| 引用本文: |
陈桑妮, 吴梅, 何薇, 孟盼, 李丹丹.百合知母汤通过改善线粒体功能与自噬保护秀丽隐杆线虫多巴胺能神经元的机制研究[J].湖南中医药大学学报,2025,45(12):2271-2279[点击复制] |
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| 百合知母汤通过改善线粒体功能与自噬保护秀丽隐杆线虫多巴胺能神经元的机制研究 |
| 陈桑妮,吴梅,何薇,孟盼,李丹丹 |
| (湖南中医药大学, 湖南 长沙 410208) |
| 摘要: |
| 目的 探讨百合知母汤对秀丽隐杆线虫（线虫）多巴胺能神经元（DN）的保护作用。方法 将同步化线虫随机分为空白组、模型组[400 μmol·L-1鱼藤酮（ROT）]、百合知母汤组（6.7 mg·mL-1百合知母汤溶液+400 μmol·L-1 ROT）和氟西汀组（100 μmol·L-1 氟西汀药物溶液+400 μmol·L-1 ROT）。根据线虫DN的绿色荧光蛋白（GFP）荧光强度和DN畸变程度筛选ROT最佳浓度；通过线虫头部摆动频率筛选百合知母汤的最佳药物浓度。利用DN荧光标记线虫（BZ555），检测其神经损伤情况；采用线粒体电子传递链（ETC）复合物荧光标记线虫（CB7272），检测其ETC复合物损伤恢复情况；采用荧光探针技术检测线粒体膜电位；采用胡桃醌诱导氧化应激线虫模型，通过生存分析评估其生存率；利用DCFH-DA探针检测线虫体内活性氧（ROS）水平，评价百合知母汤抗氧化效果。通过免疫荧光检测线粒体自噬相关基因标记线虫（RB2547），检测pink-1的表达情况。结果 ROT最佳造模剂量为400 μmol·L-1；百合知母汤最佳给药剂量为6.7 mg·mL-1。与空白组相比，模型组DN GFP荧光信号降低（P<0.01），畸变程度升高（P<0.01），ETC复合物GFP强度下降（P<0.01），ATP水平下降（P<0.01），线粒体膜电位水平下降（P<0.01），线虫存活率下降（P<0.01），ROS水平升高（P<0.01），pink-1荧光信号下降（P<0.05）。与模型组相比，百合知母汤组DN GFP荧光信号升高（P<0.05）；百合知母汤组和氟西汀组畸变程度降低（P<0.01），ETC复合物GFP强度升高（P<0.01），ATP水平升高（P<0.01），线粒体膜电位水平升高（P<0.01），线虫存活率提高（P<0.01），ROS水平降低（P<0.01），pink-1荧光信号增加（P<0.01）。结论 百合知母汤可能通过改善秀丽隐杆线虫线粒体功能障碍，增强抗氧化能力，降低ROS水平，上调pink-1表达，最终保护DN。 |
| 关键词: 百合知母汤 秀丽隐杆线虫 氧化应激 线粒体功能 多巴胺能神经元 |
| DOI：10.3969/j.issn.1674-070X.2025.12.005 |
| 投稿时间：2025-08-06 |
| 基金项目:国家自然科学基金项目（82474468）；湖湘青年英才项目（2024RC3200）；湖南省卫生健康委员会高层次人才项目（青年骨干）（20240304118）；长沙市杰出创新青年培养计划（kq2406017）。 |
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| Mechanism of Baihe Zhimu Decoction in protecting dopaminergic neurons in Caenorhabditis elegans via enhancement of mitochondrial function and autophagy |
| CHEN Sangni, WU Mei, HE Wei, MENG Pan, LI Dandan |
| (Hunan University of Chinese Medicine, Changsha, Hunan 410208, China) |
| Abstract: |
| Objective To investigate the protective effects of Baihe Zhimu Decoction (BHZMD) on dopaminergic neurons (DNs) in Caenorhabditis elegans (C. elegans). Methods Synchronized C. elegans worms were randomly divided into blank control group, model group [exposed to 400 μmol·L-1 rotenone (ROT)], BHZMD group (treated with 6.7 mg·mL-1 BHZMD solution+400 μmol·L-1 ROT), and fluoxetine group (treated with 100 μmol·L-1 fluoxetine solution+400 μmol·L-1 ROT). The optimal concentration of ROT was determined based on the fluorescence intensity of green fluorescent protein (GFP) and the degree of DN deformity of the worms. The optimal concentration of BHZMD was selected by assessing worm head thrash frequency. Dopaminergic neuronal damage was evaluated in the worms with GFP-labeled DNs (BZ555). Mitochondrial function was analyzed using worms expressing fluorescent markers for electron transport chain (ETC) complexes (CB7272) to monitor ETC recovery. Mitochondrial membrane potential was measured using a fluorescence probe. Survival analysis was performed in a juglone-induced oxidative stress model. Intracellular reactive oxygen species (ROS) levels were measured using the DCFH-DA probe. The expression of pink-1 was examined in autophagy-related gene-labeled worms (RB2547) via immunofluorescence. Results The optimal modeling concentrations of ROT and BHZMD were determined to be 400 μmol·L-1 and 6.7 mg·mL-1, respectively. Compared with the blank control group, the model group exhibited significantly decreased GFP fluorescence signals of DNs (P<0.01) and significantly increased degree of DN deformity (P<0.01). Furthermore, the GFP intensity of the mitochondrial ETC complexes was significantly reduced (P<0.01), along with decreased ATP levels (P<0.01), declined mitochondrial membrane potential (P<0.01), lower worm survival rate (P<0.01), elevated ROS levels (P<0.01), and weakened pink-1 fluorescence signals (P<0.05). Compared with the model group, the BHZMD group exhibited higher DN GFP fluorescence signals (P<0.05); both the BHZMD and fluoxetine groups demonstrated reduced degree of DN deformity (P<0.01), enhanced ETC complex GFP intensity (P<0.01), elevated ATP levels (P<0.01), improved mitochondrial membrane potential (P<0.01), higher survival rates (P<0.01), decreased ROS levels (P<0.01), and intensified pink-1 fluorescence signals (P<0.01). Conclusion BHZMD may protect DNs in C. elegans by ameliorating mitochondrial dysfunction, enhancing antioxidant capacity, reducing ROS levels, and upregulating pink-1 expression. |
| Key words: Baihe Zhimu Decoction Caenorhabditis elegans oxidative stress mitochondrial function dopaminergic neuron |
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