Protective Effect And Its Oxidation Mechanisms Of The Main Components Of Astragalus On A?_25-35-induced Alzheimer's Disease Rat Model

Part OneProtective effect of main components of Astragalus on A?25-35-induced Alzheimer's disease in ratsObjective:Alzheimer's disease (AD) is a chronic progressive disease of the central nervous system, which usually happened in the elderly. The main pathological features include neurons amyloid beta protein (amyloid protein, A?) deposited in senile plaques (SP), neuronal tau protein hyperphosphorylation formation of neurofibrillary tangles (NFTs), a large number of cholinergic neuron loss and death in the specific regions of the nervous central system. With the aging of the social population is becoming more and more seriously, the harm of AD to human health is becoming more and more seriously. According to statistics, in Europe and the United States AD has become the fourth cause of death after heart disease, cancer, stroke, and the number of AD patients in China has surpassed more than 5 million. Therefore, the prevention and treatment of AD has become one of the important medical and social problems that need to be solved urgently. However, the pathogenesis of AD is not very clear. More and more evidence supports the cascade reaction damage theory caused by abnormal deposition of A?. In this process, oxidative stress is one of the key factors for the formation and development of AD. Then, this experiment investigates the effect of the effective components of Astragalus on learning memory function on AD rats and put oxidative stress as the breakthrough point, revealing that the possible mechanisms of main effective components of Astragalus including astragaloside and astragaloside IV in prevention and treatment of AD, providing theoretical foundation and scientific basis for its use in the clinics.Methods:The rat model of Alzheimer's disease was induced by the injection of A?25-35 in the lateral ventricle of adult male Sprague Dawley (SD) rats weighing 250-300 g. Rats were randomly divided into control group (sham operation group), model group, Astragaloside group at low dose (20 mg/kg), Astragaloside group at medium dose (40 mg/kg), Astragaloside group at high dose (80 mg/kg), Astragaloside IV group (50 mg/kg), with 12 rats in each group. AD rat model was prepared by the method of lateral ventricle injection of A beta 25-35 in the model group and the treatment group rats. The A beta 25-35 was diluted to 3 nmol/L working fluid with sterile normal saline, and incubated for 1 week in a sterile incubator at 37? after sealing, which was turned into a state of aggregation. A beta 25-35 5 ?1 (15 ?g) was injected into the lateral ventricle of the rats by the brain stereotactic instrument, and the same amount of sterile saline was injected into the lateral ventricles of the sham operation group. After operation, the medicine was administered orally for 21 days, while equal volume of normal saline was given in the model group and the control group. One week after surgery Morris water maze training was performed, lasting 5 days. On day 14 test was started and recorded for 3 days. The learning and memory function changes were observed. Rats escape latency and the total distance of swimming, the number of crossing the platform and search strategies were analyzed. After Morris water maze test, a part of the rats were anesthetized and fixed with 4% formaldehyde through the heart perfusion, and the brain tissues were taken to cut frozen sections. Nissle staining was used to observe the changes of morphology and structure of neurons in the hippocampus. The changes of morphology and number of neurons in the hippocampus were observed by immunohistochemistry. The apoptosis of neurons was detected by Hoechst 33342 staining. After Morris water maze test, a part of the rats was decapitated and brain hippocampal homogenates were prepared, Enzyme linked immunosorbent assay (ELISA) was used to detect the activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in hippocampal tissue homogenate.Data are expressed as mean ± standard deviation (mean ± SD), and analyzed using SPSS 15.0 for windows statistical software for single factor variance analysis (ANOVA) analysis or non paired t-test, P< 0.05 was considered statistically significant.Results:1. Morris water maze test1) Water maze navigation experiment resultsIn the navigation experiment of Morris water maze, rats in normal group and sham operation group were more likely to search and find the safety platform than in the model group and the treatment group. On the first day of the test, the escape latency of A beta group was significantly prolonged (P< 0.05), and the total distance of swimming was significantly prolonged, and the difference was statistically significant (P< 0.01); In Astragaloside group at low dose escape latency and the total distance of swimming is shortened obviously, however, differences between groups were statistically significant. In Astragalosides group at medium and high dose and Astragalosides IV group escape latency was significantly shortened (P< 0.01), the total swimming distance was shortened obviously, but no statistical difference. From the second day of testing on, compared with the model group, in Astragaloside group at low dose, middle dose and high dose, escape latency was significantly shortened, and the difference is statistically significant. Compared with model group, in astragaloside IV group escape latency was also significantly shortened. The total swimming distance in the high dose group of Astragaloside group was significantly different from the model group (P< 0.05), while the total distance between the low dose group and the middle dose group was not significantly different. In the third day of the test, in addition to the low dose group of Astragaloside, in the rest of the treatment group, the escape latency and the total swimming distance were significantly different from the model group (P< 0.05). Compared with the model group, the average latency in Astragaloside group at middle and high dose was significantly shortened (P< 0.05), and there was no significant difference between Astragaloside group at low dose and the model group.2) Results of water maze spatial search capabilityCompared with the control group, rats in the model group the residence time in the platform was significantly prolonged. Compared with model group, in Astragaloside group at middle and high dose the residence time in the platform was significantly shortened (P< 0.05), the residence time in the platform in Astragaloside IV was also significantly shortened (P< 0.05). Compared with the control group, the number of crossing the platform in model group rats was decreased significantly. In rats of Astragalosides group at middle and high dose the number of crossing the platform increased significantly, but did not return to normal levels, the number of crossing the platform in Astragaloside IV group rats was also increased significantly. There was no significant difference found in the residence time standing in the platform and the number of crossing the platform between the model group and Astragaloside group at low dose. Compared with the control group, search strategy score in rats of model group decreased significantly. In Astragalosides group at low and high dose compared with the model group, the search strategy scores were significantly increased (P< 0.05). Compared with the model group, search strategy scores in Astragaloside IV group were also increased significantly.2. Pathological examination resultsAs Nissl staining showed, neuronal cells in the hippocampus of normal group rats arranged closely, rich in Nissl's bodies, staining deeply and the staining of the nucleus was lightly blue. Compared with the control group, in the rat hippocampal area of the model group neurons was stained lightly, the number of the nissle small body was reduced with sparse cell array and increased cell gap, the boundary is not clear. The total number of cells in the Astragaloside treatment group and Astragaloside IV group was obviously increased, while the gap became smaller and the edema of neurons decreased. As immunofluorescence staining showed, compared with the control group, the number of NeuN positive cells in the hippocampus of model group was significantly reduced.The number of NeuN positive cells in Astragalosides at middle and high dose was significantly increased comparing with model group (P< 0.05). Astragalosides IV also significantly increased the number of NeuN positive cells in the hippocampus. These results suggested that Astragalosides and Astragalosides IV protect against A?-induced hippocampal neuronal damage. 3. Oxidative stress detection resultsAs ELISA results showed, compared with the control group, glutathione peroxidase (GSH-Px) activity in hippocampus homogenate in model group rats decreased significantly. GSH-Px activity in the rat hippocampus in Astragalosides group at middle and high dose was significantly increased as compared with the model group rats (P< 0.05). In the low dose of Astragalosides group, the activity of GSH-Px in the hippocampus of rats was not increased significantly, as compared with the model group, the activity of GSH-Px in the hippocampus of rats in Astragalosides IV group was also significantly increased (P< 0.01). Compared with the control group, the activity of superoxide dismutase (SOD) in the rat hippocampus of the model group was significantly decreased. Compared with the model group, SOD activity in rat hippocampus tissues of Astragalosides group at high dose was significantly increased (P< 0.05), SOD activity in the rats hippocampal tissues of Astragalosides group at low dose and middle dose was increased, but statistics had no significant difference (P> 0.05). Astragaloside IV also didn't affect the activity of SOD in hippocampus tissues significantly. Compared with the control group, the content of Malondialdehyde (MDA) in hippocampus of model group was significantly increased. The content of MDA in the hippocampus of the rats in each dose of Astragaloside group was significantly decreased (P< 0.01). Compared with the model group, the content of MDA in the hippocampus of the rats in Astragaloside IV group was also significantly decreased (P< 0.05).Conclusion:1. The study and memory ability of induced by A?25-35 were improved by treatment with Astragaloside and Astragaloside IV.2. Rat hippocampus neuron density was significantly decreased and apoptosis cells increased obviously in the hippocampus after intracerebroventricular injection of amyloid beta 25-35. Administration with Astragaloside and Astragaloside IV for three weeks significantly reduced neuronal damage induced by amyloid beta 25-35 injection.3. A?25-35 induced oxidative stress damage in rat hippocampus. Treatment with Astragaloside significantly increased the activity of antioxidant enzymes GSH-Px and SOD, and decreased the content of MDA in the hippocampus. Astragaloside IV also significantly increased GSH-Px and SOD activity and decreased MDA conten in the hippocampus. These results suggested the protective effect of Astragaloside and Astragaloside IV against oxidative stress.In summary, Astragaloside and Astragaloside IV treatment can improve the learning and memory function and hippocampal neuronal damage in AD rats, which may be related to the anti-oxidative effect.Part TwoProtective effect of the main components of Astragalus on oxidative stress of PC 12 cells induced by A?25-35 in vitroObjective:Astragalus is a legume Astragalus genus and its main effects included with diuresis, detumescence and its strong antioxidant activity. In this study, PC 12 cells (ratpheochromocytoma cell line) was used as the experimental subjects, and the A beta 25-35 was used to induce oxidative stress injury in the cells. The purpose of this study is to detect the effect of the main effective components including Astragaloside and Astragaloside IV on A-beta induced PC 12 cell injury, and whether works through the inhibition of oxidative stress.Methods:PC 12 cells injury was induced by using the aggregated state of A beta 25-35 treatment, and the AD-like injury cell model was established in vitro. The effect on A?25-35-induced PC 12 cell damage was observed in the pretreatment of Astragaloside and Astragaloside ?. PC12 cell injury was detected by MTT ((3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide) detection assay after treatment with Astragaloside and Astragaloside IV. The effect of Astragaloside and Astragaloside IV on PC 12 cells was detected by the detection of lactate dehydrogenase (LDH). Hoechst 33342 staining was used to observe the effect of Astragaloside and Astragaloside IV on A?25-35-induced apoptosis of PC 12 cells. The effect of Astragaloside and Astragaloside IV pretreatment on the average fluorescence intensity of reactive oxygen species (ROS) was detected by flow cytometry. The effect of Astragaloside and Astragaloside IV pretreatment on GSH-Px activity, SOD activity and MDA content was detected by ELISA assay. The effect of Astragaloside and Astragaloside IV pretreatment on iNOS mRNA expression was determined by RT-PCR.Results:1. The effect of Astragaloside and Astragaloside IV pretreatment on PC12 cell injuryAs MTT detection assay showed, Astragaloside and Astragaloside IV at concentrations of 5,10,20,40,80,100 ?M can not produce toxic effects on PC12 cells, and it also does not affect cell proliferation. After 24 h treated with A?25-35 at 2.5,5,10,20,40 ?M, the cell survival rate was significantly decreased. The survival rate treated with A?25-35 at l0?M for 24 h is 65%, which concentration was chosen as the best concentration to induce injury. Pretreatment with Astragaloside at different concentrations significantly increased cell survival rate induced by A?25-35.Compared with model group, pretreatment with Astragaloside IV at different concentrations significantly improved decreased cell survival rate induced by A?25-35 (P< 0.01). LDH detection assay showed that LDH release in PC 12 cell supernatant increased significantly induced by AP25-35 at 10 ?M after 24 h. Astragaloside IV at concentrations of 5,10,20,40,80 ?M significantly inhibited LDH release in PC12 cell supernatant. Astragaloside IV at concentrations of 5,10,20,40,80?M also significantly inhibited LDH release in PC12 cell supernatant (P< 0.01).2. The effect of Astragaloside and Astragaloside IV pretreatment on apoptosis on PC12 cellsHoechst 33342 staining results showed that compared with the control group, after 24 h disposed with 10 ?M beta amyloid peptide 25-35, intracellular apoptotic bodies increased significantly, nuclear pyknosis obviously; compared with the model group, pretreatment with Astragalosides at 10,40 ?M can significantly decreased the number of apoptotic cells and nuclear pyknosis decreased obviously. Astragaloside IV at 10,40 ?M pretreatment can also obviously inhibited Abeta 25-35 induced apoptosis in PC 12 cells.3. The effect of Astragaloside and Astragaloside IV pretreatment on oxidative stress on PC12 cellsELISA results showed that compared with the control group, after 24 h treated with 10 ?M beta amyloid peptide 25-35, antioxidant enzyme activity of GSH-Px and SOD on PC 12 cells decreased significantly (P< 0.01). Astragalosides at different concentrations (5,10,20,40,80 ?m) can significantly increased the activity of GSH-Px and SOD. Astragaloside IV at concentrations of 5,10,20,40,80 ?M can also significantly inhibited the activity of GSH-Px and SOD decreased (P< 0.01). Beta amyloid 25-35 induced increased MDA content on PC 12 cells as compared with the model group.Pretreatment with Astragaloside inhibited the level of MDA in PC12 cells and Astragaloside IV pretreatment can also significantly reduced the MDA content, with significant difference (P< 0.01).4. The effect of Astragaloside and Astragaloside IV pretreatment on iNOS mRNA expression on PC12 cellsRT-PCR showed that the beta amyloid peptide 25-35 at 10 ?M for 24 hours upregulated iNOS mRNA expression in PC12 cells as compared with model group. Pretreatment with Astragalosides at 10 and 40 ?M significantly inhibited iNOS mRNA expression in PC 12 cells. Astragaloside IV at 10 and 40 ?M pretreatment can also decreased significantly iNOS mRNA expression in PC 12 cells, and the difference is statistically significant (P< 0.01).Conclusion:PC 12 cell viability induced by A?25-35 was significantly reduced, LDH release was also significantly increased, apoptosis and oxidative stress increased. Astragaloside and Astragaloside IV protected PC 12 cell injury by inhibiting the decreased cell viability and LDH release, decreasing cell apoptosis and oxidative stress. Beta amyloid 25-35 induced iNOS expression in PC12 cells, which was significantly inhibited by Astragalosides and Astragaloside IV pretreatment.Part ThreeProtective effect of the main components of Astragalus on oxidative injury of PC 12 cells induced by H2O2 in vitroObjective:To further verify the antioxidant effect of main components of Astragalus including Astragaloside and Astragaloside IV, this study aims to detect PC12 cell injury induced by H2O2 and the effect of Astragaloside and Astragaloside IV, and to preliminarily study the antioxidant effect of them.Methods:In cultured PC12 cells in vitro, H2O2 at 300 ?M was added to observe the changes of cell viability at different time to confirm the optimal conditions of H2O2 damage in PC 12 cells. After pretreatment with Astragaloside and Astragaloside IV, the effects on the cell survival rate and LDH leakage rate was determined. And the difference between Astragaloside and Astragaloside IV was performed.Results:Treated with H2O2 at 300 uM in PC 12 cells, the cell viability decreased with extended time, showing a positive correlation between them. After 4 h treatment with H2O2 at 300 ?M, cell viability decreased to about 70%, then H2O2 at 300 ?M for 4 h was selected as the establishment of H2O2 induced PC 12 cell damage model, and the appropriate conditions were used for following testing. As MTT detection assay showed, compared with the control group, cell survival rate in PC 12 cells in model group decreased significantly. As compared with PC12 cells in the model group, pretreatment with Astragaloside and Astragaloside IV at concentrations of 10,20,40 ?M significantly increased cell viability, and the difference is statistically significant (P< 0.01). LDH detection assay showed that H2O2 at 300 ?M for 4 h significantly increased LDH release. Compared with PC 12 cells in model group, Astragaloside at concentrations of 10,20,40 ?M significantly inhibited LDH release increased. Astragaloside IV at concentrations of 10,20,40 ?M also significantly decreased the level of LDH release in PC 12 cells (P< 0.01). And comparing with Astragaloside treatment, the effect of Astragalus IV at the same concentration inhibiting the level of LDH release was more obvious.Conclusion:H2O2 disposal induced PC 12 cell viability decreased significantly and the level of LDH release increased significantly. Pretreatment with Astragaloside and Astragaloside IV can significantly inhibited H2O2-induced the decreased PC 12 cells survival and increased LDH release, indicating that they has a potential protective effect on H2O2-induced PC 12 cells damage.Summary1. Pretreatment with Astragalosides and Astragaloside IV can improve the learning and memory ability on Alzheimer's disease rats induced by A beta 25-35.2. Pretreatment with Astragalosides and Astragaloside ? significantly reduced A beta 25-35-induced hippocampal neuronal damage in AD rats.3. The activity of GSH-Px and SOD in the hippocampus was significantly decreased after A beta 25-35 disposal, and MDA contents were also increased obviously. Pretreatment with Astragalosides and Astragaloside IV significantly inhibited decreased activity of GSH-Px and SOD and increased MDA contents in the hippocampus in AD rats.4. Astragalosides and Astragaloside ? protected against A beta 25-35 induced PC12 cell injury through the inhibition of cell apoptosis and oxidative stress.5. A beta 25-35 upregulated the iNOS RNA expression in PC12 cells, which was inhibited by pretreatment with Astragalosides and Astragaloside IV.6. Pretreatment with Astragalosides and Astragaloside IV also have a protective effect on H2O2-induced PC12 cell injury.