| 引用本文: |
俞瑞珊, 贺佳琦, 杜楠, 王高峰, 杨一帆, 李涛, 闫咏梅, 刘燕妮.丹星止颤方通过PINK1/Parkin通路调控自噬改善帕金森病小鼠运动功能的机制研究[J].湖南中医药大学学报,2026,46(4):669-679[点击复制] |
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| 丹星止颤方通过PINK1/Parkin通路调控自噬改善帕金森病小鼠运动功能的机制研究 |
| 俞瑞珊,贺佳琦,杜楠,王高峰,杨一帆,李涛,闫咏梅,刘燕妮 |
(1援陕西中医药大学, 陕西 咸阳 712046;
2援陕西中医药大学附属医院, 陕西 咸阳 712000) |
| 摘要: |
| 目的 探讨丹星止颤方通过调控PTEN诱导激酶1(PINK1)/E3泛素连接酶（Parkin）通路治疗帕金森病（PD）的作用及机制。方法 48只C57BL/6J小鼠随机分为空白组、模型组、美多芭组及丹星止颤方低、中、高剂量组，每组8只。除空白组外，其余各组给予鱼藤酮10 mg/（kg·d）混悬液灌胃，1次/d，连续28 d，构建PD小鼠模型。造模成功后，美多芭组灌胃美多芭混悬液112.5 mg/（kg·d），丹星止颤方低、中、高剂量组分别灌胃丹星止颤方水煎液11.1、22.2、44.4 g/（kg·d），空白组、模型组灌胃等量生理盐水。1次/d，连续灌胃28 d。末次灌胃结束，采用步态实验检测小鼠通过特定距离的时间、最大速度变化率、平均运动速度及单位时间脚步数；旷场实验检测小鼠运动总距离、运动时间、平均速度及穿过总分区次数；爬杆实验检测小鼠转头时间和爬杆时间；尼氏染色观察小鼠中脑黑质区尼氏阳性神经元数量、尼氏阳性神经元平均面积、尼氏小体平均灰度值和积分光密度（IOD）；免疫组织化学检测小鼠中脑黑质区α-突触核蛋白（α-syn）阳性表达面积占比及酪氨酸羟化酶（TH）阳性细胞平均面积占比；Western blot检测中脑黑质区α-syn及自噬相关蛋白表达水平。结果 与空白组相比，模型组小鼠通过特定距离的时间延长、最大速度变化率升高、平均运动速度降低、单位时间脚步数减少、运动总距离缩短、平均速度降低、运动时间及穿过总分区次数减少、转头时间和爬杆时间延长（P<0.05），尼氏阳性神经元数量减少、尼氏阳性神经元平均面积缩小、尼氏小体平均灰度值及IOD值降低（P<0.05）,α-syn阳性表达面积占比及α-syn蛋白表达水平均升高（P<0.05）,TH阳性细胞平均面积占比及Parkin、PINK1、Beclin-1蛋白表达水平、LC3-Ⅱ/LC3-Ⅰ蛋白比值均降低（P<0.05）；与模型组相比，美多芭组及丹星止颤方中、高剂量组小鼠通过特定距离的时间缩短、最大速度变化率降低、平均运动速度升高、单位时间脚步数增加、运动总距离延长、运动时间增加、平均速度升高、穿过总分区次数增加、转头时间和爬杆时间缩短（P<0.05），尼氏阳性神经元数量增加、尼氏阳性神经元平均面积增大、尼氏小体平均灰度值及IOD值升高（P<0.05）,α-syn阳性表达面积占比及α-syn蛋白表达水平均降低（P<0.05）,TH阳性细胞平均面积占比及Parkin、PINK1、Beclin-1蛋白表达水平、LC3-Ⅱ/LC3-Ⅰ蛋白比值均升高（P<0.05）；与丹星止颤方低剂量组相比，丹星止颤方中剂量组小鼠通过特定距离的时间缩短、最大速度变化率降低、平均运动速度升高、单位时间脚步数增加、运动总距离延长、运动时间增加、平均速度升高、转头时间和爬杆时间缩短（P<0.05），尼氏阳性神经元数量增加、尼氏阳性神经元平均面积增大、尼氏小体平均灰度值及IOD值升高（P<0.05）,α-syn阳性表达面积占比及α-syn蛋白表达水平均降低（P<0.05）,TH阳性细胞平均面积占比及Parkin、PINK1、Beclin-1蛋白表达水平、LC3-Ⅱ/LC3-Ⅰ蛋白比值均升高（P<0.05）；与丹星止颤方中剂量组相比，丹星止颤方高剂量组小鼠通过特定距离的时间延长、最大速度变化率升高、平均运动速度降低、单位时间脚步数减少、运动总距离缩短、运动时间减少、平均速度降低、转头时间及爬杆时间延长（P<0.05），尼氏阳性神经元数量减少、尼氏小体平均灰度值及IOD值降低（P<0.05）,α-syn阳性表达面积占比及α-syn蛋白表达水平均升高（P<0.05）,TH阳性细胞平均面积占比及Parkin、PINK1蛋白表达水平、LC3-Ⅱ/LC3-Ⅰ蛋白比值均降低（P<0.05）。结论 丹星止颤方立足帕金森病核心病机，以化痰祛瘀、息风止颤为治则，通过调控PINK1/Parkin通路介导的自噬相关过程，减少α-syn异常聚集，保护多巴胺能神经元及黑质区神经元结构完整性，进而改善小鼠运动功能。 |
| 关键词: 帕金森病|丹星止颤方|PINK1/Parkin通路|自噬|α-突触核蛋白 |
| DOI：10.3969/j.issn.1674-070X.2026.04.003 |
| 投稿时间：2026-01-05 |
| 基金项目:陕西省自然科学基础研究计划项目(2025SYS-SYSZD-048);研究生创新实践能力提升项目(CXS J202515);秦创原中医药产业创新聚集区项目(L2024-QCY-ZYYJJQ-Y02、L2024-QCY-ZYYJQ-Y25) |
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| Mechanistic study of Danxing Zhichan Formula in improving motor function in Parkinson's disease mice by regulating autophagy via PINK1/Parkin pathway |
| YU Ruishan, HE Jiaqi, DU Nan, WANG Gaofeng, YANG Yifan, LI Tao, YAN Yongmei, LIU Yanni |
| (Shaanxi University of Chinese Medicine, Xianyang, Shaanxi 712046, China;Affiliated Hospital of Shaanxi University of Chinese Medicine, Xianyang, Shaanxi 712000, China) |
| Abstract: |
| Objective To investigate the effects and mechanisms of Danxing Zhichan Formula(DXZCF) in treating Parkinson's disease(PD) by regulating the PTEN-induced putative kinase 1(PINK1)/E3 ubiquitin ligase(Parkin) pathway. Methods Forty-eight C57 BL/6J mice were randomly divided into blank group, model group, Madopar group, and low-, medium-, and high-dose DXZCF groups, with eight mice in each group. Except for the blank group, the other groups were intragastrically administered rotenone suspension at a dose of 10 mg/(kg·d) once daily for 28 consecutive days to establish a PD mouse model. After successful modeling,the Madopar groupwas given Madopar suspension at a dose of 112.5 mg/(kg·d) via gavage, while the low-, medium-, and high-doseDXZCF groups received DXZCF decoctions at doses of 11.1, 22.2, and 44.4 g/(kg·d) via gavage, respectively. The blank and model groups received an equal volume of normal saline. All administrations were performed once daily for 28 consecutive days. Following the final gavage, gait analysis was performed to measure the time for mice to traverse a specific distance, maximum velocity change rate, average locomotor velocity, and number of footsteps per unit time. Open-field test was used to determine total locomotor distance, locomotor duration, average velocity, and number of total zone crossings. Pole-climbing test was applied to evaluate headturning time and pole-climbing time. Nissl staining was used to observe the number of Nissl-positive neurons, average area of Nissl-positive neurons, average gray value of Nissl bodies, and integrated optical density(IOD) in the substantia nigra of the midbrain. Immunohistochemistry was performed to determine the proportion of α-synuclein(α-syn)-positive expression area and the proportion of average area of tyrosine hydroxylase(TH)-positive cells in the substantia nigra. Western blot was used to measure the expression levels of α-syn and autophagy-related proteins in the midbrain substantia nigra. Results Compared with the blank group, mice in the model group exhibited prolonged time to traverse a specific distance, elevated maximum velocity change rate,decreased average locomotor velocity, fewer footsteps per unit time, shortened total locomotor distance, reduced average velocity,decreased locomotor duration, and fewer total zone crossings, as well as prolonged head-turning time and pole-climbing time(P<0.05). Additionally, there was a reduction in the number and average area of Nissl-positive neurons, along with reduced average gray value and IOD of Nissl bodies(P<0.05). The proportion of α-syn-positive expression area and α-syn protein expression level both increased(P<0.05), while the proportion of average area of TH-positive cells, as well as the expression levels of Parkin, PINK1,and Beclin-1 proteins, and the LC3-Ⅱ/LC3-Ⅰ protein ratio, all decreased(P<0.05). Compared with the model group, mice in the Madopar group and the medium-and high-dose DXZCF groups showed shortened time to traverse a specific distance, decreased maximum velocity change rate, increased average locomotor velocity, increased number of footsteps per unit time, prolonged total locomotor distance, prolonged locomotor duration, increased average velocity, increased total zone crossings, and reduced headturning time and pole-climbing time(P<0.05). Additionally, the number and average area of Nissl-positive neurons increased, and the average gray value and IOD of Nissl bodies were elevated(P<0.05). The proportion of α-syn-positive expression area and α-syn protein expression level decreased(P<0.05), while the proportion of average area of TH-positive cells, as well as the expression levels of Parkin, PINK1, and Beclin-1 proteins, and the LC3-Ⅱ/LC3-Ⅰ protein ratio, increased(P<0.05). Compared with the lowdose DXZCF group, mice in the medium-dose DXZCF group showed shortened time to traverse a specific distance, reduced maximum velocity change rate, increased average locomotor velocity, increased number of footsteps per unit time, prolonged total locomotor distance, extended locomotor duration, increased average velocity, and shortened head-turning time and pole-climbing time(P<0.05). Additionally, the number and average area of Nissl-positive neurons increased, and the average gray value and IOD of Nissl bodies were elevated(P<0.05). The proportion of α-syn-positive expression area and α-syn protein expression level decreased(P<0.05), while the proportion of average area of TH-positive cells, as well as the expression levels of Parkin, PINK1, and Beclin-1 proteins, and the LC3-Ⅱ/LC3-Ⅰ protein ratio, increased(P<0.05). Compared with the medium-dose DXZCF group, mice in the high-dose DXZCF group exhibited prolonged time to traverse a specific distance, elevated maximum velocity change rate, reduced average locomotor velocity, fewer footsteps per unit time, shortened total locomotor distance, reduced average velocity, decreased locomotor duration, and prolonged head-turning time and pole-climbing time(P<0.05). Additionally, these mice showed fewer Nisslpositive neurons, reduced average gray value and IOD of Nissl bodies(P<0.05), increased proportion of α-syn-positive expression area and elevated α-syn protein expression level(P<0.05), as well as reduced proportion of average area of TH-positive cells, and reduced expr ession levels of Parkin and PINK1 proteins and the LC3-Ⅱ/LC 3-Ⅰ prot ein rat io (P<0.05.) Conclusion DXZCF is formulated based on the core pathogenesis of PD, adhering to the treatment principles of transforming phlegm and eliminating stasis, as well as extinguishing wind and alleviating tremors. By regulating the autophagy-related processes mediated by the PINK1/Parkin pathway, it reduces abnormal α-syn aggregation, protects the structural integrity of dopaminergic neurons and neurons in the substantia nigra, thereby improving motor function in mice. |
| Key words: Parkinson's disease|Danxing Zhichan Formula|PINK1/Parkin pathway|autophagy|α-synuclein synuclein |
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