Curcumin (Cur) induces neuroprotection against brain ischemic injury; however, the mechanism is still obscure. The aim of this study is to explore the potential neuroprotective mechanism of curcumin against oxygen-glucose deprivation/reoxygenation (OGD/R) injury in HT22 cells and investigate whether type-2 superoxide dismutase (SOD2) is involved in the curcumin-induced protection. In the present study, HT22 neuronal cells were treated with 3 h OGD plus 24 h reoxygenation to mimic ischemia/reperfusion injury. Compared with the normal cultured control group, OGD/R treatment reduced cell viability and SOD2 expression, decreased mitochondrial membrane potential (MMP) and mitochondrial complex I activity, damaged cell morphology, and increased lactic dehydrogenase (LDH) release, cell apoptosis, intracellular reactive oxygen species (ROS), and mitochondrial superoxide (
Stroke is one of the leading causes of disability and death in China and worldwide [
Curcumin is derived from seasoning curry and herbal
Therefore, in the present study, we used oxygen-glucose deprivation plus reoxygenation (OGD/R) in HT22 neuronal cells to mimic neuronal ischemia/reperfusion (I/R) injury [
HT22 cells were obtained from the Xuzhou Medical University. Curcumin, Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), and methylthiazolyldiphenyl-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lactic dehydrogenase (LDH) reagent kit was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). SOD2 activity assay kit was obtained from Trevigen (Gaithersburg, USA). The DAPI staining solution and ROS reagent kit were purchased from Beyotime Technology (Nantong, China). MitoSOX staining kits were purchased from Invitrogen Molecular Probes (San Diego, CA). The primary antibodies of anti-SOD2, anticleaved caspase-3, and anti-
The HT22 cells were cultured in the medium, containing 90% DMEM medium, 10% FBS, 100 U/ml penicillin, and 100
For the OGD treatment, the medium of the cells was changed with DMEM medium without glucose and FBS, and then the cells were cultured in a sealed container; the air of the container contained 95% N2 and 5% CO2, and the temperature of the container was 37°C. After 3 h OGD treatment, the medium of the cells was changed with normal medium, and the cells were returned to the normal incubator to mimic reperfusion.
To explore a suitable curcumin concentration, the cells were divided into 5 groups, including the normal cultured control group, 3 h OGD plus 24 h reoxygenation (OGD/R) treatment group, 10 ng/ml curcumin treatment group (10 ng/ml Cur + OGD/R), 100 ng/ml curcumin treatment group (100 ng/ml Cur + OGD/R), and 500 ng/ml curcumin treatment group (500 ng/ml Cur + OGD/R). After treatments (Figure
Experimental protocol diagram. (a) Searching a suitable curcumin concentration. The cells were divided into 5 groups, including the normal cultured control group, oxygen glucose 3 h OGD plus 24 reoxygenation treatment group (OGD/R), and 3 concentrations of curcumin treatment groups. After the treatments, cell viability and LDH release were assessed. (b) Observing curcumin-induced effect on SOD2 protein expression. The cells were divided into 4 groups, including normal cultured control group and 3 concentrations of curcumin treatment groups; after 3 h treatment, SOD2 expression was assessed by using western blot analysis. (c) Evaluating interfering effect of SOD2-siRNA. The cells were divided into 3 groups, including control group, SOD2-siRNA treatment group, and scrambled (SC)-siRNA treatment group. After the treatments, SOD2 protein expression was evaluated by using western blot analysis. (d) Exploring the role of SOD2 in curcumin-induced protection in HT22 cells. The cells were divided into 5 groups, including control, OGD/R treatment group, Cur + OGD/R group, SOD2-siRNA + Cur + OGD/R group, and SC-siRNA + Cur + OGD/R group; after the treatments, cell injury, apoptosis, SOD2 expression, cell morphology, intracellular ROS, mitochondrial functions, and superoxide were assessed.
To determine whether SOD2 mediates curcumin-induced protection against OGD/R in HT22 cells, the cells were divided into 5 groups, including control group, OGD/R treatment group, 100 ng/ml curcumin treatment group (Cur + OGD/R), SOD2-siRNA treatment group (SOD2-siRNA + Cur + OGD/R), and SC-siRNA treatment group (SC-siRNA + Cur + OGD/R). After the treatments (Figure
The HT22 cells were seeded into a 96-well cell culture plate at a density of 1 × 105 cells per well. After the treatments, 20
The cells were seeded into a 24-well cell culture plate at a density of 5 × 105 cells/well. After the treatments, the supernatants of the plate were collected to measure the LDH activity of each well, as previously described [
The cells were seeded into a 6-well cell culture plate at a density of 1 × 106 cells/well. After the treatments, the medium was removed, and the cells were collected. Then, the total protein of the treated cells was evaluated by using the Bradford method as described previously [
SOD2-siRNA and SC-siRNA were obtained from Qiangen (Germany). The siRNA oligomers, including SOD2-siRNA and SC-siRNA, were diluted in serum-free DMEM medium, and then the medium was incubated in room temperature for 5 min. The incubated oligomers were combined with diluted Lipofectamine 2000 and incubated for another 20 min. The cell culture medium was then removed from the plate, and the cells were washed twice with phosphate-buffered saline (PBS) at 37°C. Then, the complexes of 90 pmol siRNA and Lipofectamine were added into the cell culture plate, and the cells were incubated for 6 h in the incubator at 37°C. Then, the cells were washed with PBS at 37°C. The SOD2 expression was measured using western blot analysis.
HT22 cells were seeded into a 6-well cell culture plate at a density of 1 × 106 cells/well. After the treatments, as previously described in detail [
The cell apoptosis level was measured by using a flow cytometry (BD, USA). The cells were planted into a 6-well cell culture plate at a density of 5 × 105 cells/well. After the treatments, the cells were harvested by centrifugation at 1000 rpm for 10 min. Then, the supernatants of the cells were removed, and the cells were washed twice by ice-cold PBS. After the washing, the apoptotic rates of the cells were evaluated as previously described [
The mitochondria of the treated cells were isolated by using a mitochondrial isolation kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). And the mitochondrial complex I activity was measured at 30°C as previously described by Han et al. [
Mitochondrial membrane potential (MMP) of the treated HT22 cells was assessed by using the JC-1 (Sigma-Aldrich, St. Louis, MO, USA). According to the manufacturer’s instruction, mitochondrial samples were exposed to JC-1 staining buffer. At the end of the experiments, valinomycin was used as the negative control. And the fluorescence intensity of the cells was measured by using a fluorescence spectrophotometer (TECAN, CH), and the measurement temperature was 37°C. The ratio of aggregates to monomer was calculated as the MMP indicator, and the wavelengths testing the aggregates and monomer were 590 nm (red) and 525 nm (green), respectively. We followed the methods of Du et al. [
The cells were planted into a 6-well cell culture plate at a density of 5 × 105 cells/well. After the treatments, the cells were observed by using a phase-contrast microscope, and the photos of the cells were taken randomly.
HT22 cells were seeded into a confocal microscopy special dish at a density of 2 × 105 cells/well. After the treatments, a reactive oxygen species (ROS) assay kit (Beyotime Technology, Nantong, China) was taken to evaluate the intracellular ROS level. In brief, the DMEM medium without FBS was added into each well, containing 100
MitoSOX reagent was used to measure mitochondrial superoxide level. In brief, the cells were seeded into a confocal microscopy special dish at a density of 1 × 105 cells/well. After the treatments, the HT22 cells were treated with 5
The data of this study were analyzed by using SPSS 13.0 software (SPSS Inc., Chicago, USA). The values of all the experiments were expressed as means ± standard deviation (SD), and one-way ANOVA was used to assess the data. Tukey’s multiple comparison was taken to compare the differences between the groups.
To find a suitable curcumin (Cur) treatment concentration, the HT22 cells were divided into 5 groups, including control, OGD/R, and 3 concentrations of curcumin treatment groups (10, 100, and 500 ng/ml curcumin plus OGD/R respectively). After 3 h OGD and 24 h reoxygenation treatment, compared with the control, OGD/R treatment reduced cell viability (Figure
Curcumin decreased cell injury in HT22 cells exposed to OGD/R and upregulated SOD2 expression in normal condition. The HT22 cells were divided into 5 groups, including control, OGD/R, and 3 concentrations (10 ng/ml, 100 ng/ml, and 500 ng/ml) of curcumin plus OGD/R groups. After the treatments, cell viability and LDH release were measured by using the MTT method and reagent kit, respectively. Then, the cells were divided into 4 groups, including control and 3 concentrations (10 ng/ml, 100 ng/ml, and 500 ng/ml) of curcumin treatment groups; after 3 h exposure, western blot was performed to assess SOD2 expression. (a) Curcumin restored cell viability (
To explore the role of SOD2 in curcumin-induced protection against OGD/R in HT22 cells, SOD2-siRNA was taken to downregulate SOD2 protein expression (Figure
SOD2-siRNA reversed curcumin-induced cytoprotection and SOD2 upregulation in HT22 cells exposed to OGD/R. The cells were divided into 3 groups, including control, SOD2-siRNA, and scrambled (SC)-siRNA; after 6 h incubation, western blot and MTT assay were taken to assess SOD2 expression and cell viability, respectively. Then, the cells were divided into 5 groups, including control, OGD/R, Cur + OGD/R, SOD2-siRNA + Cur + OGD/R, and SC-siRNA + Cur + OGD/R; after 3 h OGD plus 24 h reoxygenation, SOD2 expression and activity, cell viability, and lactic dehydrogenase (LDH) were assessed. (a) SOD2-siRNA inhibited SOD2 protein expression (
Then, the cells were divided into 5 groups, including control, OGD/R, Cur + OGD/R, SOD2-siRNA + Cur + OGD/R, and SC-siRNA + Cur + OGD/R. Compared with the control, 3 h OGD plus 24 h reoxygenation treatment (OGD/R) decreased SOD2 protein expression, SOD2 activity and cell viability, and increased LDH activity in the medium; concurrently, coadministration with 100 ng/ml curcumin restored SOD2 expression, SOD2 activity, and cell viability and reduced LDH release (Figures
To investigate the curcumin-induced antiapoptosis in HT22 cells exposed to OGD/R, flow cytometry and western blot were used to evaluate cell apoptotic rate and apoptosis-associated protein cleaved caspase-3 expression (Figures
SOD2-siRNA reversed curcumin-induced antiapoptotic effects in HT22 cells exposed to OGD/R. The cells were divided into 5 groups, including control, OGD/R, Cur + OGD/R, SOD2-siRNA + Cur + OGD/R, and SC-siRNA + Cur + OGD/R; after 3 h OGD and 24 h reoxygenation, cell apoptotic rate and cleaved caspase-3 expression were evaluated by using flow cytometry and western blot, respectively. (a) Flow cytometry results of cells. (b) SOD2-siRNA reversed curcumin-induced antiapoptotic effect (
To further observe the curcumin-induced effects on cell morphology and mitochondrial functions in HT22 cells exposed to OGD/R, phase-contrast microscope and reagent kits were taken to assess cell morphology and cellular mitochondrial functions. The cells were divided into 5 groups as shown in the Figure
SOD2-siRNA reversed curcumin-induced ameliorations of cell morphology and mitochondrial functions in HT22 cells exposed to OGD/R. The cells were divided into 5 groups, including control, OGD/R, Cur + OGD/R, SOD2-siRNA + Cur + OGD/R, and SC-siRNA + Cur + OGD/R; after 3 h OGD and 24 h reoxygenation, cell morphology and mitochondrial functions were evaluated. (a) SOD2-siRNA reversed curcumin-induced cell morphology amelioration. (b) SOD2-siRNA reversed curcumin-induced amelioration of mitochondrial membrane potential (MMP) (
High level of intracellular ROS and mitochondrial superoxide can damage cell and mitochondria. Compared with the control group, 3 h OGD plus 24 reoxygenation (OGD/R) treatment increased ROS (Figures
SOD2-siRNA reversed curcumin-induced reduction of intracellular ROS and mitochondrial superoxide in HT22 cells exposed to OGD/R. The cells were divided into 5 groups, including control, OGD/R, Cur + OGD/R, SOD2-siRNA + Cur + OGD/R, and SC-siRNA + Cur + OGD/R; after 3 h OGD and 24 h reoxygenation, intracellular ROS and mitochondrial superoxide were evaluated. (a) Intracellular ROS fluorescence staining results. (b) SOD2-siRNA reversed curcumin-induced intracellular ROS reduction (
In the present study, the HT22 neuronal cells were exposed to OGD for 3 h and then cultured in normal medium for 24 h to imitate the neuronal I/R injury, and 100 ng/ml curcumin was coadministered to reduce the OGD/R-induced cell injury. Compared with the control, OGD/R increased the cell injury, apoptosis, intracellular ROS, and mitochondrial superoxide, reduced mitochondrial functions and intracellular SOD2, and damaged cell morphology; meanwhile, the presence of curcumin reduced cell injury, apoptosis, intracellular ROS, and mitochondrial superoxide, restored mitochondrial functions and SOD2, and maintained cell integrity. However, SOD2-siRNA, but not the SC-siRNA, significantly reversed the curcumin-induced protections above. These findings indicated that curcumin alleviates OGD/R-induced injury in HT22 cells, and SOD2 may mediate the curcumin-induced protection (Figure
Curcumin induces neuroprotection against OGD/R in neuronal cells via upregulating SOD2 protein. Oxygen-glucose deprivation and reoxygenation (OGD/R) injury can downregulate SOD2 expression, increase intracellular ROS and mitochondrial superoxide accumulations, then damage neuronal cells, increase cell apoptosis, cause mitochondrial dysfunctions, and undermine cell integrity, leading to neuronal injury ultimately. Coadministration of curcumin, however, could upregulate SOD2 expression, reduce intracellular ROS and mitochondrial superoxide accumulations, and ameliorate mitochondrial functions and cell integrity, causing neuroprotection.
Stroke is one of the leading causes of disability and death in the worldwide [
Curcumin is an extract from seasoning curry and herbal
Taken together, in the present study, we found that curcumin can reduce OGD/R-induced cell injury in HT22 cells, and SOD2 protein mediates the curcumin-induced neuroprotection.
All datasets analyzed during the current study are available from the corresponding author upon reasonable request.
The authors declare no conflicts of interest.
Yuqing Wang, Yuanyuan Zhang, and Liang Yang contributed equally to this work.
This study was supported by the Natural Science Foundation of Guangdong Province, China (no. 2016A030313613), the Science and Technology Program of Guangdong Province, China (no. 2016A020215132), and the Science and Technology Program of Guangzhou, China (nos. 201707010027 and 201707010474).