| The Puerarin is a Chinese herbal medicine of common used in clinic. As improving the cerebral and heart circulating , it extensively use in treating myocardial infarction and stroke. In recently, More and more research on the protective mechanism of Puerarin on cerebral ischemia reperfusion injury .It' s mechanism for preventing cerebral ischemia reperfusion maybe includes removing oxygen free redicals, effecting iNOS of brain, lessening activities of cortical calcineurin and calpain, increasing the expression of Heat Shock Protein 70(HSP 70), et al. In this research we are trying to study the Puerarin' s mechanism of preventing ischemic brain damage from ion channels by patch clamp technique.1. Effects of Puerarin on L-type Calcium Channel in CAl Pyramidal Neurons in Hippocampus of Adult RatsObjective: To observe the effect of Puerarin on L -type calcium channel in CAl pyramidal neurons in hippocampus of adult rats after giving several concentrations.Method: The pyramidal neurons in hippocampal CAl region were dissociated acutely from SD adult rats by the enzymatical and automatical way. The cell suspension was then placed into a 35mm petri dish .After allowing the cell to settle , selecting the cell that accord with our experimental conditions. Then recording the ion channel activity at the same holding voltage by the cell-attached of patch clamp techniques.Result: The mean unitary slop conductance for L -type calcium channel in CAl pyramidal neurons is 26pS, and single channel current is about 1. 5pA. No significant effect were seen in L -type calcium channel in CAl pyramidal neurons with the low concentrations of Puerarin (1.2, 2. 4ramol ? L ' ) . At a given holding voltage , significant change was observed in the open time of the L-type calcium channel after infusing Puerarin (4. 8, 9. 6, 19. 2mmol ? L"1 ). Open time was 12. 332 + 0. 567ms for control , 7.963 + 0. 166ms for concentration of 4.8mmol ? L"'( R0. 05, n=5 ), and 4. 523±0. 128ms ( 9. 6mmol ? L~\ R0. 05 vs control ) , then 3. 274±0. 217ms ( 19. 2mmol ? L'\ R0. 05 vs control ). The open probability was 0.0010400 + 0.0002408 in control , decrease to 0.0005620 + 0.0000259 ( 4.8mmol ? L'1, R0.05 vs control ), and 0.0003460 + 0.0000321 ( 9. 6mmol ? L~l, R0. 05 vs control ), then went down 0. 0002520±0. 0000295 ( 19. 2mmol ? L'1, R0. 05 vs control ).Conclution: Puerarin can inhibit L -type calcium in CAl pyramidal neurons, decrease the open time , reduce the open probability and depend on the concentrations.2. Effects of Cerebral Ischemia Reperfusion Injuty on L-type Calcium Channel in CAl Pyramidal Neurons in Hippocampus of Adult RatsObjective: To observe the change of L-type calcium channel in CAl Pyramidal Neurons in Hippocampus of adult rats in different time points after cerebral ischemia reperfusion injuty.Method: The experiment took the modified Pulsinelli' s tetra-vessel occlusion to prepare the global cerebral ischemia rats model. The experiment was divided into 7 groups random: (1) control group;(2) model group: there were 6 time points, that was ischemia reperfusion Oh (ischemia group)> 0. 51k lhu 6hu 12hu 24h. At above time points, the pyramidal neurons in hippocampal CAl region were dissociated acutely from SD adult rats by the enzymatical and automatical way. Then recording the ion channel activities at the same holding voltage by the cell-attached of patch clamp techniques.Result: In the time points of our observing, at 24h after reperfusion the open probability increased to 0.005667 + 0.001560, in significant difference with control group 0.001467 + 0.000398, the others time points having no significant difference with control group;at Oh after reperfusion (ischemia group), 6h, 12h> 24h, open time was prolonged to 28. 043±9.152ms, 34.850+ 7. 864ms , 21.205 + 4. 921ms ? 32.980 + 7. 228ms respectively, in significant difference with control group 12. 565 + 1. 366ms , but at 0. 5h , lh after reperfusion, it dropped back to 17. 538 + 4. 746ms and 12. 750+1. 502ms, having no significant difference with control group;at 0.5h after reperfusion, the channel current was 1. 322 + 0. 206pA, more than control group 0.931 + 0. 155pA, having significant difference with control group, the others time points having no significant difference.Conclution: At ischemia phase (Oh after reperfusion), the main factor was prolonging channel open time;at earlier reperfusion phase(0.5h), the increasing of channel current maybe was main factor;in the middle phase of reperfusion (6h> 2h), the main factor was prolonging open time;at later reperfusion phase (24h), the prolonging open time and increasing open probability were responsibility to it. Which implies that in different time points, the main mechanisms of effects of cerebral ischemia reperfusion injuty on L-type calcium channel were different.3. Effects of Puerarin on L-type Calcium Channel in CAl Pyramidal Neurons in Hippocampus of Cerebral Ischemia Reperfusion Injury RatsObjective: After cerebral ischemia reperfusion injury, researching on the effects of Puerarin on L-type calcium channel in CAl Pyramidal Neurons in Hippocampus of rats.Method: Making model was as same as the experiment 2. The experiment was divided into 13 groups random: (1) control groups;(2)model groups: subdivided into 6 time points (same as the experiment 2);(3) Puerarin groups: time points were same as model groups. In Puerarin groups, we obturated vessels with clamp after giving a Puerarin(60mg/kg) injection of abdominal cavity to rats, it interval 15minutes. The control and model groups giving a physiological saltsolutions(60mg/kg) injection of abdominal cavity to rats, after 15minuts , we we obturated vessels with clamp.Result: After injection in our observing time points, none of open probability were increased;open time was prolonged to 16. 433 +12. 351ms> 24. 423 + 2. 027ms CRQ. 05) only at Oh and 24h after reperfusion;only at 0. 5h after reperfusion, the channel current increased to 1. 311+0. 246pA, having significant difference with control group (FKQ. 05) .Compare the time point with after using Puerarin itself, we found that: open probability decreased to 0.000400 + 0.000268, 0. 000933 + 0. 000250 , 0. 001467±0. 000476 (R0.05) respectively, at lh, 12h, 24h after reperfusion;The current after using Puerarin in all time points had no significant difference with that before using Puerarin;at 0.5h, 6h, 12h, 24h after reperfusion, open time declined to 9. 565 + 2. 018ms, 15. 952 + 3. 500ms, 14.909 + 4. 140ms , 24.423 + 2.027ms after using Puerarin, having significant difference with same points before using Puerarin (fKO. 05) .Conclution: At ischemia phase(Oh after reperfusion), the Puerarin effected channel open time prolonging by decreasing open probability;at earlier reperfusion phase(0. 5h), because of Puerarin no changing channel current, we presumed that in this time point Puerarin had no effect on channel;in the middle phase of reperfusion (6h, 12h), Puerarin can abbreviate open time and make it drop back to normal;at later reperfusion phase (24h), the Puerarin can cut open time and decrease open probability in some way.Summarized from above three experiments, the following conclusion could be derived: Puerarin takes part in treatment of ischemia stoke in clinic partly due to the inhibition of L-type calcium channel in neuron, decreasing Ca2+ flowing into cell, which can protect neuron.
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