Effects Of Baicalin On Oxygen-glucose Deprivation-induced Injury In Primary Cultured Rat Cortical Neurons

For investigating ischemic-like injury in vitro, oxygen-glucose deprivation (OGD) in primary culture of neurons is a widely used model that mimics the cerebral ischemic injuries in vivo. It is also a very useful method for evaluating neuroprotective effects of drugs on cerebral ischemia in vitro. In the studies using OGD model, no clear standards are available in methodology. Therefore, many different techniques have been developed and used in the experiments. Different methods of OGD vary in hypoxic condition, reperfusion medium and duration of OGD, especially in experimental solution that is quite important to affect the activity of neurons directly. Thus, we try to find out which experimental solution is the most suitable one in Part 1 of this study.5-Lipoxygenase (5-LOX) is a key enzyme metabolizing arachidonic acid to form inflammatory mediators, such as prostaglandins and leukotrienes. Leukotrienes mainly consist of leukotriene B4 (LTB4) and cysteinyl leukotrienes (CysLTs, including LTC4, LTD4 and LTE4). 5-LOX is involved in various diseases including central nervous diseases, such as cerebral ischemia, traumatic brain injury and tumors. Inhibiting the activation of 5-LOX or reducing the increased production of leukotrienes is beneficial for neuroprotection.Baicalin is an important flavonoid and possesses various pharmacological effects, such as free radical scavenging, antioxidant, anti-inflammatory, antiviral and vasodilatoryeffects. However, its neuroprotective mechanisms are not fully clarified yet, especially its effect on 5-LOX activation.Therefore, the major aim of this study was to observe the effects of baicalin on the ischemic-like neuronal injury and 5-LOX activation induced by OGD in rat primary cortical neurons in comparison with 5-LOX inhibitor caffeic acid.Part IEstablishment of oxygen-glucose deprivation-model in neuronsInfluence of experimental solutions with different composition and pH on oxygen-glucose deprivation-induced injury in primary cultured rat cortical neuronsAIM: To determine whether the composition and pH of experimental solutions alter the oxygen-glucose deprivation (OGD) insult and the neuroprotective effect of edaravone, a positive control agent;and to find out the most suitable experimental solution for OGD in rat cortical neurons. METHODS: In the cultured rat cortical neurons, injury was induced by OGD in different experimental solutions, and cell viability was evaluated by MTT and LDH assay. Edaravone was added to the neuron medium to observe its neuroprotection on OGD-induced injury in different experimental solutions. RESULTS: (1) MTT assay showed that OGD injured neurons are in a time-dependent manner;the injury became severer gradually with OGD treatment elongation. (2) After OGD for 3 h followed by reperfusion for 12 h, neuron insult was most severe in S4 (pH 7.8), and was mildest in S2 (pH 6.5). Meanwhile, neuron viability was decreased by 31% and 21%, LDH release increased by 66% and 65% in S3 (pH 7.4) and SI (pH 6.1) respectively. Edaravone (1 nmol/L) significantly protected neurons against OGD insult in the solutions with pH 6.1, 7.4 and 7.8, but not in S2 (pH 6.5), and showed its strongest protective effects in S3 (pH 7.4). (3) In S3 and S5, OGD for 3 h and reperfusion for 12 h significantly decreased neuron viability and increased LDH release. However, its effect was milder in S5 (containing 1.8 mmoI/L Mg2+). Edaravone (1 umol/L) protected neurons from OGD insult in S3 (containing 0.8 mmol/L Mg2+), but notin S5. (4) In S3 and S6, OGD for 3 h and reperfusion for 12 h significantly decreased neuron viability and increased LDH release. However, its effect was milder in S3 (without glycine). Edaravone (1 umol/L) protected neurons from OGD insult in S3 and S6 (containing 0.01 mmol/L glycine), but it was more effective in S3. (5) In S3 and S7, OGD for 3 h and reperfusion for 12 h significantly decreased neuron viability and increased LDH release. However, its effect was milder in S7 (containing 0.9 mmol/L Ca2+). Edaravone (1 ujnol/L) protected neurons from OGD insult in S3 (containing 1.8 mmol/L Ca2+), but not in S7. CONCLUSION: (1) The experimental solutions with different composition and pH can alter the OGD insult and the neuroprotection of positive control agent edaravone. (2) The most suitable experimental solution should be the Earle solution with pH 1.2-1 A for inducing OGD insult in rat cortical neurons. Thus, OGD injury model in neurons has been greatly improved in our experiment, especially improving the composition and pH of the experimental solutions, and this condition will more effectively mimic brain ischemic insult and evaluate drug effects in vitro.Part IIBaicalin attenuates oxygen-glucose deprivation-induced injury in ratcortical neurons and inhibites 5-lipoxygenase activationAIM: To determine whether baicalin protects rat cortical neurons from oxygen-glucose deprivation (OGD)-induced ischemic-like injury;if so, whether its effect is related to inhibition of 5-lipoxygenase (5-LOX) activation. METHODS: Neurons were exposed to OGD for 1.5 h followed by reperfusion for 24 h, baicalin, 5-LOX inhibitor caffeic acid and NMDA receptor antagonist MK-801 at various concentrations were added into the culture media 30 min before OGD, and maintained during OGD and1. Oxygen-glucose deprivation-induced neuron injury model have been largely improved, and the most suitable experimental solution is the Earle solution with pH 7.2-7.4.2. Baicalin concentration-dependently attenuates oxygen-glucose deprivation-induced injury in primary cultured rat cortical neurons.3. Baicalin effectively inhibits 5-LOX translocation and production of CysLTs induced by OGD, which is related to its neuroprotection.4. Baicalin significantly inhibits glutamate-increased intracellular calcium, which is similar to NMDA receptor antagonist MK-801 but distinct from caffeic acid, a direct inhibitor of 5-LOX. Thus, baicalin may inhibit 5-LOX activity via down-regulating NMDA receptor responses.