毛蕊花糖苷对MPTP制备的帕金森病模型小鼠的神经保护作用及机制研究

Research on the protective effect and mechanisms of verbascoside on Parkinson disease model mice prepared by MPTP

目的探讨毛蕊花糖苷对帕金森病(PD)模型小鼠脑的神经保护作用及其可能的机制。方法选取75只C57/BL小鼠(SPF级、健康雄性、24~26 g)为实验对象,随机分为空白对照组、模型组(1-甲基-4-苯基-1,2,3,6-四氢吡啶/MPTP组)、实验组(低剂量组:MPTP+30 mg/kg毛蕊花糖苷;中剂量组:MPTP+60 mg/kg毛蕊花糖苷;高剂量组:MPTP+120 mg/kg毛蕊花糖苷),每组15只。造模完成后采用爬杆、悬挂实验检测各组小鼠的行动能力,采用超微电镜检测多巴胺能神经元细胞亚细胞结构变化,采用免疫组织化学染色法检测酪氨酸羟化酶(TH)阳性细胞数量,采用Western blotting检测TH、α-突出核蛋白(α-syn)、核因子红细胞-2相关因子2(Nrf2)、血红素加氧酶-1(HO-1)、谷胱甘肽过氧化物酶4(GPX4)蛋白的表达情况,采用酶联免疫吸附试验(ELISA)检测各组小鼠黑质的谷胱甘肽、总铁离子、超氧化物歧化酶(SOD)、脑组织丙二醛等。结果模型组小鼠完成爬杆实验的时间较空白对照组长,实验组小鼠完成爬杆实验的时间较模型组短,差异均有统计学意义(均P<0.05);模型组小鼠的悬挂实验评分低于空白对照组,实验组小鼠的悬挂实验评分高于模型组,差异均有统计学意义(均P<0.05)。电镜下模型组较实验组小鼠中脑黑质区多巴胺能神经元细胞的变性、坏死更明显;免疫组织化学染色结果显示,模型组小鼠黑质TH阳性神经元数目较空白对照组减少,实验组小鼠黑质TH阳性细胞数目较模型组增加,差异均有统计学意义(均P<0.05)。Western blotting结果显示,模型组小鼠黑质TH、Nrf2、HO-1、GPX4的蛋白表达量低于空白对照组,实验组小鼠黑质TH、Nrf2、HO-1、GPX4的蛋白表达量高于模型组,差异均有统计学意义(均P<0.05);模型组小鼠黑质α-syn表达量高于空白对照组,实验组小鼠黑质α-syn表达量低于模型组,差异均有统计学意义(均P<0.05)。ELISA结果显示,模型组小鼠黑质的谷胱甘肽、SOD表达量低于空白对照组,实验组小鼠黑质的谷胱甘肽、SOD表达量高于模型组,差异均有统计学意义(均P<0.05);模型组小鼠黑质的的丙二醛、总铁离子表达量高于空白对照组,实验组的丙二醛、总铁离子表达量低于模型组,差异均有统计学意义(均P<0.05)。结论毛蕊花糖苷可能是通过抑制PD模型小鼠中脑黑质区的多巴胺能神经元细胞的铁死亡起到神经保护作用。

Objective To investigate the neuroprotective effect of verbascoside(VB)on the brain of Parkinson's disease(PD)model mice and its possible mechanism.Methods A total of 75 C57/BL mice(SPF grade,healthy male,24-26 g)were randomly divided into blank control group,model group(MPTP group),and experimental group(low dose group:MPTP+30 mg/kg VB;medium dose group:MPTP+60 mg/kg VB;high dose group:MPTP+120 mg/kg VB),with 15 mice in each group.After the establishment of the model,the movement ability of mice in different groups was detected by rod climbing and hanging experiments;dopaminergic neurons were detected by ultramicroelectron microscopy;the number of tyrosine hydroxylase(TH)positive cells was detected by immunohistochemical staining;the expression of tyrosine hydroxylase(TH),α-synuclein(α-syn),nuclear factor erythroid 2-related factor 2(Nrf2),heme oxygenase-1(HO-1),glutathione peroxidase 4(GPX4)signaling pathway related proteins was detected by Western blotting;glutathione,total iron ion,superoxide dismutase(SOD),and malondialdehyde in brain tissue was detected by enzyme-linked immunosorbent assay.Results The time of rod climbing experiment of mice in model group was significantly longer than that of blank control group,the time of rod climbing experiment of mice in experimental group was significantly shorter than that of model group,with statistical significance(P<0.05);the score of hanging experiment of mice in model group was lower than that of blank control group,the score of hanging experiment of mice in experimental group was higher than that of model group,with statistical significance(P<0.05).The degeneration and necrosis of dopaminergic neurons in substantia nigra of the midbrain of mice in the model group(MPTP group)were more obvious than those in the experimental group under electron microscope;immunohistochemical staining experiment showed that the number of TH positive cells in the model group was much lower than that in the blank control group,the number of TH positive cells in the experimental group was much higher than that in the model group,with statistical significance(P<0.05).Western blotting experiment showed that the expression of TH,Nrf2,HO-1,and GPX4 in the model group was lower than that in the blank control group,the expression of TH,Nrf2,HO-1,and GPX4 in the experimental group was higher than that in the model group,with statistical significance(P<0.05);the expression ofα-syn in the model group was higher than that in the blank control group,the expression ofα-syn in the experimental group was lower than that in the model group,with statistical significance(P<0.05).The results of ELISA showed that the expression of glutathione and SOD in the model group was significantly lower than that in the blank control group,the expression of glutathione and SOD in the experimental group was significantly higher than that in the model group,with statistical significance(P<0.05);the expression of malondialdehyde and total iron ions in the model group was significantly lower than that in the blank control group,the expression of malondialdehyde and total iron ions in the experimental group was significantly lower than that in the model group,with statistical significance(P<0.05).Conclusions Verminoside may play a protective role by inhibiting iron death of dopaminergic neurons in the substantia nigra of Parkinson's disease model mice.