Effects and mechanism of dexmedetomidine on neuronal cell injury induced by hypoxia-ischemia

PC12 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in DMEM containing L-glutamine, 10% fetal bovine serum, 100 U/mL penicillin, 5% horse serum, and 100 μg/mL streptomycin (all from Invitrogen). Cell culture of PC12 cells was performed in humidified atmosphere of 5% CO₂ and 95% air at 37 °C. Primary hippocampal neuronal cells were isolated from neonatal Wistar rats (2–3 d old, male) following the methods described previously [19]. In brief, bilateral hippocampi were isolated carefully, followed by mechanical fragmentation and digestion with 0.25% trypsin at 37 °C for 20 min. After centrifugation at 200×g for 5 min, the isolated cells were collected and re-suspended in Dulbecco’s modified Eagle medium and Ham’s F-12 medium (DMEM/F12; Invitrogen, Burlington, ON, Canada) supplemented with 20% fetal bovine serum (Invitrogen). Then, the re-suspension containing neurons at a density of 1.0 × 10⁶ cells/mL was planted into a cell culture flask, which was pre-coated with 0.1 mg/mL polyL-lysine (Sigma-Aldrich, St. Louis, MO, USA), and these neurons were cultured at 37 °C in humidified air with 5% CO₂. The culture medium was exchanged with neurobasal medium supplemented with 2% B27, 10 mM glutamine and 1 mM sodium pyruvate. All the procedures were approved by the Ethical Committee of Qilu Hospital of Shandong University.

To establish OGD-induced hypoxic-ischemic model, the cells were cultured in the DMEM culture medium without glucose, and then incubated in an oxygen-free chamber with 95% N₂ and 5% CO₂ for 4 h at 37 °C. There were three different groups in the experiments: (1) control, (2) OGD exposure and (3) OGD exposure plus treatment with DMED. The control group was always maintained in normal DMEM and put in the incubator under normoxic conditions. To verify the possible involved signaling pathway, DAPT (Notch1 inhibitor, 10 μM; Sigma-Aldrich) and SN50 (NF-κB inhibitor, 25 μg/mL; Sigma-Aldrich) were respectively added into PC12 cells, which were treated with OGD and DMED, followed by measurement of cell viability, apoptosis and oxidative stress.

Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [20]. After a period of incubation, PC12 cells (100 μL), diluted to 1 × 10⁵ cells/mL, were seeded into each well of a 96-well micro-plate. After treatments, MTT solution was added to the cells with a final concentration of 1 mg/mL and the mixture was allowed to incubate at 37 °C for 4 h. Then the supernatant was removed by centrifuging, and the pellets were dissolved in dimethyl sulfoxide (DMSO). The absorbance was measured by spectrophotometry at 570 nm using an Elx-800uv reader (Bio-Tek Instruments, Winooski, VT, USA).

Cell apoptosis was assessed by Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining using an in situ cell death detection kit (KeyGEN, Nanjing, China). After treatment, PC12 cells were fixed by 4% paraformaldehyde at 4 °C for 30 min, followed by rinsing with phosphate-buffered saline (PBS). Then, PC12 cells were stained according to the manufacture’s information. The number of cells was counted under microscopic observation. Cell apoptosis was expressed as the ratio of the number of TUNEL-positive cells to the total number of cells, which were counted in five randomly chosen fields.

The total RNA of PC12 cells after treatment was isolated using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. RNA quality and quantity was measured using a nanodrop spectrophotometer (ND-1000, Nanodrop Technologies, Wilmington, DE, USA). RNA was reversely transcribed into cDNA using a Multiscribe RT kit (Applied Biosystems, Foster City, CA, USA) in accordance with the recommendation of supplier. Quantification of mRNA expression was performed with Fast SYBR Green (Applied Biosystems) following the supplier’s protocol. Primers were designed and synthesized by Sangon Biotech (Shanghai, China). On the basis of the method described previously [21], relative expression of genes were calculated, normalizing to GAPDH.

Oxidative stress-induced cell injury was evaluated according to the alterations of lactate dehydrogenase (LDH), malordiaolehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). The release of LDH into the medium was measured by LDH assay kit (Bio Vision Inc., Milpitas, CA, USA) at 440 nm. The percentage of LDH leakage was expressed as culture medium OD values / (culture medium OD values + cells homogenate OD values) × 100%. For assay of free radicals, the cells were washed with ice-cold PBS. The content of MDA was determined with a Cell MDA assay kit (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacture’s protocol. The absorbance of each sample was measured at 530 nm. SOD activity was measured by the xanthine-xanthine oxidase method. The supernatant was added to xanthine-xanthine oxidase reagent and the mixture was incubated for 40 min at 37 °C. SDS was added to stop the reaction, followed by measurement of absorbance at 550 nm. GSH-Px activity was measured with Glutathione Peroxidase Assay Kit (Jiang Lai biology, Shanghai, China) following the protocol of supplier.

Primary neuronal cells were divided into three groups, namely control, OGD and OGD + DMED groups. After treatments, culture media was collected for measurements of TNF-α and IL-6 using commercially available enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA). The concentration of Nox2 in primary neuronal cells was determined by using an ELISA kit purchased from MyBioSource (San Diego, CA, USA). CAT activity was assessed by using chemical assay kits (Jiancheng Bioengineering Institute).

The results in Fig. 1a showed that OGD treatment time-dependently decreased the viability of PC12 cells. Compared with control group, cell viability was significantly decreased at 3 h (p < 0.01), 6 h (p < 0.01) and 9 h (p < 0.05) after OGD treatment. Interestingly, the OGD-induced reduction of cell viability was significantly alleviated by DMED at 3 h (p < 0.01), 6 h (p < 0.05) and 9 h (p < 0.05) after OGD. Apoptosis is considered to play a critical role in brain ischemia injury [22]. TUNEL assay was used to analyze apoptosis in PC12 cells. As shown in Fig. 1b, OGD significantly promoted cell apoptosis compared to control group (p < 0.01), while DMED obviously alleviated the OGD-induced increase of TUNEL-positive cells in PC12 cells (p < 0.05). The qRT-PCR and western blot analysis were respectively used to detect the mRNA and protein levels of Bax and Bcl-2. The results showed that the expression of Bax was significantly increased and the expression of Bcl-2 was significantly decreased after the treatment of OGD at both mRNA and protein levels (p < 0.001). However the administration of DMED obviously alleviated the effect of OGD on PC12 cells compared with OGD group (p < 0.05 or p < 0.01; Fig. 1c-d).

Oxidative stress induced by OGD can lead to the death of PC12 cells [23], thus the related indicators of oxidative stress, including LDH, MDA, SOD and GSH-Px were all evaluated. As shown in Fig. 2a-b, OGD treatment significantly increased LDH leakage into the medium and the content of MDA compared with control group (p < 0.01 or p < 0.001), while DMED markedly attenuated the increases compared with OGD group (p < 0.05 or p < 0.01). Conversely, the activity of SOD and GSH-Px were both reduced by OGD compared with control group (p < 0.05 or p < 0.01), whereas these reductions were ameliorated by DMED compared with OGD group (p < 0.05, Fig. 2c-d).

The expressions of BDNF, NGF, and Nestin were analyzed by qRT-PCR and western blot analysis. The results showed that OGD treatment significantly decreased the mRNA levels of BDNF, NGF and Nestin in PC12 cells compared with control group (p < 0.01). However, DMED alleviated the OGD-induced decreases of BDNF, NGF and Nestin levels compared with OGD group (p < 0.05), indicating that DMED promoted the repair of neuronal impairment induced by OGD (Fig. 3a-c). Results in Fig. 3d also proved the protective effect of DMED on neuronal impairment.

The expressions of key kinases involved in Notch and NF-κB signaling pathways were analyzed by qRT-PCR and western blot analysis. The results showed that OGD treatment significantly increased the mRNA and protein levels of Notch1 and NICD compared with control group (p < 0.001), but the OGD-induced up-regulations of Notch1 and NICD were alleviated by DMED (p < 0.05 or p < 0.01), indicating that DMED inhibited the OGD-induced activation of Notch pathway (Fig. 4a-d). Similarly, the marked up-regulations of NF-κB and IκBα at mRNA and protein levels after treatment of OGD (p < 0.01 or p < 0.001) were both significantly alleviated by DMED treatment (p < 0.05 or p < 0.01), indicating that DMED inhibited the OGD-induced activation of NF-κB pathway (Fig. 4e-h).

The effects of DMED on OGD-induced cell injury were assessed again with or without the presence of Notch inhibitor (DAPT) or NF-κB inhibitor (SN50). Results in Fig. 5a and e-f showed cell viability at 6 h after OGD as well as activities of SOD and GSH-Px was enhanced by treatment of DAPT or SN50 compared with OGD + DMED group (p < 0.05). Cell apoptosis as well as levels of LDH and MDA was markedly reduced by treatment of DAPT or SN50 compared with OGD + DMED group (p < 0.05, Fig. 5b-d). Results indicated that inhibition of Notch or NF-κB pathway could enhanced the protective effects of DMED on OGD-induced cell injury in PC12 cells.

The protective effects of DMED on OGD-induced cell injury were verified in primary neuronal cells. The cell injury was evaluated according to the alterations of IL-6, TNF-α, Nox2 and CAT. Results in Fig. 6a-c showed levels of IL-6, TNF-α and Nox2 were elevated by OGD (p < 0.01 or p < 0.001), whereas the increases were alleviated by DMED (p < 0.05 or p < 0.01). Conversely, OGD decreased activity of CAT (p < 0.01) but the decrease was alleviated by DMED (p < 0.05).