铅暴露对斑马鱼胚胎心脏和脊柱发育的影响
Effects of lead exposure on development of heart and spine in zebrafish embryos
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摘要:
背景 铅分布广泛。铅暴露会干扰斑马鱼生命早期发育，但铅暴露影响斑马鱼骨骼发育和心脏发育的机制尚不清楚。
目的 探究醋酸铅暴露诱发的骨发育和心脏发育毒性的分子机制。
方法 将受精后3 h（3 hpf）斑马鱼胚胎暴露于不同浓度的醋酸铅（0、6、12、24和48 μmol·L−1），直至受精后5天（5 dpf）。统计5dpf的畸形表型，通过荧光定量PCR方法检测脊柱发育相关基因（
bmp2b 、bmp4 、bmp9 、runx2a 、runx2b ）和心脏发育相关基因（nkx2.5 、myh6 、myh7 ）的mRNA表达情况，分析发育相关调控通路Wnt/β-catenin通路（wnt5a 、wnt8a 、wnt10a 、β -catenin ）、TGF-β通路（tgf -β 1 、tgf -β 2 ）相关基因和Eph-Ephrin信号中关键分子eph 的mRNA表达情况。结果 5dpf时与对照组相比，醋酸铅处理组斑马鱼出现包括脊柱弯曲和心包囊水肿的畸形表型。24、48 μmol·L−1 醋酸铅组脊柱弯曲畸形率分别达到 26.47%和 71.52%，高于对照组（
P <0.01）。qPCR结果发现，与对照组相比，48 μmol·L−1暴露组中，脊柱发育相关基因bmp2b 、bmp4 、bmp9 、runx2a 、runx2b 的mRNA表达分别下调82.8%、58.0%、88.7%、85.5%、69.2%（P <0.05或P <0.01），心脏发育相关基因myh6 、myh7 、nkx2.5 的mRNA表达分别下调63.7%、58.9%、55.2%（P <0.01），Wnt/β-catenin通路中wnt8a 、β -catenin 的mRNA表达分别下调了71.5%、47.3%（P <0.05或P <0.01），TGF-β通路中tgf -β 1 的mRNA表达下调了67.5%（P <0.01），eph 的mRNA表达下调了86.9%（P <0.01）。结论 醋酸铅通过下调脊柱发育和心脏发育相关基因的mRNA表达，以及抑制发育相关Wnt/β-catenin、TGF-β通路和Eph-Ephrin信号，引起脊柱弯曲和心包囊水肿等畸形表型，从而对斑马鱼心脏和骨骼产生发育毒性作用。
Abstract:Background Lead is widely distributed. Lead exposure interferes with early life development in zebrafish, but the mechanisms by which lead exposure affects skeletal development and cardiac development are not clear as yet.
Objective To investigate the molecular mechanisms of bone development and cardiac development toxicity induced by lead acetate exposure.
Methods Zebrafish embryos were exposed to different concentrations of lead acetate (0, 6, 12, 24, and 48 μmol·L−1) for 3 h post-fertilization (3 hpf) until 5 d post-fertilization (5 dpf). The malformation phenotypes of 5 dpf were counted, and the mRNA expressions of spinal development-related genes (
bmp2b ,bmp4 ,bmp9 ,runx2a ,runx2b ) and heart development-related genes (nkx2.5 ,myh6 ,myh7 ) were detected by quantitative PCR (qPCR). Expressions of genes of development-related regulatory pathways including Wnt/β-catenin pathway (wnt5a ,wnt8a ,wnt10a ,β -catenin ) and TGF-β pathway (tgf -β 1 ,tgf -β 2 ) as well as key moleculeeph of Eph-Ephrin signaling were analyzed.Results At 5 dpf, the zebrafish in the lead acetate treated groups showed deformed phenotypes including spinal curvature and pericardial sac edema compared to the control group. In the lead acetate groups at 24 and 48 μmol·L−1, the spinal curvature deformity rates reached 26.47% and 71.52% (
P <0.01) respectively. The qPCR results revealed that the expression levels of spinal development-related genesbmp2b ,bmp4 ,bmp9 ,runx2a , andrunx2b were downregulated in the 48 μmol·L−1 exposure group compared to the control group by 82.8%, 58.0%, 88.7%, 85.5%, and 69.2%, respectively (P <0.05 orP <0.01); the expression levels of heart development-related genesmyh6 ,myh7 , andnkx2.5 were down-regulated by 63.7%, 58.9%, and 55.2%, respectively (P <0.01); the expression levels ofwnt8a andβ -catenin in the Wnt/β-catenin pathway were down-regulated by 71.5% and 47.3% (P < 0.05 orP < 0.01), respectively; the expression level oftgf -β 1 in the TGF-β pathway was down-regulated by 67.5% (P <0.01); the expression level ofeph was down-regulated by 86.9% (P <0.01).Conclusion Lead acetate exerts developmental toxic effects on zebrafish heart and bone by down-regulating the expressions of genes related to spinal development and heart development, as well as inhibiting development-related Wnt/β-catenin and TGF-β pathways and Eph-Ephrin signaling, causing malformed phenotypes such as spinal curvature and pericardial sac edema.
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