Effects Of Vitamin D On The Expression Of CaSR And PI3k/Akt Pathway In Hippocampal Neuronsincubated With Magnesiu-Free Extracellular Fluid

Objective: To investigate the effects of vitamin D(Vit D)on the expression of calciumsensitive receptor(Ca SR)and phosphoinositol 3 kinase/serine/threonine signaling pathways in primary hippocampal neurons of SD rats induced epileptic discharge in vitro.Hope to provide a new ideal for the clinical treatment of epilepsy.Methods: Hippocampal neurons of SD rats under 3 days of age were cultured in vitro until10 days later,and were divided into 7 groups by random number table method: normal control group,magnesium free group,magnesium free and sodium valproate group,magnesium free and vitamin D group,magnesium free and vitamin D and spermine group,magnesium free and vitamin D and NPS2390 group,magnesium free and vitamin D and LY294002 group.The model of patchy discharge was established by extracellular fluid induction without magnesium.The number and morphology of hippocampal neurons cultured for different days were observed under inverted microscope.Cell viability was detected by MTT assay,cell morphology was detected by HE staining and TUNEL staining,and protein expressions of intracellular hippocampal neurons were detected by Western-blot assay: Ca SR,VDR,PI3 K,P-PI3 K,Akt,PAkt,Bcl-2 and Bax.Results: Inverted microscope observation showed that the hippocampal neurons cultured for 10 days were mature with plump cell body,strong refractive power and dense neural network structure between axons,which could be used for experimental drug intervention.HE staining showed that after magnesium free intervention,hippocampal neurons became round,synapses became thinner and shorter,and there were signs of atrophy.A large number of hippocampal neurons in magnesium free and vitamin D and spermine group,magnesium free and vitamin D and LY294002 group showed pyknosis,swelling and rupture.TUNEL staining showed that compared with magnesium-free group,apoptosis rate of magnesium free and vitamin D group was decreased(P<0.05),magnesium free and vitamin D and spermine group,magnesium free and vitamin D group and LY294002 group were significantly increased(P<0.05).Western-blot analysis showed that compared with the normal group,the expressions of VDR and Bcl-2 were decreased in the magnesia-free group,while the expressions of Bax and Ca SR were increased.Compared with magnesium-free group,the expressions of VDR and Bcl-2 were increased in magnesium free and vitamin D group,the phosphorylation levels of PI3 K and Akt were increased,and the protein expressions of Bax and Ca SR were decreased.Compared with magnesium free and vitamin D group,the expression of VDR and Bcl-2 was decreased in magnesium free and vitamin D and spermine group and magnesium free and vitamin D and LY294002 group,and the expression of Bax and Ca SR was increased.Conclusion: Magnesium-free extracellular fluid induced hippocampal neuron injury and increased the expression of Ca SR.Vit D can alleviate magnesium-free hippocampal neuron injury,inhibit cell apoptosis and decrease Ca SR expression.Vit D can regulate PI3k/Akt signaling pathway,reduce epileptic hippocampal neuron injury,inhibit neuronal apoptosis,and protect nerve cells.