activation, MMP decrease, Bax increase, Bcl-2 decrease, and cytochrome c release. In addition, curcumin elevated antioxidant enzymes, GSH and SOD, but decreased the production of MDA and ROS caused by Mn. Importantly, induction of HO-1 is a critical event in the protective effect of curcumin in microglial cells.

Acknowledgment

This study was supported by research funds from Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2015R1D1A1A01059448).

Conflict of Interest

The authors declare no conflict of interest.

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