砷、镉慢性雾化吸入通过NF-κB/NLRP3信号通路引起小鼠纹状体多巴胺能神经元丢失

Chronic aerosol inhalation of arsenic and cadmium induces dopaminergic neuron loss in mouse striatum via NF-κB/NLRP3 signaling pathway

目的组织原位表征砷、镉慢性雾化吸入暴露小鼠纹状体神经病理学特征,从氧化应激—NF-κB/NLRP3通路切入,探讨其病理机制。方法选用8周龄C57BL/6J雄性小鼠,分为对照组(n=6)、亚砷酸钠雾化吸入组(n=6)和氯化镉雾化吸入组(n=4),每天经超声雾化吸入双重去离子水、6 mg/kg亚砷酸钠溶液和10 mg/kg氯化镉溶液4 h,染毒6个月。麻醉下脱颈处死取脑,HE染色和尼氏染色观察纹状体病理学改变;免疫组织化学法检测8-羟基脱氧鸟苷(8-OHdG)、酪氨酸羟化酶(TH)、p-NF-κB-p65和NLRP3水平;GFAP与C3、Iba-1与CD68免疫荧光共标法分别检测星形胶质细胞和小胶质细胞活化。结果与对照组相比,砷、镉雾化吸入组纹状体受损神经元明显增多(尼氏染色:t_(As)=4.920,t_(Cd)=6.185,P<0.01;HE染色:t_(As)=4.150,t_(Cd)=6.761,P<0.01),TH水平降低(t_(As)=2.145,t_(Cd)=4.603,P<0.05或P<0.01),8-OHdG、p-NF-κB-p65和NLRP3水平提高(8-OHdG:t_(As)=3.993,t_(Cd)=2.396,P<0.01或P<0.05;p-NF-κB-p65:t_(As)=7.117,t_(Cd)=9.352,P<0.01;NLRP3:t_(As)=6.967,t_(Cd)=7.306,P<0.01),炎性活化的星形胶质细胞(A1型)与小胶质细胞(M1型)比例明显升高,差异有统计学意义(A1型星形胶质细胞:t_(As)=4.586,t_(Cd)=3.003,P<0.01或P<0.05;M1型小胶质细胞:t_(As)=6.135,t_(Cd)=6.245,P<0.05)。结论慢性砷、镉吸入暴露会损害纹状体多巴胺能神经元,机制可能与诱导氧化应激,激活NF-κB/NLRP3炎性小体通路,引起胶质细胞炎性响应有关。

Objective The neuropathological characteristics of striatum of mice chronically exposed to arsenic and cadmium by aerosol inhalation were characterized in situ,and the pathological mechanism was investigated from the oxidative stress-NF-κb/NLRP3 pathway.Methods 8-week-old C57BL/6J male mice were selected and divided into control group(n=6),sodium arsenite atomization inhalation group(n=6)and cadmium chloride atomization inhalation group(n=4).The mice were subjected to ultrasonic atomization inhalation of double deionized water,6 mg/kg sodium arsenite solution and 10 mg/kg cadmium chloride solution respectively for 4 h every day,lasting for 6 months.Mouse brains were extracted under anesthesia.The pathological changes of striatum were observed by HE staining and Nissl staining.The levels of 8-hydroxy-deoxyguanosine(8-OHdG),tyrosine hydroxylase(TH),p-NF-κB-p65 and NLRP3 were detected by immunohistochemical method.The activation of astrocytes and microglia was detected by GFAP co-labeled with C3 and Iba-1 colabeled with CD68 by immunofluorescence staining.Results Compared with the control group,more neurons were damaged in the striatum in the arsenic and cadmium atomization inhalation groups(Nissl staining:t_(As)=4.920,t_(Cd)=6.185,P<0.01;HE staining:t_(As)=4.150,t_(Cd)=6.761,P<0.01),TH levels decreased(t_(As)=2.145,t_(Cd)=4.603,P<0.05 or P<0.01),the levels of 8-OHdG,p-NF-κB-p65 and NLRP3 increased(8-OHdG:t_(As)=3.993,t_(Cd)=2.396,P<0.01 or P<0.05;p-NF-κB-p65:t_(As)=7.117,t_(Cd)=9.352,P<0.01;NLRP3:t_(As)=6.967,t_(Cd)=7.306,P<0.01),the ratios of inflammatory activated A1 astrocytes and M1 microglia increased significantly(A1 astrocytes:t_(As)=4.586,t_(Cd)=3.003,P<0.01 or P<0.05;M1 microglia:t_(As)=6.135,t_(Cd)=6.245,P<0.05).Conclusion Inhalation exposure to arsenic and cadmium can damage striatal dopaminergic neurons,and the mechanism may be related to the activation of NF-κB/NLRP3 inflammasome pathway by inducing oxidative stress and causing inflammatory response of glial cells.