Effects And Mechanisms Of N-butanol Fraction Of Potentilla Anserine L. Against Hypoxia Injury On Hippocampus Neuron Of Rats

Objective: To study the protective effects and its mechanisms of n-butanol fraction of Potentilla anserine L on Hippocampus neuron of neonatal rats in hypoxia injury .Methods:①Hippocampus neurons of Sprague-Dawley neonatal rats (24h) were cultured in vitro, and then were identified by immunohistochemistry with anti-MAP2.②We ascertained the best time of hypoxia through LDH method and then established the model of hypoxia.③The dose of n-butanol fraction of Potentilla anserine L was ascertained through MTT and LDH methods. The cultured hippocampus neurons were divided randomly into control group,hypoxia injury model group and intervention group of n-butanol fraction of Potentilla anserine L (0.25mg/ml,0.0625mg/ml,0.0156mg/ml).④The effect of different doses of Potentilla anserine L on acute hypoxia injury of hippocampus neurons were examined by trypan blue staining, transmission electron microscope and biochemical indicators (LDH).⑤The possible mechanisms of acute hyoxia injury on hippocampus neurons were approached by biochemical indicators(SOD,MDA), RT-PCR (caspase-9, caspase-3), immuno-histochemistry stain and Western Blot (Cyt-c,Caspase-9,Caspase-3).Results: 1 The Morphological findings: After cultured 7d, mature hippocampal neurons showed abundant cytoplasm, good refraction, nuclei were often located in the center of the cell or leaned to one side, nucleus and nucleolus were clear, neurite and dendrite of neurons interlaced into reticulate.2 Identification of neurons: immunohistochemistry staining with anti-MAP2 demonstrated that 75.2±8.1% of the cultured cells were neurons.3 During three hours of hypoxia injury, the cells viability in model group degraded in the largest extent, so we ascertained three hours as the best time of hypoxia. we ascertained 0.25mg/ml,0.0625mg/ml,0.0156mg/ml as the high,intermediate and low dose of n-butanol fraction of Potentilla anserine L through MTT and LDH method.4 the effects of n-butanol fraction of Potentilla anserine L on Hippocampus neuron of neonatal rats in hypoxia injury: (1) MTT: the high and intermediate dose of n-butanol fraction of Potentilla anserine L increase the survival rate of neuron subjected to hypoxia(P<0.01), no significant difference was observed between low dose group and model group(P>0.05). (2) trypan blue staining :Each dose of Potentilla anserine L could decrease the death rate of neuron subjected to hypoxia. (3) Transmission Electron Microscope (TEM): after the treatment of n-butanol fraction of Potentilla anserine L, ultramicrostructure changes,such as the rupture of membrane, the membrane and cristae of mitochondria dissolved, the degranulation of rough endoplasmic reticulum, and the interstitial substance edemae were lessened.(4) Biochemical Indicators: each dose of Potentilla anserine L could decreased the activities of LDH(P<0.01).5 The possible mechanisms of n-butanol fraction of Potentilla anserine L on Hippocampus neuron of neonatal rats in hypoxia injury: (1) Biochemical Indicators: each dose of Potentilla anserine L could raise the activities of SOD (P<0.01), no significant difference of MDA was observed between control group, model group and intervention group. (P>0.05).(2) Immuno-histochemistry Stain :the high, intermediate and low doses of Potentilla anserine L could depress the express levels of cyt-c on hypoxia injury neuron compared with model group(P<0.01), the high, intermediate and low doses of Potentilla anserine L could depress the express levels of caspase-9 on hypoxia injury neuron compared with model group(the high and intermediate doses P<0.01;the low dose P<0.05), the high and intermediate doses of Potentilla anserine L could depress the express levels of caspase-3 (P<0.01) on hypoxia injury neuron compared with model group,no significant difference was observed between low dose of Potentilla anserine L group and model group. (3)RT-PCR: each dose of Potentilla anserine L could depress the express level of caspase-9 mRNA on hypoxia injury neuron compared with model group(the high and intermediate doses P<0.01;the low dose P<0.05). The high, intermediate and low doses of Potentilla anserine L could depress the express levels of caspase-3 mRNA on hypoxia injury neuron compared with model group (P<0.01).(4)Western Blot:each dose of Potentilla anserine L could depress the protein level of Cyt-c on hypoxia injury neuron compared with model group (P<0.01). Each dose of Potentilla anserine L could depress the express level of Caspase-9,Caspase-3 on hypoxia injury neuron compared with model group (the high and intermediate doses P<0.01).Conclusions: n-butanol fraction of Potentilla anserine L could improve the injury of neuron induced by hypoxia, lessen the degree of ultramicrostructure changes, and reduce the release of LDH, indicating its protection effect on Hippocampus neuron of neonatal rats in hypoxia injury. The possible mechanisms of this protection were: to elevate the cleaning ability to oxygen free radical, improve the function of mitochondria ,depress cyt-c level, then weaken caspase cascade reaction and intercept the happeness of death.