Dexmedetomidine provides protection to neurons against OGD/R-induced oxidative stress and neuronal apoptosis

Abstract Dexmedetomidine, a potent α2-adrenoceptor (α2-AR) agonist, is extensively used in the operating room (OR) and intensive care unit (ICU) and has been applied for the treatment of several diseases. Western blotting has been routinely used to investigate the protein levels of α-adrenergic receptor (α-AR), apoptosis related proteins (Bcl-2, Bax and Cleaved Caspase 3) and a range of proteins associated with the Nrf2/ARE pathway (Nrf2, HO-1, NQO-1, SOD) in neurons. The CCK-8 assay was used to determine cell survival rates while the Co-IP assay was used to investigate the binding ability between α2-AR and Nrf2. The TUNEL assay was used to detect cell apoptosis in neurons. OGD/R treatment reduced the level of α2-AR protein in neurons and reduced neuronal survival in a time-dependent manner. However, treatment with dexmedetomidine led to the increased protein expression of α2-AR in OGD/R-treated neurons and enhanced survival rate of OGD/R-treated neurons. From a mechanistic point-of-view, Nrf2 can effectively bind with α2-AR. Silencing Nrf2 reversed the effects of dexmedetomidine on cell viability, oxidative stress, and neuronal apoptosis in OGD/R-treated neurons. The activation of α2-AR by dexmedetomidine had a protective effect in neurons against OGD/R-triggered oxidative stress and neuronal apoptosis by modulating the Nrf2/ARE pathway.


Introduction
Ischemic stroke is a type of neurological disorder that is caused by an insufficiency in oxygen supply and blood flow restoration, frequently resulting in permanent disability (Murphy and Corbett 2009;Sun et al. 2018). Following neuronal injury, post-cerebral ischemia can be a serious problem. Over recent years, there have been some advances in the treatment of ischemia and evaluating the progression of patients affected by post-cerebral ischemia. Studies have revealed that a range of mechanisms are involved in postcerebral ischemia, including oxidative stress, neuronal apoptosis and excitatory toxicity; collectively, these processes can lead to irreversible brain damage (George and Steinberg 2015). Although strategies for reducing neuronal injury have been investigated, at least to some extent, the prognosis of patients affected by this condition is still not satisfactory. Therefore, it is vital to investigate the pathological process underlying neuronal injury so that we may identify new treatment strategies for ischemic stroke.
Dexmedetomidine is a potent a2-adrenoceptor (a2-AR) agonist that has been extensively applied in the intensive care unit (ICU) and operating room (OR) because it is more efficient than most other sedatives or anesthetics (Gurbet et al. 2006;Lam and Alexander 2008). Several lines of evidence have shown that dexmedetomidine is able to play a pivotal part in the treatment of several diseases. For example, dexmedetomidine is known to inhibit inflammation in lipopolysaccharide-induced endotoxemia by regulating the cholinergic anti-inflammatory pathway (Xiang et al. 2014). Furthermore, dexmedetomidine has been shown to suppress inflammation following renal ischemia reperfusion injury (Gu et al. 2011). Moreover, dexmedetomidine regulates BDNF signaling to alleviate the apoptosis of neurons in kainic acidinduced excitotoxicity (Chiu et al. 2019). Other research has shown that dexmedetomidine attenuates the apoptosis of neurons following cerebral ischemia-reperfusion injury (Zhai et al. 2019). Nonetheless, the exact function of dexmedetomidine in glucose deprivation/reoxygenation (OGD/R)-treated neurons and its related mechanisms, have yet to be investigated.
NF-E2-related factor 2 (Nrf2) is a pivotal modulator of antioxidants and can balance oxygen free radicals and inflammation within cells (Sandberg et al. 2014). Under conditions involving inflammation and oxidative stress, the levels of reactive oxygen species (ROS) begin to increase. In turn, this causes increased oxidation of Keap1, thus leading to the release of Nrf2. Following translocation to the nucleus, Nrf2 is capable of binding to the antioxidant response element (ARE) in order to activate the expression of antioxidant genes, including superoxide dismutase (SOD), heme oxygenase-1 (HO-1), and NAD(P)H: quinone oxidoreductase-1 (NQO-1), so as to eliminate excessive ROS (Xue et al. 2016;Wasik et al. 2017). As oxidative stress can be induced by OGD/R treatment, and can therefore result in neuronal injury, studies have begun to focus on biological molecules that can reduce oxidative stress in OGD/R-treated neurons. Interestingly, a significant body of evidence now indicates that the Nrf2/ARE signaling pathway is a vital regulator of oxidative stress in a variety of diseases. For instance, the upregulation of CKIP-1 attenuates high-glucose triggered oxidative stress in human retinal endothelial cells by regulating the Nrf2/ARE signaling pathway . Another study demonstrated that the activation of the Nrf2/ARE pathway suppressed cognitive deficits in a mouse model of Alzheimer's disease by modulating oxidative stress (Tian et al. 2019). Other research has indicated that the Nrf2/ARE pathway plays a pivotal role in Parkinson's disease and could therefore represent a promising target for the development of new therapeutic options for Parkinson's disease (Gureev and Popov 2019). However, the regulatory mechanisms associated with Nrf2/ARE pathway activation in OGD/R-treated neurons has not been investigated specifically.
In the present study, we confirmed that dexmedetomidine exerted a protective protect in neurons against OGD/R-triggered oxidative stress and neuronal apoptosis by modulating the Nrf2/ARE pathway. The findings of our study may provide novel insight with which to facilitate the development of new therapies for the reduction of oxidative stress after ischemic stroke.

Cell culture and transfection
The SH-SY5Y cell line (EY-X0726) was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and then cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. Cells were grown at 37 C in 95% air and 5% CO 2 . Transfection was carried out by transfecting neuronal cells with siRNA designed against Nrf2 (si-Nrf2). Transfection was carried out by using Lipofectamine2000 (Thermo Fisher Scientific, Waltham, MA, USA). The primer sequences were as follows, si-Nrf2-forward-

Construction of a model for OGD/R
The OGD/R model was constructed in accordance with previous studies (Agrawal et al. 2013). SH-SY5Y cells (5 Â 10 5 cells/ ml) were then incubated at 37 C in 95% air and 5% CO 2 for 24 h. Thereafter, cells were maintained in glucose-free DMEM under hypoxic conditions (3% O 2 , 5% CO 2 and 92% N 2 ) for 8 h. Thereafter, the medium was replaced with fresh medium containing glucose (20%). Cells were then cultured for 24 h under normal conditions. Other neurons were cultured in normal media and under normal conditions to form the control group.

The cell counting kit-8 (CCK-8) assay
Cell survival was evaluated at 24 h post-OGD/R induction by performing a CCK-8 assay. In brief, 1 Â 10 4 cells were seeded into 96-well plates and cultured overnight at 37 C. Then, 10 ml of CCK-8 solution (Beyotime Biotechnology) was added into each well and cultured at 37 C for another 2 h. Finally, cell survival was assessed by detecting the absorbance at 450 nm with a microplate reader (BMG Labtech GmbH).

Co-immunoprecipitation (Co-IP)
Co-IP assays were conducted by using a method that was described previously (Liu et al. 2014). In order to perform Co-IP for a2-AR and/or Nrf2 proteins, we used anti-a2-AR and anti-Nrf2 agarose beads to pull down a2-AR and Nrf2, respectively. Rabbit purified IgGs were used as negative controls. The supernatants were transferred to new microcentrifuge tubes for western blotting analysis.

The detection of MDA, SOD and GSH-PX
Specific Activity Assay Kits (Cayman Chemical, USA) were used to determine the levels of MDA, SOD and GSH-PX in neurons. When the assays were complete, the absorbance at 450 nm was read via a spectrophotometric assay and a microplate reader (Syntron, USA).

Immunofluorescence assay
Cells were fixed with 4% paraformaldehyde and then permeabilized by Triton X-100 dissolved in PBS. After blocking, the cells were incubated overnight with anti-MAP2 antibody (ab32454, Abcam) at 4 C. Next, the cells were incubated with secondary antibodies. Cell nuclei were then counterstained by using DAPI and the samples were examined by using a fluorescence microscope.

ROS detection
ROS generation was detected by using a commercial ROS assay kit (Beyotime Reagent Co., China). In brief, 10 lM of DCFH-DA was mixed with the cells and incubated. After washing, the fluorescence intensity of DCFH-DA was observed under a laser scanning confocal microscope (LSCM) at 488 nm excitation and 525 nm emission wavelengths. ROS levels were then analyzed by Image-Pro Plus 6.0 software (Media cybernetics, USA).

TdT-mediated dUTP Nick-End labeling (TUNEL) staining
Apoptosis was detected in SH-SY5Y cells by an In Situ Cell Death Detection Kit (TUNEL fluorescence FITC kit, Roche). Cells were grown in 24-well culture plates. The cells were then fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 5 min on ice. Next, 50 ll of the TUNEL solution was added to the samples and cultured for 1 h at 37 C. The nuclei were then stained with DAPI. TUNEL staining was then visualized by fluorescence microscopy (Leica, Germany).

Statistical analysis
All data analysis was conducted by SPSS version 20.0 (Chicago, IL, USA) and all experiments were carried out in triplicate. Data are shown as mean ± standard deviation (SD). For comparisons between two independent groups, the Student's t-test was applied. When comparing differences among three or more groups, the one-way analysis of variance (ANOVA) was applied. Statistical significance was set to p < 0.05.

The identification of neurons
First, we used electron microscopy to identify specific neuronal morphological characteristics (Figure 1(A)). In addition, we used immunofluorescence assays to detect MAP2, an established neuronal marker (Figure 1(B)).

Dexmedetomidine promotes the cell viability of OGD/Rtreated neurons
Dexmedetomidine was previously identified to play an essential role in the treatment of several diseases. We attempted to ascertain whether dexmedetomidine could help protect neurons against oxidative damage. First, we investigated the expression of a2-AR in OGD/R-treated neurons at different time points. Western blotting demonstrated that the levels of a2-AR protein decreased significantly in response to OGD/ R treatment in a time-dependent manner (@p < 0.05 vs. control; Figure 2(A)). CCK-8 assays further showed that cell survival was also decreased significantly following OGD/R treatment (@p < 0.05 vs. control; Figure 2(B)). ROS generation was enhanced following OGD/R treatment (@p < 0.05 vs. control; Supplementary Figure 1S). Next, we used different concentrations of dexmedetomidine to treat the OGD/R-treated cells and discovered that dexmedetomidine led to a significant increase in the expression of a2-AR protein in OGD/R-treated neurons at different time points (@p < 0.05 vs. 0 mg/ml; Figure 2(C)). In addition, the survival rate of OGD/R-treated cells was also increased significantly following treatment with dexmedetomidine (@p < 0.05 vs. 0 mg/ml; Figure 2(D)). Thus, dexmedetomidine treatment promoted the cell viability of OGD/R-treated neurons.
Dexmedetomidine inhibits OGD/R-induced oxidative stress and neuronal apoptosis in OGD/Rtreated neurons Next, we investigated whether dexmedetomidine exerted an effect on oxidative stress and neuronal apoptosis in OGD/Rtreated neurons. First, the levels of MDA, SOD and GSH-PX were determined and found that OGD/R-treatment significantly increased the levels of MDA but decreased the levels of SOD and GSH-PX; however, these effects were abrogated by dexmedetomidine treatment (@p < 0.05 vs. control; # p < 0.05 vs. OGD/R þ saline; Figure 3(A), thus indicating that dexmedetomidine inhibited OGD/R-induced oxidative stress. TUNEL assays further revealed that dexmedetomidine treatment reversed the enhanced cell apoptosis mediated by OGD/R (@p < 0.05 vs. control; #p < 0.05 vs. OGD/R þ saline; Figure 3(B). In addition, we examined the levels of several proteins associated with apoptosis (Bcl-2, Bax and Cleaved Caspase-3) by western blotting. Analysis showed that OGD/R triggered the down-regulation of Bcl-2 and the upregulation of Bax and Cleaved Caspase-3. However, dexmedetomidine treatment abolished these effects (@p < 0.05 vs. control; #p < 0.05 vs. OGD/R þ saline; Figure 3(C). OGD/R-treatment markedly increased the generation of ROS; however, these effects were abrogated by dexmedetomidine treatment (@p < 0.05 vs. control; #p < 0.05 vs. OGD/R þ saline; Supplementary Figure 2S). Thus, dexmedetomidine inhibited OGD/R-triggered oxidative stress and neuronal apoptosis in OGD/R-treated neurons.

Dexmedetomidine modulates the Nrf2/ARE pathway in OGD/R-treated neurons
Next, we investigated the mechanisms underlying the effects of dexmedetomidine on OGD/R-treated neurons. The Nrf2/ ARE pathway, a pathway shown to play a key role in the inhibition of oxidative stress, was previously implicated with numerous diseases, including OGD/R-induced neuronal injury. We attempted to ascertain whether dexmedetomidine could affect the Nrf2/ARE pathway in OGD/R-induced neuronal injury. The CCK-8 assay showed that treatment with Lir (an activator of the Nrf2/ARE pathway) could reverse the poor cell viability of OGD/R-treated neurons (@p < 0.05 vs. control; #p < 0.05 vs. OGD/R þ DMSO; Figure 4(A), implying that the Nrf2/ARE pathway might play a role in OGD/Rinduced neuronal injury. Furthermore, the Co-IP assay revealed that a2-AR was able to bind with Nrf2 (Figure 4(B). Next, we investigated the levels of a2-AR protein and a range of proteins associated with the Nrf2/ARE pathway by western blotting. We found that dexmedetomidine treatment significantly increased the protein levels of a2-AR, n-Nrf2, HO-1, NQO-1 and SOD but reduced the protein level of c-Nrf2 in OGD/R-treated neurons (@p < 0.05 vs. control; #p < 0.05 vs. OGD/R þ saline; Figure 4(C), illustrating that dexmedetomidine treatment can activate the Nrf2/ARE pathway. Consequently, these results revealed that dexmedetomidine can modulate the Nrf2/ARE pathway in OGD/Rtreated neurons.
Nrf2 silencing reverses the influence of dexmedetomidine on cell viability, oxidative stress and neuronal apoptosis in OGD/R-treated neurons Rescue assays were carried out to ensure that dexmedetomidine was able to modulate cell viability, oxidative stress and neuronal apoptosis in OGD/R-treated neurons by activating the Nrf2/ARE pathway. First, we knocked down Nrf2 in neurons; the protein levels of Nrf2 were significantly reduced following the transfection of neurons with the si-Nrf2 vectors ( Ã p < 0.05 vs. NC; Figure 5(A). Furthermore, the protein levels of HO-1, NQO-1 and SOD were increased following treatment with dexmedetomidine but decreased in line with Nrf2 depletion. We also found that Nrf2 deficiency reversed the effects of dexmedetomidine treatment and caused an upregulation in the protein levels of HO-1, NQO-1 and SOD (@p < 0.05 vs. OGD/R þ saline þ NC; #p < 0.05 vs. OGD/ R þ Dex þ NC; $p < 0.05 vs. OGD/R þ saline þ si-Nrf2; Figure  5(B). In addition, cell survival was increased in response to dexmedetomidine treatment but decreased when Nrf2 was downregulated. The downregulation of Nrf2 was able to counteract the effects of the dexmedetomidine treatmentinduced enhancement of cell survival (@p < 0.05 vs. Figure 3. Dexmedetomidine inhibits OGD/R-triggered oxidative stress and neuronal apoptosis in OGD/R-treated neurons. (A) OGD/R treatment upregulated the levels of MDA but downregulated the levels of SOD and GSH-PX in neurons, but these effects were abrogated by dexmedetomidine treatment. (B) TUNEL assays revealed that dexmedetomidine treatment reversed cellular apoptosis induced by OGD/R treatment. (C) Western blotting showed that OGD/R triggered a downregulation in Bcl-2 protein levels but upregulated the levels of Bax and Cleaved Caspase-3. Dexmedetomidine treatment abolished these effects. @p < 0.05 vs. control; #p < 0.05 vs. OGD/R þ saline.

Discussion
Ischemic stroke is a terrible disease that affects millions of people across the world. A large body of evidence now indicates that the pathophysiology of neuronal injury following ischemic stroke is associated with increased levels of cell apoptosis (Min et al. 2017). Consequently, it is very important to investigate mechanisms that may be used to inhibit apoptosis in neurons. Previous studies demonstrated that oxidative stress can be induced by OGD/R treatment and that this can subsequently result in neuronal injury (Liu et al. 2016); consequently, a range of studies are focusing on identifying biomarkers that might be able to reduce oxidative stress in OGD/R-treated neurons. In the present study, the cell model of OGD/R was constructed to help us to investigate potential biomarkers associated with neuronal injury. This model demonstrated that cell survival was decreased significantly as a result of OGD/R treatment in a time-dependent manner, thus suggesting that the OGD/R cell model had been successfully established and could be used for further studies.
Dexmedetomidine is widely used in the operating room and ICU and plays a vital role in the treatment of several diseases, including acute lung injury after kidney ischemia reperfusion injury (Gu et al. 2011), kainic acid-induced excitotoxicity (Chiu et al. 2019), and cerebral ischemia-reperfusion injury (Zhai et al. 2019). However, the exact effect of dexmedetomidine on OGD/R-treated neurons, and the mechanisms involved, had not been investigated prior to the present study. In this study, we discovered that the levels of a2-AR protein were decreased in response to OGD/R treatment in a time-dependent manner, implying that dexmedetomidine might play an essential role in neuronal injury. In addition, dexmedetomidine promoted the viability of OGD/R-treated neurons. More importantly, dexmedetomidine was confirmed to inhibit OGD/R-induced oxidative stress and apoptosis in OGD/R-treated neurons. Taken together, these data indicated that dexmedetomidine is able to protect neurons against OGD/R-triggered oxidative stress and neuronal apoptosis.
We further investigated the mechanisms that might be involved with these observations. Numerous previous studies have reported that the Nrf2/ARE signaling pathway is a vital regulator of oxidative stress in a range of diseases, including high-glucose triggered oxidative stress in human retinal endothelial cells ) and Alzheimer's disease (Tian et al. 2019). The Nrf2/ARE pathway is also known to play a pivotal role in Parkinson disease (Gureev and Popov 2019). However, the exact mechanisms underlying how dexmedetomidine and the Nrf2/ARE pathway affect OGD/Rtreated neurons have not been fully explored previously. In the present study, we found that Liraglutide (Lir), an activator of the Nrf2/ARE pathway, could improve cell survival after OGD/R treatment. Moreover, we showed that Nrf2 was able to bind with a2-AR. We also showed that dexmedetomidine was able to modulate the Nrf2/ARE pathway in OGD/Rinduced neurons. Finally, we showed that Nrf2 depletion reversed dexmedetomidine-mediated cell viability, oxidative stress, and neuronal apoptosis in OGD/R-treated neurons.
However, some deficiencies still existed in our study. We only investigated the protective effects of dexmedetomidine on OGD/R-triggered oxidative stress and neuronal apoptosis, further research on other aspects (such as macrophage polarization, autophagy and exosomes) is still needed. In the future, more experiments were done to further explore dexmedetomidine in neuronal injury. Figure 6. A schematic of the proposed mechanism in our work. The current research suggests that the activation of a2-AR by dexmedetomidine had a protective effect in neurons against OGD/R-triggered oxidative stress and neuronal apoptosis by modulating the Nrf2/ARE pathway.

Conclusions
In summary, this study proved that the activation of a2-AR by dexmedetomidine exerted a protective effect on neurons against OGD/R-triggered oxidative stress and neuronal apoptosis by modulating the Nrf2/ARE pathway. Our findings may therefore help to find ways to reduce the effects of neuronal injury induced by oxidative stress.