光甘草定对MPTP致帕金森病小鼠认知障碍的防治作用及其机制研究

Effect and mechanism of Glabridin on cognitive impairment from MPTP to Parkinson's disease in mice

目的探讨中草药光甘草定(GLA)对1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)所致帕金森病(PD)小鼠认知障碍的改善作用及其作用机制,以及通过参与神经可塑性调节的细胞外信号调节蛋白激酶(ERK)信号通路影响对脑皮质、海马组织的保护作用。方法选择120只C57BL//6N小鼠,按随机数字表法分为对照组、模型组、GLA对照组和MPTP+GLA低、中、高剂量组,每组20只。采用腹腔注射MPTP 20 mg/kg的方法复制小鼠PD模型,对照组注射相同剂量的生理盐水。GLA对照组给予50 mg/kg GLA灌胃;MPTP+GLA低、中、高剂量组分别于每次MPTP给药1 h后用20、30和50 mg/kg的GLA灌胃,均每日1次,连用7 d。观察各组小鼠学习、记忆能力和海马组织的病理学改变,以及大脑皮质中肿瘤坏死因子-α(TNF-α)、白细胞介素-18(IL-18)、丙二醛(MDA)、超氧化物歧化酶(SOD)含量、酪氨酸羟化酶(TH)和磷酸化-ERK1/2(p-ERK1/2)的蛋白表达水平的变化。结果与对照组比较,模型组大脑皮质中MDA、TNF-α、IL-18含量均明显升高〔MDA(nmol/mg):4.68±0.51比2.05±0.22,TNF-α(μg/L):116.87±15.65比48.52±7.83,IL-18(μg/L):57.52±6.89比24.26±1.89,均P<0.05〕,而SOD活性明显降低(U/mg:77.84±7.84比130.89±18.56,P<0.05);与模型组比较,MPTP+GLA各组TNF-α、IL-18、MDA含量均明显降低,而SOD的活性明显增高(均P<0.05),以MPTP+GLA高剂量组的变化更明显;MPTP+GLA高剂量组小鼠学习和记忆能力明显改善,海马组织中神经元减少、神经变性改善,胶质细胞增生减少。免疫组化结果显示:与模型组比较,MPTP+GLA高剂量组小鼠皮质和海马组织TH阳性细胞的缺失明显减轻〔吸光度(A值):大脑皮质组织为39.14±3.25比23.14±3.10,海马组织为72.14±4.25比52.16±3.32,均P<0.05〕,p-ERK1/2的阳性细胞表达受到明显抑制(A值:大脑皮质组织为63.91±3.55比88.35±6.41,海马组织为73.36±3.52比93.25±6.73,均P<0.05);蛋白质免疫印迹试验(Western blotting)显示:与模型组比较,MPTP+GLA高剂量组皮质和海马组织TH的蛋白表达水平明显增加(灰度值:皮质组织为0.71±0.09比0.53±0.06,海马组织为0.68±0.06比0.45±0.03,均P<0.05),p-ERK1/2的蛋白表达水平明显受到抑制(灰度值:皮质组织为0.76±0.10比0.96±0.08,海马组织为0.74±0.06比0.96±0.12,均P<0.05)。结论中药GLA可改善MPTP诱导的PD小鼠学习和记忆能力,防治MPTP诱导的神经元损伤及胶质细胞增生;ERK信号通路与PD脑组织中神经损伤存在关联性;GLA通过抑制ERK信号通路发挥对MPTP所致PD小鼠学习和记忆能力的保护作用。

Objective To investigate the improving effect and mechanism of the traditional Chinese herbal medicine Glabridin(GLA)on cognitive impairment in Parkinson's disease(PD)mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP),and its protective effect on the cerebral cortex and hippocampus through affecting the extracellular regulated protein kinases(ERK)signaling pathway involved in the regulation of neural plasticity.Methods A total of 120 C57BL//6N mice were randomly divided into control group,model group,GLA control group and MPTP+GLA low,medium and high-dose groups,with 20 mice in each group.The mouse PD model was established by intraperitoneal injection of MPTP 20 mg/kg,and the control group was injected with the same dose of normal saline.The GLA control group was given 50 mg/kg GLA by gavage;the MPTP+GLA low,medium and high dose groups were given 20,30 and 50 mg/kg GLA by gavage 1 hour after each MPTP administration,once a day for 7 consecutive days.The learning and memory abilities of the mice in each group and the pathological changes of hippocampal tissue were observed,as well as the changes in the contents of tumor necrosis factor-α(TNF-α),interleukin-18(IL-18),malondialdehyde(MDA),superoxide dismutase(SOD),tyrosine hydroxylase(TH)and protein expression levels of phosphorylated-ERK 1/2(p-ERK1/2)in the cerebral cortex.Results Compared with the control group,the levels of MDA and TNF-α,IL-18 in the cerebral cortex of the model group were significantly increased[MDA(nmol/mg):4.68±0.51 vs.2.05±0.22,TNF-α(μg/L):116.87±15.65 vs.48.52±7.83,IL-18(μg/L):57.52±6.89 vs.24.26±1.89,all P<0.05],while the activity of SOD was significantly decreased(U/mg:77.84±7.84 vs.130.89±18.56,P<0.05).Compared with the model group,the contents of TNF-α,IL-18,and MDA in each MPTP+GLA group were significantly decreased,while the activity of SOD was significantly increased(all P<0.05),and the change in the high-dose MPTP+GLA group was more obvious.The learning and memory ability of the high-dose MPTP+GLA group was significantly improved,the number of neurons in the hippocampus tissue decreased,the neurodegeneration was improved,and the glial cell proliferation was reduced.The results of immunohistochemistry showed that compared with the model group,the loss of TH positive cells in the high-dose MPTP+GLA group was significantly reduced[absorbance(A value):cerebral cortex was 39.14±3.25 vs.23.14±3.1,hippocampus was 72.14±4.25 vs.52.16±3.32,both P<0.05],and the expression of p-ERK1/2 positive cells was significantly inhibited(A value:cerebral cortex was 63.91±3.55 vs.88.35±6.41,hippocampus was 73.36±3.52 vs.93.25±6.73,both P<0.05).Western blotting showed that compared with the model group,the protein expression level of cerebral cortex and hippocampus TH in the high-dose MPTP+GLA group was significantly increased(gray value:cerebral cortex was 0.71±0.09 vs.0.53±0.06,hippocampus was 0.68±0.06 vs.0.45±0.03,both P<0.05),and the protein expression level of p-ERK1/2 was significantly inhibited(gray value:cerebral cortex was 0.76±0.10 vs.0.96±0.08,hippocampus was 0.74±0.06 vs.0.96±0.12,both P<0.05).Conclusions The traditional Chinese medicine GLA can improve the learning and memory ability of MPTPinduced PD mice,as well as prevent and treat MPTP-induced neuronal damage and glial cell proliferation.The ERK signaling pathway is associated with neural damage in PD brain tissue.GLA protects the learning and memory ability of MPTP-induced PD mice by inhibiting the ERK signaling pathway.