Mechanism Research Of Marginal Vitamin A Deficiency During Pregnancy Affect Offspring’s Learning And Memory By Rar Alpha, SRC And NR1 Signaling Pathway

Objective This study aimed to explore the role of RARalpha in affecting NR1 via regulating Src in learning and memory function of adult off-spring rat, who had marginal vitamin A deficiency(MVAD) since pregrancy. And to explore the correcation among RARa,Src and NR1.Methods Establish the model of VAN and MVAD during pregnancy (VAN and MVAD group); Culture the primary hippocampus neurons from two groups of newborn rat, and use the cell immunofluorescence staining to identify; Real-time RCR and western blot are used to detect the mRNA and protein expression of RAR alpha, SFKs and NR1, from the newborn rat hippocampus and neurons before and after the RA treatment; Using immunofluorescence staining to detect the change of fluorescence about the three indicators (RAR alpha, SFKs and NR1) after treating with RA; Using the silence and overexpression RAR alpha and no-load adenovirus transfect neurons and test the mRNA expression of three indicators (RAR alpha, Src and NR1); Using western blot to test three target protein expression of the neurons which was treated by Src family inhibitor PP2;Using Co-Immunoprecipitation to probe whether there is interactions between RAR alpha and Src or NR1;Using the calcium imaging instrument to detect the calcium excitability of neurons from two groups and RA treatment; Using Morris water maze to detect the spatial learning and memory ability of 7 weeks old rat from two groups.Results① Successfully establish the rat model of MVAD and VAN during prangancy;② The immunofluorescence results showed that 95% of the isolated cells expressed the neuron maker neuron-specific enolase (NSE);③The expressions of RARα, Src and NR1 in the hippocampus and neurons of MVAD newborn rats during pregnancy were significantly decreased compared with VAN group (P<0.05).④The calcium excitatory of MVAD group neurons was obviously lower than that in VAN group (P< 0.05);⑤The Morris water maze experiment found the function of learning and memory about the 7weeks old offspring from MVAD groups was lower than that in VAN group(P<0.01);⑥After 24h treatment by 1umol/L RA, the mRNA and protein expression of RAR alpha, SFKs and NR1 in neurons were significantly increased (P<0.001), immunofluorescence staining showed that the dendritic growth significantly and the RAR alpha expression in the dendritic was increased significantly, and the calcium excitability of RA treated neurons was significantly increased (P<0.01);⑦ By transfect the silence and overexpression RAR alpha and no-load adenovirus to neurons, the mRNA expression changes of Src was consistent with RAR alpha, and the change was statistically difference (P<0.05), and NR1 have no significant change; ⑧After treated by the family of the Src kinase inhibitor PP2, the protein expression of NR1 have significantly decreased, with no significant change of RAR alpha protein;⑨Co-immunoprecipitation found that RAR alpha interacted with Src, and there was no interaction between RAR alpha and NR1;⑩ Water maze training found that, after water maze training, the serum VA concentration and mRNA expressions of RAR alpha, Src and NR1 were stable, while water maze experiments can increase the protein expression of RAR alpha, Src and NR1.Conclusion MVAD during pregnancy can affect learning and memory function of offspring rat, and the mechanism may be related to RA signaling pathway, RAR alpha in RA signaling pathway may influence the expression of Src through transcriptional and non-transcriptional regulation, and then affect the expression of NR1 and inflow of calcium, thereby affect the hippocampal synaptic plasticity and learning and memory function.