Transgenerational Inheritance Of Hippocampal Synaptic Damage Induced By Maternal Di(2-ethylhexyl)phthalate Exposure Via Long Non-coding RNA MALAT1 Regulating CREB In Paternal Male Offspring

Objective: Di-2-ethylhexyl phthalate(DEHP)is a common plasticizer,widely used in medical equipment,plastic containers and toys,food packaging,cosmetics,drugs and food supplements,etc.In recent years,due to the large amount of exposure of DEHP in the environment,it has become one of the main issues of human concern.DEHP can have many adverse effects on human health.Brain tissue is one of the most important organs threatened by DEHP.A large number of studies have shown that maternal exposure to DEHP can inhibit brain growth and affect memory and cognition of offspring.Hippocampus plays a vital role in learning and memory.Some evidence shows that in the early stage of development,the hippocampus is highly sensitive to phthalates,which will cause damage to the growth and neural structure of the hippocampus.In recent years,some studies have proposed that endocrine disruptors have cross-generational effects on brain development and behavior.Transgenerational effect may be mediated by epigenetic mechanism.Metastasis associated lung adenocarcinoma transcript 1(MALAT1)is a long-chain non-codingRNA that plays an important role in regulating the formation of synapses.In this study,the effects of maternal DEHP exposure on hippocampal dendrite development and synaptic morphological plasticity of F3 generation male offspring were investigated by establishing a paternal transgenerational genetic animal model of rats exposed to DEHP during pregnancy and feeding.We also explored the mechanism of lncRNA MALAT1 regulating synaptic development through c AMP response element binding protein(CREB),in order to clarify the transgenerational genetic effect of maternal DEHP exposure on hippocampal synapses of F3 male offspring and its related mechanism.Research methods: Healthy female adult Wistar rats(Beijing Sibeifu Biotechnology Co.,Ltd.)were randomly divided into 4 groups according to body weight: 0mg/kg/day group,30 mg/kg/day group,300 mg/kg/day group and 750 mg/kg/day group.From the first day of pregnancy(GD1)to the 21 st day after birth(PND21),the corresponding dose of DEHP corn oil solution was used to gavage each group,and the offspring of the female rats exposed to DEHP were propagated,and the F3 generation of male offspring were taken for subsequent experiments.When the F3 offspring PND21,the hippocampus was taken for Golgi staining to analyze the total length of the basal and apical dendrites of the pyramidal neurons in the CA1 area and the complexity of the dendritic branches;The changes of synaptic structure in hippocampal CA1 area of offspring rats were observed by transmission electron microscope,and the width of synaptic gap,the thickness of postsynaptic dense substance and the curvature of synaptic interface were measured;The expression of lncRNA MALAT1 and CREB in the hippocampus of offspring rats was detected by q RT-PCR;Western blot was used to detect the protein expression levels of CREB and postsynaptic density 95(PSD95)in the hippocampus of male offspring.The F3 generation male offspring were injected with lncRNA MALAT1 gene interfering adenovirus into the lateral ventricle,and the expression of CREB after lncRNA MALAT1 knockdown was detected by q RT-PCR;Western blot was used to detect the protein levels of CREB and PSD-95 after MALAT1 knockdown;Golgi staining and transmission electron microscopy were used to observe the changes of hippocampal dendritic length,branch complexity and synaptic structure.Results:(1)Golgi staining results showed that after exposure to DEHP,the length of basal dendrites in hippocampal CA1 area of F3 generation male offspring became shorter,the number of dendritic branches decreased,and the complexity of dendritic branches decreased(p<0.05).(2)The results of transmission electron microscopy showed that,compared with the control group,the synaptic gap in the hippocampus of F3 generation male offspring rats after DEHP exposure significantly widened,the thickness of PSD became thinner,and the curvature of the synaptic interface decreased(p<0.05).(3)The results of q RT-PCR showed that the expression of lncRNA MALAT1 in the hippocampus of F3 generation male offspring rats after DEHP exposure increased,while the expression of CREB decreased(p<0.05).(4)The results of Western blot showed that the levels of CREB and PSD95 protein in the hippocampus of F3 generation male offspring rats significantly decreased after DEHP exposure(p<0.05).(5)After the treatment of lncRNA MALAT1 knockdown,the expression of MALAT1 decreased significantly,while the expression of CREB increased significantly.The results of Western blot showed that the level of CREB and PSD95 protein increased significantly(p<0.05).(6)Golgi staining and transmission electron microscopy showed that MALAT1 knockdown restored the damage caused by maternal DEHP exposure to the hippocampal dendrites and synapses of F3 generation male offspring.Conclusion:(1)Maternal DEHP exposure resulted in the reduction of the length and branching points of the basal dendrites of the pyramidal neurons in the hippocampal CA1 area of the F3 generation male offspring,and the reduction of the complexity of the dendrites and branches(2)Maternal DEHP exposure could damage the synaptic morphology of the hippocampal CA1 area of the F3 generation male offspring.(3)The mechanism of maternal DEHP exposure leading to hippocampal synaptic transgenerational damage in F3 paternal male offspring may be related to the downregulation of CREB/PSD95 mediated by lncRNA MALAT1.