| Objective:Bisphenol A(BPA)is a high production volume(HPV)chemical,which is commonly used in food packaging materials,dental sealants,medical devices and heat carriers.BPA intake,inhalation and skin contact are common phenomena.The Centers for Disease Control and Prevention(CDC)reports that more than 90%of the U.S.population's urine samples may contain a certain amount of BPA.BPA has been shown to interact with a variety of physiological receptors.For example,estrogen receptor?/?(ER?/?)has been shown to bind to BPA,estrogen related receptor?(ER?/?)has also been found to interact with BPA,and androgen receptor(ER?/?)has been shown to bind to BPA Receptors(AR)have been shown to bind to BPA and thyroid hormone receptors have also been found to interact with BPA.BPA can bind to them and show endocrine disrupting effects.Many studies have pointed out the reproductive toxicity of BPA and conducted extensive reviews to address the specific mode of action and concentration of BPA toxicity.In 2006,the National Institute of Environmental Health Sciences(NIEHS),National Institute of dental Craniofacial Research(NIDCR)and US Environmental Protection Agency(US Environmental Protection Agency)were established An expert group composed of agency(EPA)and public welfare organizations reviewed human exposure to BPA in vivo and in vitro[15].A team of experts focusing on in vivo animal research found that the findings contradicted the findings of the survey.However,researchers remain convinced that BPA affects the reproductive systems of both men and women.In addition,recent epidemiological studies suggest that BPA exposure may be associated with changes in hormone levels,impairment of ovarian and uterine functions,and decreased sperm quality.The latest data of experimental studies show that in animal models,BPA exposure has adverse effects on oocyte quality and maturation,sperm production and quality decline,testicular cell damage,hormone level disorder,ovarian function and uterine morphology interruption.Recently,the prevalence of BPA analogues in environment,food,consumer goods and human urine samples has been reported.Because of the high similarity with BPA in structure,we infer that these analogues may also have endocrine disrupting capacity similar to BPA,and may also have adverse effects on the reproductive system.New evidence suggests that BPA analogues interact with various physiological receptors,such as estrogen receptor?and?,androgen receptor and aromatic hydrocarbon receptor.In this study,we established a BPA pregnant rat model by giving different concentrations of BPA during pregnancy,and studied the effects of different doses of BPA on the learning and memory function of offspring rats,and explored its possible mechanism.Methods:1.Animal model and groupingThirty Wistar rats were selected.Female adult rats about 10 weeks old were selected.The average weight of rats was 200-220g.All rats should be fed adaptively for one week after purchase before formal feeding.Then the rats were randomly divided into three groups:0 mg/kg/day group,5 mg/kg/day group and 50 mg/kg/day group,with 10female rats in each group.Female rats in each group were mated with normal male rats in a ratio of 1:2,and the first day was gestational day 0(gd0).Starting from gd0,different concentrations of BPA were dissolved in ethanol(1%of the final solution)by tube feeding,and diluted to the specified concentration with well rinsed tap water.The 0 mg/kg/day group received water(vehicle)containing 1%ethanol.The administration lasted until the 21st day after birth(pnd21).On the fourth day after birth(pnd4),each litter of pups is sorted.In order to ensure that each offspring can get better nutrition from the mother and maintain normal growth and development,8-10 pups are kept in each litter to ensure the same sex ratio.2.Weight records of pregnant rats,offspring rats and organs of offspring ratsThe weight of pregnant rats was recorded at the beginning,middle and late stages of pregnancy.The weight of newborn rats was recorded.At PND21 day,cerebellum,left hippocampus,right hippocampus,spleen,thyroid and pancreas of offspring were weighed and recorded.3.Measurement of long-term synaptic plasticity in hippocampusRats were anesthetized by intravenous injection of 20%urethane.The anesthetized rats were fixed on the stereotactic apparatus.According to the brain atlas of paxions,the bipolar stimulation electrode was inserted into the pyramidal cell layer of CA3 area.The recording electrode was inserted into the pyramidal cell layer of CA1 area.A single stimulus evoked a population spike.After 30 minutes of baseline recording,high frequency stimulation with the same parameters was given for 5 seconds.The changes of synaptic plasticity in hippocampal CA3-CA1 region after stimulation were observed.The amplitude of spike wave and the slope of field excitatory postsynaptic potential were observed by monopulse experiment.4.ImmunofluorescenceAt pnd7,pnd14 and pnd21,the rats were killed by cardiac perfusion and the brains were removed.The brain tissue was fixed in 4%paraformaldehyde fixative overnight.5?m coronal section was made.Firstly,the antigen was repaired by microwave after dewaxing to water with gradient xylene and gradient alcohol.Then,the blocking serum in the kit was used for blocking.The first antibody was cultured overnight at 4?.After reheating at 37?for 30 minutes,PBS was soaked and then incubated with fluorescent antibody for 2 hours without light.The second antibody was eluted with PBS and dripped into the anti quench block containing DAPI.Typical photos were observed and collected under inverted fluorescence microscope.5.TUNEL assayFor TUNEL,the slides were dewaxed and rehydrated.The slides were immersed in10 mm citrate at p H 6 in 95°water bath for 30 minutes,and further digested with 1?g/ml protease K at 37°for 10 minutes.Next,according to the manufacturer's instructions,the TUNEL reagent fluorescein in Roche in situ cell death detection kit was applied to the slide.Under the light microscope.6.Reverse transcription and quantitative real-time PCRAccording to the manufacturer's protocol,Trizol was used to isolate the total RNA of the tissues treated with different factors for 24 hours.Complementary DNA was synthesized by reverse transcription of total RNA using RT reaction kit.Quantitative real-time PCR was used to determine m RNA levels using on-demand Taq Man gene expression analysis according to the procedure described previously.The results were performed three times in at least five independent experiments.SYBR premix ex Taq was used as a DNA specific fluorescent dye.All reactions were repeated at least three times.The expression levels of target protein and gene related to glyceraldehyde-3-phosphate dehydrogenase(GAPDH)were calculated by software.7.Western blotThe homogenate was broken by ultrasound,then centrifuged,and the protein concentration was determined by BCA kit.Electrophoretic separation and transfer to PVDF membrane.5%skimmed milk powder sealed for 1 hour.The first antibody was cultured overnight.ECL chemiluminescence reaction was observed after the second antibody was incubated at room temperature for 1 h.GAPDH was used as internal reference to analyze protein concentration.8.StatisticsAll the experiments in this study were repeated three times,and the results of three independent experiments were expressed in the form of mean±standard deviation.The data in this study were statistically analyzed by SPSS17.0 software.P<0.05 means there is difference.Results:1.Effects of BPA on physiological function of pregnant and offspring ratsIn this study,5 mg/kg/d BPA and 50 mg/kg/d BPA were applied to pregnant rats respectively,and the weight of pregnant rats was measured at different periods.The results showed that BPA did not affect the body weight of pregnant rats.To determine whether BPA exposure can affect the biological behavior of offspring by inhibiting organ development,we weighed and recorded the weight of cerebellum,left hippocampus,right hippocampus,spleen,thyroid and pancreas of offspring.The results showed that BPA had no significant effect on the weight of offspring.The results showed that BPA had no significant effect on organ weight of offspring.Typical field potential changes in pyramidal cell layer of hippocampal CA1 region were observed before and after exposure to different concentrations of BPA.The results showed that LTP did not occur in female offspring and male offspring exposed to BPA at low concentrations.After high concentration BPA exposure,the increase of PS amplitude and f-EPSP slope in hippocampal CA1 area of male offspring was lower than that of control group,and the difference was statistically significant.It is suggested that high concentration of BPA exposure may cause mild LTP damage in male offspring.2.Effects of BPA on the proliferation of hippocampal neurons in offspring ratsImmunofluorescence staining results showed that high concentration of BPA could inhibit the expression of Nestin in neurons of hippocampal dentate gyrus(DG)region in male offspring rats.But there was no significant change in female offspring.The results of Western blot and real-time PCR showed that the expression of Nestin and Cyclin D1in the hippocampus of male offspring was significantly down-regulated by BPA at 50mg/kg/d,however not at 5 mg/kg/d BPA.The results of real-time PCR also showed that Nestin expression in PND14 and 21 was slightly down regulated compared with that in PND7.Furthermore,BPA had no significant effect on the expression of Nestin and Cyclin D1 in the hippocampus of female offspring.3.Effects of BPA on the apoptosis of hippocampal neurons in offspring ratsTunel assay results showed that high concentration of BPA could promote the apoptosis of CA1 hippocampal neurons of male offspring rat,but had little effect on female offspring.The results of Western blot and real-time PCR showed that the expression of bcl-2 was down regulated and the expression of bax was up regulated in the hippocampus of male offspring by BPA at 50 mg/kg/d,however not at 5 mg/kg/d BPA.For female offspring,BPA had no significant effect on the expression of bcl-2 and bax in the hippocampus.4.Effects of BPA on the expression of Rho A/Rac-1 in hippocampal neurons in offspring ratsBecause Rho A and Rac-1 play an important role in hippocampal nerve development,we examined the effects of BPA on Rho A and Rac-1 in hippocampal neurons.The results of fluorescence staining showed that BPA at 50 mg/kg/d could significantly up-regulate the expression of Rho A and down-regulate the expression of Rac-1 in the hippocampal CA1 region of male offspring.However,the effect of low concentration BPA was not significant,and the effect of BPA on female offspring was not significant.The results of Western blot and real-time PCR showed that the expression of Rho A was up regulated and the expression of Rac-1 was down regulated in the hippocampus of male offspring by BPA at 50 mg/kg/d,however not at 5 mg/kg/d BPA.The results of Western blot and real-time PCR showed that BPA had no significant effect on the expression of Rho A and Rac-1 in the hippocampus of female offspring.Conclusion:1.After high concentration BPA exposure,the increase of PS amplitude and f-EPSP slope in hippocampal CA1 area of male offspring was lower than that of control group.2.BPA exposure during pregnancy and lactation could impair learning and memory in hippocampus of male offspring by affecting the growth and apoptosis of hippocampal neurons.3.High concentration of BPA could inhibit Nestin,Cyclin D1,bcl-2 and Rac-1 in male offspring rats and the expression of bax and Rho A was promoted by BPA. |
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