1. Effect Of (–)-epigallocatechin-3-gallate (EGCG) On Amino Acid Transmitters Release After Oxygen-glucose Deprivation In Primary Cultured Cortical Neurons Of Rats 2. Electrophysiology Research Of L-type Cav1.2 Channel On HEK-293 Transfected Cell

First Part Effect of (–)-Epigallocatechin-3-gallate (EGCG) on Amino Acid Transmitters Release after Oxygen–glucose Deprivation in Primary Cultured Cortical Neurons of RatsObjective: To investigate the underlying protective mechanisms of EGCG in the cerebral ischemia injury in cultured cortical neurons. Methods: 1. EGCG(25μmol/L,50μmol/L and 100μmol/L) were added before 4 h oxygen-glucose deprivation (OGD) initiated. The effects of EGCG on cell viability of primary cultured rat cortical neurons exposed to 4 h oxygen-glucose deprivation were evaluated with lactate dehydrogenase (LDH) release and 3-[4,5-dimethylthia -2-yl]-2,5-diphenyl- tetrazolium bromide (MTT). 2. Furthermore, the concentration of aspartic (Asp), glutamate (Glu), glycine (Gly),Taurine (Tau) and gamma- aminobutyric acid (GABA) in the extracellular fluid from primary cultured rat cortical neurons were measured by high-performance liquid chromatographic(HPLC). Results: 1. Low concentration (25μmol/L and 50μmol/L) of EGCG showed protective effect to oxygen–glucose deprivation of cortical neurons while high concentration (100μmol/L) did not show such effect. 2. EGCG could regulate glutamate (Glu), glycine (Gly) and gamma- aminobutyric acid (GABA) level on both hippocampal neurons and cortical neurons suffered from oxygen–glucose deprivation. Conclusion: 1.EGCG could increase neuronal viability in vitro. 2. Furthermore, EGCG could attenuate extracellular Glu level and increase Gly andγ-GABA levels. These results indicated that the neuroprotective effects of EGCG were attributed to the regulation of excitatory and inhibitory amino acid transmitters'balance during the cerebral ischemia injury. Second Part Electrophysiology Research of L-type Cav1.2 Channel on HEK-293 Transfected CellObjective: To determine the difference between mutated and normal human embryonic kidney cells through electrophysiology on L-type Cav1.2 channel. Methods: 1. Transfected HEK-293 cell. 2. Electrophysiology: HEK-293 cells were depolarized from a holding potential (HP) of -80 mV for 400 ms to membrane potentials from -40 to +60mVin 10 mV increments for Ca_V1.2 current. Results: C-terminal domain of Ca_V1.2 channels at S1905 were deleted, which were defined as'STOP'mutation. Currents were elicited from -40 to +60mV for 400 ms to membrane potentialsin 10-mV increments for Ca_V1.2 current, which was activated at -20mV. The current amplitude of mutation group is decreased compared with Control group(P < 0.05),which is significantly different. The current amplitude of Control group was decreased, when added the drug of Isradipine (the blocker of Ca_V1.2 channels). This confirmed that the currents we recorded was Ca_V1.2 currents. Conclusion: The current amplitude of mutation group is significantly decreased compared with Control group(P < 0.05),which means the residues at S1905 on C-terminal domain of Ca_V1.2 channels is important for the Ca_V1.2 current amplitude.