| Di-(2-ethylhexyl) phthalate (DEHP) is a typical phthalates, a widely used plasticizer. It is easy to break out from the plastic polluting the environment. Humans and wildlife have been exposed to them continuously. A large number of animal experiments show that, DEHP is a kind of environmental endocrine disruptors, leading to produce reproductive and developmental toxicity, genotoxicity and multiple organ damage such as cancer. But now little is known for its neurodevelopmental toxicity. Endogenous sex hormones play an important role on the development of neuronal dendrites and occurrence of synapses, which thereby affecting the development and function of the brain. As DEHP has anti-androgen and estrogenic activity, it may interfere with the body’s normal physiological activity of the sex hormones, affecting the developing nervous system of the individual. Our previous results suggest that perinatal exposure to DEHP causes learning and memory deficits and anxiety and depression-like behavior, which indicated DEHP may affect neurobehavioral development of animals. Given the morphological structure is the basis of neuronal activity, we hypothesized that DEHP may affect neuronal development.In the early development of neurons, there were many growth cones and filopodias on dendritic shafts, after which the dendritic filopodia gradually being replaced by thick and short, relatively stable form of dendritic spines. While more than 90% of excitability synaptogenesis occurred in dendritic spines. The dendritic spine, which is maturation and stability, is important structural basis to generate links between synapses. Greater synaptic density represents the nervous system which has a very complex network of synaptic connections and greater plasticity potential. Therefore, the development of dendritic spines of neurons, the formation of synapses and neural circuits’establishment constitute the key process to brain development. Estrogen binds to receptors, activating a series of signaling pathways involved in the regulation of dendritic growth and synaptic plasticity. Changes on key proteins affect the effectiveness of transmit between synapses in neurons directly. Synapsin I is located on the presynaptic membrane as a marker. After extracellular regulated protein kinase (ERK) pathway was activated, Synapsin I can be phosphorylated which involved in the regulating synaptic release of neurotransmitters. PSD95 is a postsynaptic scaffolding protein on postsynaptic density, links and anchor N-methyl-D-aspartic acid receptor(NMDAR), a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor(AMPAR) and downstream signaling molecules in the postsynaptic membrane, whose close contact on this structure plays an important role in synaptic plasticity. Whether DEHP affects dendritic and synaptic development by estrogen similar way, we do not know.In this study, DEHP was exposured to cultured hippocampal neuronal dendrites and synaptic development in vitro, we used estrogen receptor blocker ICI 182,780 and western blot techniques to explore the molecular mechanisms of exposure to DEHP.Method:Neonatal SD rats within 24 h of the hippocampus were cultured in vitro. Then we added DEHP to the culture on the fifth day (DIV 5) for five days. The exposure time is 5 days. First, we setted the control group (DMSO), DEHP group (1、 10、100 nM,1 and 10μM) to explore the dose effect of DEHP on neuron. When cells were DIV10, we used immunofluorescence staining to observe neuronal dendrites and synaptic morphology. Secondly, choose a very significant dose effect (1 nM or 1 μM) as further study group, setting up the control group, DEHP group, estradiol (E2) group,’ DEHP+E2 group to explore the relationship between DEHP and E2. Then, estrogen receptor antagonist ICI 182,780 was used to explore whether the effect of DEHP on morphological development in neurons via estrogen receptor.We used rhodamine-conjuncted phalloidin to labele fibros actin (F-actin) specificaly, observing dendritic morphology via laser scanning confocal microscope, detectting density of dendrite filopodias and dendritic spine. In order to explore the impact of chronic exposure to DEHP on synapse density, we used immune double staining to label a presynaptic protein(Synapsin I) and cytoskeletal protein(F-actin), where Synapsin I (green fluorescence) and F-actin (red fluorescence) colocalized were defined as synapses (yellow fluorescence). Then we analyse the impact of long-term exposure to DEHP on synapse development.In order to further investigate the molecular mechanisms of DEHP exposure on neuronal morphology, Western blot method was used to detect the protein expression of Synapsin I, PSD95 and NR2B subunit of NMDAR in neurons, analysising the alterations of synaptic proteins and excitatory amino acid receptors. Then we detected protein expression of ERK and p-ERK to explore whether NMDAR and ERK signaling pathway were involved in long-term exposure of DEHP on neuronal development.The results:1. DEHP (1 nM-10 μM) exposure to neurons for five days, a significant reduction in hippocampal neuronal dendrite filopodias and dendritic spine density (P<0.05, P<0.01) were exibited, the effect curve was "U" type, suggesting the inhibition of DEHP on neuronal dendrites development. Compared to E2 group, (1 nM or 1 μM) DEHP+E2 group was significantly inhibited(P<0.01), prompting that DEHP displayed an antagonistic effect with estrogen; compared with DEHP group, the density of filopodias and dendritic spines were significantly increased (P<0.01) in DEHP+ICI group, but still decreased to ICI group (P<0.01), suggesting the estrogen receptor blockers eliminate the inhibition of DEHP partly, indicating that estrogen receptors may be involved in the inhibitory effect of DEHP on the growth of neuronal dendrites.2. (1 nM-10μM) DEHP exposure to cultures for five days, a significant reduction in hippocampal synaptic density (P<0.01) was occurred. Conpared to control group,10 nM DEHP group had no effect on the postsynaptic density; the effect curve was "U" type, suggesting that DEHP inhibits neuronal synapse development; compared with the E2 group, which increased synaptic density(P<0.01), 1 nM or 1 μM DEHP+E2 group was significantly deduced(P<0.01), prompted DEHP showed antagonistic effects interacting with estrogen; compared to DEHP group, synaptic density in DEHP+ICI group were significantly increased (P<0.01), but DEHP+ICI group still exhibited significant declined(P<0.01) to ICI group, which suggested that estrogen receptor blockers can partially relieve the inhibitory effect of DEHP, porting that estrogen receptor may be involved in adverse effects of DEHP on synaptic development.3. Long-term exposure to DEHP (1 nM and 1 μM), significantly reduced NMDAR subunit NR2B (P<0.05 or P<0.01), synapsin I (P<0.01 or P<0.01) and PSD95 (P<0.01 or P<0.01) in the protein expression level, significantly reduced the activity of p-ERK (P<0.01 or P<0.01).Conclusion:Long-term exposure to DEHP down regulated dendrite filopodias, dendritic spines and synaptic density in cultured rat hippocampal neurons; while reducing the expression of synaptic proteins PSD95, SynapsinI and NMDAR NR2B subunit, p-ERK activity decreased as well, suggesting that DEHP inhibit the development of dendrite and synapse in neurons which probably binds to the estrogen receptor bind, deduced protein expression via ERK pathway to affect development of dendrites and synapses in neurons. Furthermore, DEHP exhibit antagonistic effect. Long-term exposure to DEHP induced inhibition on dendrites development and synaptic occurrence in hippocampal neurons, which may be one reason why children, who were exposed to DEHP, suffered the emergence of a variety of neurobehavioral defects. |
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