Protective Effects Of Phenolic Compounds Extracted From Gastrodia Elata Blume On The Primary Cortical Neurons After OGD/R Injury

Ischemic cerebral vascular disease(ICVD) is one of the diseases with high mortality and disability rate. Ischemia-reperfusion injury is the main pathological process of ICVD and is often followed by the ischemic central necrosis and the ischemic penumbra neuronal cells apoptosis. Therefore, seeking drugs which can prevent the apoptosis of nerve cells will be a positive effect on the repairment of the ischemia or reperfusion injury. Studies have shown that the recovery of neurological function is limited after cerebral ischemia and the sprouts of neuronal axon and synaptic plasticity occur after the ischemia injury. Promoting axonal sprouting and synaptogenesis will be helpful to the recovery of neural function. Therefore, inhibiting apoptosis of nerve cells and promoting synaptogenesis are of great significance to the treatment and prognosis of cerebral ischemia reperfusion injury. Based on previous studies, we reproduced the model of OGD/R with rat primary cortical neurons. Concerning the neuron mitochondrial apoptotic pathway and synaptic plasticity, we discussed the roles of the effective components C and D, which were extracted from Gastrodia elata with the ability to pass through the blood-brain barrier, in the protection of neuron from the cerebral ischemia reperfusion injury. This study provides a theoretical basis for the clinical treatment of ischemic cerebrovascular disease with Gastrodia elata. 1. Effects of components C, D from Gastrodia elata on the growth of primary cortical neurons.Objective: To primarily culure the rat cortical neurons and to identify the effects of components C, D on the growth of cortical neurons.Methods:1.1 Primary culture and identification of the rat cortical neurons with NSE and MAP-2 immunofluorescence.1.2 Primary cortical neurons were treated with different concentrations of components C, D on the 7th day of culture. After 24 h, the survival rate of neurons was measured by performing the MTT method.Results:1.1 The purity of the primarily cultured cortical neurons reached more than 90% by performing NSE and MAP-2 immunofluorescence identification. Thereforethe cells can be used in the following experimental studies.1.2 Phenolic compound C had no effect on the growth of neurons when the concentrations were lower than 100μg/m L(P>0.05), and the cell survival rate decreased significantly when the concentration reached 200μg/m L(P<0.05); Phenolic compound D had no effect on the neuronal growth at the concentration of 50μg/m L(P>0.05), and the cell survival rate decreased significantly when the concentration reached 100μg/m L(P<0.05).Conclusion: The purity of primary cortical neurons reached 90% by NSE and MAP-2 identification. Phenolic compound C had no effect on the growth of neurons at concentration of 100μg/m L(P>0.05). Phenolic compound D had no effect on the growth of neurons at the concentration of 50μg/m L(P>0.05). 2. Protective of phenolic compounds C and D on the OGD/R injury in cortical neurons.Objective: To investigate the protection effects of phenolic compounds C and D by using primary cortical neurons OGD/R model.Methods:2.1The model of primary cortical neurons OGD/R injury Primary cortical neurons were cultured for 7d. Treated with different concentrations of sugar-free Na2S2O4 Earle ’s for 1h, 3h and 4h and followed by cultures for 1h with normal neurons culture medium, the survival rate of neurons was measured by performing MTT assay.2.2 The primary cortical neurons were treated with different concentrations of phenolic components C and D for 24 h after cultured for 7d to build the OGD/R injury model. The survival rate of neurons was measured by performing MTT assay.2.3 The cell culture medium was collected for detecting the NO concentration and SOD activity. Cell lysate was collected for measuring the MDA concentration and the leakage rate of LDH.Results:2.1 The model was established successfully under the condition of Na2S2O4 at a concentration of 40mmol/L with the oxygen glucose deprivation for 1h, reperfusion injury for 1h and the survival rate of cells of 50%~60%.2.2 The phenolic component C at the concentration of 1μg/mL~12.5μg/m L and the component D at the concentration of 0.05μg/mL~1.6μg/m L significantly increased the survival rate of OGD/R injury model(P<0.05).2.3 Compared with the control group, phenolic component C(3.125μg/mL~25μg/m L) and D(0.2μg/m L~1.6μg/m L) reduced the release of NO and MDA formation but increased the intracellular SOD activity in OGD /Rep model(P<0.05). In addition, phenolic component C showed no inhibition effect on leakage rate of LDH.Conclusion: The phenolic components C and D reduced the morphology damage and decreased the releasing of NO and the concentration of MDA but increased cell survival rate and SOD activity in primary cortical neurons OGD/R model. 3. The effects of compounds C and D on the apoptosis protein expressions in primary cortical neurons.Objective: To investigate whether phenolic compounds C and D have the anti-apoptosis effects concerning the neuron mitochondrial apoptotic pathways.Methods: The effects of phenolic components C and D on the protein expressions of Bax, Bcl-2 and Caspase-3 were tested by using western blot analysis.Results: Phenolic compound C(3.125μg/m L~12.5μg/m L), compound D(0.2μg/m L~1.6μg/m L)(P<0.05) inhibited the protein expressions of Bax and caspase-3but up-regulated the protein expression of Bcl-2and the Bcl-2/Bax ratio.Conclusion: Phenolic components C and D inhibit the protein expressions of Bax and caspase-3but up-regulate the protein expression of Bcl-2. 4. Effects of compounds C and D on the synaptic plasticity in primary cortical neurons.Objective: To explore the effects of compounds of C and D on the synaptic plasticity in the primary cortical neuron.Methods: After building the primary cortical neurons OGD/R injury model, the synaptic length was measured by software of Image J. The effect of compounds C and D on the protein expressions of GAP-43 and SYP by Using Western blot to test theResults: Compound C(3.125μg/mL~25μg/m L) and compound D(0.2μg/m~1.6μg/m L) significantly promoted the development of synapse with up-regulating the expression of GAP-43 and SYP(P<0.05).Conclusion: Phenolic components C and D can reduce the damage in synaptic structure by promoting the expressions of protein GAP-43 and SYP.