ABSTRACT
Objective To explore the effects of H 2 S on neuronal injuries induced by oxygen glucose deprivation /reoxygenation ( OGD/R) in cortical neurons .Methods For OGD, the primary cultured cortical neurons were incubated with glucose-free EBSS media for 4h in N2/CO2/O2 (93%/5%/2%) atmosphere.Thereafter, the media were replaced by Neurobasal/B27 culture media and the neurons were incubated for 12 h in a 5%CO2 incubator at 37℃.NaHS was used as a H2S donor and cell survival rate was determined by cell counting kit 8(CCk-8).[Ca2+]i was determined using fura-2/AM and fluorescence microscopic imaging systems .The release rate of lactate dehydrogenase ( LDH) was determined by lactate dehydrogenase assay kit , and cell damage was analyzed by staining of propidium iodide ( PI ) .Results After pretreated with 200, 300 and 600μmol/L sodium hydrosulfide ( NaHS) for 30min before OGD/R, the cell survival rate of neurons significantly increased (n=4).[Ca2+]I(n=5), LDH release rate (n=4) and cell damage percentage (n=6) in the neuron pretreated with 300 μM NaHS were significantly lower than those in ODG/R cells.Treatment with 10μmol/L calcium chelator BAPTA also reduced the LDH release rate and cell damage percentage induced by ODG /R in neurons . Conclusion The results indicate that H 2 S may inhibit the OGD/R induced damage in cortical neurons via reducing calcium overload of neurons .
ABSTRACT
BACKGROUND: Ginkgetin has been widely acknowledged as having multiple pharmaceutical values in domestic and abroad. In many western countries, ginaton is imported in large amount. Domestic production of ginkgetin is great, however, seldom applied and there is no ginaton agent for injection.OBJECTIVE: To observe the effects of the increased contents of total flavonoid glycoside and quercetin glycoside of ginkgetin injection on memory function of mice, superoxide dismutase (SOD) activity in myocardial tissues and hemorrheological indexes of rabbits with ischemia-reperfusion injury.DESIGN: Randomized control animal experiment.SETTING: Hebei Medical College of Employees.MATERIALS: Ninety Kunming mice (3-4 months old), weight (25±1) g and thirty-two Japanese rabbits (3-4 months old) were selected. Self-made high-purity ginkgetin injection [5 mL containing 17.5 mg ginaton, of which there were 8.7 mg ginkgo flavonoid glycoside (49.8%) and 4.61% lactone];Ginkgetin injection made in German (Jinnaduo): Manufactured by Schwabe Germany [5 mL containing 17.5 mg ginaton, of which there were 4.2 mg ginkgo flavonoid glycoside (24%)].METHODS: The experiment was conducted in the Central Department of Experimental Animal, Hebei Medical College of Employees from September to November 2002. ①Ninety mice were randomly divided into 6 groups:1, 2 mL/kg self-made ginkgetin injection group, 1, 2 mL/kg German ginkgetin injection group, model group and control group with 15 mice in each group. Mice in the model group and control group were intraperitoneally injected with normal saline, mice in each group were intraperitoneally injected respectively with testing medicine for 10 continuous days,One hour after the 10th day of administration, mice were intraperitoneally injected with 2 mL/kg scopolamine hydrobromide and dysmnesy models were duplicated. Ten minutes after that, mice were put in the step-down instrument for 36V-voltage-stimulus after accommodation. Measurements were re-performed respectively at 5 minutes and 24 hours after training.Latency and times of electric shock within 5 minutes were recorded.②Thirty-two Japanese rabbits were selected and randomly divided into normal control group, German ginkgetin injection group, 1 mL/kg, 0.5 mL/kg self-made ginkgetin injection group with 8 rabbits in each group. Medicineswere continuously injected into aural veins. Three days after administration, blood was collected to detect the hemorheological indexes. ③Thirtytwo rabbits were randomized into 4 groups: 1 mL/kg, 2 mL/kg self-made ginkgetin injection group, 2 mL/kg German ginkgetin injection group and normal saline group with 8 ones in each group. Rabbit models with ischemia-reperfusion injury in myocardial tissues were established, rabbits were ligated for 30 minutes and then unclamped to get ischemic reperfusion injury in myocardial tissues, testing drugs were injected via carotid artery at the moment of reperfusion according to different groups. Before reperfusion and 30, 60 minutes after reperfusion, blood was drawn from the arteria femoralis, activity of SOD in plasma was measured. Animals were executed to obtain myocardial tissues so as to measure SOD activity.MAIN OUTCOME MEASURES: ①Latency and times of shock within 5 minutes in the experiment were recorded. ②Hemorheological indexes and determination of SOD activity in myocardial tissues.RESULTS: All experimental animals were involved in the analysis of results and no one died. ①Test for memory: Latency and times of errors in step down test in the injection group were better than those in the control group, and differences were significant (P ＜ 0.01 or 0.05). Times of errors within 5 minutes in 1 mL/kg self-made ginkgetin irjection group was less than that in the German ginkgetin injection group and the differences were obvious.②Hemorheological indexes: Whole-blood viscosity low shear value,rigid index of red cells and gathering index of red cells etc. in injection groups decreased. ③SOD activity: Compared with control group, that was significantly increased in 1, 2 mL/kg self-made ginkgetin injection group and were in a dose-dependent manner. Those were obviously increased in the 1 mL/kg German ginkgetin injection group. Increase in SOD activity of ischemic myocardial tissues in the 1 mL/kg self-made ginkgetin injection group was more significant than that in the 2 mL/kg German ginkgetin injection group.CONCLUSION: Both self-made and German ginkgetin injections can enhance the ability of memory; While at the basis of same dose, self-made high-purity ginkgetin injection is superior to German ginkgetin injection.