徐利,李莹莹,张锦军,丁剑
XU Li,LI Yingying,ZHANG Jinjun,DING Jian

• 1:山东省泰山医院神经内科

摘要(Abstract)：

目的 探讨毛钩藤碱对帕金森病(Parkinson′s disease, PD)模型大鼠的神经保护作用及机制。方法 采用右侧中脑黑质致密部注射6-羟基多巴胺(6-hydroxydopamine, 6-OHDA)制备大鼠PD模型，将建模成功的PD大鼠随机分为模型组、美多芭组、毛钩藤碱低剂量和高剂量组，同时设置正常对照组，每组12只。给予相应药物灌胃干预，每天1次，连续1个月。采用旋转试验、转棒试验和Morris水迷宫试验检测大鼠行为学变化；HE染色法观察大鼠右侧中脑黑质病理学变化；高效液相色谱法(HPLC)检测大鼠右侧纹状体中多巴胺(dopamine, DA)水平；免疫组化法检测大鼠右侧中脑黑质中多巴胺能神经元标志物酪氨酸羟化酶(tyrosine hydroxylase, TH)表达；ELISA法检测大鼠右侧中脑黑质IL-6、IL-1β和TNF-α水平；Western blot检测大鼠右侧中脑黑质中NF-κB p65、NF-κB抑制因子α(IκBα)、p-IκBα等蛋白表达水平。结果 毛钩藤碱能显著改善PD大鼠行为学障碍及中脑黑质损伤，升高纹状体DA含量及中脑黑质中TH阳性神经元数量和IκBα蛋白表达水平，降低中脑黑质中IL-6、IL-1β和TNF-α水平及p-IκBα、NF-κB p65蛋白表达水平(P<0.05)。结论 毛钩藤碱可通过减少多巴胺能神经元丢失，提高脑内DA含量，抑制炎症反应，改善PD大鼠行为学障碍，其机制可能与抑制NF-κB信号通路活化有关。
Objective To investigate the mechanism of hirsutine on the nerve of rats with Parkinson′s disease(PD).Methods The PD rat model was established by injecting 6-hydroxydopamine(6-OHDA)into the dense part of the right midbrain substantia nigra.The successful PD-modeled rats were randomly divided into model group, madopar group, hirsutine low-dose and high-dose groups, and normal control group, 12 in each group.Each group was given relevant drug gavage intervention, once a day for 1 month.Rotation test, rotating rod test and Morris water maze test were used to detect the behavioral changes of rats.The pathological changes of the right midbrain substantia nigra of rats were observed by HE staining.The level of dopamine(DA)in the right striatum of rats was detected high performance liquid chromatography(HPLC).The expression of dopaminergic neurons marker tyrosine hydroxylase(TH)in the right midbrain substantia nigra was detected by immunohistochemistry.The levels of IL-6,IL-1β and TNF-α in the right midbrain substantia nigra of rats were detected by ELISA.The protein expression levels of NF-κB p65,NF-κB inhibitor α(IκBα)and p-IκBα in the right midbrain substantia nigra of rats were detected by Western blot.Results Hirsutine could significantly improve the behavioral impairment and midbrain substantia nigra injury of PD rats, increase the DA content in striatum, the number of TH positive neurons and the expression of IκBα protein in midbrain substantia nigra, and decrease the levels of IL-6,IL-1β and TNF-α in the midbrain substantia nigra and the protein expressions of p-IκBα and NF-κB p65(P<0.05).Conclusion Hirsutine has improved the behavioral disorder of PD rats by reducing the loss of dopaminergic neurons, increasing the content of DA in the brain, and inhibiting the inflammatory response.The mechanism may be related to the inhibiting of NF-κB signaling pathway activation.

关键词(KeyWords)： 毛钩藤碱;帕金森病;神经保护;NF-κB信号通路
hirsutine;Parkinson′s disease;neuroprotection;NF-κB signaling pathway

Abstract:

Keywords:

基金项目(Foundation): 吴阶平医学基金会临床科研专项资助基金(320.6750.18328)

作者(Author): 徐利,李莹莹,张锦军,丁剑
XU Li,LI Yingying,ZHANG Jinjun,DING Jian

DOI: 10.14066/j.cnki.cn21-1349/r.2021.0836

参考文献(References)：

• [1] GUO M,WANG J,ZHAO Y,et al.Microglial exosomes facilitate α-synuclein transmission in Parkinson′s disease[J].Brain,2020,143(5):1476-1497.
• [2] SAINI N,AKHTAR A,CHAUHAN M,et al.Protective effect of indole-3-carbinol,an NF-κB inhibitor in experimental paradigm of Parkinson′s disease:in silico and in vivo studies[J].Brain Behav Immun,2020,90:108-137.
• [3] LAN YL,ZHOU J J,LIU J,et al.Uncaria rhynchophylla ameliorates Parkinson′s disease by inhibiting HSP90 expression:insights from quantitative proteomics[J].Cell Physiol Biochem,2018,47(4):1453-1464.
• [4] WANG M,GUO J,WANG Z,et al.Simultaneous separation and determination of hirsutine and hirsuteine by cyclodextrin-modified micellar electrokinetic capillary chromatography[J].Phytochem Anal,2020,31(1):112-118.
• [5] WU L X,GU X F,ZHU Y C,et al.Protective effects of novel single compound,hirsutine on hypoxic neonatal rat cardiomyocytes[J].Eur J Pharmacol,2011,650(1):290-297.
• [6] WANG W W,GAO H,LI W T.Study on the protective effects of bushen zhichan prescription on dopaminergic neurons in 6-hydroxydopamine-induced Parkinson′s disease model[J].Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology(世界科学技术-中医药现代化),2020,22(9):3270-3276.
• [7] ZHENG L,WANG H,BA Y Y,et al.Protective effect of alkaloids from Piper longum in rat dopaminergic neuron injury of 6-OHDA-induced Parkinson′s disease[J].China Journal of Chinese Materia Medica(中国中药杂志),2014,39(9):1660-1665.
• [8] SUN S N,LI F.The effect of Bifidobacterium lactis on anxiety and depression in patients with Parkinson′s disease[J].Journal of Shenyang Pharmaceutical University(沈阳药科大学学报),2020,37(4):344-349.
• [9] JIANG X,NIU C,WANG J B,et al.Neuroprotective effect of Ethyl rosmarinate in rats with Parkinson′s disease induced by 6-hydroxydopamine[J].Journal of Liaoning University of Traditional Chinese Medicine(辽宁中医药大学学报),2019,21(11):57-60.
• [10] SURMEIER D J.Determinants of dopaminergic neuron loss in Parkinson′s disease[J].FEBS J,2018,285(19):3657-3668.
• [11] MASATO A,PLOTEGHER N,BOASSA D,et al.Impaired dopamine metabolism in Parkinson′s disease pathogenesis[J].Mol Neurodegener,2019,14(1):35.
• [12] QIAN H,KANG X,HU J,et al.Reversing a model of Parkinson′s disease with in situ converted nigral neurons[J].Nature,2020,582(7813):550-556.
• [13] KAWAKAMI Z,IKARASHI Y,KASE Y.Isoliquiritigenin is a novel NMDA receptor antagonist in kampo medicine yokukansan[J].Cell Mol Neurobiol,2011,31(8):1203-1212.
• [14] JUNG H Y,NAM K N,WOO B C,et al.Hirsutine,an indole alkaloid of Uncaria rhynchophylla,inhibits inflammation-mediated neurotoxicity and microglial activation[J].Mol Med Rep,2013,7(1):154-158.
• [15] SINGH S S,RAI S N,BIRLA H,et al.NF-κB-Mediated neuroinflammation in Parkinson′s disease and potential therapeutic effect of polyphenols[J].Neurotox Res,2020,37(3):491-507.
• [16] LI Z H,CAI R L,SUN J,et al.Effect of shenling baizhusan on protein and mRNA expression of NF-κB p65,IκBα,IκKβ in ulcerative colitis rats with syndrome of dampness stagnancy due to spleen deficiency[J].Chinese Journal of Experimental Traditional Medical Formulae(中国实验方剂学杂志),2020,26(19):108-113.
• [17] WU J,NIU P,ZHAO Y,et al.Impact of miR-223-3p and miR-2909 on inflammatory factors IL-6,IL-1β,and TNF-α,and the TLR4/TLR2/NF-κB/STAT3 signaling pathway induced by lipopolysaccharide in human adipose stem cells[J].PLoS One,2019,14(2):e0212063.
• [18] HUANG B,LIU J,MENG T,et al.Polydatin prevents lipopolysaccharide(LPS)-induced Parkinson′s disease via regulation of the AKT/GSK3β-Nrf2/NF-κB signaling axis[J].Front Immunol,2018,9:2527.
• [19] LOU C,TAKAHASHI K,IRIMURA T,et al.Identification of hirsutine as an anti-metastatic phytochemical by targeting NF-κB activation[J].Int J Oncol,2014,45(5):2085-2091.
• [20] LOU C,YOKOYAMA S,SAIKI I,et al.Selective anticancer activity of hirsutine against HER2positive breast cancer cells by inducing DNA damage[J].Oncol Rep,2015,33(4):2072-2076.
• [21] BI Y,QU P C,WANG Q S,et al.Neuroprotective effects of alkaloids from Piper longum in a MPTP-induced mouse model of Parkinson′s disease[J].Pharm Biol,2015,53(10):1516-24.
• [22] LI C,SUN Y,YANG W,et al.Pilose antler extracts(PAEs)protect against neurodegeneration in 6-OHDA-induced Parkinson′s disease rat models[J].Evid Based Complement Alternat Med,2019,2019:7276407.
• [23] ZHANG H,MA H Q,WANG X J.The protective effect of Ginkgo biloba extract on the brain of Parkinson′s disease rats through the activation of the Nrf2-ARE pathway[J].Journal of Shenyang Pharmaceutical Universitys(沈阳药科大学学报),2018,35(8):675-679.