| Objective:Di-(2-ethylhexyl)phthalate(DEHP)is an ubiquitous environmental contaminant because of its extensive use in plastics and its persistence.As an environmental endocrine disruptor,DEHP is found in soil,water and air,and can enter the human body through a variety of ways.Epidemiological investigations have shown that DEHP exposure during early development is related to cerebellar-related adverse neurodevelopmental outcomes.The cerebellum is an important brain area in neurodevelopment,which is related to the motor function,coordination and exercise learning of the body.Cerebellar granule cells are the most abundant in the cerebellum and their normal development is the prerequisite to ensure cerebellar function.However,it is unclear whether maternal exposure to DEHP during pregnancy and lactation influences the development of cerebellar granule cells in offspring,and its related mechanism needs to be studied.In this study,we established an animal model of DEHP exposure by intragastrium during pregnancy and lactation,to investigate the effect of maternal DEHP exposure on the proliferation and apoptosis of cerebellar granule cells,and to explore the possible mechanism.Methods:In this study,Wistar pregnant mice and primary cerebellar granule cells were used as research object.Female Wistar rats were assigned into four groups:0,30,300 and 750 mg/kg/day DEHP exposure groups.An animal model of DEHP exposure was established by intragastric administration of DEHP according to the corresponding dose from the first day of pregnancy to 21 days after birth.Five metabolites of DHEP including MEHP,MEHHP,MEOHP,MECPP and MCMHP in the urine of female rats at GD19 were determined by gas chromatography-mass spectrometry to preliminarily prove whether the DEHP exposed animal model is established.Corresponding cerebellar-related neurobehavioral tests were performed on the pups at various time points after birth,including surface righting reflex,negative geotaxis reflex and grip test.The proliferation rate of cerebellar granule cells in each group was observed by immunofluorescence staining at PN 7 and PN 14,and the expression of related proteins in the Shh signaling pathway was detected by Realtime PCR and Western blot.The estrogen levels in the cerebellum of the offspring were detected by Elisa and the expression of aromatase was detected by Western blot.In addition,TUNEL was used to detect the apoptosis of cerebellar granule cells at PN 7 and PN14,and the expression of related proteins in the PI3K/Akt signaling pathway of cerebellum was detected by Western blot.At the same time,the primary cerebellar granule neurons were extracted in vitro,and the MEHP exposure model of primary cerebellar granule neurons was then established.The tolerance of MEHP to cerebellar granule neurons was detected by CCK8.The apoptosis of cerebellar granule neurons were detected by TUNEL staining after MEHP exposure,and the expression of Cleaved caspase 3 was detected by immunofluorescence staining.Meanwhile,Western blot was used to detect the expression of the proteins in the PI3K/Akt signaling pathway of cerebellar granule neurons infected with MEHP.The level of apoptosis of the cerebellar granule cells and the changes of PI3K/Akt signaling pathway-related proteins in each group were determined by using the activator IGF1 and the inhibitor LY294002 of the PI3K/Akt signaling pathway.Results:1.Effect of DEHP exposure during pregnancy and lactation on the weight of offspring and cerebellumThe weight of offspring and cerebellum were measured on PN 7 and PN 14.The results showed that there was no statistical difference in the weight of offspring and cerebellum in each group.2.Concentration of five metabolites of DEHP in urine of pregnant rats exposed to DEHPThe concentrations of the five DEHP metabolites were measured in the four groups by gas chromatography-mass spectrometry(GC-MS).The results showed that the concentrations of the five DEHP metabolites in the control group were below limit of detection(LOD).In addition,significantly higher concentrations of the five DEHP metabolites in the rats of 30,300 and 750 mg/kg/d exposure groups were observed compared to untreated control rats(P<10.05).Concentrations of metabolites increased as the dose of DEHP contamination rose.These results demonstrated that we were successful in establishing an animal model for studying DEHP exposed3.Effect of maternal DEHP exposure on cerebellar granule cell proliferation in pups.Immunofluorescence results showed that the proliferation rate of granule cells(Pax6 PCNA+/Pax6+)in the cerebellar layer of male offspring exposed to 300 mg/kg/d and 750 mg/kg/d DEHP at PN 7 and PN 14 was significantly decreased compared with the control group(P<0.05).However,there was no significant difference in the proliferation rate of cerebellar granule cells between the control group and the 30 mg/kg/d DEHP exposure group(P>0.05).In addition,there was no significant difference in the proliferation rate between the groups for female offspring(P>0.05).4.Effect of maternal DEHP exposure on the neuromotor development of pups.For the righting reflex,the latency times in male pups from the 300 and 750mg/kg/d exposure groups were significantly increased compared with those of the control group at PN 3,PN 5 and PN 7(P<0.05).Subsequently,forelimb grip strength was measured from PN14 to PN16,and the hanging times in male offspring of the 300 and 750mg/kg/d exposure groups were significantly reduced relative to those of the control group at PN 14,PN 15 and PN 16(P<0.05).However,a difference in these four groups for female offspring was not evident.In addition,the negative geotaxis reflex test was conducted at PN 7,PN 8 and PN 9,but significant variations were not found in male and female offspring of the four groups.5.Effect of maternal DEHP exposure on the Shh signaling pathway.Real time RCR results showed that the levels of Shh,Glil,N-myc and Cyclindl mRNA in the cerebellum of male offspring exposed to 300 and 750 mg/kg/d DEHP were significantly decreased at PN 7 and PN 14 compared with the control(P<0.05).Western blot results also indicated that the protein expressions of Shh,Glil,N-myc and Cyclindl in the cerebellum of male offspring exposed to 300 and 750 mg/kg/d DEHP were also obvious lower than those in the control group(P<0.05),while the mRNA levels and protein expressions of Shh,Glil,N-myc and Cyclindl in the cerebellum of female offspring showed no significant changes in each group at PN 7 and PN 14(P>0.05).6.Effect of maternal DEHP exposure on the estrogen levels and aromatase expressions of offspring.The protein expression of the aromatase in the cerebellum of the male offspring exposed to 300 and 750 mg/kg/d DEHP at each time point was significantly lower than that of the control group(P<0.05),while the protein expression of the aromatase in the cerebellum of the female offspring showed no significant difference at each time point(P>0.05).In addition,Elisa results showed that compared with the control group,the cerebellar estrogen levels of male offspring exposed to 300 and 750 mg/kg/d DEHP doses also decreased significantly(P<0.05),similarly,the cerebellar estrogen levels of female offspring showed no significant difference at all time points in the four dose groups(P>0.05).7.Effects of maternal DEHP exposure on cerebellar granule cell apoptosis in offspringTUNEL results showed that the TUNEL positive cell rate in the 300 mg/kg/d and 750 mg/kg/d DEHP exposure group was significantly higher than that in the control group(P<0.05),while there was no significant effect on the TUNEL positive cell rate in the female offspring(P>0.05).8.Effect of maternal DEHP exposure on cerebellar Total caspase 3 and Cleaved caspase 3 protein expression in offspring.For male offspring,the Cleaved caspase-3 expression levels were significantly higher in the 300 mg/kg/d and 750 mg/kg/d DEHP exposure groups than the control group(P<0.05),while the Total caspase-3 expression levels were decreased compared with the control group(P<0.05)at PN 7 and PN 14.However,there was no significant difference in the Total caspase 3 and Cleaved caspase-3 expression between the exposed groups for female pups(P>0.05).9.Effect of maternal DEHP exposure on the PI3K/AKT signaling pathway in offspring.In this investigation,for male pups,it was found that the expression of p-PI3K,p-AKT and Bcl-2 was significantly decreased in 300 and 750 mg/kg/d exposure groups at PN 7 and PN 14 in comparison with the control,and the protein expression of Bax from 300 and 750 mg/kg/d exposure groups was elevated.In addition,for female pups,no evident differences were found in the expression of p-PI3K,p-AKT,Bcl-2 and Bax at PN 7 and PN 14.Our results indicated that DEHP exposure during pregnancy and latency inhibited the PI3K/AKT signaling pathway of male offspring.10.Effect of MEHP on the activity of primary cerebellar granule cell.CCK8 results showed that cell viability decreased with an increase in MEHP dose and exposure time.Primary cerebellar granule cells treated with 25 ?M MEHP did not noticeably affect the cell viability of CGCs after 12 and 24 h,but decreased after 48 h.Moreover,exposure to 250 ?M MEHP for 24 h led to an approximately 20%reduction in cell viability when compared to the control(P<0.05).The cell viability was reduced to 50%when cerebellar granule cell were exposed to 1000 ?M MEHP for 24 h.11.MEHP promoted the apoptosis of primary cerebellar granule neuronsPrimary CGCs were stained by TUNEL and cleaved caspase-3 to evaluate apoptosis induced by MEHP in vitro.The percentage of TUNEL positive cells gradually increased in CGCs as the MEHP exposure concentration increased(P<0.05).We also found that the percentage of Cleaved caspase-3 positive cells significantly increased in MEHP treatment groups in comparison with the control(P<0.05).Furthermore,the protein expression of Cleaved caspase-3 was also upregulated in the three MEHP exposure groups when compared with the control(P<0.05),while the level of Total caspase-3 was downregulated(P<0.05).12 MEHP interferes with the expression of PI3K/AKT signaling pathway protein moleculesThe results showed that compared with the control group,the protein expression levels of p-Pi3k,p-Akt and Bcl-2 in the MEHP exposure groups were significantly decreased(P<0.05),while the protein expression levels of Bax were significantly increased(P<0.05).In addition,there was no marked difference in total PI3K and AKT protein levels of the four independent groups(P>0.05).13 IGF1 reduces the apoptosis of cerebellar granule cells by activating the PI3K/AKT signaling pathway,and LY294002 aggravates the mehp-induced apoptosis of cerebellar granule cells by inhibiting the PI3K/AKT signaling pathwayCompared with the control group,the protein expression of Bax and Cleaved caspase-3 increased in the MEHP only treatment group when compared with the control(P<0.05),while the levels of p-PI3K,p-Akt,Bcl-2 and Total caspase 3proteins decreased(P<0.05).In addition,combined treatment with IGF1 led to the recovery of levels of p-PI3K,p-AKT and Bcl-2 proteins,but decreased the expression of Cleaved caspase-3 and Bax when compared to MEHP only treatment.Moreover,there was no obvious difference in the expression of these proteins between the IGF1 alone treatment and the control.Furthermore,we found that the number of cells that were TUNEL positive was reduced upon pretreatment with 100 ng/ml IGF1 in comparison with the MEHP only treatment group,and there was no evident difference between the control and IGF1only treatment groups.We also found that DEHP had a similar effect to that of LY294002 in that it inhibited the PI3K/AKT signaling pathway(P<0.05)and increased the TUNEL positive rate in cerebellar granule cells(P<0.05).Moreover,the inhibition effect is more obvious when applying both MEHP and LY294002.Conclusion:1.DEHP exposure during gestation can inhibit the proliferation of cerebellar granule cells in male offspring by down-regulating Shh signaling pathway,and lead to neurodevelopmental disorder2.DEHP exposure during gestation can induce excessive apoptosis of cerebellar granule cells by inhibiting the PI3K/Akt signaling pathway.3.The sex difference of cerebellar granule cells damaged by DEHP may be related to the level of estrogen and the expression of aromatase in the cerebellar of offspring rats... |
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