目的: 研究中药香青兰总黄酮(TFD)改善帕金森病模型小鼠神经损伤的作用机制。方法: 120只小鼠被随机分为正常对照组20只和观察组100只,观察组小鼠腹腔注射30 mg/(kg·d)MPTP以制备PD小鼠模型,对照组小鼠腹腔注射等体积生理盐水,连续7 d后再将观察组小鼠随机分为5组,分别为模型组和TFD治疗组[25、50、75、100 mg/(kg·d)]。治疗组按照小鼠的体重灌胃给药,正常对照组和模型组小鼠给予等容量的生理盐水,连续1个月。通过行为学实验评价小鼠运动功能,通过免疫荧光组织化学技术检测小鼠中脑黑质区组织细胞酪氨酸羟化酶(TH)和选择性自噬接头蛋白(P62)阳性染色的细胞数,采用荧光定量PCR和Western-blot技术检测小鼠黑质区组织的微管相关蛋白1轻链3β(LC3B)、P62、自噬效应蛋白(Beclin1)和TH mRNA和蛋白表达水平。结果: TFD治疗显著增加PD模型小鼠的悬挂运动评分、运动距离和下落潜伏期(P＜0.05);TFD治疗增加PD模型小鼠黑质区组织表达P62和TH的阳性染色细胞数,TFD治疗增加PD模型小鼠黑质区组织表达PINK1、Parkin、P62、和TH蛋白的阳性染色细胞数(P＜0.05)。结论: TFD可能通过上调LC3B、P62、Beclin1改善自噬,促进多巴胺能神经元受损线粒体及其他细胞器的清除,从而对MPTP诱导的PD模型小鼠产生神经保护作用。
Objective: To study the mechanism of total flavonoids of Dracocephalummoldavica L. (TFD) on improving nerve injury in Parkinson's disease model mice. Methods: A total of 120 mice were randomly divided into normal control group (n=20) and observation group (n=100). The mice in the observation group were intraperitoneally injected with 30 mg/(kg·d) PTP to prepare PD mouse model, and the mice in the control group were intraperitoneally injected with the same volume of normal saline. After 7 days, the mice in the observation group were randomly divided into 5 groups : model group and TFD treatment group [25, 50, 75, 100 mg/(kg·d )]. The mice in the treatment group were given intragastric administration according to the body weight of the mice, and the mice in the normal control group and the model group were given an equal volume of normal saline for 1 month. The motor function of mice was evaluated by behavioral experiments. The number of positive staining cells of tyrosine hydroxylase (TH) and selective autophagy adaptor protein (P62) in the substantia nigra of mice was detected by immunofluorescence histochemistry. The expression levels of microtubule-associated protein 1 light chain 3β (LC3B), P62, autophagy effector protein (Beclin1) and TH mRNA and protein in the substantia nigra of mice were detected by fluorescence quantitative PCR and Western-blot. Results: TFD treatment significantly increased the suspension motion score, movement distance and fall latency of PD model mice (P＜0.05). TFD treatment increased the number of positive staining cells expressing P62 and TH in the substantia nigra of PD model mice, and TFD treatment increased the number of positive staining cells expressing PINK1, Parkin, P62 and TH proteins in the substantia nigra of PD model mice (P＜0.05). Conclusion: TFD may improve autophagy by up-regulating LC3B, P62 and Beclin1, and promote the clearance of damaged mitochondria and other organelle of dopaminergic neurons, thus producing neuroprotective effects on MPTP-induced PD model mice.
[1] Dorsey ER, Sherer T, Okun MS, et al. The emerging evidence of the parkinson pandemic[J]. J Parkinsons Dis, 2018,8(s1):S3-S8.
[2] Deuschl G, Beghi E, Fazeks F, et al. The burden of neurological diseases in Europe: an analysis for the Global Burden of Disease Study 2017[J]. Lancet Public Health, 2020,5(10):e551-e567.
[3] Kalia LV, Lang AE. Parkinson’s disease[J].Lancet,2015,386(9996):896-912.
[4] BurbullaL F, Song PP, Mazzulli JR, et al. Dopamine oxidation mediates mitochondrial and lysosomal dysfunction in Parkinson’s disease[J].Science, 2017,357(6357):1255-1261.
[5] Galluzzi L, Baehrecke EH, Ballabio A, et al. Molecular definitions of autophagy and related processes[J]. EMBO J, 2017,36(13):1811-1836.
[6] Perfeito R,Cunha-oliveira,Rego AC. Reprint of: Revisiting oxidative stress and mitochondrial dysfunction in the pathogenesis of Parkinson disease-resemblance to the effect of amphetamine drugs of abuse[J].Radic Biol Med, 2013,62:186-201.
[7] Shpilka T, Haynes CM. The mitochondrial UPR: mechanisms, physiological functions and implications in ageing[J]. Nat Rev Mol Cell Biol, 2018,19(2):109-120.
[8] Med Collaborative PD,Gray R, Ives N, et al. Long-term effectiveness of dopamine agonists and monoamine oxidase B inhibitors compared with levodopa as initial treatment for Parkinson’s disease (PD MED):a large, open-label, pragmatic randomised trial[J]. Lancet, 2014,384(9949):1196-1205.
[9] 冯长根,李琼. 香青兰化学成分与药理活性研究综述[J]. 中成药,2003,25(2):155-157.
[10] 奇玲,罗达尚. 中国少数民族传统医药大系[M].呼和浩特:内蒙古科学技术出版社,2000:397-398.
[11] Wu P, Yan XS, Zhou LL, et al. Involvement of apoptosis in the protective effects of Dracocephalum moldavaica in cerebral ischemia reperfusion rat model[J].Toxicol Environ Health A, 2019,82(19):1036-1044.
[12] 周丽丽,莫超然,刘心朗,等. 蒙药香青兰总黄酮对帕金森病模型小鼠的神经保护作用[J].中国比较医学杂志,2021,31(5):41-46.
[13] Klionsky DJ,Al ZME.Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)[J].Autophagy,2016,1(12):1-222.
