| [Objective]To study the effect of prenatal exposure to B[a]P on the neurodevelopment and the ability of learning and memory of pup rats,and explore the effect of HDAC1,HDAC2 and BDNF in the B[a]P-induced neurons apoptosis in vivo and in vitro.[Methods]1.Animal experiment :1)Pregnat adult rats were randomly assigned to five groups and given B[a]P by intraperitoneal injection on the day 17 of gestation by daily for three consecutive days at the dosages of 25,50,100 mg/kg B[a]P,olive oil as a solvent group and normal control group respectively,and then expectant naturally.2)①Weighed on days 1,4,7,14 and 28 respectively after pup rats birth, detectd early development indicators of pup rats;②With the Morris water maze and Open-field test detect learning and memory ability and the adaptability to new environment;③Three pup rats of each group were sacrificed after the birth on days 1,4,7,14 and 28 respectively, neurons loss in the CA3 hippocampus and apoptosis of hippocampus neurons were observed by thionin and TUNEL;④The expression levels of HDAC1,HDAC2,caspase-3 and BDNF mRNA and protein were detected by QRT-PCR,immunohistochemistry and Elisa respectively.2. In vitro study:1) The primary cultured neurons of rat were assigned into two lots. The first lot was separated into 4 groups and treated with B[a]P at final concentrations as 0μmol/L (DMSO group),10μmol/L(low-dose group), 20μmol/L(medium-dose group), and 40μmol/L(high-dose group) respectively, and added S9 at the same time, then incubated for 48 hours. The second lot was separated into 5 groups, all treated with 20μmol/L B[a]P, then incubated for 0,6,12,24 and 48h respectively. 2)①The cell viability and apoptosis rates were evaluated with MTT and flow cytometry respectively;②HAT/HDAC activity were detected by HAT/HDAC activity assay kit;③The gene and protein levels of HDAC1,HDAC2,caspase-3 and BDNF were detected by QRT-PCR,western-blot and Elisa respectively.[Results]1.①Neurodevelopment of neonatal rats test: compared to the control and solvent group, offspring weight of B[a]P exposure group reduced gradually(P<0.01,P<0.05);The time of ear opening in middle and high-dose group[(4.1 0.4),(5.0 0.4)d)] posterior to the control and solvent group(P<0.01).Compared to the control group(36.1%) ,the attainment rate of the surface righting reflex test in high-dose on PND4 (6.5%)decreased, and on PND7 (50.0%)decreased significantly compared to the control and solvent group(80.3%,79.3%)(P<0.01).Compared to the control group, time of forelimb hanging test decreased in high-dose on PND12, 14 and decreased significantly on PND14 compared to the solvent group(P<0.01).The attainment rate of olfactory discrimination significantly lower than the control group (94.3%)in high-dose group(61.9%) on PND12(P<0.05);②Learning and memory capability test: Morris water maze test showed that compared to the untreated and solvent group the escape latency of different dose groups were significantly increased, and the time of spatial probe and numbers of traversing flat in high-dose group were shortened significantly(P<0.01).Open-field test showed that center retention time in middle and high-dose group were prolonged compared to control, numbers through lattice reduced, and rearing decreased in high-dose group, there were statistical significance(P<0.05).Compared to the solvent group numbers through lattice in different doses were reduced significantly(P<0.01,P<0.05);③The thionine staining showed: hippocampal neurons of the control group and solvent group were more numerous, connected more closely, lining up in order, neurons nissl staining was more deep, but with the increasing of doses of B[a]P exposed , number of neurons were gradually decreased ,the neurocytes displayed disorderly, the cell junction get loose, neurons nissl staining became lighter than the control, the karyon shrinked,some rings around the neurocyte were observed;④TUNEL results: at the same time point, the AI of hippocampus neurons increased significantly with the increasing dose (P<0.01, P<0.05);⑤Gene and protein results: the expression of HDAC1,HDAC2,caspase-3 mRNA and protein levels were increased as the dose of B[a]P exposed at the same time point,while BDNF decreased(P<0.01, P<0.05).The BDNF protein level in high significantly reduced (50%) as compared to control(P<0.01, P<0.05);PND28 BDNF protein levels in plasma in high dose group was decaeased significantly (68%,75%)compared with the control and the solvent (P<0.01, P<0.05).2.①MTT and Flow cytometry results: neurons viability was significantly decreased at different dose and time group (P<0.01). Cell viability of 40μmol/L group significantly reduced (50%) as compared to DMSO (P<0.01); which at 48h group reduced (50%)compared with 0h(P<0.01).Compared with DMSO group, apoptosis rates of neurons in 20,40μmol/L group were significantly increased(P<0.05), compared with 10μmol/L group, 40μmol/L group also increased (P<0.05);the apoptosis rates at 24,48h after exposure were significantly increased compared with that at 0h (P<0.05);②HAT/HDAC activity results: compared with DMSO group,HAT activity of 40μmol/L group reduced (47%) as compared to DMSO(P<0.05), while HDAC increased(59%)(P<0.01);HAT activity at 48h after exposure was decline (68%)compared with that at 0h,HDAC ascend (142%)(P<0.01);③QRT-PCR results: compared with DMSO group ,HDAC1 mRNA expression increased ,but there were no statistical significance. The caspase-3 and HDAC2 mRNA expression showed significant increments in 40μmol/L group compared with DMSO group(P<0.01), while the expressions of BDNF mRNA of 20,40μmol/L significantly decreased(P<0.01),the caspase-3 mRNA expression in 40μmol/L group increased significantly compared with the 10μmol/L group (P<0.05).The expressions of HDAC1,HDAC2 and caspase-3 mRNA at 48h after exposure increased significantly compared with that at 0h,whereas, BDNF mRNA at 24,48h decreased (P<0.01, P<0.05);④Protein examination results:the HDAC1,HDAC2 and caspase-3 protein levels in 40μmol/L group showed significant increments compared with DMSO group(P<0.05); The expressions of caspase-3 protein at 24h,48h ,HDAC1 and HDAC2 at 48h after exposure increased significantly compared with that at 0h(P<0.05). The gene and protein levels of BDNF decreased significantly as different dose and time (P<0.05).[Conclusion]The prenatal exposure to B[a]P would suppress neonatal rats'neurodevelopment ,and induce the morphology changes of pups'brain nerve cells and hippocampus neural cells apoptosis, the higher dose the more severe injury. From both in vitro study and the animal experiment, it is discovered that HDAC1, HDAC2 may play an important role in B[a]P-induced neuronal apoptosis. After expose to B[a]P, HDAC1 and HDAC2 increased, levels of histone acetylation decreased, which may lead to the expression of BDNF decreased and they may influence neuronal apoptosis. |
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