Effects Of N-Acetylcysteine Interfere BDE-209 On The Hippocampal Neurons Of Newborn Rat In Vitro And Its Related Mechanism

【Background】1.About Polybrominated Diphenly Ethers (PBDEs)PBDEs are flame retardant brominated compounds. PBDEs have a total of 209 kinds of isomers according to the different number and location of bromine atoms on the benzene ring.PBDEs are often used as flame retardant additives to be added into electron equipment or electrical appliance, automatic controller, building materials , textile product and other fields. PBDEs are difficult to be dissolved, long residence time, having strong lipotropic and hydrophobic property, bioaccumulation and biological amplification. PBDEs can enter the human body through food, breast-feeding and respiratory.PBDEs can be formed persistent organic pollutants,which cause potential enormous harm on the environment and human. Therefore, experts have also called that PBDEs is the "chemical time bomb."Previous research has shown that low-brominated PBDES can lead to secretion of thyroid hormone disorders, neurological and behavioral changes and malignant tumors. The current greatest concern for potential adverse health effects of PBDEs relates to their developmental neurotoxicity. Such concern is motivated by the following consideration: 1) Animal studies, carried out with different PBDEs, have indicated that pre-and post-natal exposures to PBDEs may cause long-lasting behavioral alterations, particularly in the domains of motor activity and cognitive behavior. Neurochemical changes have also been found following developmental exposure to PBDEs; 2) PBDEs affect thyroid hormone homeostasis, which may result in developmental neurotoxicity; 3) There is indication that young animals may have a reduced ability to excrete PBDEs, and pups have higher tissue (including brain) concentrations than the dams; 4) PBDEs are excreted in milk, and relatively high concentrations are found in North America; 5) Dust has been found to be a major source of exposure. Therefore, in the United States and Europe, low-brominated PBDEs such as BDE-47,99,153 and so on have been limitted to production and use.2.About brominated diphenly ethers-209(BDE-209,decaBDE)BDE-209 is a kind of high bromine PBDES containing 10 bromine atoms, which has good thermal stability, less addition amount, cheap and other advantages. It is also the most widely used PBDEs. due to its biological accumulation effect, BDE-209 concentration in the blood will continue to rise. Some Researchers think PBDEs exposure in the perinatal period can effect the capacity spontaneous behavior and learning and memory of next generation adult rats. Some research also proved that BDE-209 has a neural developmental toxicity, immunotoxicity, endocrine toxic and carcinogenecity. However, some researchers also believe that the metabolism of BDE-209 in the body is very fast, having low accumulation, and will not bring impact on the body under normal exposure level. Due to inconsistency of the results, researches on the biological toxicity of BDE-20 are becoming necessary.Our research group has demonstrated that exposure to BDE-209 during the pregnancy and lactation period can cause learning and memory barriers and immune dysfunction of the rats. And further study of its mechanism may be associated with oxidative stress enhanced, apoptosis increased.3.About N-acetyl-L-cysteine (NAC)NAC can increase the glutathion(GSH) content of the body. NAC as a small molecule material, are easy to enter the cells.NAC can change into precursors of GSH synthesis after deacetylation, then promote the synthesis of GSH,so it can improve GSH content of organization which decrease the damage from free radicals, drugs and poisons. Glutathion is three peptides that is a combination of oglutamic acid, cysteine and glycine. Sulfhydryl of the cysteine is its active groups. GSH has a variety of important functions as the antidote, anti-oxidation, etc. In vivo and in vitro experimental evidence prompted that NAC can enhance intracellular GSH biosynthesis can be interpreted the process of protection in a variety of diseases. A lot of studies have shown that NAC can inhibit oxidative stress of a variety of toxicant, which reduce apoptosis and injury of cell or tissue in vivo and in vitro.In this study, from the molecular level, the hippocampal neurons of newborn rat is expose to BDE-209 in vitro and protective effects of NAC were observed by observing the form of nerve cells and synapse development under light microscope, by detection of apoptosis, the expression of P38MAPK, calcium ion content and ROS oxidation stress indicators. The nerve toxicity mechanism of BDE-209 and protective effects of NAC is explored into. The study is divided into four parts for the specific experiments. Part IEFFECTS OF N-ACETYLCYSTEINE INTERFERE BDE-209 ON MORPHOLOGY IN NEWBORN RAT HIPPOCAMPAL NEURONS[Objective]:The hippocampal neurons of primary culture newborn SD rat,Identification of neurons in the purity.Observation of BDE-209 exposed on structure and cell survival rate in newborn rat hippocampal neurons.[Material and Methods]:1. The SD newborn rats,From newborn rat hippocampus neurons in a primary cell culture.2. Nerve cells purity of identification that the hippocampus Neurons of training 7 d .3. The BDE-209 Exposure on the hippocampal neurons of primary culture newborn rat after 7d .Experiment is divided into ten groups,That is, the blank control group ,the DMSO control group and experimental A, B, C Group(BDE-209 concentrations were 10.0,30.0 and 50.0 ug / ml),the NAC control group, the NAC+DMSO control group and intervention D, E, F group(NAC concentrations were 0.1 mmol/l added in at the same time).Each section will parallel three.The control group joined with the 1‰DMSO medium.The inverted microscope under the observation of cells afer 24h.4. MTT colorimetric analysis detecting neuron survival. [Results]: 1. The observation of Primary neurons in cell culture of seven days neurons were clear and bright , Was significantly raised, Halo around-obviously,Nuclei and the nucleolus clearly visible, longer processes and branches, Edge clear, Obviously the formation of neural networks.2. Immuocytochemistry identified neurons and glial cells. Results from that Neurons is the main account for 95%.3. Observation of different doses of nerve cells form under Inverted microscope.With the increasing concentration of BDE-209,Emerging deep into the nuclear chromatin, Nuclear enrichment, Agglutination , Processes was reduce and disappeared.4. MTT colorimetric analysis detecting neuron survival. The neuron survival in hippocampus was decreased with increased exposure to BDE-209,and The experimentB,Cgroups had significant difference from control group(P< 0.0 1). The intervention group compared with the experimental group, there is statistical significance(P<0.01).[Conclusions]:The BDE-209 can lead to apoptosis in primary culture of rat hippocampal neurons during some range of dose and time. The BDE-209 can cause newborn rat hippocampal nerve cells and synapses change in the structure. The neuron survival in hippocampus was decreased with increased exposure to BDE-209. NAC can interfere with the toxicity of BDE-209, making significant improvement in cell morphology and cell survival.Part II EFFECTS OF N-ACETYLCYSTEINE INTERFERE BDE-209 ON APOPTOSLS AND EXPRESSION OF P38MAPK IN NEWBORN RAT HIPPOCAMPAL NEURONS [Objective]:To investigate the efects of BDE-209 on apoptosis ,expression of P38MAPK and protection efects of NAC in newborn rat hippocampal neurons.[Material and Methods]:1. The BDE-209 Exposure on the hippocampal neurons of primary culture newborn rat after 7d .Experiment is divided into ten groups,That is, the blank control group ,the DMSO control group and experimental A, B, C Group(BDE-209 concentrations were 10.0,30.0 and 50.0 ug / ml),the NAC control group, the NAC+DMSO control group and intervention D, E, F group(NAC concentrations were 0.1 mmol/l added in at the same time).Each section will parallel three.The control group joined with the 1‰DMSO medium. The inverted microscope under the observation of cells afer 24h.2. Detect by flow cytometry hippocampus apoptosis.3. Confocal laser scanning microscopy immunofluorescence used to detect BDE-209 on expression of P38MAPK in newborn rat hippocampal neurons.[Results]:1. Detect by flow cytometry hippocampus apoptosis:The control group apoptosis rate 2.35 percent. Different concentrations of BDE-209 for 24 h Hippocampal nerve cells can be induced apoptosis,experimental D, E group apoptosis rate can be as high as 20.28 percent and 29.81 percent after 24 h. Show the BDE-209 can lead to apoptosis of hippocampal nerve cells, and The experiment groups had significant difference from control group(P< 0.0 1). The intervention group compared with the experimental group, there is statistical significance(P<0.05).2. Immunofluorescence and image analysis showed normal hippocampal neurons cytoplasmic has weak expression of P38MAPK, the fluorescence intensity: 1.38±0.31; expression of P38MAPK shows concentration-dependent trend. The experimental C group is strongest fluorescence intensity, which reached 8.71±0.80 (P <0.01). Intervention group compared with the experimental group, there is statistically significant (P <0.05).[Conclusions]:The BDE-209 can lead to apoptosis and positive expression of P38MAPK in primary culture of rat hippocampal neurons during some range of dose and time. The NAC can inhibit apoptosis by depressing the expression of P38MAPK in hippocampal neurons in vitro, which would protect hippocampal neurons.Part IIIEFFECTS OF N-ACETYLCYSTEINE INTERFERE BDE-209 ON ROS IN NEWBORN RAT HIPPOCAMPAL NEURONS[Objective]:To evaluate the effect of BDE-209 on Reactive Oxygen Species (ROS) content and protection efects of NAC in newborn Rat Hippocampal Neurons in vitro.[Material and Methods]:1. The BDE-209 Exposure on the hippocampal neurons of primary culture newborn rat after 7d .Experiment is divided into ten groups,That is, the blank control group ,the DMSO control group and experimental A, B, C Group(BDE-209 concentrations were 10.0,30.0 and 50.0 ug / ml),the NAC control group, the NAC+DMSO control group and intervention D, E, F group(NAC concentrations were 0.1 mmol/l added in at the same time).Each section will parallel three.The control group joined with the 1‰DMSO medium. The inverted microscope under the observation of cells afer 24h.2. Confocal laser scanning microscopy immunofluorescence used to detect BDE-209 on ROS content in newborn rat hippocampal neurons.[Results]:Experimental group and control group have significant differences in the expression of ROS(P<0.01),and dose-dependent trend, ROS expression heighten ,accompany with the concentration of BDE-209 increase; the expression of ROS between the intervention group and control group are no significant difference(P>0.05).[Conclusions]:The BDE-209 can lead to incease the expression of ROS in primary culture of rat hippocampal neurons during some range of dose and time. And NAC can inhibit the expression of ROS, which would protect hippocampal neurons.PartⅣEFFECTS OF N-ACETYLCYSTEINE INTERFERE BDE-209 ON CALCIUM OVERLOAD IN NEWBORN RAT HIPPOCAMPAL NEURONS[Objective]:To test the BDE-209 exposure on calcium overload and protection efects of NAC in newborn Rat Hippocampal Neurons . [Material and Methods]:1. The BDE-209 Exposure on the hippocampal neurons of primary culture newborn rat after 7d .Experiment is divided into ten groups,That is, the blank control group ,the DMSO control group and experimental A, B, C Group(BDE-209 concentrations were 10.0,30.0 and 50.0 ug / ml),the NAC control group, the NAC+DMSO control group and intervention D, E, F group(NAC concentrations were 0.1 mmol/l added in at the same time).Each section will parallel three.The control group joined with the 1‰DMSO medium. The inverted microscope under the observation of cells afer 24h.2. Detect by flow cytometry hippocampus calcium ion content.[Results]:Experimental group and control group have significant differences in the calcium ion content (P<0.01),and dose-dependent trend, calcium ion content heighten ,accompany with the concentration of BDE-209 increase; the calcium ion content between the intervention group and control group are no significant difference(P>0.05).[Conclusions]:BDE-209 exposure cause calcium overload in newborn culture of rat hippocampal neurons during some range of dose and time. And NAC can inhibit the calcium overload, which would protect hippocampal neurons.