|
Objective: To develop vascular dementia rat model, proanthocyanidins on learning and memory in rats with vascular dementia, and its mechanism is detected; cultured PC12 cells in vitro, OGD injury model, observed proanthocyanidins on cell morphology and cell activity of the two proanthocyanidins as vascular dementia drugs provide experimental basis. Methods: The in vivo experiments using two different modeling methods to establish vascular dementia rat model, detection of proanthocyanidins on learning and memory in rats with vascular dementia and study its mechanism: (1) the use of the middle cerebral artery occlusion ( MCAO) Preparation of vascular dementia rat model, 50 SD rats were randomly divided into sham operation group, model group, positive control group Ginkgo biloba extract the object 24 mg / kg and grape seed proanthocyanidins 50mg/kg, 150mg/kg dose group, n = 10, continuous oral administration of 29 days. Administration 21 days to 26 days by Morris water maze test in vascular dementia rats spatial learning and memory, 27 and 28 days to step down test, observe the different doses of proanthocyanidins learning and memory abilities of rats with vascular dementia affected. Behavioral assay after 29 Tianshui chloral hydrate anesthesia rats were sacrificed, heart blood serum to detect the level of vascular dementia rat serum inflammatory factors M1 after extraction ELISA kit for the determination of serum tumor necrosis factor-alpha (TNF -alpha), interleukin-1 beta (IL-1beta) content. Kit to detect the group in ischemic brain tissue superoxide dismutase (SOD), cholinesterase (ChE) activity and total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content. (2) the use of modified Pulsinelli vascular occlusion (4-VO) established vascular dementia rat model, after the completion of the four-vessel ligation, daily oral administration, dose packet Ibid. Administered for 15 days to 20 days using the Morris water maze in rats with vascular dementia spatial learning and memory, 21 and 22 days dark avoidance assay with different doses of proanthocyanidins learning and memory in rats with vascular dementia the impact. The behavioral experiments Detection Tianshui 23 after chloral hydrate anesthesia rats were sacrificed, and the brain was isolated hippocampus and cortex, colorimetric detection of brain tissue superoxide dismutase (SOD) activity, total antioxidant capacity (T-AOC ), glutathione (GSH), malondialdehyde (MDA) content. Real-time quantitative PCR (Real-time PCR) assay rat hippocampal Bax, bcl-2 mRNA of the expression. Hippocampus, TNF-α, IL-1β protein expression was determined by Western blot. The Nissl staining observed pathological changes of vascular dementia rat hippocampal CA1 neurons. Vitro experiments, in vitro cultured PC12 cells, Applied Chemistry established cell OGD injury model. Edaravone regarded as a positive drug observed oxygen-glucose deprivation injury morphological changes, and the application of tetrazolium (MTT) (methyl thiazolyl tetrazolium MTT) to observe the different concentrations of proanthocyanidins on cell viability. Kit to detect extracellular fluid lactate dehydrogenase (lactate dehydrogenase, LDH) activity, cell culture supernatant super oxide dismutase (SOD) activity and malondialdehyde (MDA) content. Results: middle cerebral artery occlusion (MCAO)-induced vascular dementia rat model experiments: (1) proanthocyanidins on Morris water maze learning and memory in rats: the fifth day of the water maze test, the model group and the sham-operated group compared take longer to find the platform (P = 0.000); proanthocyanidins low-dose and high-dose groups could significantly shorten the escape latency of rats with vascular dementia (P = 0.020, P = 0.000). In space removed platform to explore the experiment, compared with the model group, the proanthocyanidins low-dose and high dose group through the platform frequency increased significantly (P = 0.047, P = 0.002). (2) proanthocyanidins on learning and memory in rats step down test: compared with the sham group, model group significantly increased the number of learning (P = 0.000), latency was significantly shorter (P = 0.001), and show that the learning and memory damaged. Proanthocyanidins low-dose and high-dose group could significantly reduce the number of learning (P = 0.000, P = -0.000), increase in vascular dementia rats incubation period (P = 0.023, P = -0.025); above behavioral experiments results show that proanthocyanidins can improve the learning and memory abilities of rats with vascular dementia. (3) of proanthocyanidins on vascular dementia rat ischemic brain tissue biochemical indicators: Compared with the sham that rats, brain tissue SOD content was significantly reduced (P = 0.001), total antioxidant capacity was significantly reduced (P = 0.000), MDA content was significantly increased (P = 0.000). Proanthocyanidins low dose and high dose group may improve vascular dementia rat brain superoxide dismutase activity (P = 0.033, P = 0.001); decreased MDA content (P = 0.000, P = 0.000); model group phase ratio, the proanthocyanidins high dose group can significantly improve the total antioxidant capacity (P = 0.014), indicating that proanthocyanidins antioxidant system to protect the brain, reducing free radical damage. Cholinesterase activity was no significant difference between the groups. (4) the proanthocyanidins administration group serum TNF-alpha, IL-1beta content decreased, there was a significant difference compared with the model group, that proanthocyanidins can reduce the inflammatory response of rats with vascular dementia peripheral system. Improved Pulsinelli vascular occlusion method: (1) proanthocyanidins induced vascular dementia rat model of experimental learning and memory in the rat Morris water maze: Day 5 model group compared with the sham group, in the water maze test the rat takes longer to find the platform (P = 0.000); the proanthocyanidins administration can significantly shorten the escape latency of rats with vascular dementia (P = 0.039, P = 0.002). In space exploration experiments, compared with the model group, proanthocyanidins administration can increase the number of platforms and the original platform quadrant swimming time in vascular dementia rats through the percentage value of the total swimming time (P = 0.015, P = 0.010 ; P = 0.015, P = 0.000). In the dark avoidance experiments, rats with vascular dementia stay in the light box for a short time (P = 0.000), and proanthocyanidins in rats treated residence time increased (P = 0.000, P = 0.000), indicating that the administration rats solid memory; the camera obscura inescapable electrical stimulation of the above behavioral experiments results show that procyanidins can improve learning and memory abilities of rats with vascular dementia. (2) proanthocyanidins vascular dementia rat brain tissue antioxidant capacity: compared with the sham group, vascular dementia rat brain tissue GSH content decreased SOD activity decreased, T-AOC reduce MDA content increased ( P = 0.000, P = 0.000, P = 0.000, P = 0.000). Proanthocyanidins (50 and 150 mg / kg) can improve vascular dementia rat brain tissue GSH content (P = 0.035, P = 0.012), SOD activity (P = 0.037, P = 0.001) and total antioxidant capacity (P = 0.044, P = 0.010), decreased MDA content (P = 0.044, P = 0.010), indicating that proanthocyanidins antioxidant system to protect the brain, reducing free radical damage. (3) proanthocyanidins on vascular dementia hippocampus Bax, bcl-2 mRNA expression of: compared with the sham group, VD of rat hippocampus Bax mRNA expression increased (P = 0.000), bcl-2 mRNA downregulation ( P = 0.003). Proanthocyanidins (50 and 150 mg / kg) of reduction in vascular dementia rat hippocampus Bax mRNA expression (P = 0.004, P = 0.000), raised the bcl-2 mRNA expression (P = 0.000, P = 0.000), the bcl-2 / Bax mRNA expression ratio increased significantly (P = 0.001, P = 0.000), indicating that procyanidins may inhibit vascular dementia rat hippocampal neurons apoptosis. (4) proanthocyanidins on the hippocampus of rats with TNF-alpha and IL-1beta protein levels: compared with the sham group, model group hippocampus tissue TNF-alpha (P = 0.000), IL-1 beta (P = 0.000) protein was significantly improved; the original anthocyanins administration group can significantly reduce TNF-alpha (p 0.000, p 0.000), IL-1 (P = 0.000, P = 0.000) protein expression, showed that proanthocyanidins can reduce the hippocampus of rats with vascular dementia expression of inflammatory factors in the organization. (5) proanthocyanidins pathological vascular dementia rat hippocampal CA1 region: Nissl staining showed that the sham-operated group hippocampal CA1 neurons arranged in neat rows, the cell body, clear cell level, the cytoplasm within Nissl number . The fuzzy model hippocampal CA1 neurons structure, reducing the number of neurons arranged obviously loose, wrinkled into a triangle or cone, neuronal cell bodies and dendrites Nissl bodies dissolved disappear. Compared with the model group, the treatment group proanthocyanidins in hippocampal CA1 neurons arranged in neat rows, the Nissl bodies within the cytoplasm of neurons compared with model group increased, indicating that the the proanthocyanidins administration significantly reduced hippocampus of rats with vascular dementia nerve injury . Oxygen glucose deprivation, PC12 cell morphology change, proanthocyanidins (5mg / L, 25mg / L, 100mg / L) three doses can reduce oxygen glucose deprivation injury of PC12 cells into which 25 mg / L, 100mg / L cell viability of the two-dose group with model group, a significant difference (P = 0.007, P = 0.044), indicating that with the increase of the concentration of administration, each group cell damage slightly reduced, showing a certain drug form protection performance. Compared with the model group, the application of proanthocyanidins PC12 cells in each group of extracellular fluid LDH activity also decreased, and there is a significant difference (P = 0.000, P = 0.000, P = 0.000). Compared with model group, PC12 cells after the application of proanthocyanidins cell supernatant MDA content decreased concentration-dependent trend, and there is a significant difference (P = 0.002, P = 0.000, P = 0.000). Compared with model group, PC12 cells after the application of proanthocyanidins the cell supernatant SOD activity gradually increased in a dose-dependent trend and a significant difference (P = 0.000, P = 0.000, P = 0.000). The above results suggest that proanthocyanidins have a protective effect on PC12 cells Ischemia in, which may be related proanthocyanidins increased SOD activity and reduce the activity of oxygen free radicals. Conclusions (1) water maze, step down test and passive avoidance avoidance behavior such as experimental science experimental results, the proanthocyanidins can significantly improve vascular dementia rats learning and memory, the mechanism of this protective effect may be: procyanidins can improve vascular dementia rat brain antioxidant system capabilities; reduce vascular serum levels of inflammatory factors and hippocampal expression of inflammatory cytokines, a significant anti-inflammatory effects; lowered the apoptotic gene bax expression, raised the anti-apoptotic gene bcl-2 expression and production of anti- apoptosis, thus contributing to the survival of hippocampal neurons. (2) proanthocyanidins able to maintain damaged PC12 cell membrane integrity, to protect the cell's mitochondria, increase cell viability. This neuroprotective effect may be related to proanthocyanidins improve SOD oxidase activity, reduce the activity of oxygen free radicals.
|