Protective Effects On Cerebral Ischemia/Reperfusion Injury Of Astragalosides And Its Mechanism

Astragalus is a traditional Chinese medicine, as legumes Mongolia astragalus or membranaceus (Fisch) Bge's dry pods root, which contains saponins, flavonoid glycosides category, sugars, amino acids and trace elements. Astragalosides(AST) is the main component of astragalus with the function of antioxidant, immune regulation, promotes intelligence, which was commonly used in the prevention and treatment of cardiovascular and cerebrovascular diseases, aging, immune function disorders and other diseases. In this paper, the focal cerebral ischemia reperfusion (I/R) model and hippocampal neurons anoxia-reoxygenation model were established to explore the protective effects of AST on the nerve function and learning and memory function after cerebral ischemia-reperfusion injury and its mechanism.PartⅠEffect of astragalosides on the learning and memory after brain ischemia/ reperfusion injury and its mechanismTo explore the effect of AST on the learning and memory function and its mechanism on MCAO rats. Morris water maze was used to observe the effect of AST on the escape latencies and swimming distance. NGF and BDNF protein expression were tested by immunohistochemistry, while the activity of p-ERK,p-JNK and p-Akt were measured by western blot. The results showed that AST(80mg/kg) could significantly shorten escape latencies and swimming distance at days5, 6 and 7 after I/R; AST(40mg/kg) could significantly shorten escape latencies at days 6 and 7, prolong swimming distance at days 5, 6 and 7 after I/R; AST (20mg/kg) could significantly shorten escape latencies of at day 7 and swimming distance at days 6 and 7. Compared with model group, AST (20,40,80mg/kg) increased the expression of NGF and BDNF; AST(40,80mg/kg) increased the expression of p-ERK, p-Akt and decreased the expression of p-JNK.PartⅡProtective Effect of Astragalosides against Focal Cerebral Ischemia-reperfusion in ratMale SD rats received right middle cerebral artery occlusion of 120 minutes'duration. The rats were decapitated at different reperfusion time points: days 1, 3, 7, and 14 of recirculation. The effect of AST on the weight of rats, neurological function score, the index of brain, the water content and infarction volume of brain were measured; the activity of superoxide dismutase (SOD), lactate dehydrogenase (LDH) and nitric oxide synthase (NOS), and the content of malondialdehyde (MDA), lactate (LD) and a nitric oxide (NO) of brain tissue were detected too. Using hematoxylin-eosine (HE) staining to observe brain tissue pathological changes, iNOSmRNA, NGFmRNA, BDNFmRNA and p75NTR mRNA expression were measured by RT-PCR; the expression of TrkAmRNA and TrkB mRNA were measured by real-time PCR.The results are as follows:1 The body weight reduced at days1, 3, 7, and 14 on MCAO rats after I/R, and AST (40mg/kg) could significantly increase body weight at days 3, 7and 14.2 The neuroolgical function score peaked at day 1, then began to decline, and returned almost to the sham level at day 14; AST (40mg/kg) could obviously reduce the neurological function score at day 3.3 The brain index increased beginning at day 1, peaked at day 3, and returned almost to the sham level at day 14; AST (40mg/kg) could significantly decrease of brain index at days 3 and 7.4 The water content increased beginning at day 1, and returned almost to the sham level at day 14; AST (40mg/kg) could significantly decrease the water content at days 3 and 7.5 The brain infarct volume increased beginning at day 1, peaked at day 3, then began to decline; AST (40mg/kg) could significantly reduce the infarct volume at days 3, 7 and 14.6 The activity of SOD decreased and the content of MDA increased at days 1, 3 and 7; AST (40mg/kg) could significantly increase SOD activity and decrease brain tissue MDA content at days 3 and 7.7 The activity of LDH significantly decreased and the content of LD increased at days 1, 3 and 7; AST (40mg/kg) could significantly increase LDH activity and reduce the level of LD at days 3 and 7.8 The activity of NOS and the content of NO increased beginning at day 1, peaked at day 3, and returned almost to the sham level at day 14; AST (40mg/kg) could significantly decrease NO content and NOS activity at days 3and 7.9 The expression of iNOSmRNA in the rat brain tissue significantly increased at day 1, peaked at day 3, and returned almost to the sham level at day 14; AST (40mg/kg) could reduce the expression of iNOSmRNA at days 3 and 7. 10 AST (40mg/kg) could relieve the brain tissue histopathologic injury at days 3,7 and 14.11 The expression of NGFmRNA in the rat brain significantly increased at days 1,3,7 and 14, peaked at day 7; AST (40mg/kg) could increase the expression of NGFmRNA at days 3,7 and 14.12 The expression of BDNFmRNA in the rat brain increased at days 1,3,7 and 14, peaked at day 3; AST (40mg/kg) could significantly increase the expression of BDNFmRNA at days 3,7 and 14. 13 The expression of p75NTRmRNA in the brain tissue significantly increased at days 1,3 and 7, peaked at day 3, and returned almost to the sham level at day 14; AST (40mg/kg) could obviously decrease the expression of p75NTRmRNA at days 3 and 7.14 The expression of TrkAmRNA in the brain tissue significantly increased at days 1,3 and 7, peaked at day 7, and returned almost to the sham level at day 14; AST (40mg/kg) could increase the expression of TrkAmRNA at days 7 and 14.15 The expression of TrkBmRNA increased at days 1,3 and 7; AST (40mg/kg) could increase the expression of TrkBmRNA at days 3 and 7.PartⅢProtective effect of astragalosides against injury induced by anoxia/reoxygenation on hippocampal neuron and its mechanismTo observe the protective effects and its mechanism of AST on primary cultured hippocampal neurons after anoxia and reoxygenation (A/R). An A/R model was established using cultured primary hippocampal neurons which derived from 18 day embryonic rat. MTT assay method and LDH releasing rate were used to measure the cell viability, which evaluated the cellular injury. We detected the activity of SOD and the content of MDA and NO at the same time. The apoptosis rate of hippocampal neurons after A/R was measured by hoechst 33258 staining and flow cytometry with AnnexinV-PI staining. The intracellular calcium was observed with a cofocal laser - scanning microscope and determined by mean fluorescent value with Fluo-3/AM. The results showed that the cell viability of A/R group was reduced markedly, the AST(10, 20, 40μg/ml) significantly enhanced the cell viability of hippocampal neurons before anoxia and AST(40μg/ml) also enhanced it after anoxia. The LDH releasing rate of model group increased markedly, AST(10, 20, 40μg/ml) significantly reduced the LDH releasing rate. The activity of SOD decreased and the content of MDA and NO increased significantly after A/R, but AST (10, 20, 40μg/ml) could enhance the SOD activity and reduce the MDA and NO contents markedly. Compared with the A/R group, AST(10, 20, and 40μg/ml) could reduce the A/R -induced apoptosis and decrease the fluorescence intensity value of [Ca2+]i.Conclusions: It was found that AST could improve the rats learning and memory function after brain ischemia/reperfusion injury, the mechanism may related with anti-oxidation, increasing the expression of NGF, BDNF, TrkA and TrkB, decreasing the expression of p75NTR, inhibiting the hippocampal neurons apoptosis, up-regulating the expression of p-ERK and p-Akt, down-regulating the expression of p-JNK.