Neuroprotective Effects of Curcumin against Oxygen-Glucose Deprivation/Reoxygenation-Induced Injury in Cultured Primary Rat Astrocyte by Improving Mitochondrial Function and Regulating the ERK Signaling Pathway

Objectives Curcumin (Cur) is a natural polyphenol isolated from turmeric and has potent anti-inflammatory and antioxidant activities. This study aimed to explore the effects and possible mechanisms of curcumin on oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury in cultured rat astrocyte primary cells. Methods After screening for effective doses, the cultured rat astrocyte primary cells were divided into three groups: control, OGD/R, and OGD/R + curcumin (10 μM, 20 μM, and 40 μM). Cell viability was detected using CCK8 assays. The level of malondialdehyde and superoxide dismutase activity was determined using commercial kits. The endothelial nitric oxide synthase and adenosine triphosphate concentrations were determined by enzyme-linked immunosorbent assay. The mRNA levels of the inflammatory indexes interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1β were evaluated by quantitative reverse-transcription polymerase chain reaction. Annexin V-fluorescein isothiocyanate/propidium iodide was used to detect apoptosis. JC-1 was used to assess the mitochondrial membrane potential. The protein expression of apoptosis-related proteins (B-cell lymphoma-2 (Bcl-2), BCL-2-associated X (Bax), and cleaved caspase 3), mitochondria-related proteins (dynamin-related protein 1 (DRP1), phosphorylated DRP1 (p-DRP1), and mitofusin 2), and essential proteins of the extracellular signal-regulated kinase (ERK) signaling pathway (ERK1/2, p-ERK1/2) were analyzed by western blot. Results Our data indicated that curcumin reversed OGD/R-induced cell viability loss, oxidative stress, inflammatory cytokine production, and cell apoptosis in a dose-dependent manner. Furthermore, curcumin attenuated OGD/R-induced mitochondrial dysfunction and ERK1/2 phosphorylation in a dose-dependent manner. Conclusions Curcumin protected against OGD/R-induced injury in rat astrocyte primary cells through improving mitochondrial function and regulating the ERK signaling pathway.


Introduction
Spinal cord injury (SCI) is a devastating problem that leads to sensory and functional disabilities in some of the body's internal organs below the level of the injury [1]. e original spinal cord injury is a primary damage that triggers secondary injury [2]. Secondary injury produces a cascade of biochemical, mechanical, and physiological changes, leading to further spinal cord damage [3]. Secondary injury plays a astrocytic mitochondria, which are closely related to the pathogenesis of secondary SCI [9]. e extracellular signalregulated kinase (ERK)/dynamin-related protein 1 (DRP1) signaling pathway mediates mitochondrial dysfunction during secondary SCI injury [10,11].
Curcumin (Cur), a hydrophobic polyphenol derived from the dried rhizomes of Curcuma longa L. (turmeric), has long been used in traditional Chinese medicine [12]. It was initially used as a cooking spice in some Asian countries [13] and has received a great deal of attention for the therapy of several diseases, including cardiovascular disease [14], cancer [15], and conditions of the CNS [16]. As a natural product, curcumin has been shown to have potent antiinflammatory and antioxidant activities, which are closely related to its therapeutic effects [17]. It also plays a protective role in SCI [18][19][20]. However, the underlying mechanisms of curcumin in the treatment of SCI are not entirely understood.
In the present study, we explored the effects of curcumin on primary rat astrocyte damaged by oxygen-glucose deprivation/reoxygenation (OGD/R) and its possible mechanism of action on mitochondrial function and the ERK signaling pathway.

Astrocyte
Culture. All animals were treated according to the National Institutes of Health guidelines for the Care and Use of Laboratory Animals. Primary spinal cord astrocyte was isolated from six newborn Sprague Dawley rats, as previously described [21]. Briefly, the newborn rats were deeply anesthetized with isoflurane and then decapitated. e spinal column was cut and placed on ice in a glass culture dish. e spinal cord above the T10 level was removed with sterile phosphate-buffered saline (PBS). Spinal cord tissues were cut into small pieces and digested with 0.125% trypsin. e mixture was filtered with 70 μm sterile sieves and placed in a complete DMEM/high-glucose medium ( # SH30022.01, Hyclone, USA) containing 10% fetal bovine serum ( # 10270-106, fetal bovine serum, Gibco, USA). Cells were incubated at 37°C with 5% CO 2 for five days, and then the mixed microglial cells and oligodendrocytes were removed by shaking at 37°C overnight at 200 rpm.

OGD/R Protocol.
e cultured rat astrocyte primary cells were washed three times with PBS and incubated with serum-free DMEM. en, the cells were placed in an anaerobic environment (1% O 2 , 5% CO 2 , and 94% N 2 ) at 37°C for 6 h, namely, OGD. Astrocytes were washed once with PBS after 6 h of OGD and placed in the normal conditions (reoxygenation) and complete DMEM culture with 10% BSA.

CCK8.
e isolated astrocyte was seeded in a 96-well plate at a density of 8,000 cells per well and incubated at room temperature overnight. Curcumin ( # C110685, Aladdin, China) was added at various concentrations (1 μM, 5 μM, 10 μM, 20 μM, and 40 μM) into the medium 30 min before OGD and at the time of reoxygenation. Approximately 24 h after oxygen-glucose deprivation/reoxygenation (OGD/R), 10 mL of CCK8 solution ( # CA1210, Solarbio, China) was added and incubated in the dark for 1 h. e optical density values were measured at a wavelength of 450 nm.

Immunofluorescence Staining.
e astrocyte was grown on coverslips overnight and then fixed with 4% paraformaldehyde. Cells were permeabilized with 0.3% Triton X-100 and blocked with 10% BSA. Astrocyte was incubated individually with astrocytic markers (GFAP and S100β) and antibodies ( # PAB32097 and # PAB30176, Bioswamp, China) at 4°C overnight. en, fluorescein isothiocyanate (FITC, red) or Alexa Fluor 488 (green)-conjugated secondary antibodies were added. e protein expression of GFAP and S100β was observed using a fluorescence microscope (Leica, Germany).

Reactive Oxygen Species (ROS) Production Assay.
e intracellular ROS levels were determined with a commercial ROS kit ( # S0033, Beyotime, China) by following the manufacturer's manuals. After treatment of curcumin and OGD/ R for 24 h, astrocyte was incubated with 2′-7′-dichlorofluorescein diacetate. After washing, the cells were suspended in PBS, and the fluorescence was detected by flow cytometry (ACEA Biosciences, USA).

Annexin V-FITC Flow Cytometry.
e early cell apoptosis was detected by Annexin V-FITC/propidium iodide (PI) apoptosis detection kit ( # 556547, BD Biosciences, USA). e procedure for curcumin treatment and OGD/R modeling was the same as described above. e cells were washed twice after all groups of cells were harvested. Annexin V-FITC and PI were added by following the manufacturer's protocols.
e cell apoptosis was determined by flow cytometry (ACEA Biosciences, USA).

Mitochondrial Membrane Potential Detection.
e mitochondrial membrane potential (ΔΨm) was detected by JC-1 detection kit ( # C2006; Beyotime, China) according to the supplier's protocol. In brief, astrocyte in all groups was stained with JC-1, which presented potential-dependent aggregation in mitochondria. JC-1 exhibits green fluorescence (527 nm) at low membrane potential while producing red fluorescence (590 nm) at high membrane potential.

Transmission Electron Microscopy.
e astrocyte cells were digested and centrifuged. en, the cells were collected and fixed with 2.5% glutaraldehyde and 1% osmic acid. After dehydration, the cells were embedded in epoxy resin. e cell slices were stained with 2% uranyl acetate and lead citrate. e cellular mitochondria were observed with a transmission electron microscope (TEM; Hitachi HT7700, Japan).

Statistical Analysis.
e results are expressed as mean ± standard deviation. e differences among groups were analyzed by one-way analysis of variance. e statistical significance was established at P < 0.05. Statistical analysis was performed by the SPSS software. e graphs were produced by using GraphPad Prism software.

Purity Identification of Isolated Primary Rat Astrocyte.
Immunofluorescence staining of astrocytic markers (GFAP and S100β) was performed to determine the purity of the astrocyte. After 24 h of incubation, cells were uniformly stained with anti-GFAP and anti-S100β antibodies. e high purity of isolated primary rat astrocyte is shown in Figures 1(a) and 1(b).

Screening for Effective Concentration of Curcumin against OGD/R Injury in Astrocyte.
e cell viability of cultured astrocyte was determined through CCK8 assay, which was used to evaluate the protective effects of curcumin against OGD/R-induced cytotoxicity. Compared to the control group, there is a significant reduction in cell viability in the OGD/R group, which was restored by curcumin pretreatment (1, 5, 10, and 20 μM) in a dose-dependent way ( Figure 2).

Curcumin Pretreatments Attenuated Loss of Cell Viability and Increased Oxidative Stress Indexes in Rat Astrocyte
Primary Cells Injured with OGD/R. Cell viability and oxidative stress indices were investigated to evaluate the protective effects of curcumin on rat astrocyte primary cells injured by OGD/R. e results showed that the pretreatments with curcumin (10, 20, and 40 μM) increased cell viability after exposure in a dose-dependent way (Figure 3(a)). e intracellular concentrations of MDA, SOD, eNOS, and ROS were determined to evaluate the antioxidative effects of curcumin. In OGD/R group, the MDA concentration was increased significantly, but markedly reduced by pretreatment with curcumin (10, 20, and 40 μM) (Figure 3(b)). SOD activity and eNOS levels decreased after OGD/R exposure, which was restored by pretreatment with curcumin (10, 20, and 40 μM) (Figures 3(c) and 3(d)). Additionally, ROS levels were significantly elevated after exposure to OGD/R, and curcumin pretreatment reduced ROS production in the dose-dependent way (Figure 3(e)).

Curcumin Pretreatments Inhibited Cell Apoptosis in OGD/R-Injured Rat Astrocyte Primary Cells.
e rate of apoptosis and the expression of apoptosis-related proteins (Bax, Bcl-2, and cleaved caspase 3) were detected to evaluate the curcumin effects on OGD/R-induced cell apoptosis in rat astrocyte primary cells. e apoptosis rate was observably elevated in OGD/R group, while the curcumin pretreatment alleviated the apoptosis (Figures 5(a) and 5(b)). Western blot assay revealed that OGD/R-induced cell apoptosis was concomitant with increased expression of Bax and cleaved Evidence-Based Complementary and Alternative Medicine caspase 3 and decreased expression of Bcl-2 in cultured rat astrocyte primary cells. Apoptosis was reversed through pretreatment with curcumin (10 μM, 20 μM, and 40 μM) in the dose-dependent way (Figures 5(c)-5(g)).

Curcumin Pretreatments Alleviated Mitochondrial Damage in OGD/R-Injured Primary Rat Astrocyte.
e degree of mitochondrial depolarization in cultured astrocyte was evaluated by JC-1. After exposure to OGD/R, the ΔΨm of astrocyte increased, which was reduced by pretreatment with curcumin (10, 20, and 40 μM) (Figures 6(a) and 6(b)). e results showed that ATP production decreased significantly in the OGD/R group, whereas curcumin (10, 20, and 40 μM) pretreatment restored ATP production ( Figure 6(c)). e expression of mitochondrial-related proteins (DRP1, p-DRP1, and MFN2) was detected to evaluate mitochondrial function. DRP1 expression was significantly increased in the OGD/R group and decreased by curcumin (10, 20, and 40 μM) pretreatment. e presentation of p-DRP1 and MFN2 was markedly reduced in the OGD/R group, which was restored by pretreatment with curcumin (10, 20, and 40 μM) (Figures 6(d)-6(g)). In the OGD/R group, mitochondrial swelling and loss of cristae were observed using a TEM, while pretreatment with curcumin (10, 20, and 40 μM) attenuated these morphological changes in mitochondria ( Figure 6(h)).

Discussion
SCI, an incurable disease, often results in devastating sequelae [24]. Astrocytes are the most abundant glial cells in the central nervous system (CNS) and have been known to protect neurons and assist their survival under pathological conditions [25]. Astrocyte dysfunction contributes to the secondary injury in SCI [26]. erefore, astrocyte may be potential targets for SCI therapy. Curcumin was reported to be effective in treating SCI [19,27]. Curcumin's therapeutic effects have been closely related to its own anti-inflammatory and antioxidant activities [28], although the potential mechanisms are still only partly understood. e present study demonstrates that curcumin exerts potent antioxidant, anti-inflammatory, and neuroprotective effects on primary rat astrocyte damaged by OGD/R. ese effects are mediated by improving mitochondrial function and regulating the ERK signaling pathway. Mitochondria produce ATP, which is essential for cell viability [29]. Mitochondria are also vulnerable to various injuries [30]. Dysfunction of astrocytic mitochondria can cause deleterious actions on neurons via astrocyte-neuron interactions [31]. Mitochondria have emerged as potential therapeutic targets for treating SCI [30]. DRP1 is a key protein that promotes mitochondrial fission [32]. Excessive mitochondrial fission is correlated with DRP1 dephosphorylation at Ser 637, resulting in mitochondria morphological changes [32]. MFN2, a dynamin-like GTPase, is involved in mitochondrial fusion [33]. e absence of MFN2 leads to mitochondrial fragmentation and neurodegeneration [34]. Curcumin pretreatment not only attenuated mitochondrial swelling and loss of cristae in injured astrocyte induced by (c-g) Representative images and summary of data for relative protein expressions of BCL-2-associated X (Bax), B-cell lymphoma 2 (Bcl-2), and cleaved caspase 3 through western blot analysis. Versus control group, * P < 0.05, * * P < 0.01, * * * P < 0.001, respectively; versus OGD/R group, # P < 0.05, ## P < 0.01, and ### P < 0.001 (n � 3).

Control
OGD/R OGD/R + Cur 10 µM OGD/R + Cur 20 µM OGD/R + Cur 40 µM  Evidence-Based Complementary and Alternative Medicine OGD/R, but also improved DRP1 phosphorylation in Ser 637 and MFN2 expression in the current study. e findings are consistent with the results of previously published studies [32,35,36]. ERK is essential for the regulation of cell proliferation, differentiation, and survival [37], and its signaling pathway which is closely related to mitochondrial function and cell viability [38,39] is critical to the survival of astrocyte [40]. ERK phosphorylation is elevated after SCI [22], and overactivation of the ERK signaling pathway is involved in persistent inflammation and neuropathic pain sequela [41]. In this study, ERK phosphorylation was increased in the OGD/R group, while curcumin pretreatment decreased p-ERK expression.
Data Availability e data sets used and/or analyzed in the study are available from the corresponding author on reasonable request.