摘要
目的探讨亚精胺降低脂质过氧化物积累、抑制神经元铁死亡、缓解帕金森病(Parkinson disease,PD)的作用机制。方法30只7~8周龄雄性C57BL/6小鼠随机分为对照组、PD模型组和亚精胺治疗组,每组10只。PD模型组与亚精胺治疗组小鼠连续5 d腹腔注射1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP,30 mg/kg)建立PD小鼠模型;对照组小鼠腹腔注射等体积生理盐水;亚精胺治疗组自PD造模第1天起腹腔注射10 mg/kg亚精胺治疗,连续14 d。采用旷场实验测定小鼠运动总距离和平均运动速度,评价亚精胺治疗对PD小鼠模型行为学的影响。取小鼠黑质,经透射电子显微镜观察,并通过ELISA试剂盒测定黑质中丙二醛(MDA)、活性氧自由基(ROS)和Fe^(2+)水平。Western blot法测定小鼠黑质铁死亡相关蛋白核因子E2相关因子2(Nrf2)、谷胱甘肽过氧化物酶4(GPX4)和血红素氧合酶-1(HO-1)的表达水平。Neuro2A细胞分为vehicle组、LPS组、亚精胺+LPS组、亚精胺+LPS+激活转录因子4短发夹RNA(ATF4 shRNA)组和亚精胺+LPS+非靶向短发夹RNA(shRNA NT)组。vehicle组不做任何处理,LPS组细胞加入100μg/mL LPS,亚精胺+LPS组细胞加入100μg/mL LPS和50μg/mL亚精胺,亚精胺+LPS+ATF4 shRNA组和亚精胺+LPS+shRNA NT组细胞分别转染ATF4 shRNA或shRNA NT后,加入100μg/mL LPS和50μg/mL亚精胺。采用RT-qPCR方法检测ATF4 mRNA;CCK-8法测定细胞增殖能力;ELISA法检测Fe^(2+)、MDA水平;Western blot测定细胞自噬相关蛋白LC3-Ⅰ/LC3-Ⅱ、Beclin和p62的表达水平。结果与对照组相比,PD模型组小鼠旷场实验运动总距离和平均运动速度降低(均P<0.05);PD模型组小鼠黑质神经元出现肿胀、坏死或凋亡,神经元内线粒体内膜皱缩,嵴间隙变宽,膜密度增加;MDA、ROS、Fe^(2+)水平升高(均P<0.05);Nrf2、GPX4和HO-1蛋白相对表达降低(均P<0.05)。与PD模型组相比,亚精胺治疗组上述指标显著逆转(均P<0.05)。体外细胞实验,与vehicle组相比,LPS组细胞增殖能力降低(P<0.05);Fe^(2+)、MDA水平升高(均P<0.05);LC3-Ⅰ/LC3-Ⅱ、Beclin相对表达水平降低(均P<0.05),p62相对表达水平升高(P<0.05);ATF4 mRNA相对表达水平差异无统计学意义(P>0.05)。与LPS组相比,亚精胺+LPS组细胞增殖能力增强(P<0.05);Fe^(2+)、MDA水平降低(均P<0.05);LC3-Ⅰ/LC3-Ⅱ、Beclin相对表达水平升高(均P<0.05),p62相对表达水平降低(P<0.05);ATF4 mRNA相对表达水平升高(P<0.05)。与亚精胺+LPS组相比,亚精胺+LPS+ATF4 shRNA组的ATF4 mRNA相对表达水平降低(P<0.05),且部分抵消了亚精胺处理对上述指标的逆转效果(均P<0.05)。结论亚精胺通过上调ATF4的表达促进神经元自噬,逆转神经元内脂质过氧化物积累,抑制神经元铁死亡,从而缓解帕金森氏病。
Objective To investigate whether Spermidine alleviates Parkinson′s disease(PD)by reducing lipid peroxide accumula⁃tion and inhibiting neuronal ferroptosis.Methods Thirty male C57BL/6 mice(7 to 8 weeks old)were randomly divided into three groups(n=10 per group):control group,PD model group,and Spermidine group.Mice in PD model group and Spermidine group received intraperitoneal injection of MPTP(30 mg/kg)for 5 consecutive days.Control mice received an equal volume of normal saline.From the first day of MPTP administration,mice in Spermidine group were administered with Spermidine(10 mg/kg,i.p.)for 14 consecutive days.The behavioral effects of Spermidine on PD model mice were assessed using the open field test,with total distance traveled and average speed analyzed.The substantia nigra tissues of mice were collected and observed by transmission electron microscopy(TEM).The levels of malondialdehyde(MDA),reactive oxygen species(ROS),and Fe^(2+)in the substantia nigra were determined using corresponding ELISA kits.The protein expression levels of ferroptosis-related factors(Nrf2,GPX4,and HO-1)in the substantia nigra tissues were measured by Western blot analysis.Neuro2A cells were divided into five groups:vehicle group,lipopolysaccharide(LPS)group,Spermidine+LPS group,Spermidine+LPS+ATF4 shRNA group,and Spermidine+LPS+shRNA NT group.The cells in vehicle group received no treatment;the cells in LPS group were treated with 100μg/mL LPS;the cells in Spermidine+LPS group were treated with 100μg/mL LPS and 50μg/mL Spermidine;and the cells in both Spermidine+LPS+ATF4 shRNA group and Spermidine+LPS+shRNA NT group were first transfected with ATF4 shRNA or shRNA NT,respectively,and then treated with 100μg/mL LPS and 50μg/mL Spermidine.ATF4 mRNA levels were qualified by RT-qPCR.Cell proliferation was assessed using CCK-8 assay.Fe^(2+)and MDA levels were measured by ELISA.The expressions of autophagy-related proteins(LC3-Ⅰ/LC3-Ⅱ,Beclin and p62)were analyzed by Western blotting.Results Compared with control group,mice in PD model group exhibited a significant reduction in both the total movement distance and the average speed in the open field test(both P<0.05);TEM revealed neurons in the substantia nigra with signs of swelling,necrosis,or apoptosis;the mitochondrial inner membrane of the neurons was shrunken,the crista gaps were wi⁃dened,and the membrane density was increased;in addition,the levels of MDA,ROS,and Fe^(2+)in the substantia nigra were signifi⁃cantly elevated in PD model group(all P<0.05),while the relative protein expression levels of Nrf2,GPX4 and HO-1 were signifi⁃cantly decreased(all P<0.05).Spermidine treatment significantly reversed the above-mentioned changes in PD model mice(all P<0.05).In vitro cell experiments,compared with vehicle group,the cell proliferation ability was significantly decreased in LPS group(P<0.05),the levels of Fe^(2+)and MDA were significantly increased(all P<0.05),the relative protein expression levels of autophagy-related proteins LC3-Ⅰ/LC3-Ⅱand Beclin were significantly decreased(all P<0.05),and the relative protein expression level of p62 was sig⁃nificantly increased(P<0.05);the ATF4 mRNA expression levels showed no significant change(P>0.05).Compared with LPS group,the cells in Spermidine+LPS group showed enhanced cell proliferation(P<0.05),reduced levels of Fe^(2+)and MDA(both P<0.05),upregulated relative protein expression of LC3-Ⅰ/LC3-Ⅱand Beclin(both P<0.05),and downregulated relative protein expression of p62(P<0.05).Furthermore,Spermidine treatment significantly increased the mRNA expression of ATF4(P<0.05).Compared with Spermidine+LPS group,ATF4 mRNA expression decreased in Spermidine+LPS+ATF4 shRNA group(P<0.05),and the reversing effect of Spermidine was partially abolished(all P<0.05).Conclusion Spermidine can alleviate Parkinson′s disease by up⁃regulating ATF4 expression,thereby promoting neuronal autophagy,reversing lipid peroxide accumulation,and inhibiting neuronal ferroptosis.
作者
范春娜
迟杰骏
陶静
FAN Chunna;CHI Jiejun;TAO Jing(Department of Rehabilitation Medicine,Xinjiang Uygur Autonomous Region People′s Hospital,Urumqi 830001,China)
出处
《山西医科大学学报》
2026年第2期160-167,共8页
Journal of Shanxi Medical University
基金
新疆维吾尔自治区自然科学基金项目(2022D01C125)。
