MATERIALS AND METHODS

Materials: Berberine chloride was obtained from Sigma Chemical Co. (St. Louis, MO, USA). The stock solution was prepared at 1 mmol L¯¹ in deionized water and kept at -20°C. Dulbecco’s Modified Eagle’s medium (DMEM) with glutamine, glucose/glutamine-free DMEM, Fetal Bovine Serum (FBS) were purchased from GIBCO. Penicillin-streptomycin, poly-L-lysine, Dizocilpine (MK-801), N^G nitro-L-arginine methyl ester (L-NAME) and modified Griess reagent were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals are of the analytical grade available from regular commercial sources.

Cell culture: OLN-93 cell line was purchased from Pasteur Institute of Iran (Tehran, Iran). The cells were grown in DMEM medium supplemented with 10% FBS and 100 IU mL¯¹ penicillin and100 μg mL¯¹ streptomycin in a humidified 95% air and 5% CO₂ incubator at 37°C. The cells were sub cultured twice a week by gentle scraping and were cultured on poly-L-lysine-coated 12-well plates at a density of 5<×10⁴ mg L¯¹. Culture dishes were coated with poly-L-lysine 24 h before the experiment. Poly-L-lysine (150 000-300 000 MW) was dissolved in deionized water and the plate wells were filled with this solution (20 μg mL¯¹). After 5 min standing at room temperature, the solution was aspirated and the plates were left to dry in a laminar flow hood overnight. Cells were used for experiments 24 h after seeding.

Oxygen-glucose deprivation and drug exposure: The Oxygen-Glucose Deprivation (OGD) condition was performed as described previously (Frantseva et al., 1999). Briefly, the culture medium was replaced with glucose/glutamine-free DMEM and cells were exposed to hypoxia for 30 min in a small anaerobic chamber filled with 95% (v/v) N₂ and 5% (v/v) CO₂ at 37°C. To terminate the oxygen-glucose deprivation, the chamber was opened and the medium was replaced with DMEM and the cultures were then placed in an incubator with 5% CO₂ for 24 h. To examine the drug effects, cell cultures were treated 3 h before oxygen-glucose deprivation with berberine (2 μM). This concentration was selected based on the previous study by our group and berberine presented the peak of protective effect at this concentration (Nadjafi et al., 2014). The pre-incubation time of 3 h was selected based upon previous studies (Rahbar-Roshandel et al., 2008). To investigate the effects of the inhibitors on oxygen-glucose deprivation-induced cell death, MK-801, a non-competitive antagonist of the NMDA receptor and L-NMAE, a NOS inhibitor, was added to the medium 3 h before oxygen-glucose deprivation (Tavakoli-Far et al., 2009). Plates not exposed to oxygen-glucose deprivation were used as external control and those exposed to oxygen-glucose deprivation conditions without the addition of any drug were used as internal control (OGD control). Each experiment was repeated at least three times, so every data point is therefore the mean of at least three measurements.

Measurement of nitrite: The level of nitrite as a measure of NO production in the culture medium was quantified using modified Griess reagent. In brief, the medium in each well was removed and centrifuged at 10,000 rpm for 10 min at 4°C.

Fig. 1:

Effect of berberine on Nitric Oxide (NO) production during ischemia/reperfusion-induced injury in OLN-93 oligodendrocytes. The effects of berberine (2 μM), MK-801 (10 μM) and L-NAME (100 μM) were evaluated on NO production during a 30 min Oxygen-Glucose Deprivation (OGD)/24 h reperfusion-induced cell injury in cultured OLN-93 oligodendrocytes. The plates not exposed to oxygen-glucose deprivation were considered as external control and those exposed to oxygen-glucose deprivation conditions without the addition of any drug were used as OGD control. The values are presented as Mean"SE compared with OGD control (*p<0.05; **p<0.01)

Then, the supernatant were mixed with an equal volume of modified Griess reagent at room temperature for 10 min and the absorbance was measured at 540 nm using an Ultrospec ^®3000 UV/Visible spectrophotometer (Pharmacia Biotech, Cambridge, England). The nitrite concentration was determined from a sodium nitrite standard curve.

Statistical analysis: All results are expressed as Mean±SE. The significance of differences was evaluated using Student’s t-test by Excel 2007 (Microsoft; Seattle, WA, USA) software. The p<0.05 was considered statistically significant.

RESULTS

Effects of berberine, L-NAME and MK-801 on nitric oxide production during 30 min oxygen-glucose deprivation/reperfusion in OLN-93 cells: NO production in the culture medium was measured by modified Griess reagent after 30 min oxygen-glucose deprivation/reperfusion. Exposure of OLN-93 cells to 30 min of oxygen-glucose deprivation/reperfusion significantly increased nitrite production compared with the external control (p<0.01). Pretreatment of cultures with berberine (2 μM), significantly decreased NO production, as compared with the oxygen-glucose deprivation control (p<0.05). The effects of L-NAME (100 μM), a NOS inhibitor and MK-801 (10 μM), a non-competitive antagonist of the NMDA receptor, on NO production were also evaluated and both compounds reduced the NO production, significantly (p<0.01; p<0.05, respectively) (Fig. 1).

DISCUSSION

Berberine, an isoquinoline alkaloid, presents anti-apoptotic effects on the neurons suffering ischemia in addition to its other pharmacologic properties (Cui et al., 2009; Zhang et al., 2012; Chai et al., 2014). The previous report showed that berebrine could present protective effects and attenuate the intracellular Ca²⁺ elevation during oxygen-glucose deprivation/reperfusion-induced injury in OLN-93 oligodendrocytes (Nadjafi et al., 2014).

The results of this study revealed that berberine could attenuate the NO production during a short-term ischemic/reoxygenation-induced injury in OLN-93 oligodendrocytes. Moreover, L-NAME, a NOS inhibitor and MK-801, the non-competitive NMDA receptor antagonist, decreased the NO production through same experiments.

Glutamate-mediated injury to oligodendrocytes contributes to mental or physical impairment in acute and chronic diseases as periventricular leukomalacia (prenatal white matter injury leading to cerebral palsy), spinal cord injury, multiple sclerosis and stroke (Karadottir et al., 2005; Matute et al., 2007). It is known that, oligodendrocytes express Ca²⁺-permeable ionotropic glutamate receptors including NMDA, AMPA and kainate receptors (Salter and Fern, 2005; Matute, 2006; Wang and Qin, 2010) whereas, AMPA/kainate receptors are expressed on somata and NMDA receptors on processes (Salter and Fern, 2005).

Some evidences demonstrated a direct link between ischemic damage to oligodendrocytes and excitotoxicity (Matute, 2010). Also, the intracellular Ca²⁺ overload, is the key mediator of the excitotoxic oligodendroglial injury (Matute et al., 2001, 2007; Matute, 2010; Wang and Qin, 2010). The subsequent excitotoxic oligodendroglial death due to overactivation of AMPA and kainate receptors had been demonstrated, earlier (Matute et al., 2001). Now it is well known that, the expressed NMDA receptors in the oligodendrocytes are also activated in ischemia (Karadottir et al., 2005).

In the case of excitotoxicity and/or ischemia, it is considered that there is a relation between intracellular Ca²⁺ overload and NO production (Szydlowska and Tymianski, 2010; Wang and Qin, 2010). Hereof, calcium ions can enter into the neuronal cell through various mechanisms; these include the over-activation of ionotropic glutamate receptors or of a range of channels and transporters (Szydlowska and Tymianski, 2010). Potentially toxic cytoplasmic calcium concentrations can also occur due to release from intracellular sources (Szydlowska and Tymianski, 2010). Such increases of cytoplasmic calcium concentrations can trigger a range of downstream neurotoxic events such as activation of nitric oxide synthase (Szydlowska and Tymianski, 2010). Notably, in neurons, NMDA receptor is linked to the neurotoxic molecules such as nNOS (Szydlowska and Tymianski, 2010). After ischemia, over-activation of NMDA receptor results in activation of nNOS leads to the production of NO and reactive oxygen species that can end to neuronal cell death (Szydlowska and Tymianski, 2010). Over all, through glutamate-induced neuronal death, NMDA receptor activation leads to increased cytosolic calcium and the intracellular Ca²⁺ elevation may stimulate nNOS production of NO and peroxynitrite which take part in the events that end to cell death (Brown, 2010; Szydlowska and Tymianski, 2010).

The results of present study showed the attenuation of NO production by MK-801, the non-competitive NMDA receptor antagonist and L-NAME, a NOS inhibitor, in OLN-93 oligodendrocytes during short-term ischemia/reperfusion. Thus, it revealed that NO production might contribute in ischemic-induced excitotoxicity in oligodendrocyte similar to neuron. On this subject, one study has also suggested the involvement of nitric oxide and excitotoxicity in hypoxic damage to the periventricular white matter in rat neonatal brain (Kaur et al., 2006).

Recently, neuroprotective effect of berberine against cerebral ischemia and reperfusion has been demonstrated by many studies (Cui et al., 2009; Hu et al., 2012; Chai et al., 2014). Briefly, the effects of berberine on this condition are due to its anti-oxidative and the anti-apoptotic effects (Hu et al., 2012; Chai et al., 2013). Also, berberine can be neuroprotective against high glucose-induced damage (Hsu et al., 2013). Interestingly, it seems that the facilitation of glucose metabolism by berberine may contribute to the neuroprotective effect of this alkaloid against in vitro ischemia-induced neuronal cell damage in mouse organotypic hippocampal slice cultures (Cui et al., 2009).

CONCLUSION

In conclusion, it seems that reduction of NO production may also be one of the mechanisms by which berberine protects the OLN-93 oligodendrocytes against ischemic/reperfusion-induced excitotoxic injury.