Inhibitory Effects And Mechanisms Of N-butanol Fraction Of Potentilla Anserine L. Against Hypoxia-induced Calcium Overload On Hippocampus Neuron

Objective: To study the inhibitory effects and mechanisms of n-butanol fraction of Potentilla anserine L. against Hypoxia- induced calcium overload on hippocampus neuron in rats.Methods: 1. Hipocampus neurons of Sprague-Dawley neonatal rats were cultured in vitro. 2. The best time of hypoxia for establishing of the model of hypoxia was determined through [Ca2+]i detection. 3. The cultured hippocampus neurons were divided randomly into control group, hypoxia injury model group,intervention group of Nimodipine (2umol/l)and intervention group of NP (0.2500mg/ml, 0.0625 mg/ml, 0.0156mg/ml). 4. The inhibition of calcium increasing happening in acute hyoxia injury on hippocampus neurons were detected by [Ca2+]i. 5.The effect of NP on acute hypoxia injury of cell morphology were observed through LSCM. 6.The effect of different dosage of NP on acute hypoxia injury of free radicals released were examined by biochemical indicators (NO). 7.Changes in expression of Calpain 1, Calpain 2, nNOS, NMDAR1, Cav1.3 mRNA were detected by PR-PCR. 8.Changes in expression of Calpain 1, nNOS protein were detected by immuno-histochemistry stain and Western Blot. Results: 1 The Morphological findings: after having been cultured for 7d, hippocampal neurons showed abundant cytoplasm, good refraction, cell wall was intergrity and smooth, the majority of neurons showed a multipolar morphology,axons and dendrites of neuron interlaced into reticulate.2 During three hours of hypoxia injury, the [Ca2+]i in model group degraded to the largest extent, and three hours were determined as the best time of hypoxia.3 The inhibitory effects of n-butanol fraction of Potentilla anserine L. on Hippocampus neuron of neonatal rats in hypoxia injury: (1) [Ca2+]i: NP at different dosage depressed [Ca2+]i (the high and intermediate doses P<0.01;the low dose P<0.05). (2) LSCM: after the treatment with NP, MAP2 hydrolization and fragmentation were lessened. (3) Biochemical Indicators: NP at different dosage decreased NO(the high and intermediate doses P<0.01;the low dose P<0.05). (4)RT-PCR: 1)NP at different dosage depressed the express levels of Calpain 1 mRNA on hypoxia injury neuron compared with model group (the high and intermediate doses P<0.01;the low dose P<0.05).2)no significant difference of the Calpain 2 mRNA level was observed between the control group and the model group (P>0.05). no significant difference of express level of Calpain 2 mRNA on hypoxia injury neuron was observed between the model group and NP intervention groups. (P>0.05). 3)The high and intermediate dose of NP depressed the express level of nNOS mRNA on hypoxia injury neuron significantly with comparing with the model group(P<0.01), low dose group didn't. (P>0.05). (5) Immuno-histochemistry Stain and Western Blot: : 1) Each dose of NP depressed the express levels of Calpain 1 protein on hypoxia injury neuron compared with model group(the high and intermediate doses P<0.01;the low dose P<0.05). 2)The high and intermediate dose of NP depressed the express levels of nNOS protein on hypoxia injury neuron compared with model group(P<0.01), no significant difference was observed between low dose group and model group(P>0.05).4 The possible protection mechanisms of n-butanol fraction of Potentilla anserine L. against calcium overload in Hippocampus neuron of neonatal rats: (1)Each dose of NP depressed the express level of NMDAR1 mRNA on hypoxia injury neuron compared with model group(the high and intermediate doses P<0.01;the low dose P<0.05). (2)Each dose of NP depressed the express levels of Cav1.3 mRNA on hypoxia injury neuron compared with model group (P<0.01).Conclusions: That n-butanol fraction of Potentilla anserine L. can attenuate the calcium overload induced by hypoxia 3h on Hippocampus neuron of neonatal rats. The possible mechanisms of this inhibitory effects might be: depressing the express levels of Calpain 1, depress MAP2 hydrolization, through inhibition the calcium overload of neurons, cell sytoskeleton preotection , depressing the express levels of nNOS and NO generation. The possible inhibition mechanisms of calcium overload might be : depressing the express levels of NMDAR1 and Cav1.3 mRNA.