| Objective: Vascular dementia (called VD as follows) is a clinical syndrome caused by cerebrovascular disease which results from a series of cerebrovascular blood-supply disorder. With the aging of the population, VD, disability rate and mortality are increased, has become one of important disease threatening the elder people's health, so it's an important medical value and social significance to study and explore actively the pathogenesis and treatment. QingNao capsule is a trituration based on TCM intervention treatment principles which includes removing blood stasis and eliminating phlegm. It is based on Traditional Chinese medicine (TCM) theory and combined with years clinical medication and clinical experience of treating VD. And QingNao capsule obtain the very good curative effect. To approach the mechanism of QingNao capsule in treating VD, we adopted VD model of mice and PC12 hypoxia model and studied its effect on and PC12 cytopathic hypoxia model, and discuss the preliminary mechanism of VD treatment of QingNao capsule.Methods:1 The influence of QingNao capsule to monoamine neural transmitter in brain of miceWe adopted the VD model of mice with the operation of repeated ischemia-reperfusion method preparation. The successful model maker were divided into five groups at random,model control group, low dose of QingNao capsule group (3.4 g crude drug /kg), the middle dose of QingNao capsule group (6.8 g crude drug /kg), the high dose of QingNao capsule group (13.6 g crude drug /kg) and Hydergine group (1.5 mg/kg), another set sham-operation group. We duplicated the model of VD mice with the operation of cerebral ischem reperfusion through separating and ligating both side carotid arteries with putting blood from coda except sham-operation group.And then 5 groups were continuously filled medicine or water through the stomach from the second day after the operation of cerebral ischem reperfusion. Sham-operation group and model group were given water and other groups were given medicine, with 10 mice in each group, given medicine or water through the stomach is the capacity of 0.2 mL.10g-1, continuous four weeks.1.1 Detection of learning and memory abilityWe detected the learning and memory ability of mice by Morris Water Maze which include hidden-platform acquisition training and spatial probe test after operation one week, two weeks, three weeks and four weeks, the experiment of Morris water maze was done after given medcine one hour, the escape latencies, times of going through the platform, the average of swimming path, orientation angle, the ratio of platform and the total swimming time and the ratio of platform and the total swimming path were recorded and compared.1.2 Monoamine Neural transmitter detectionThe animals were decapitated Afrer behavioral testing finished. Monoamine Neural Transmitter Determination, including Norepinephrine (NE), Dopamine (DA) and 5-hydroxy tryptamine (5-HT) within brain by fluorescence spectrophotometry.2 The effect of QingNao capsule and the blood serum of QingNao capsule on PC12 hypoxia damage2.1 The effect of QingNao capsule on PC12 hypoxia damage2.1.1 Detection of QingNao capsule on PC12 hypoxia damageConcentration gradient of QingNao capsule was set to 1μg.L-1, 10μg.L-1, 100μg.L-1, 500μg.L-1, 1000μg.L-1, 5000μg.L-1, 10000μg.L-1, 50000μg.L-1, and the action time was 24h; Persistence of each hole was detected by MTT method, denoting by optical density.2.1.2 Interference of QingNao capsule to PC12 proliferationInterference gradient concentrations was set as follows: 10μg.L-1, 50μg.L-1, 100μg.L-1, 250μg.L-1, 500μg.L-1, 1000μg.L-1, 2500μg.L-1, the incubation times were 1h, 4h, 24h, the final concentration of Sodium Dithionite was 5mmol/L which was co-cultured with QingNao capsule for 16h; the optimal concentration and action time were detected and determined by MTT method.2.2 The effect of the blood serum of QingNao capsule on PC12 hypoxia damagePC12 with logarithmic growth divided into model control group, low dose group of 5% medicated serum, middle dose group of 5% medicated serum, high dose group of 5% medicated serum and blank serum control group, total 5 groups. After damaged by Sodium Dithionite for 16h, we observed each cells form by MTT colorimetric method and LDH vigor measurement, compared to the effect of the blood serum of QingNao capsule each group on model protective.Results:1 The influence of QingNao capsule to monoamine neural transmitter in brain of mice1.1 Hidden-platform acquisition trainingIn the same time point, the escape latencies, the average of swimming path and orientation angle of model control group were apparently longer than sham-operation(P < 0.01); In the same time point, Compared with the model group, the escape latencies of Hydergine group, the high dose and the middle dose of QingNao capsule group were apparently shorter(P < 0.05 or P < 0.01), the average of swimming path and orientation Angle were apparently lower (P<0.05) ; All Parameters of low dose of QingNao capsule group were improved, but not statistically significant (P > 0.05).Spatial probe experiment: In the same time point, the times of going through the platform of model control group were apparently fewer than sham-operation(P < 0.01), the ratio of platform and the total swimming time path were apparently fewer than sham-operation(P < 0.01), orientation Angle were apparently more than sham-operation(P < 0.01); In the same time point, Compared with the model group, the times of going through the platform of Hydergine group, the high dose and the middle dose of QingNao capsule group were apparently more(P < 0.05 or P < 0.01), the ratio of platform and the total swimming time path were apparently more,orientation Angle were apparently fewew than sham-operation(P < 0.01); All Parameters of low dose of QingNao capsule group were improved, but not statistically significant (P > 0.05).1.2 Monoamine neural transmitter detectionThe content of monoamine neural transmitter in model control group were apparently fewer than sham-operation (P < 0.01), Compared with model group, the content of monoamine neural transmitter in Hydergine group, the high dose and the middle dose of QingNao capsule group were apparently increased (P < 0.05 or P < 0.01), the content of NE and DA in low dose of QingNao capsule group tended to promote, but had no statistically significant (P > 0.05), the content of 5-HT in low dose of QingNao capsule group were apparently increased (P < 0.05).2 The effect of QingNao capsule and the blood serum of QingNao capsule on PC12 hypoxia damage2.1 The effect of QingNao capsule on PC12 hypoxia damage2.1.1 Detection of QingNao capsule on PC12 cytotoxicity OD value was decreased significantly (P<0.01) when the drug concentration exceeded 10mg.L-1 and obvious cytotoxicity emerged (P<0.01). Most cells were shrinked to round shape and few of dead cells were floating in the holes.2.1.2 Interference of QingNao capsule to PC12 proliferation Compared with the control group, serum-free model group OD value significantly reduced (P < 0.01); And compared with the model group, 50μg.L-1 than gets 1h, 4h, 24h preincubated; 100μg.L-1 and 250μg.L-1 preincubated 1h; that the OD value were increased significantly(P < 0.05), 100μg.L-1 and 250μg.L-1 preincubated 4h, 24h could increase the OD value significantly (P < 0.01); 10μg.L-1 and 500μg.L-1group preincubated 1h, 4h, 24h, that the OD value tended to promote, but had no significant difference (P>0.05). In conclusion, the optimal concentrations of QingNao capsule were 50μg.L-1, 100μg.L-1, 250μg.L-1and the optimal time was 24h. 2.1.3 PC12 cell morphology observationThe PC12 cells of the control group were the circular, the refractive sex of is stronger; the most cells of model group were shrinked to round shape and few of dead cells were floating in the holes.2.1.4 MTT assay for cell viabilityCompared with the control group, the OD value of model group was significantly reduced (P < 0.01); Compared with model group, the OD value of 50μg.L-1, 100μg.L-1, 250μg.L-1 group preincubated 24h was increased significantly (P < 0.05 or P < 0.01).2.2 The effect of the blood serum of QingNao capsule on PC12 hypoxia damage2.2.1 Determination by MTT : Compared with blank serum model group, the OD value of model group was significantly reduced (P < 0.01); Compared with model group, the OD value of the middle and high dose of 5% Medicated serum group was significantly increased (P < 0.01), the OD value of low dose of 5% Medicated serum group wasn't significantly increased (P < 0.05).2.2.2 LDH activity assay Compared with the blank serum control group, LDH leakage quantity of model group is significantly increased (P < 0.01); Compared with model group, LDH leakage quantity of the middle and high dose of 5% Medicated serum group was significantly decreased (P < 0.01), LDH leakage quantity of low dose of 5% Medicated serum group was significantly decreased (P < 0.05), consistent with the MTT results.Conclusion:1 QingNao capsule can significantly improve the ability of learning and memory in VD mice, the possible mechanism is that QingNao capsule can heighten the content of NE DAand 5-HT in the brain tissue of VD mice. 2 QingNao capsule and its medicated serum all can enhance the vitality of PC12 cells hypoxia damage caused by Sodium Dithionite, inhibit the LDH activity in the culture medium; And has the protective effect on PC12 cells hypoxia damage caused by Sodium Dithionite.
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