Gingerol ameliorates neuronal damage induced by hypoxia‐reoxygenation via the miR‐210/brain‐derived neurotrophic factor axis

1 INTRODUCTION

Cerebral ischemia, which is caused by dysfunctional blood flow to the brain, is a common vascular disease worldwide, leading to deadly and disabling diseases such as cerebral infarction or global hypoxic–ischemic encephalopathy without appropriate therapy.1 The most perilous risk of cerebral ischemia occurs in China (approximately 40%), according to a previous report.2 Acute reperfusion therapy is the most common treatment for cerebral ischemia to dissolve the thrombus and restore blood flow to the brain.1, 3 However, an obvious increase in neuronal necroptosis is stimulated by inflammation in the presence of reperfusion. A previous investigation4 suggested that reperfusion led to an obvious increase in the number of edematous neurons in rats with cerebral ischemia. During cerebral ischemia and reperfusion, signal transducer and activator of transcription 3 (STAT3) fails to interact with manganese-superoxide dismutase (SOD2), leading to elevated levels of neuronal death.5 Moreover, excess reactive oxygen species production following reperfusion aggravates oxidative stress in the ischemic region, resulting in increased neuronal death and further brain damage.6 Neuroprotection has been the major holy grail of reperfusion treatment. Collectively, neuroprotective drugs are needed to enhance the effectiveness of reperfusion.

Gingerol is extracted from ginger and contributes to chemotherapy or chemoprevention of chronic diseases through multiple activities, such as antioxidant, anti-inflammatory and neuroprotective activities. Choi et al.7 found that gingerol reduces the levels of c-Jun N-terminal kinase, IκB-β, and AP-1 to inhibit inflammation in RAW264.7 cells. A previous investigation by Joshi et al.8 revealed that gingerol represses mercuric chloride-stimulated oxidative stress by inhibiting aggressive behaviors of metal-mediated free radicals that promote the oxidative degradation of biological membranes. Gingerol plays a protective role in cerebral ischemia by reducing apoptosis and autophagy in hypoxia-stimulated PC-12 cells, according to a previous investigation by Kang et al.9 This evidence suggests that gingerol has potential medicinal value in cerebral ischemia therapy. Gingerol attenuates neuroinflammation in microglia following cerebral ischemia by decreasing the levels of the Akt/mTOR/STAT3 pathway.10 The characteristics of neuroprotective drugs should be determined, including molecular mechanisms of action, molecular targets, and toxicity, before clinical trials are conducted. However, the specific mechanism of gingerol in cerebral ischemia reperfusion remains unknown.

miRNAs have emerged as potential biomarkers for determining the diagnosis and prognosis of cerebral ischemia. Hamzei Taj et al.11 concluded that miR-124 functioned as a therapeutic target in cerebral ischemia because it increases neuronal survival and induces an anti-inflammatory phenotype. As shown in the study by Bernstein et al.,12 miR-98 mediates the recovery of neurological function and the permeability of the blood–brain barrier to ameliorate cerebral ischemia. miR-210, which is located on chromosome 1 with a length of approximately 110 nucleotides, plays different roles in cerebral ischemia. Two investigations by Zhang et al.13 and Lou et al.14 revealed that miR-210 contributes to angiogenesis in cerebral ischemia via the integrin-vascular endothelial growth factor pathway and Notch pathway, respectively. Huang et al. concluded that downregulation of miR-210 exerts an anti-inflammatory effect on cerebral ischemia by reducing macrophage infiltration and microglial activation and inactivating proinflammatory cytokines.15 Moreover, serum miR-210 expression contributed to predicting the levels of S100B and neuron-specific enolase, which are biomarkers of hypoxic–ischemic encephalopathy.16

Brain-derived neurotrophic factor (BDNF) is extensively involved in the development of the nervous system and the subsequent formation of cognitive function as a member of the nerve growth factor family.17-19 BDNF plays a therapeutic role in cerebral ischemia. A previous investigation20 showed that the interaction between BDNF and laminin reduces neuronal degeneration and the infarct volume to ameliorate neuronal damage induced by cerebral ischemia. The BDNF-tyrosine kinase receptor B (TrkB) pathway mediates the function of the mesolimbic circuitry, cognitive function and social/depressive-like behaviors following cerebral ischemia.21-23 Activation of the BDNF/TrkB pathway attenuates ischemia-stimulated neuronal apoptosis and inflammation to reduce the infarct volume and ameliorate neurological function, which potentially contribute to neuronal recovery after cerebral ischemia–reperfusion injury.24, 25

An elevated level of BDNF and reduced expression of miR-210 were observed after gingerol treatment in hypoxia/reoxygenation-stimulated neuronal cells in our investigation, indicating that the molecular mechanism of gingerol in cerebral ischemia-induced neuronal damage might depend on miR-210 and BDNF. This investigation aimed to determine the role of gingerol in cerebral ischemia-induced neuronal damage and to elucidate whether miR-210 mediated gingerol-induced neuroprotection by targeting BDNF.

2 METHODS 2.1 Cell culture

Mouse neuroblastoma Neuro 2A (N2a) cells, which are capable of differentiating into various types of neurons, and HEK293T cells used to express dual luciferase reporter genes were purchased from American Type Culture Collection (ATCC). N2a cells and HEK293T cells were incubated with Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% streptomycin–penicillin (Sigma–Aldrich) in 5% CO2 at 37°C. In this investigation, 6-gingerol (MedChemExpress, purity = 99.54%) was prepared as a stock solution with a concentration of 1 mmol/L in dimethyl sulfoxide (DMSO, MedChemExpress). The working concentrations were prepared by dilution in DMSO before experiments. Modeling was based on a previous investigation26 with modifications according to the study requirements in this article. First, N2a cells were subjected to hypoxia by an incubation with 5% CO2 and 95% N2 in deoxygenated glucose-free Hanks' balanced salt solution (Invitrogen) for 2 h. Then, the cells were treated with 5, 10, or 20 μmol/L gingerol, followed by hypoxia for 10 h. Finally, the medium was replaced with DMEM supplemented with 10% FBS and 1% streptomycin–penicillin, and the cells were subjected to reoxygenation with 5% CO2 and 95% air for 4 h.

2.2 Cell transfection

The BDNF siRNA, NC siRNA, miR-210 mimic, NC mimic, NC inhibitor, and miR-210 inhibitor were synthesized by Sangon Company (Shanghai, China). N2a cells were seeded onto six-well plates at a density of 100,000 cells per ml. Cells were transfected with 1 μg of miR-210 mimic, 1 μg of BDNF siRNA, or 1 μg of miR-210 inhibitor using Lipofectamine 3000 (Invitrogen). The sequences were as follows: miR-210 mimic-sense, 5′-CUG UGC GUG UGA CAG CGG CUG A-3′; miR-210 mimic-antisense, 5′-AGC CGC UGU CAC ACG CAC AGU U-3′; NC mimic-sense, 5′-UUC UCC GAA CGU GUC ACG UTT-3′; NC mimic-antisense, 5′-ACG UGA CAC GUU CGG AGA ATT-3′; miR-210 inhibitor, 5′-UCA GCC GCU GUC ACA CGC ACA G-3′; miR-210 inhibitor NC, 5′-CAG UAC UUU UGU GUA GUA CAA-3′; BDNF SiRNA, sense: 5′-GCUACAUGUUGGUGGUUUAUG-3′, anti-sense: 5′-UAAACCACCAACAUGUAGCUA-3′; SiRNA NC, sense: 5′- GCUAAGUUGACGUUUGUGUUG-3′, antisense: 5′-ACACAAACGUCAACUUAGCUA-3′.

2.3 Western blot

The concentration of total protein extracted from N2a cells using RIPA lysis buffer (Solarbio, China) was determined using a BCA protein kit (Abcam, UK), followed by SDS–PAGE. The separated proteins were transferred to PVDF membranes, followed by incubations with primary antibodies purchased from Abcam, including anti-BDNF (ab108319, 1:1000), anti-TrkB (ab187041, 1:5000), anti-Bax (ab182734, 1:1000), anti-Bcl-2 (ab194583, 1:2000), anti-cleaved caspase 3 (ab214430, 1:5000), and anti-β-actin (ab8226, 1:10000) antibodies, at 4 °C overnight. Then, the membrane was incubated with an HRP-conjugated secondary antibody for 4 h at 4°C. Pierce™ ECL Western Blotting Substrate (Thermo Scientific, China) and a Bio-Rad XR gel imaging analysis system (Bio-Rad) were used to determine the protein levels.

2.4 Quantitative polymerase chain reaction

The purity of total RNA extracted from N2a cells using TRIzol reagent (Invitrogen) was determined using a spectrophotometer at 260/280 nm, followed by reverse transcription and fluorescence quantification using a fasting one-step RT–qPCR kit (SYBR) (Tiangen, China) and ABI 7000 instrument (Applied Biosystems). The primers for miR-210 and the BDNF mRNA were synthesized by Sangon Company (Shanghai, China):

miR-210-forward: 5′-GCT GCC CAG GCA CAG AT-3′; miR-210-reverse: 5′-TGC CCA CCG CAC ACT G-3′; BDNF-forward: 5′-TCA CTT AGC AAG AGT CTC AGG T-3′; and BDNF-reverse: 5′-GGT ACC CTT CTG TGT GCC AA-3′. U6 and GAPDH served as internal reference genes for normalization. The relative expression was normalized using the 2−ΔΔCq equation.

2.5 Apoptosis analysis using flow cytometry

N2a cells were seeded onto six-well plates at a density of 100,000 cells per well and then exposed to different treatments. After 48 h, cells were harvested using trypsin (Gibco) and incubated with Annexin V-FITC and propidium iodide (PI), which were purchased from Procell Company (China), for 25 min in the dark. The apoptosis rate of N2a cells was determined using flow cytometry (BD Biosciences). Cell apoptosis (%) = (cell count in Q2 phase + cell count in Q2 phase Q4)/total cell count × 100%.

2.6 MTT assay of cell viability

A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay kit was purchased from Abcam Company (UK). First, N2a cells were seeded in 96-well plates and incubated with DMEM containing 10% FBS and 1% streptomycin–penicillin. After exposure to different treatments, the viability of N2a cells was determined by staining with MTT for 10 min, followed by measurement with an enzyme reader at 570 nm.

2.7 Dual luciferase reporter gene assay

A complementary fragment of miR-210 that binds to the 3′UTR of BDNF mRNA was predicted by TargetScan (http://www.targetscan.org/vert_72/). Wild type BDNF containing the complementary fragment and the BDNF mutant without the complementary fragment were inserted into the plasmid pmirGLO. HEK293T cells were cotransfected with 100 pmol/L miR-210 mimic and 400 ng of pmirGLO-BDNF wt/pmirGLO BDNF mut in a 24-well plate using Lipofectamine 3000 (Invitrogen). The relative luciferase activity was determined using a dual luciferase reporter gene detection system (Promega) according to the manufacturer's instructions. The relative luciferase activity was calculated as the ratio of firefly luciferase activity/Renilla luciferase activity. The BDNF WT and BDNF mutant sequences were as follows: BDNF WT, 5′-GGAAACAGUCAUUUGCGCACAAC-3′ and BDNF MUT, 5′-GGAAACAGUCAUUUGGCGUGUUC-3′.

2.8 Statistics and analysis

SPSS 24.0 (IBM) and GraphPad 8.0 (GraphPad) software were used for statistical analyses and data visualization. One-way ANOVA was used to analyze the differences among multiple groups, and Dunnett's multiple comparisons test was then used for pairwise comparisons. In this investigation, the difference was considered statistically significant when p < 0.05 at the 95% confidence interval. All comparisons were two-sided tests. All experiments were conducted at least three times, and triplicates samples from each group were analyzed in each experiment. Error bars indicate the SD of experiments. Experimental data are presented as the means ± SD of three independent experiments.

3 RESULTS 3.1 Gingerol contributed to the ameliorating damage to N2a cells induced by hypoxia/reoxygenation

N2a cells subjected to hypoxia and reoxygenation were treated with 5, 10, or 20 μmol/L gingerol to determine the effect of gingerol on neuronal damage caused by hypoxia/reoxygenation (Figure 1A). An obvious decrease in N2a cell viability was detected in response to hypoxia/reoxygenation compared with the control group without hypoxia/reoxygenation, which was reversed by gingerol treatment (Figure 1B). N2a cells stimulated with hypoxia/reoxygenation showed increased apoptosis compared with unstimulated cells, which was suppressed by gingerol (Figure 1C). Proteins associated with apoptosis were analyzed to determine apoptosis of N2a cells at the molecular level. The levels of Bax and cleaved caspase 3 were significantly increased, but the Bcl-2 level was decreased in hypoxia/reoxygenation-stimulated N2a cells. In contrast, gingerol obviously reduced the levels of Bax and cleaved caspase 3 and increased Bcl-2 levels in hypoxia/reoxygenation-stimulated N2a cells (Figure 1D). The BDNF/TrkB pathway mediates apoptosis in response to cerebral ischemia. Levels of the BDNF and TrkB proteins were determined in this investigation. Compared with unstimulated N2a cells, the levels of BDNF and TrkB were markedly attenuated in response to hypoxia/reoxygenation. However, gingerol significantly increased the levels of BDNF and TrkB (Figure 1E). As illustrated in Figure 1F, miR-210 expression was substantially increased in hypoxia/reoxygenation-stimulated N2a cells, whereas it was decreased in response to gingerol treatment. Thus, gingerol contributed to ameliorating the damage to N2a cells induced by hypoxia/reoxygenation by inhibiting apoptosis and increasing viability.

image

Gingerol contributed to ameliorating the damage to N2a cells induced by hypoxia-reoxygenation. Cells were exposed to 2 h of hypoxia before gingerol treatment and then subjected to 10 h of hypoxia and 4 h of reoxygenation. Note: *p < 0.05 and **p < 0.01. H, hypoxia. R, reoxygenation. 6-G, 6-gingerol. (A) Flowchart illustrating gingerol treatment and hypoxia-reoxygenation incubation. (B) Cell activity determined using the MTT assay. Gingerol reversed the decrease in the viability of N2a cells exposed to hypoxia/reoxygenation. (C) Apoptosis measured using flow cytometry. Gingerol reduced the increased apoptosis of N2a cells exposed to hypoxia-reoxygenation. (D) Levels of the Bax, Bcl-2, and cleaved caspase 3 proteins determined using western blotting. Gingerol reversed the elevated levels of Bax and cleaved caspase 3 and the reduced level of Bcl-2 induced by hypoxia/reoxygenation. (E) Levels of the BDNF and TrkB proteins determined using western blotting. Gingerol reversed the decrease in BDNF and TrkB levels in response to hypoxia/reoxygenation. (F) The expression of miR-210 quantified using qPCR. Gingerol inhibited the increased expression of miR-210 induced by hypoxia/reoxygenation

3.2 miR-210 inhibition ameliorated neuronal damage induced by hypoxia/reoxygenation

N2a cells were transfected with the miR-210 inhibitor to downregulate miR-210 and investigate the role of miR-210 in hypoxia/reoxygenation-stimulated neuronal damage (Figure 2A). Silencing of miR-210 reversed the decrease in the viability of N2a cells exposed to hypoxia/reoxygenation (Figure 2B). The miR-210 inhibitor reduced the increased apoptosis of hypoxia/reoxygenation-stimulated N2a cells (Figure 2C). Moreover, downregulated miR-210 increased Bcl-2 levels but attenuated Bax and cleaved caspase 3 levels in hypoxia/reoxygenation-stimulated N2a cells (Figure 2D). According to Figure 2E, downregulated miR-210 reversed the decreases in BDNF and TrkB levels observed in response to hypoxia/reoxygenation in N2a cells. Therefore, miR-210 inhibition ameliorated neuronal damage induced by hypoxia/reoxygenation.

image

Silencing of miR-210 ameliorated neuronal damage induced by hypoxia-reoxygenation. Cells were subjected to hypoxia/reoxygenation, and the expression of miR-210 was repressed by the miR-210 inhibitor. Note: *p < 0.05, **p < 0.01, and ***p < 0.001. H, hypoxia. R, reoxygenation. 6-G, 6-gingerol. (A) The expression of miR-210 quantified using qPCR. The miR-210 inhibitor repressed the elevated expression of miR-210 induced by hypoxia-reoxygenation. (B) Cell activity determined using the MTT assay. The miR-210 inhibitor reversed the decrease in the viability of N2a cells induced by hypoxia-reoxygenation. (C) Apoptosis measured using flow cytometry. The miR-210 inhibitor reduced the increased apoptosis of N2a cells induced by hypoxia/reoxygenation. (D) Levels of the Bax, Bcl-2, and cleaved caspase 3 proteins determined using western blotting. The miR-210 inhibitor reversed the elevated levels of Bax and cleaved caspase 3 and the reduced level of Bcl-2 induced by hypoxia/reoxygenation. (E) Levels of the BDNF and TrkB proteins determined using western blotting. The miR-210 inhibitor reversed the decreased levels of BDNF and TrkB in response to hypoxia/reoxygenation

3.3 miR-210 repressed the expression of BDNF via binding with the 3′UTR of BDNF mRNA

The aforementioned results suggested a role for miR-210 in hypoxia/reoxygenation-stimulated N2a cells, but its specific mechanism remained unclear. According to the prediction of the bioinformatics tool TargetScan 7.2, a complementary fragment of miR-210 binds to the 3′ untranslated region of the BDNF mRNA (Figure 3A). A luciferase reporter assay was used to determine the targeting relationship between miR-210 and BDNF. The results revealed that miR-210 decreased the luciferase activity of the wild type BDNF construct, with a nonsignificant change in the luciferase activity of the BDNF mutant (Figure 3B), implying that BDNF was the target of miR-210. The level of the BDNF mRNA was decreased in response to miR-210 overexpression, while miR-210 inhibition attenuated the decrease in the BDNF mRNA level (Figure 3C). Furthermore, miR-210 overexpression reduced the level of the BDNF protein, but a significant increase in the BDNF protein level was observed after the transfection of the miR-210 inhibitor (Figure 3D). Thus, miR-210 suppressed BDNF expression by targeting the 3′UTR of BDNF.

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miR-210 repressed the expression of BDNF via targeting the 3′UTR of BDNF mRNA. Note: *p < 0.05. MUT, BDNF mutant. WT, BDNF wild type. (A) The binding site between miR-210 and BDNF identified using TargetScan 7.2. (B) Dual luciferase reporter gene assay. Cotransfection of miR-210 mimic and BDNF WT into cells led to a decrease in luciferase activity. (C) The expression of the BDNF mRNA quantified using qPCR. The miR-210 mimic decreased the expression of BDNF, which was increased by the miR-210 inhibitor, as shown by RT–qPCR. (D) BDNF protein level determined using western blotting. The miR-210 mimic decreased the level of the BDNF protein, which was increased by the miR-210 inhibitor according to western blot data

3.4 Gingerol exerts a neuroprotective effect by downregulating miR-210

While the neuroprotective role of gingerol in hypoxia/reoxygenation was determined in the previous experiment, we had not yet determined whether miR-210 mediated the effect of gingerol on neuronal damage induced by hypoxia/reoxygenation. Before hypoxia/reoxygenation and gingerol treatment, the miR-210 mimic was transfected into N2a cells to increase miR-210 expression (Figure 4A). Overexpressed miR-210 significantly reduced the increase in N2a cell viability observed in response to gingerol treatment (Figure 4B). Overexpression of miR-210 obviously reversed gingerol-inhibited apoptosis in N2a cells (Figure 4C). Furthermore, the miR-210 mimic reduced the gingerol-induced increase in Bcl-2 levels and reversed the gingerol-induced decrease in Bax and cleaved caspase 3 levels (Figure 4D). Overexpressed miR-210 suppressed the increased levels of BDNF and TrkB in response to gingerol treatment (Figure 4E). Therefore, gingerol exerted a neuroprotective effect by downregulating miR-210.

image

Gingerol exerted a neuroprotective effect by downregulating miR-210. Cells were subjected to 2 h of hypoxia before gingerol treatment and then exposed to 10 h of hypoxia and 4 h of reoxygenation. The expression of miR-210 was upregulated by the miR-210 mimic. Note: *p < 0.05, **p < 0.01, and ***p < 0.001. H, hypoxia. R, reoxygenation. 6-G, 6-gingerol. (A) The expression of miR-210 determined using qPCR. The miR-210 mimic reversed the gingerol-induced decrease in miR-210 expression. (B) Cell viability measured using the MTT assay. The miR-210 mimic reduced the gingerol-induced increase in the viability of N2a cells. (C) Apoptosis measured using flow cytometry. The miR-210 mimic reversed the gingerol-induced decrease in the apoptosis of N2a cells. (D) Protein levels of Bax, Bcl-2 and cleaved caspase 3 determined using western blotting. The miR-210 mimic reversed the gingerol-induced decreases in the levels of Bax and cleaved caspase 3 and the increase in the level of Bcl-2. (E) Protein levels of BDNF and TrkB measured using western blotting. The miR-210 mimic inhibited the gingerol-induced increases in the levels of BDNF and TrkB

3.5 Inhibition of miR-210 ameliorated neuronal damage induced by hypoxia/reoxygenation by increasing BDNF expression

We hypothesized that miR-210 might exert a detrimental effect on neurons by inhibiting BDNF expression. A BDNF siRNA was transfected into N2a cells to repress the expression of BDNF and confirm this hypothesis. The miR-210 inhibition-induced increase in cell viability was repressed after transfection with the BDNF siRNA (Figure 5A). The miR-210 inhibition-induced decrease in apoptosis was reversed by the BDNF siRNA (Figure 5B). Additionally, transfection of the BDNF siRNA obviously reversed the increase in Bcl-2 levels and the reductions in Bax and cleaved caspase 3 levels caused by miR-210 inhibition (Figure 5C). The BDNF siRNA reduced the significant increases in BDNF and TrkB levels induced by miR-210 inhibition. Collectively, these results implied that miR-210 inhibition ameliorated hypoxia/reoxygenation-induced neuronal damage by increasing BDNF expression.

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Silencing of miR-210 reversed neuronal damage induced by hypoxia-reoxygenation by increasing BDNF expression. The expression of BDNF and miR-210 was suppressed by the BDNF siRNA and miR-210 inhibitor, respectively. Cells were exposed to hypoxia/reoxygenation. Note: *p < 0.05 and **p < 0.01. H, hypoxia. R, reoxygenation. 6-G, 6-gingerol. (A) Cell viability measured using the MTT assay. BDNF silencing reversed the increase in the viability of N2a cells observed in response to miR-210 inhibition. (B) Apoptosis determined using flow cytometry. BDNF silencing reversed the decreased apoptosis of N2a cells observed in response to miR-210 inhibition. (C) Levels of the Bax, Bcl-2, and cleaved caspase 3 proteins determined using western blotting. BDNF silencing reversed the decreased levels of Bax and cleaved caspase 3 and the increased level of Bcl-2 observed in response to miR-210 inhibition. (D) Levels of the BDNF and TrkB proteins determined using western blotting. BDNF silencing suppressed the increased levels of BDNF and TrkB observed in response to miR-210 inhibition. (E) Graphical representation illustrated the main findings of the present work

4 DISCUSSION

Ischemia/reperfusion-induced neuronal damage is a complex process. Reoxygenation following hypoxia induces excess ROS production through the cooperation of xanthine oxidase, NADPH oxidase, mitochondria, and uncoupled nitric oxide synthase, leading to NLRP3-mediated inflammation and neuronal apoptosis, which are the causes of neuronal damage. Two investigations by Ryou et al.27 and Zhang et al.28 suggested that methylene blue and propofol exert protective effects on neuronal cells subjected to hypoxia/reoxygenation by increasing hypoxia-inducible factor-1α (HIF-1α)-involved energy metabolism and improving the mitochondrial redistribution, respectively, indicating that neuroprotective drugs are needed to prevent neuronal complications of reperfusion. Our investigation found that gingerol significantly ameliorated neuronal damage in response to hypoxia/reoxygenation by decreasing miR-210 expression and subsequently increasing the levels of BDNF and TrkB, which reduced caspase-3-mediated apoptosis and increased the viability of neuronal cells (Figure 5E).

Gingerol exerts a significant neuroprotective effect. A previous investigation29 showed that gingerol decreased astrocyte activation to ameliorate neuroinflammation and subsequent cognitive dysfunction. Gingerol increased the level of HIF-1α to repress prion protein-evoked neuronal apoptosis.30 Moreover, gingerol suppressed the accumulation of ROS and reactive nitrogen species to protect Aβ-stimulated SH-SY5Y cells from oxidative stress.31 Our investigation revealed that gingerol increased the viability and decreased apoptosis of hypoxia/reoxygenation-exposed N2a cells and induced proteolysis of caspase 3, a decrease in Bax levels and an increase in Bcl-2 levels. Based on these results, gingerol exerted a neuroprotective effect on cerebral ischemia–reperfusion injury. Gingerol has therapeutic value in myocardial ischemia–reperfusion injury by inhibiting inflammation, autophagy and apoptosis in cardiomyocytes.32-34 Nevertheless, the protective mechanism of gingerol in cerebral ischemia–reperfusion injury remains unclear. Two reports by Kang et al.9 and Liu et al.10 suggested that gingerol represses the elevated levels of inflammation, apoptosis and autophagy in neuronal cells induced by hypoxia/reoxygenation by targeting signaling pathways or miRNAs.

Gingerol regulates the expression of miRNAs to manipulate cellular processes involved in the development of disease. Importantly, our investigation showed that gingerol inhibited the increase in miR-210 expression observed in response to hypoxia/reoxygenation, indicating that miR-210 might be the mediator of the protective effect of gingerol on hypoxia/reoxygenation-stimulated neuronal cells. Two reports35, 36 indicate that the potential mechanism by which gingerol alters miR-210 expression is mediated by upstream factors such as lncRNAs or transcription factors, which should be investigated in future studies. miR-210 is upregulated in acute cerebral ischemia (ACI), indicating the potential role of this miRNA in ACI diagnosis, according to a previous investigation.37 As shown in the study by Ma et al.,38 the hypoxia-induced increase in miR-210 expression contributes to blood–brain barrier dysfunction. The expression of miR-210 was decreased by a miR-210 inhibitor to determine the role of miR-210 in cerebral ischemia–reperfusion injury. Downregulation of miR-210 reduced the increased apoptosis observed in response to hypoxia/reoxygenation; moreover, it reversed the reduction in cell viability evoked by hypoxia/reoxygenation. Based on our investigation, gingerol exerts a neuroprotective effect on cerebral ischemia–reperfusion injury by decreasing the expression of miR-210. Upregulation of miR-210 repressed the increased viability and decreased apoptosis of gingerol-treated neurons.

Interestingly, our investigation showed that gingerol reversed the decreases in the levels of BDNF and TrkB in response to hypoxia/reoxygenation. BDNF was negatively regulated by miR-210 in our study, indicating that the increased level of BDNF might be mediated by the gingerol-induced reduction in miR-210 expression to increase neuronal survival. BDNF is a critical contributor to neuronal processes and has emerged as a key therapeutic target in neurodegenerative diseases. Intravenous administration of BDNF contributes to the recovery of the neuronal system following focal cerebral ischemia. Moreover, the neuroprotective effect of BDNF has been reported to mediate the effectiveness of drugs, including dexmedetomidine and extract of yokukansan, in cerebral ischemia.39, 40 Our investigation identified BDNF as the target of miR-210. Notably, miR-210 inhibited BDNF expression via complementary binding to the 3′UTR of BDNF mRNA, reducing the activation of the BDNF/TrkB pathway and subsequent neuronal apoptosis. A BDNF siRNA was used to reverse the increased expression of BDNF observed in response to miR-210 downregulation. In contrast to the downregulation of miR-210, the reduction in BDNF expression decreased the levels of BDNF and TrkB, increased apoptosis and decreased viability. Thus, the downregulation of miR-210 increased the levels of BDNF and TrkB, resulting in the repression of neuronal apoptosis stimulated by hypoxia/reoxygenation.

5 CONCLUSION

Gingerol significantly attenuated neuronal damage in response to hypoxia/reoxygenation via the miR-210/BDNF axis. In our investigation, gingerol negatively regulated the expression of miR-210, leading to increased levels of BDNF and TrkB, which markedly reduced caspase 3-mediated apoptosis and increased cell viability to increase cell survival following hypoxia/reoxygenation. Gingerol chemically prevents neuronal damage stimulated by cerebral ischemia and reperfusion.

CONFLICT OF INTEREST

The authors have no conflicts of interest to declare.

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