Research Of Mechanisms Of Salidroside Against Hypoxia Injury On Hippocampus Neuron Of Rats

Objective: To study the effects and mechanisms of salidroside against hypoxia injury on Hippocampus neuron of rats.Methods: 1 Hippocampus neurons of Sprague-Dawley neonatal rats (24h) were cultured in vitro, and then identified by immunocytochemistry with anti-MAP2. 2 With the best time ascertain of physical hypoxia and chemistry hypoxia by method of [Ca2+]i detection and MTT individually, the models of physical and chemistry hypoxia were established. 3 The dose of salidroside was ascertained through MTT method. The groups were divided into control group, model group( physical and chemistry),salidroside group of high concentration (1mM), salidroside group of intermediate concentration (0.25 mM), salidroside group of low concentration (15.6μM), positive control group of physical hypoxia (Nimodipine (0.2μM)) and positive control group of chemisty hypoxia (diazoxide (100μM)). 4 The effects of salidroside at different concentration on hypoxia injury of hippocampus neurons were examined by trypan blue stain, hematoxylin and eosin stain. 5 The effects of salidroside at different concentration on calcium concentration of hippocampus neurons injuried by physical hyoxia on were researched by [Ca2+]i detected with fluorospectrophotometry. 6 The effects of salidroside at different concentration on cell morphology of hippocampus neurons injuried by physical hypoxia injury were observed through LSCM. 7 Changes in expression of NMDAR1, Cav1.3 mRNA in physical hypoxia injury and Kir6.2, SUR1 mRNA in chemistry hypoxia injury were detected by real-time PCR. 8 Changes in expression of Calpain 1 protein in physical hypoxia injury were detected by immunocytochemistry stain, changes in expression of Kir6.2, SUR1 protein in chemistry hypoxia injury were detected by immunocytochemistry stain. Results: 1 The Morphological findings: after having been cultured for 7d, hippocampal neurons showed abundant cytoplasm, good refraction, the cell wall was intergrity and smooth, the majority of neurons showed a multipolar morphology,axons and dendrites of neuron interlaced into reticulate.Immunocytochemistry staining with anti-MAP2 demonstrated that 82.3±7.1% of the cultured cells were neurons.2 Establishing the model of hypoxia: The [Ca2+]i upgraded following the prolongation of the hypoxia time. The [Ca2+]i after hypoxia for three hours was higher than that for two hours(P<0.01), but it had no significant difference between it and that for 4 hours(P>0.05). Then three hours were determined as the best time of physical hypoxia. 10 minutes was ascertained in chemistry hypoxia by MTT method.3 1 mM,0.25 mM,15.6μM was ascertained as the high, intermediate and low dose of salidroside through MTT method.4 Effects of salidroside against hypoxia injury on Hippocampus neuron of rats: (1) The consequence of MTT method showed that the high, intermediate and low dose of salidroside increased the survival rate of neuron subjected to hypoxia (the high and intermediate doses P<0.01; the low dose P<0.05). (2) The consequence of trypan blue staining showed that every dose of salidroside decreased the death rate of neuron subjected to hypoxia. (3) The consequence of HE staining showed that each dose of salidroside decreased the injury extent of neuron subjected to hypoxia. (4) Salidroside at different dosage depressed [Ca2+]i (the high and intermediate doses P<0.01; the low dose P<0.05). (5) The consequence of LSCM showed that salidroside at different dosage depressed [Ca2+]i in Hippocampus neuron sujected to physical hypoxia. (6) The consequence of immunocytochemistry stain showed that the expression level of Calpain 1 was increased in model group of physical hypoxia injury compared with that of the normal group(P<0.01), the high, intermediate and low doses of salidroside depressed the expression level of Calpain 1 compared with that of the model group (the high and intermediate doses P<0.01; the low dose P<0.05). 5 The mechanisms of salidroside on Hippocampus neuron of neonatal rats in hypoxia injury: (1) The results of Real-time PCR detection showed that in the physical hypoxia injury, the expression level of NMDAR1 mRNA was increased in the model group compared with that in the normal group (P<0.01), every dose of salidroside depressed the express level of NMDAR1 mRNA on hypoxia injury neuron compared with model group (P<0.05). The expression level of Cav1.3 mRNA was increased in model group compared with that in the normal group(P<0.01), the high and intermediate doses of salidroside depressed the expression level of Cav1.3 mRNA on hypoxia injury neuron compared with that in the model group (P<0.01). In the chemistry hypoxia injury, with giving the sodium sulphoxylate for 1h, the expression levels of Kir6.2 mRNA and SUR1 mRNA were depressed in the model group compared with that in the normal group (P<0.01), each dose of salidroside increased the expression level of Kir6.2 mRNA on hypoxia injury neuron compared with that in the model group (P<0.01), each dose of salidroside increased the expression level of SUR1 mRNA on hypoxia injury neuron compared with that in the model group (P<0.05). (2) The consequence of immunocytochemistry stain showed that each dose of salidroside increased the expression level of Kir6.2 protein on chemistry hypoxia injury neuron compared with that in the model group (P<0.01). Each dose of salidroside increased the express level of SUR1 protein on chemistry hypoxia injury neuron compared with that in the model group (P<0.05).Conclusions: Salidroside has the protective effect on Hippocampus neuron of neonatal rats injured by physical hypoxia and chemistry hypoxia. Salidroside attenuates the calcium overload induced by physical hypoxia by depressing the expression level of NMDAR1 and Cav1.3 mRNA. Salidroside protects neuron injured by chemistry hypoxia through increasing the expression level of Kir6.2 and SUR1 mRNA.