| 吴忧,朱虹,张厚文,等.菖蒲郁金汤对帕金森病小鼠的神经保护作用及机制探讨[J].浙江中医药大学学报,2022,46(10):1070-1076, 1085. |
| 菖蒲郁金汤对帕金森病小鼠的神经保护作用及机制探讨 |
| The Neuroprotective Effect and Mechanism of Changpu Yujin Decoction on Mice of Parkinson‘s Disease |
| DOI：10.16466/j.issn1005-5509.2022.10.003 |
| 中文关键词: 帕金森病 菖蒲郁金汤 多巴胺能神经元 自噬 α-突触核蛋白 神经保护 纹状体 中脑黑质 |
| 英文关键词: Parkinson‘s disease Changpu Yujin Decoction dopaminergic neurons autophagy α-synuclein neuroprotection striatum substantia nigra of midbrain |
| 基金项目:浙江省卫生健康科技计划项目(2021KY841、2022KY929)；浙江中医药大学校级科研基金项目(2020ZG47) |
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| 中文摘要: |
| [目的] 观察菖蒲郁金汤对帕金森病(Parkinson‘s disease，PD)小鼠的治疗作用以及对多巴胺(dopamine，DA)能神经元的保护作用，并探讨其可能的作用机制。[方法] 将36只C57BL/6雄性小鼠随机分为正常对照组、MPTP模型组、菖蒲郁金汤组，每组12只。MPTP模型组和菖蒲郁金汤组采用1-甲基-4-苯基-1，2，3，6-四氢吡啶(1-methyl-4-phenyl-1，2，3，6-tetrahydropyridine，MPTP)腹腔注射，30 mg/(kg·d)，连续7 d，建立PD小鼠模型，正常对照组给予等量0.9%氯化钠溶液腹腔注射，连续7 d。菖蒲郁金汤组在MPTP注射的同时开始给予20 g/(kg·d)菖蒲郁金汤灌胃21 d，MPTP模型组和正常对照组分别在MPTP、0.9%氯化钠溶液注射的同时给予20 g/(kg·d)的0.9%氯化钠溶液灌胃21 d。最后给药结束后1、7和14 d时对各组小鼠分别进行行为学评价。14 d时处死小鼠，取小鼠中脑黑质部位脑组织，应用酪氨酸羟化酶免疫组化染色检测DA能神经元存活数目，免疫印迹法检测α-突触核蛋白(α-synuclein，α-Syn)和自噬特异性蛋白微管相关蛋白1轻链3B(microtubule-associated protein 1 light chain 3 Beta，LC3B)的蛋白表达；取纹状体脑组织，电镜观察神经元超微结构变化及自噬小体。[结果] 与MPTP模型组比较，菖蒲郁金汤组小鼠1、7、14 d时爬杆时间缩短(均P＜0.05)，旷场试验的总距离和旷场平均速度均增加(均P＜0.01)，7、14 d时悬挂实验分值增加(均P＜0.05)；菖蒲郁金汤组小鼠中脑黑质DA神经元存活数目增加(P＜0.01)，而且染色浓度增加，α-Syn蛋白表达下调(P＜0.01)，微管相关蛋白1轻链3B-Ⅱ(microtubule-associated protein 1 light chain 3 Beta-Ⅱ，LC3B-Ⅱ)/微管相关蛋白1轻链3B-Ⅰ(microtubule-associated protein 1 light chain 3 Beta-Ⅰ，LC3B-Ⅰ)比值增加(P＜0.01)。电镜显示，与MPTP模型组比较，菖蒲郁金汤组纹状体神经元胞质水肿、空泡变性现象减轻，线粒体膜结构相对完整，可见自噬溶酶体。 [结论] 菖蒲郁金汤可有效减轻PD小鼠行为症状，对DA能神经元具有保护作用，通过增强神经元细胞的自噬活性从而促进异常聚集α-Syn的自噬性清除可能是其主要作用机制。 |
| 英文摘要: |
| [Objective] To observe the therapeutic effect of Changpu Yujin Decoction on Parkinson‘s disease(PD) mice and its protective effect on dopaminergic(DA) neurons， and discuss its possible mechanism. [Methods] Thirty-six C57BL/6 male mice were divided into normal control group， MPTP model group and ChangpuYujin Decoction group randomly， each group contained 12 mice. Mice in Changpu Yujin Decoction group and MPTP model group were dealt with 30 mg/(kg·d) 1-methyl-4-phenyl-1，2，3，6-tetrahydropyridine(MPTP) intraperitoneal injection continuously for 7 days to establish PD models. Mice in normal control group were dealt with 30 mg/(kg·d) 0.9% sodium chloride solution intraperitoneally injection continuously for 7 days. Mice in Changpu Yujin Decoction group were given 20 g/(kg·d) Changpu Yujin Decoction by gavageat the same time as MPTP injection for a total of 21 days， while mice in MPTP model group and normal control group were given 20 g/(kg·d) 0.9% sodium chloride solution by gavage at the same time as MPTP injection or 0.9% sodium chloride solution injection for a total of 21 days. Behavioral evaluation of mice was performed on the 1st， 7th and 14th day， after the last drug or 0.9% sodium chloride solution intraperitoneal injection. Then mice were killed， brain tissue in the substantia nigra of midbrain was taken for tyrosine hydroxylase(TH) immunohistochemical staining to detect the survival number of DA neurons and the protein expressions of α-synuclein(α-Syn) and autophagy protein microtubule-associated protein 1 light chain 3 Beta(LC3B) were detected by Western blot. Striatum brain tissue was taken for electron microscopy to observe ultrastructural changes of neurons and autophagosomes. [Results] Compared with MPTP model group， Changpu Yujin Decoction shortened the time of pole climbing(P＜0.05)， increased the score of hanging test(P＜0.05)， the total distance and average speed of open field test(P＜0.01)， increased the number of surviving DA neurons(P＜0.01) and cytoplasmic staining concentration in substantia nigra， down-regulated the protein expression of α-Syn(P＜0.01) and increased the ratio of microtubule-associated protein 1 light chain 3 Beta-Ⅱ(LC3B-Ⅱ) and microtubule-associated protein 1 light chain 3 Beta-Ⅰ(LC3B-Ⅰ)(P＜0.01). Electron microscopy showed that compared with MPTP model group， the striatum neurons in Changpu Yujin Decoction group had less cytoplasmic edema and vacuolar degeneration， the mitochondrial membrane structure was relatively intact， and autophagic lysosomes could be seen. [Conclusion] Changpu Yujin Decoction could significantly relieve the behavioral symptoms of PD mice， and has a neuroprotective effect on PD. Enhancing neuronal autophagy to promote the clearance of α-Syn may be one of the main mechanisms. |
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