摘要
目的 在动物水平探讨铁死亡脂质过氧化损伤调控通路与帕金森病(Parkinson's disease, PD)的发病机制。方法 建立PD斑马鱼动物模型,取受精后48 h斑马鱼胚胎为对照组(n=100),200μmol/L的1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP)为MPTP组(n=100),200μmol/L的MPTP+1.5μg/ml诺米芬辛为诺米芬辛组(n=100)。对受精后5 d斑马鱼进行行为学分析(检测运动能力、运动速率及运动距离);免疫荧光检测斑马鱼脑内α突触核蛋白和酪氨酸羟化酶表达,评估多巴胺能神经元(dopaminergic neurons, DA)活性;检测铁、谷胱甘肽(glutathione, GSH)及脂质过氧化水平,包括过氧化氢酶(catalase, CAT)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、脂质过氧化物(lipid peroxide, LPO)、丙二醛、活性氧,验证铁沉积可能通过铁死亡的脂质过氧化代谢调控机制引起DA损伤。结果 与对照组比较,MPTP组运动速率和运动距离明显降低,差异有统计学意义(P<0.01);与MPTP组比较,诺米芬辛组运动速率和运动距离明显升高,差异有统计学意义(P<0.01)。免疫荧光检测显示,与对照组比较,MPTP组DA损伤明显增加,诺米芬辛组DA损伤明显轻于MPTP组。各组CAT水平比较,差异无统计学意义(P>0.05)。与对照组比较,MPTP组铁、LPO和丙二醛水平明显升高,GSH、GPX4水平明显降低,差异有统计学意义(838.18±143.42 vs 478.53±112.58,P<0.05;1.71±0.11 vs 1.48±0.14,P<0.05;4.50±0.64 vs 4.23±0.13,P<0.05;38.93±1.72 vs 45.97±2.32,P<0.05;0.17±0.03 vs 0.39±0.04,P<0.05);与MPTP组比较,诺米芬辛组GSH、GPX4水平明显升高,铁、LPO水平明显降低,差异有统计学意义(40.79±1.02 vs 38.93±1.72,P<0.05;0.26±0.05 vs 0.17±0.03,P<0.05;326.75±110.95 vs 838.18±143.42,P<0.05;1.01±0.27 vs 1.71±0.11,P<0.05)。活性氧流式细胞检测结果显示,对照组、MPTP组及诺米芬辛组活性氧阳性细胞占比分别为直方图:31.6%、46.2%、31.3%,散点图:62.4%、73.0%、65.7%。结论 在MPTP诱导的斑马鱼基础上,PD斑马鱼存在铁死亡。从动物水平阐明铁沉积引起DA损伤与铁死亡脂质过氧化代谢机制有关。
Objective To investigate the regulatory pathway of lipid peroxidation in ferroptosis and the pathogenesis of Parkinson's disease(PD)at animal level.Methods Zebrafish PD model was established by exposure to 200μmol/L 1-methyl-4-pheny-1,2,3,6-tetrahydropyridine(MPTP).Zebrafish embryo in 48 h after fertilisation were grouped into a control group(n=100),a MPTP group(n=100),and a MPTP+Nomi group(1.5 ug/ml nomifensine,n=100).Behavioral analysis was performed on zebrafish in 5 d after fertilization for movement ability,rate and distance.The expression of a-synuclein and tyrosine hydroxylase in the zebrafish brain was detected by immu-nofluorescence assay to evaluate the activity of dopaminergic neurons(DA).The levels of iron,glutathione(GSH)and lipid peroxidation,which includes catalase(CAT),glutathione peroxidase 4(GPX4),lipid peroxide(LPO),malondialdehyde(MDA)and reactive oxygen species(ROS),were measured to verify whether iron deposition may cause damage to DA through the metabolic regulation mechanism of lipid peroxidation in iron death.Results Compared with the control group,the movement rate and distance were significantly reduced in the MPTP group(P<0.01),while Nomi treatment obviously increased the movement rate and distance when compared with the MPTP group(P<0.01).The MPTP group exhibited obvious DA damage in comparison with the control group,and Nomi treatment reversed the damage caused by MPTP.There was no sta-tistical difference in the CAT level in the three group(P>0.05).The MPTP group had signifi-cantly increased levels of iron,LPO and MDA and decreased levels of GSH and GPX4 when com-pared with the control group(838.18±143.42 us 478.53±112.58,P<0.05;1.71±0.11 us 1.48±.0.14,P<0.05;4.50±0.64 us 4.23±0.13,P<0.05;38.93±1.72 us 45.97±2.32,P<0.05;0.17±0.03 us 0.39±0,04,P<0.05).The MPTP+Nomi group had significantly increased levels of GSH and GPX4 and decreased levels of iron and LPO when compared with the MPTP group(40.79±.1.02 us 38.93±1.72,P<0.05;0.26±0.05 us 0.17±0.03,P<0.05;326.75±110.95 us 838.18±143.42,P<0.05;1.01±0.27 us 1.71±0.11,P<0.05).The results of the ROS flow cytometry analysis showed that the percentages of ROS positive cells in the control group,MPTP group,and Nomi group were as follows:Histogram:31.6%,46.2%and 31.3%,Scatter plot:62.4%,73.0%and 65.7%,respectively.Conclusion Ferroptosis is found in PD zebrafish induced by MPTP exposure.Our study has clarified at animal level that the damage of DA caused by iron deposition is related to the metabolic mechanism of lipid peroxidation caused by iron death.
作者
姚亚妮
夏欢
冯婷婷
杨新玲
Yao Yani;Xia Huan;Feng Tingting;Yang Xinling(Fourth De partment of Com prehensive Internal Medicine,The First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,Xinjiang Uygur Autonomous Region,China)
出处
《中华老年心脑血管病杂志》
北大核心
2025年第6期793-797,共5页
Chinese Journal of Geriatric Heart,Brain and Vessel Diseases
基金
国家自然科学基金(82160232)
新疆维吾尔自治区杰出青年基金(2022D01E68)。
