Protective Effects Of Astragalosides Against Synergistic Neuron Injury Induced By Glucocorticoids And Aβ And Its Mechanism

Background:Alzheimer's disease(AD) is one of the major neurodegenerative disorder of the elderly, and is characterized clinically by progressive loss of memory, cognitive deficits, behavioral disturbances and human communication disorders.With the aging of the social population, the incidence of AD is increasing rapidly leading to aserious of social problem. Thus, it is very important to study the pathogenesis of AD and prevention drugs for this disease.At present, the pathogenesis of Alzheimer's disease is still not entirely clear. It may be interactive result of multiplicity factors such as gene mutation, environmental effects and neuroendocrine etc. In recent years, it has been paid more and more attention to the occurrence and progress of AD induced by chronic stress or glucocorticoids overexposure. Many studies confirm that chronic stress and high level of glucocorticoids can contribute to hippocampal neurons damage, leading to HPA axis dysfunction and further increase the damage of hippocampus, forming a vicious circle. Clinical trials also confirmed that the basis plasma levels of glucocorticoids increase in older people. Older people with AD have higher levels of glucocorticoids than those without dementia. Therefore, glucocorticoids as stress hormones is closely related to the incidence of AD.Alzheimer's disease (AD) is characterized by senile plaques composed ofβ-amyloid (Aβ) peptide. Aβhas neurotoxicity and can enhance or enlarge neuronal injury induced by a variety of noxious stimulation. Chronic stress and glucocorticoids exposure may also lead to generation and deposition of Aβand formation of senile plaques in brain tissue of transgenic AD mice. Therefore, it is great significance to study the synergistic injury of glucocorticoids and Aβto neuron and the role in the occurrence and development of AD.Astragalus is a traditional Chinese medicine. Astragalosides (AST), an effective part extracted from Astragalus, have anti-inflammatory, immune regulation, promote intelligence, anti-aging and neuroprotective effect. AST was commonly used in the prevention and treatment of cardiovascular, cerebrovascular diseases,aging,immune function disorders, anti-tumor and other diseases. The subject which based on our previous research is further to study the synergetic neuronal injury of glucocorticoids and Aβ, observe the protective effects of AST and its mechanismMethods and ResultsPart I Protective effects of Astragalosides on learning and memory impairment induced by glucocorticoids in mice and its mechanismIn the study, dexamethasone (DEX, 5mg/kg, ig 21d) was used to induce learning and memory impairment and neuronal damage in 12-month-old mice. Spontaneous motor activity was measured (at 10 d and 20 d after DEX treatment) by a computer-controlled spontaneous motor activity apparatus. Morris water maze was used to observe the learning and memory function of mice. HE staining was used to detect brain tissue pathological changes. The neurons apoptosis in brain cortex and hippocampus stained by Hoechst 33258 was observed by fluorescence microscopy. RT– PCR was used to detect the mRNA levels ofβ-amyloid precursor protein (APP),α-secretase,β-secretase and caspase-3. Immunohistochemical detection was used to observe the expression level of APP, cytochrome c and Caspase– 3. Ac-DEVD-pNA and Ac-LEHD-pNA cleavage methods were used to detect the activity of Caspase-3 and Caspase-9. The results showed that DEX could reduce the autonomy activities of mice, AST (40mg/kg) could increase the spontaneous activity at 10d after DEX treatment, DEX could reduce the mice swimming time in the platform quadrant and the number of crossing the platform, AST(20, 40mg/kg) could increase the platform quadrant swimming time, and AST(40mg/kg) could increase the number of acrossing platform. HE and Hoechst 33258 staining results showed that DEX could lead to hippocampal CA1, CA3 region and cortical neuronal injury and apoptosis, AST could improve the neuronal damage and apoptosis. RT-PCR analysis showed that DEX could increase the APP695, APP751,β-secretase and Caspase-3 mRNA levels and reduceα-secretase mRNA level. AST (20,40 mg/kg) could reduce the APP695, APP751,β-secretase and Caspase-3 mRNA levels and increaseα-secretase mRNA level. Immunohistochemistry results showed that DEX could increase the mice brain APP, cytochrome c and Caspase-3 expression in cortex and hippocampus CA1, CA3 areas, AST could reduce APP, cytochrome c and Caspase-3 expression. And DEX could increase mice brain tissue caspase-3 and caspase-9 activity, AST(20, 40mg/kg) could reduce the mice brain tissue caspase-3 and caspase-9 activity.Part II Protective effects of Astragalosides on learning and memory impairment induced by glucocorticoids and Aβin rat and its mechanismIn this study, the learning and memory injury model rats were established by injecting Aβ25-35 in the hippocampal CAl area and intragastric administration dexamethasone (DEX) for 14 days. Morris water maze was used to test the learning and memory function of rats. The brain tissue pathological changes of hippocampal CA1 area was detected by HE stain. Fluorescence microscopy was used to observe the neuronal apoptosis in hippocampal CA1 area by Hoechst 33258 stain. Electron microscopy was used to observe the ultra-structure of hippocampal neurons. RT-PCR was used to detect the brain APP,α-secretase,β-secretase, Caspase-3 mRNA levels. Immunohistochemistry was used to detect APP, Aβ1-40, cytochrome c and Caspase-3 expression levels in hippocampal CA1 area. Western blot was used to detect the APP protein expression levels, and the Aβ1-40 level was analyzed by ELISA.The water maze results showed that DEX and Aβwere able to extend the average escape latency, reduce the swimming time in platform quadrant and the number of crossing the platform. Ginsenoside Rg1 and AST (20, 40mg/kg) could increase the swimming time in platform quadrant and the number of crossing the platform. HE and Hoechst 33258 stain results showed that DEX and Aβcould lead to neuronal injury and apoptosis in hippocampal CA1 area, Rg1 and AST had protective effect to damage of neurons. Electron microscope examination showed that DEX and Aβcould induce the chromatin margination phenomenon, cytoplasm vacuoles, mitochondrial swelling and ridges disappear, and reduce the number of ribosomes in hippocampal neurons. Rg1 and AST (20,40mg/kg) could improve the ultra-structure. RT-PCR analysis showed that, DEX and Aβcould increase the APP695, APP751,β-secretase and Caspase-3 mRNA levels, reduceα-secretase mRNA level. AST (20,40 mg/kg) and Rg1 could decrease the APP695, APP751,β-secretase and Caspase-3 mRNA levels and increaseα-secretase mRNA level. Immunohistochemistry results showed that DEX and Aβcould increase the APP, Aβ1-40, cytochrome c and Caspase-3 expression in hippocampal CA1 area. AST and Rg1 could decrease the APP, Aβ1-40, cytochrome c and Caspase-3 expression. The Western blot and ELISA results showed that DEX and Aβcould increase the APP, Aβ1-40 expression and AST and Rg1 could decrease the APP, Aβ1-40 levels.Part III Protective effects of Astragalosides on PC12 cells injury induced by glucocorticoids and Aβand its mechanismIn this experiment we observed the injuries of DEX, Aβ, DEX and Aβco-application on PC12 cells and the protective effect of AST and its mechanism. MTT assay was used to detect the PC12 cells activity. PC12 cells apoptosis was detected by flow cytometer with Annexin-Ⅴ/PI double stain and PI single stain; RT-PCR was used to detect the APP,α-secretase,β-secretase and Caspase-3 mRNA level of PC12 cells. Western blot was used to detect APP protein expression of PC12 cells.MTT results showed that, DEX (0.1,1μmol/L) had no significant effect on PC12 cells activity, while DEX (5,10μmol/L) could significantly reduce PC12 cells activity (D5, P <0.05; D10, P <0.01). Aβ(1,5,10μmol/L) could decrease the PC12 cells activity after 24h treatment (P <0.05). DEX and Aβco-application could significantly decrease PC12 cells activity (P <0.01). Ginsenoside Rg1 (16μmol/L) and AST (10,20 mg/L) could increase PC12 cells activity induced by DEX and Aβ. The flow cytometer results showed that DEX and Aβtreated 24h could significantly increase the apoptotic rate of PC12 cells, Rg1 and AST could significantly decrease the apoptotic rate of PC12 cells. RT-PCR analysis showed that DEX and Aβco-application could significantly increase the APP770,β-secretase and Caspase-3 mRNA levels, lowerα-secretase mRNA levels. Rg1 and AST could decrease the elevated APP770,β-secretase and Caspase-3 mRNA levels and increase theα-secretase mRNA level of PC12 cells. Western blot analysis result showed that DEX and Aβcould increase the APP expression, AST and Rg1 could decrease the APP expression of PC12 cells.ConclusionStress level glucocorticoids could decrease the learning and memory function, impair the brain cortex and hippocampal neurons in mice. DEX and Aβco-application could significantly promote learning and memory impairment, increase neurons injury of hippocampal CA1 area in rats and promote the toxicity to PC12 cells. Its mechanism may be related to increase APP,β-secretase expression, decreaseα-secretase expression and lead to generate more Aβ. The mechanism may also relate to promote cytochrome c release and Caspase-9, Caspase-3 expression and activity which induced neuronal apoptosis. The study indicate that the ynergistic effects of GCs and Aβhave important role in the generation and progress of AD.AST could improve the learning and memory impairment and have protective effects on neuron and PC12 cells damage induced by DEX and Aβ. Its mechanism may be related to inhibit APP expression, decrease the formation of Aβ. And it may be related to decrease the neurotoxic effect of GCs and Aβ, inhibit neuronal apoptosis. The study indicate that AST may have prevent effects on the generation and progress of AD.