长期氯化铜暴露致C57BL/6J小鼠神经毒性的研究

Neurotoxicity induced by long-term exposure to copper chloride in C57BL/6J mice

目的探究不同剂量的氯化铜暴露对C57BL/6J雌性小鼠运动功能和黑质内神经细胞发育的影响。方法30只12周龄C57BL/6J小鼠随机分为对照组、10 mg/kg氯化铜处理组、20 mg/kg氯化铜处理组,每组各10只,灌喂处理8周。实验结束后,对小鼠进行行为学测试,包括转棒、爬杆和旷场实验,检测小鼠的运动能力,再通过酶联免疫吸附法和免疫印迹法探究小鼠神经细胞和炎症因子的表达情况。结果行为学实验结果显示,与对照组相比,经铜处理组小鼠的在杆时长均高于对照组,并且随着毒物浓度增加,在杆时长也相应增加(F=27.03,P<0.05);经铜暴露后的小鼠在棒时长均减少,其中20 mg/kg氯化铜处理组小鼠的在棒时长减少(F=3.35,P=0.05);旷场实验结果显示,与对照组相比,铜处理组的小鼠在旷场中运动的总路程减少(F=13.93,P<0.05)。Western blot结果显示,与对照组相比,经铜处理后的小鼠黑质中酪氨酸羟化酶(F=8.74,P<0.05)、多巴胺转运体(F=13.09,P<0.05)、Bcl-2(F=9.23,P<0.05)和PGC-1α(F=5.04,P=0.030)蛋白表达量和黑质内甲酚紫阳性细胞数量(F=63.52,P<0.05)均减少。酶联免疫吸附测定显示,与对照组相比,铜处理后小鼠血清铜(F=5.93,P=0.020)和黑质铜(F=7.48,P=0.010)含量增加;铜处理后小鼠的促炎因子白细胞介素-6(F=28.89,P<0.05)、肿瘤坏死因子α(F=22.73,P<0.05)、白细胞介素-1β(F=11.77,P=0.002)水平均升高,并且随着毒物浓度升高,白细胞介素-6和肿瘤坏死因子α水平也相应升高;铜处理后小鼠的促炎因子白细胞介素-4(F=4.79,P=0.040)、IL-10(F=14.70,P=0.001)和转化生长因子β1(F=6.19,P=0.020)水平均降低。结论氯化铜长期暴露增加了小鼠血清和黑质内铜水平,诱导了小鼠神经元细胞的丢失,激活了小鼠神经炎症,最终可导致小鼠运动功能障碍。

Objective To explore the effects of different doses of copper chloride exposure on motor function and development of neurons in substantia nigra in female C57BL/6J mice.Methods Thirty 12-week-old C57BL/6J mice were randomly divided into the control group,the 10 mg/Kg copper chloride treated group and the 20 mg/Kg copper chloride treated group(each n=10),and the mouse models were made by instillation for 8 weeks.At the end of the experiment,the mice were subjected to behavioral tests,including stick-turning,pole-climbing and open-field experiments,to detect their locomotor ability,and then the expression of neurons and inflammatory factors was probed by enzyme-linked immunosorbent assay and Western blot.Results The results of behavioral experiments displayed that the length of time on the rod was significantly higher in the copper-treated groups compared with the control group,and the length of time on the rod increased with the increase of toxicant concentration(F=27.03,P<0.05).The length of time on the rod in the copper-exposed mice decreased,and that in the 20 mg/Kg copper chloride treated group declined significantly(F=3.35,P=0.05).The results of open-field experiments revealed that the total distance traveled by mice in the open field decreased in the copper-treated groups compared with the control group(F=13.93,P<0.05).The results of Western blot showed that compared with the control group,the expression levels of tyrosine hydroxylase(F=8.74,P<0.05),dopamine transporter(F=13.09,P<0.05),Bcl-2(F=9.23,P<0.05)and PGC-1α(F=5.04,P=0.03)protein in substantia nigra and the number of Nissl+cells in substantia nigra(F=63.52,P<0.05)all decreased after copper treatment.Enzyme-linked immunosorbent assay presented that the copper levels in serum(F=5.93,P=0.02)and substantia nigra(F=7.48,P=0.01)in the copper-treated mice increased compared with the control group.The levels of pro-inflammatory factors IL-6(F=28.89,P<0.05),TNF-α(F=22.73,P<0.05)and IL-1β(F=11.77,P=0.002)all increased in the copper-treated mice,and the levels of IL-6 and TNF-αalso increased correspondingly with the increase of toxicant concentration.The copper-treated mice showed decreased levels of pro-inflammatory factors IL-4(F=4.79,P=0.04),IL-10(F=14.70,P=0.001)and TGF-β1(F=6.19,P=0.02).Conclusion Long-term exposure to copper chloride increases the copper levels in serum and substantia nigra of the mice,induces neuron loss,activates neuroinflammation in the mice,and eventually leads to motor dysfunction.