| Objective: Di(2-ethylhexyl)phthalate(DEHP)is a relatively common type of Phthalates(PAEs).Food packaging and other aspects can increase the flexibility and plasticity of plastic products.Studies have shown that maternal exposure to DEHP during pregnancy can affect children's brain development,leading to decreased learning and memory skills and even decreased intelligence and attention deficits.Dendritic spine density and morphology can reflect the function of dendritic spines.Impaired dendritic spine function can affect learning and memory.At present,whether the maternal DEHP exposure affects the development of dendritic spines in the hippocampal CA1 pyramidal neurons is not distinct,and its related mechanism remains to be researched.This study aims to establish a model of DEHP exposure in pregnant and lactation to explore whether maternal DEHP exposure during pregnancy and lactation affects the development of dendritic spines in the hippocampal CA1 pyramidal neurons.Methods: Wistar pregnant mice were randomized into four dose groups: 0,30,300 and 750 mg/kg/day.Each group was intragastrically administered with a corresponding concentration of DEHP corn oil solution.In the pups PN7,PN14 and PN21,Evaluation of dendritic spine development in pyramidal neurons in the hippocampal CA1 region of the hippocampus by Golgi-Cox staining.G-actin and F-actin were separated by ultracentrifugation,and the ratio of G-actin/F-actin was analyzed.Western blot was used to detect the expression of dendritic spine development-related proteins in hippocampal.The activity of Rac1 protein was detected by pull-down method.Results: 1.Golgi-Cox staining of dendritic spines of pyramidal neurons in the hippocampal CA1 region of the pups,In male rats,dendritic spine density in hippocampal CA1 pyramidal neurons of 300 mg/kg/day and 750 mg/kg/day group was significantly decreased than that the control group(P<0.05);Compared with the control group,the dendritic spine density of the hippocampal CA1 pyramidal neurons in the 30 mg/kg/day group was significantly decreased only at PN21(P<0.05);Compared with the 30 mg/kg/day group,the dendritic spine density of the hippocampal CA1 pyramidal neurons in 300 mg/kg/day and 750 mg/kg/day group was significantly decreased(P<0.05);Compared with the 300 mg/kg/day group,dendritic spine density in the hippocampal CA1 pyramidal neurons of 750 mg/kg/day group was significantly decreased in PN14 and PN21(P<0.05).In female pups,there was no significant difference in dendritic spine density in the hippocampal CA1 pyramidal neurons at each time point(P>0.05).Statistical analysis of neuronal dendritic spine morphology,In the hippocampus of male pups,the proportion of slender spines increased relatively,the proportion of mushroom-type spines decreased,and the short-throated spines and bifurcations did not difference significantly.There was no significant difference in the morphology of dendritic spines(P>0.05);There was no significant difference in the morphological changes of dendritic spines in the hippocampus of female rats(P>0.05).2.Detection of cytoskeletal protein expression in hippocampus of rats by ultracentrifugation showed.In male pups,At each time point,the ratio of G-actin/F-actin in the hippocampus of 300 mg/kg/day group and 750 mg/kg/day group was increased compared with the control group,except for PN21 of 300 mg/kg/day,the rest were statistically significant(P<0.05);The ratio of G-actin/F-actin in the hippocampus of 750 mg/kg/day group was significantly increased than 30 mg/kg/day group(P<0.05).In female pups,there was no significant difference in the ratio of G-actin/F-actin in hippocampus of each exposed group at each time point(P>0.05).3.Western blot analysis showed that the developmental proteins related to dendritic spine in hippocampus of pups showed.There was no significant difference in the expression of cofilin,LIMK1 and PAK1/2/3 protein in hippocampus of male rats(P>0.05).Compared with the control group,the expression levels of p-cofilin,p-LIMK1,p-PAK1/2/3,and drebrin in the hippocampus of 300 mg/kg/day and 750 mg/kg/day group were significantly decreased(P<0.05);Compared with the 30 mg/kg/day group,the expression levels of p-cofilin,p-LIMK1,p-PAK1/2/3,and drebrin were decreased in the hippocampus of 750 mg/kg/day group,except p-cofilin in PN21.The decrease of p-PAK1/2/3 in PN14 was not significant,and the others were statistically significant(P<0.05).In female pups,there was no significant difference in the expression of dendritic spine-related protein in hippocampus of each exposed group at each time point(P>0.05).4.Detection of Rac1 protein activity was showed by western blot.At each time point,there was no significant difference in the expression of Rac1 protein in hippocampus of each exposed group(P>0.05).After GTP-Rac1 was isolated by pull-down assay,the expression of GTP-Rac1 was detected by western blot assay.In male pups,the expression of GTP-Rac1 in 300 mg/kg/day and 750 mg/kg/day group was significantly decrease than that in the control group(P<0.05);Compared with 30 mg/kg/day group,the expression of GTP-Rac1 in hippocampus of 750 mg/kg/day group was significantly decreased(P<0.05).In female pups,the expression level of GTP-Rac1 in hippocampus of each exposed group was consistent with Rac1,and there was no significant difference(P>0.05).Conclusion: 1.Dendritic spine development in the hippocampal CA1 pyramidal neurons of male rats was injured after DEHP exposure.2.Exposure to DEHP during pregnancy and lactation resulted in decreased dendritic spine density in hippocampal CA1 pyramidal neurons of male rats,which may be affected the expression of PAK/LIMK1/cofilin signaling pathway-related proteins in hippocampus and interfere with the construction of cytoskeletal actin. |
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