孕期双酚A染毒对子代大鼠海马中诱导型一氧化氮合酶表达及活性的影响

Effects of bisphenol A exposure during pregnancy on expression and activity of inducible nitric oxide synthase in hippocampus of rat offspring at different developmental stages

[背景]现有研究表明双酚A(BPA)具有一定的神经毒性。一氧化氮(NO)是神经系统的重要逆行信使,其含量改变在神经毒性机制中发挥一定作用。当外源性化学物作用于机体时,诱导型一氧化氮合酶(iNOS)表达与活性变化对于NO的生成具有一定影响。[目的]探讨孕期BPA染毒对各发育阶段子代大鼠海马中iNOS表达及活性的影响。[方法]将60只SPF级健康SD孕鼠随机分为对照组和0.05、0.5、5、50 mg·kg^-1·d^-1的BPA染毒组,每组12只。于妊娠期第5天(GD5)-GD19以灌胃的方式进行染毒,以玉米油为溶剂配制相应浓度的BPA试剂,对照组母鼠灌胃玉米油。于GD20每组各处死6只孕鼠,剥离胎盘,取胎鼠脑组织,分离海马;剩余6只孕鼠继续饲养,仔鼠于出生后第21天(PND21)断乳,分别于PND21和PND56时,每组取6只仔鼠,经乙醚麻醉后处死,取仔鼠的海马组织。计算GD20、PND21、PND56这3个发育阶段仔鼠脑组织脏器系数,采用试剂盒检测海马组织中的NO含量、iNOS活性,采用实时定量PCR法测定各组iNOSmRNA水平,通过Western blotting检测iNOS的蛋白表达。[结果]NO检测结果显示,GD20各BPA染毒组仔鼠海马组织中NO含量均低于对照组(均P <0.05);而PND21和PND56的各BPA染毒组仔鼠(除PND21的0.05 mg·kg^-1组外)海马组织中NO含量均高于对照组,但PND56的0.5、5、50 mg·kg^-1BPA组仔鼠海马中NO含量低于0.05 mg·kg^-1组(均P <0.05)。iNOS mRNA表达结果显示,各发育阶段的BPA组仔鼠海马中iNOS mRNA含量均高于对照组(均P <0.05);其中GD20的50 mg·kg^-1 BPA组仔鼠海马中iNOS mRNA含量低于0.5 mg·kg^-1组(P <0.05);PND21的0.5、5、50 mg·kg^-1 BPA组仔鼠海马中iNOS mRNA含量均低于0.05 mg·kg^-1 BPA组,5、50 mg·kg^-1组仔鼠海马中iNOS mRNA含量也低于0.5 mg·kg^-1 BPA组(均P <0.05);PND56的5 mg·kg^-1 BPA组仔鼠海马中iNOS mRNA含量高于0.05、0.5 mg·kg^-1组,而50 mg·kg^-1组的iNOS mRNA含量低于5 mg·kg^-1组(均P <0.05)。iNOS蛋白表达结果显示,各发育阶段的0.5、5、50 mg·kg^-1 BPA组仔鼠海马中iNOS蛋白相对表达量均高于对照组和0.05 mg·kg^-1 BPA组(均P <0.05),其中GD20的50 mg·kg^-1 BPA组仔鼠海马中的iNOS蛋白相对表达量也高于5 mg·kg^-1组(P <0.05);各发育阶段对照组和0.05 mg·kg^-1BPA组iNOS的蛋白相对表达量相比较,差异无统计学意义(均P> 0.05)。而iNOS活性的检测结果显示,GD20的50 mg·kg^-1 BPA组仔鼠海马中iNOS活性高于对照组及其他各剂量BPA组(均P <0.05);而各BPA染毒组的PND21和PND56仔鼠海马中iNOS活性均高于各自对照组,其中PND21的0.5 mg·kg^-1 BPA组仔鼠海马中iNOS活性也高于0.05 mg·kg^-1组(均P <0.05)。[结论]孕期BPA染毒可促进不同发育阶段仔鼠海马组织中iNOS的基因及蛋白表达,增加iNOS酶活性,该过程在孕期BPA染毒所致仔鼠出生后NO含量升高以及由于NO含量升高所可能导致的神经毒性损伤中发挥一定作用。

[Background] Available studies have shown that bisphenol A(BPA) could induce neurotoxicity. Nitric oxide(NO) is an important retrograde messenger in the nervous system, and the alternations of its level play an important role in the mechanism underlying neurotoxicity. When exogenous chemicals are applied to the body, changes in the expression and activity of inducible nitric oxide synthase(iNOS) may have an impact on the production of NO.[Objective] This experiment investigates the effects of BPA exposure during pregnancy on the expression and activity of iNOS in the hippocampus of rat offspring at different developmental stages. [Methods] Sixty SPF-grade healthy SD pregnant rats were randomly divided into control group and 0.05, 0.5, 5, and 50 mg·kg^-1·d^-1 BPA exposure groups, with 12 rats in each group. The pregnant rats in the exposure groups were exposed to BPA(dissolved in corn oil) via oral gavage daily from gestational day(GD) 5 to GD19, and the rats in the control group were fed with corn oil. Six pregnant rats were sacrificed in each group on GD20, the placenta was harvested and the hippocampus was separated from brain tissues. The remaining six pregnant rats of each group were fed continuously, and the infant rats were weaned on postnatal day(PND) 21. Six rat offspring of each group were sacrificed on PND21 and PND56 respectively after ether anesthesia, and the hippocampus was also taken. The organ coefficients of brain tissues of rat offspring at different developmental stages(GD20, PND21, and PND56) were calculated. The levels of NO and the activity of iNOS in the hippocampus were detected using biochemical kits. The expression levels of iNOS m RNA and protein in the hippocampus was detected by real-time quantitative polymerase chain reaction and Western Blotting respectively. [Results] The hippocampal NO levels of GD20 rat offspring in the BPA exposure groups were lower than that in the control group(Ps < 0.05);the hippocampal NO levels of PND21 and PND56 rat offspring in the BPA exposure groups(except PND21 rat offspring in the 0.05 mg·kg^-1 BAP exposure group) were significantly higher than those in the control group;however, the hippocampal NO levels of PND56 rat offspring in the 0.5, 5, and 50 mg·kg^-1 BPA exposure groups were lower than that in the 0.05 mg·kg^-1 BPA exposure group(Ps < 0.05). Besides, the iNOS m RNA expression levels in the hippocampus of rat offspring in the BPA exposure groups at all developmental stages were higher than that in the control group(Ps < 0.05). The iNOS m RNA expression level in the hippocampus of GD20 rat offspring in the 50 mg·kg^-1 BPA exposure group was lower than that in the 0.5 mg·kg^-1 BPA exposure group(P < 0.05). Similar to the results on GD20, the iNOS m RNA expression levels in the hippocampus of PND21 rat offspring exposed to 0.5, 5 and 50 mg·kg^-1 BPA were lower than that in the 0.05 mg·kg^-1 BAP exposure group, and the iNOS m RNA expression level of rat offspring in the 5 and 50 mg·kg^-1 BPA exposure groups were lower than that in the 0.5 mg·kg^-1 BAP exposure group(Ps < 0.05). The iNOS m RNA level in the hippocampus of PND56 rat offspring in the 5 mg·kg^-1 BPA exposure group was higher than those in the 0.05 mg·kg^-1 and 0.5 mg·kg^-1 BPA exposure groups, and the level in the 50 mg·kg^-1 BPA exposure group was lower than that in the 5 mg·kg^-1 BPA exposure group(Ps < 0.05). The relative expression levels of iNOS protein in the hippocampus of rat offspring exposed to 0.5, 5, and 50 mg·kg^-1 BPA at different developmental stages were higher than those in the control group and the 0.05 mg·kg^-1 BPA exposure group(Ps < 0.05). The relative expression level of iNOS protein in the hippocampus of GD20 rat offspring exposed to 50 mg·kg^-1 BPA was also higher than that in the 5 mg·kg^-1 BPA exposure group(P < 0.05). There was no significant difference in iNOS protein relative expression level between rat offspring in the control group and the 0.05 mg·kg^-1 BPA exposure group at different developmental stages(Ps > 0.05). The activity of iNOS in the hippocampus of GD20 rat offspring in the 50 mg·kg^-1 BPA exposure group was higher than those in the control group and the other BPA exposure groups(Ps < 0.05). The iNOS activities in the hippocampus of PND21 and PND56 rat offspring in the BPA exposure groups were higher than that in the control group(Ps < 0.05). The iNOS activity in the hippocampus of PND21 rat offspring in the 0.5 mg·kg^-1 BPA exposure group was also higher than that in the 0.05 mg·kg^-1 BPA exposure group(Ps < 0.05). [Conclusion] BPA exposure during pregnancy can promote the m RNA and protein expressions and the activity of iNOS in the hippocampus of rat offspring at different developmental stages, which may play roles in the increase of NO level after birth and the neurotoxicity caused by the NO upregulation.