Curcumin Inhibits Mitochondrial Injury and Apoptosis from the Early Stage in EAE Mice

The exact pathophysiological change concerning mitochondrial injury and oligodendrocyte apoptosis in MS and EAE model is still unknown. Whether curcumin is able to inhibit mitochondrial injury and suppress the apoptosis in the early stages of MS/EAE is still unclear. We first explored mitochondrial injury and apoptosis at different time points p.i. in C57 BL/6 EAE mice. We then explored the effects of curcumin on mitochondria and apoptosis. Results showed that mitochondrial injury can be observed 3 days p.i. Apoptosis in the spinal cord occurred 3 days p.i. and the apoptotic cells were shown to be oligodendrocytes and neuronal cells. Curcumin significantly reduced the number of apoptotic cells and inhibited the upregulation of cyt-c, caspase-9, and caspase-3 at 7 days p.i. in the EAE mice. These observations demonstrate that mitochondrial injury and oligodendrocyte/neuronal apoptosis occur in the early stages of EAE. Curcumin can inhibit apoptosis in EAE mice which maybe act through protection of mitochondrial injury and inhibition of the intrinsic apoptotic pathway.


Introduction
The fundamental pathophysiology of newly formed lesions in multiple sclerosis (MS) is still not very clear, though it is generally attributed to demyelination events caused by the T-cell-mediated immune response [1]. Recently, studies on autopsy specimen found that the initial pathological findings with the formation of new lesions are different from the traditional MS pathological findings. Oligodendrocyte apoptosis is the earliest pathological change in new lesions [2][3][4]. However, the mechanism for oligodendrocyte apoptosis is still unknown. The experimental autoimmune encephalomyelitis (EAE) model is a classical animal model for MS, but the relationship between apoptosis and the typical pathological changes in the EAE model is still unclear. Mitochondrial injury has been identified as an important nonimmune mechanism in the pathology of MS [5]. Mitochondrial dysfunction and a higher demand of energy have been found in MS [6,7]. Whether mitochondrial injury is involved in the early pathology of MS/EAE is still unknown.
Curcumin is a polyphenolic phytochemical isolated from the rhizome of the plant Curcuma longa which naturally is not present in the normal brain. It has been shown to have a neuroprotective role in EAE [8,9], but it is unknown whether curcumin is able to protect mitochondria from injury and subsequently suppress apoptosis in the early pathology of MS/EAE.
In this study, we explored apoptosis and mitochondrial injury in C57 BL/6 EAE mice at different time points postimmunization (p.i.). We also explored the mechanism by which curcumin inhibits apoptosis, which occurs through the protection of mitochondrial injury during the early stages of EAE. This study aimed to elucidate the early pathological events of MS/EAE and to find an effective intervention for the early stages of MS.

Induction and Clinical Evaluation of EAE.
To induce EAE, mice were subcutaneously immunized with a 0.2 mL emulsion consisting of 300 g/mL of MOG 33-55 peptide and an equal volume of complete Freund's adjuvant (CFA). The mice were also intraperitoneally injected with 500 ng of pertussis toxins on days 0 and 2 p.i. All procedures were conducted under anesthesia with 3.5% chloral hydrate. The clinical score was evaluated each day using the standard 0-5 scale [10,11]; 0: unaffected, 1: tail limpness, 2: failure to right on attempt to roll over, 3: partial paralysis, 4: complete paralysis, and 5: moribund.

Groups and Treatments.
To access the early pathological events of EAE, mice were divided into normal, 3-day, 7-day, 16-day (acute stage), and 32-day (chronic stage) p.i. groups. No other special treatments were given to any of the 4 experimental groups after EAE was induced. In order to explore the role of curcumin, mice were divided into normal, EAE, control, and curcumin groups. The last 3 groups were divided into 3-day, 7-day, 16-day, and 32day groups p.i. The mice received i.p. injection of curcumin at 200 mg/kg/d dissolved in 0.5% methylcellulose (curcumin group) or 0.5% methylcellulose (control group) when they were immunized [12].   Death Detection Kit, POD). The protocols were followed according to the manufacturer's instructions. Paraffin-embedded samples (3 m thick sections) were deparaffinized and rehydrated with xylene and ethanol and permeabilized with 20 g/mL of proteinase K for 30 min. Endogenous peroxidase was inactivated by coating the samples with 3% H 2 O 2 . Sections were rinsed with PBS and then immersed for 60 min in TdT buffer at 37 ∘ C and then incubated for 30 min with the antidigoxigenin peroxidase conjugate, followed by the peroxidase substrate (3diaminobenzidine tetrahydrochloride [DAB]). Finally, sections were counterstained with 0.5% (wt/vol) methyl green.
The apoptosis index was evaluated using a light microscope. Results were expressed as a percentage of TUNEL-positive cells to the total cells [13].

Transmission Electron Microscope Examination (TEM).
All biopsy specimens were fixed in glutaraldehyde solution followed by 1% osmium tetroxide and then embedded in resin block. Semithin 500 nm sections were cut by the ultramicrotome and stained with toluidine blue. After light microscopic orientation, 70 nm ultrathin sections were cut and stained by uranyl acetate and lead citrate [14]. The ultrathin sections  were then examined by a transmission electron microscope (Hitachi-7500).

Immunofluorescence Double Labeling.
Paraffin-embedded sections were deparaffinized, rehydrated in a graded series of ethanol, and then incubated in H 2 O 2 (0.3%, 15 min). For antigen retrieval, sections were treated with 10 mmol/L sodium citrate buffer (pH 6.0) and heated with a microwave oven for 20 min. Tissue was permeabilized with 0.5% Triton X-100, and then sections were incubated in 5% goat serum for 30 min at room temperature. Sections were then incubated with MAP2 (1 : 500) or CNPase (1 : 1000) primary antibodies at 4 ∘ C overnight. Next, sections were washed and incubated with tetramethylrhodamine isothiocyanate-(TRITC-) conjugated goat anti-mouse IgG (1 : 100) in the dark for 60 min, and then DAPI was added for 5 min. After washing with PBS, TUNEL mixture solution was added [15]. The results were observed with a fluorescence microscope (Lecia, Germany).
TUNEL-positive cells were labeled with green fluorescence, and MAP2 (+) or CNPase (+) cells were labeled with red fluorescence. Images were processed with Image-Pro 6.0 software.

Statistical Analysis.
All results were presented as means ± standard deviation (SD). Statistical differences between groups were compared using one-way analysis of variance (ANOVA). Statistical analysis was performed using SPSS 18.0. A value < 0.05 was considered statistically significant.

Mitochondrial Injury Occurred in the Early Stages after EAE Was Immunized.
To explore the early pathological changes after EAE was immunized, the morphological characteristics of mitochondria, myelin sheath, and axons in the spinal cord were observed by TEM. Both mitochondrial crista structure and membranes were clear when observed in the normal group. The morphology of mitochondria, myelin sheath, and axons was normal. At 3 days p.i, swollen mitochondria and compressed crista were seen, while the structure of the myelin sheath and axons was still normal. At 16 days p.i, the pathological changes were more severe. Mitochondria were swollen and there was either reduction of mitochondrial crest and matrix density or vacuolation. Demyelination and axoplasmic atrophy were also observed. The lesions in the 30-day group were similar, with mild alleviation compared to the 16-day group (Figure 1).

Cell Apoptosis Occurred in the Early Stages after EAE
Was Immunized. Analysis of apoptotic cells at different time points post-EAE was performed using TUNEL. No obviously TUNEL (+) cells were observed in the normal group. After EAE was immunized, TUNEL (+) cells were observed as early as 3 days p.i. However, no significant differences were observed in the number of TUNEL (+) cells between the 3-day and 7-day groups ( > 0.05). The TUNEL (+) cells reached maximum levels at 16 days p.i. ( < 0.05) and largely expressed around the blood vessels. The expression level in the 30-day group was lower than in the 16-day group ( > 0.05) (Figure 2 and Table 1).

Localization of Apoptotic Cells in the Early Stages after EAE Was Immunized.
We localized the TUNEL-positive cells with CNPase/MAP-2-positive cells (a specific marker of neural/oligodendroglia) via immunofluorescence double labeling. The results showed that CNPase/MAP-2 (+) cells were colocalized with TUNEL (+) cell in the spinal cord 3 days after EAE was immunized. The results suggested that the apoptotic cells in the early stages post-EAE were oligodendroglia and neurons (Figure 3).

Curcumin Inhibits Cell Apoptosis in EAE.
The effect of curcumin on apoptosis in the EAE model was evaluated by TUNEL. The results showed that, in both the acute stage (16 days) and chronic stage (30 days) of EAE, the number of TUNEL (+) cell in the curcumin group was significantly lower than the EAE group and control group at the same time points (Figure 4 and Table 2).

MBP Expression Level Detected by Western Blot.
The MBP protein expression level in the spinal cord was evaluated by western blot. No significant differences were observed at 3 days or 7 days between the normal and the EAE groups ( > 0.05). The expression of MBP in the EAE group was significantly lower than the normal group at the 16-day and 30-day time points ( < 0.05). The expression of MBP in the curcumin group was significantly higher than in the EAE group at the same time points ( < 0.05) ( Figure 5).

Cleaved-Caspase-3 Expression Levels Detected by Western Blot.
The expression of caspase-3, a specific apoptosis marker, was evaluated at different time points using western blot. There was a relatively low expression of caspase-3 in the normal group. The expression levels remained low with no significant differences observed between any of the groups 3 days p.i ( > 0.05). In the EAE group, the expression of caspase-3 increased at 7 days p.i. and reached peak levels at 16 days ( < 0.01) and then dropped a little at 30 days ( < 0.01). The expression of caspase-3 in the curcumin group was significantly lower at the same time points than the EAE group for the 7-day, 16-day, and 30-day groups ( < 0.05) ( Figure 6).

Cyt-c Expression Levels Detected by Western Blot.
The expression tendency of cyt-c was consistent with caspase-9. No significant difference was observed between the 3-day groups and the normal group ( > 0.05). The expression of cyt-c increased at 7 days p.i. and reached peak levels at 16 days ( < 0.01). The expression at 30 days was lower than at 16 days ( < 0.01). The expression in the curcumin group was significantly lower than in the EAE group at the same time points at 7, 16, and 30 days ( < 0.05) (Figure 7).

Cleaved-Caspase-9 Expression Levels Detected by Western Blot.
Caspase-9 is a specific marker for mitochondrial apoptosis. The expression of cleaved caspase-9 was evaluated by western blot. No significant differences were observed between any of the three groups at 3 days p.i. and the normal group ( > 0.05). In the EAE group, expression of caspase-9 increased at 7 days ( < 0.01) and reached peak levels at 16 days ( < 0.01). The expression in the curcumin group was significantly lower than in the EAE group at the same time point by 7, 16, and 30 days (Figure 8).

Discussion
There is a paucity of reports concerning the pathological characteristics of the whole course post-EAE. In this study, for the first time, we explored apoptosis and mitochondrial injury at different stages (early stage: 3d and 7d; acute stage: 16d; chronic stage: 30d) of EAE. Barnett et al. found that oligodendrocyte apoptosis occurred in the early stages when new lesions were formed in MS [3,4]. However, whether oligodendrocyte apoptosis occurs in the early stages of EAE is unknown. We found that TUNEL-positive cells occurred as early as 3 days after the EAE model was conducted. The symptomatic severity of EAE was consistent with the number of TUNEL-positive cells. The apoptotic cells in the early stages of EAE were confirmed to be oligodendrocytes and neurons. These apoptotic cells were accompanied by intact myelin sheaths and axons, without the typical morphology of EAE lesions. The results suggest that oligodendrocyte and neuronal apoptosis is one of the earliest pathological events in EAE, occurring in the early stages of EAE/MS pathology.
It has been shown that hypoxia-like tissue injury happens in the early stages of MS, which suggests that mitochondrial injury might be involved in the pathogenesis of MS [17]. Mitochondrial injury mediates the intrinsic apoptosis pathway [18]. However, mitochondrial injury in EAE and MS is still under exploration. We found that mitochondrial injury occurred 3 days after EAE was conducted and continued through the rest of the stages of EAE. The results indicate that mitochondrial injury may be one of the main causes for the apoptosis in the early stage of EAE. Mitochondrial injury may be responsible for the chain reaction of demyelination and inflammation after EAE. Clarifying this pathway would require further study.
Curcumin has been shown to ameliorate EAE. The mechanism by which this is done include but is not limited to the inhibition of IL-17 production [12], modulation of CD4 + T cell and toll-like receptors (TLRs) [19], and downregulation of IL-12 [20], IFN-, IL-12, and IL-23 [8]. However, the mechanism and effects of curcumin on apoptosis are unclear. In this study, we first demonstrated that curcumin inhibits apoptosis in the spinal cord in both the acute stage (16 days) and chronic stage (30 days) of EAE, and then we explored the manner in which this occurred. We demonstrated that curcumin can inhibit the expression of activated caspase-3, caspase-9, and cyt-c starting from 7 days p.i. We also demonstrate that curcumin inhibits the decrease of MBP protein, which inhibits demyelination in EAE. Activation of caspase-3 is the key event in the course of intrinsic apoptosis [21]. Although apoptosis occurred at 3 days p.i, the expression of activated caspase-3 significantly increased until 7 days p.i. The expression of MBP was significantly decreased after 16 days p.i, which manifested in demyelination not occurring until 16 days p.i. This suggested that neuronal apoptosis occurred earlier in the course of the disease than demyelination.
Mitochondria are responsible for the intrinsic apoptosis pathway [22]. Cytochrome-c (cyt-c) release is an essential step in the intrinsic apoptotic cascade [23,24]. The release of cyt-c into the cytosol triggers the formation of the apoptosome and the activation of the caspase cascade [25]. Caspase-9, known as the initiator of intrinsic apoptosis, binds to the apoptosome and activates the effector caspases-3, which is considered the major enzyme in a cell's commitment to apoptosis [26]. In this study, the expression of activated cytc and caspase-9 was elevated from 7 days after the EAE mice were immunized, and the high expression persisted throughout the entire course of EAE. This phenomenon is consistent with the variation of mitochondrial injury and the severity of EAE we observed. We found that curcumin inhibits the expression of activated cyt-c and caspase-9 from 7 days p.i. and also inhibits mitochondrial injury and reduces the number of apoptotic cells. This result illuminates the way that curcumin suppresses the intrinsic apoptotic pathwayit inhibits the release of cyt-c into the cytosol and then reduces the activation of caspase-9. This may be one of the mechanisms by which curcumin inhibits apoptosis in EAE mice. Whether the exogenous apoptotic pathway is also affected by curcumin needs to be investigated in a further study.
This study demonstrated that mitochondrial injury and oligodendrocyte/neuronal apoptosis occur in the early stages of EAE pathology, which then proceeds to demyelination and axonal injury. We also found that curcumin inhibits the apoptosis of EAE from the early stages of EAE. This inhibitory effect occurs through mitochondrial protection and inhibition of the intrinsic apoptotic pathway.