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汪辛强, 胡国恒, 谢丹, 王焱, 陈亚, 何飘.基于Nrf2/NQO1/GCL信号通路探讨益肾健脑方对血管性痴呆大鼠神经元凋亡的影响[J].湖南中医药大学学报,2024,44(11):1975-1983[点击复制] |
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| 基于Nrf2/NQO1/GCL信号通路探讨益肾健脑方对血管性痴呆大鼠神经元凋亡的影响 |
| 汪辛强,胡国恒,谢丹,王焱,陈亚,何飘 |
| (湖南中医药大学第一附属医院, 湖南 长沙 410007;湖南中医药大学, 湖南 长沙 410208) |
| 摘要: |
| 目的 研究益肾健脑方对血管性痴呆（vascular dementia, VD）大鼠神经元凋亡的影响及作用机制。方法 将60只SPF级大鼠随机分为模型制备组50只和空白组10只，模型制备组分时段结扎双侧颈总动脉，空白组仅分离双侧颈总动脉但不结扎。术后4周通过Morris水迷宫筛选出模型制备成功大鼠28只，随机分为模型组、多奈哌齐组（0.45 mg·kg-1·d-1）、益肾健脑方高剂量组（24.3 g·kg-1·d-1）、益肾健脑方低剂量组（12.15 g·kg-1·d-1）各7只，加上空白组10只，共5组。给药4周后，行Morris水迷宫检测大鼠学习记忆能力，HE染色观察大脑皮质和海马CA1区组织病理变化，TUNEL染色观察大脑皮质和海马CA1区神经细胞凋亡情况，免疫组织化学法检测大脑皮质和海马CA1区天冬氨酸特异性半胱氨酸蛋白酶-3（cysteinyl aspartate-specific proteinase-3, Caspase-3）、天冬氨酸特异性半胱氨酸蛋白酶-7（cysteinyl aspartate-specific proteinase-7, Caspase-7）蛋白表达水平，Western blot检测海马组织核因子E2相关因子2（nuclear factor-E2-related factor 2, Nrf2）、醌氧化还原酶1（quinone oxidoreductase 1, NQO1）、谷氨酰半胱氨酸连接酶（glutamate-cysteine ligase, GCL）蛋白表达水平。结果 与空白组比较，模型组定位航行实验第5天逃避潜伏期明显增加，空间探索实验穿越平台次数明显减少，目标象限停留时间明显缩短（P<0.01）；模型组脑组织病理损伤明显，神经细胞凋亡率显著升高（P<0.01）；模型组大脑皮质区和海马CA1区Caspase-3、Caspase-7阳性表达显著升高（P<0.01）；模型组海马组织Nrf2、NQO1、GCL蛋白表达显著降低（P<0.01）。与模型组比较，多奈哌齐组和益肾健脑方高、低剂量组定位航行实验中第5天逃避潜伏期明显缩短，空间探索实验中穿越平台次数增加，目标象限停留时间明显延长（P<0.05，P<0.01）；多奈哌齐组和益肾健脑方高、低剂量组脑组织病理损伤减少，神经细胞凋亡率均显著降低（P<0.01）；多奈哌齐组和益肾健脑方高、低剂量组Caspase-3、Caspase-7阳性表达均显著降低（P<0.01）；多奈哌齐组和益肾健脑方高、低剂量组海马组织Nrf2、NQO1、GCL蛋白表达均显著升高（P<0.01）。结论 益肾健脑方可以改善VD大鼠学习记忆能力、抑制Caspase-3和Caspase-7蛋白表达及减少神经元凋亡，其机制可能与激活Nrf2/NQO1/GCL信号通路有关。 |
| 关键词: 益肾健脑方 血管性痴呆 氧化应激 凋亡 核因子E2相关因子2 醌氧化还原酶1 谷氨酰半胱氨酸连接酶 |
| DOI：10.3969/j.issn.1674－070X.2024.11.006 |
| 投稿时间：2023-07-25 |
| 基金项目:全国名老中医药专家传承工作室建设项目（国中医药函人教函〔2022〕75号）;湖南省自然科学基金项目（2022JJ40334）;湖南省卫生健康委员会科研计划项目（202203074417）;湖南中医药大学校级科研基金项目（2021XJJJ044）。 |
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| Effects of Yishen Jiannao Formula on neuronal apoptosis in vascular dementia rats based on Nrf2/NQO1/GCL pathway |
| WANG Xinqiang, HU Guoheng, XIE Dan, WANG Yan, CHEN Ya, HE Piao |
| (The First Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410007, China;Hunan University of Chinese Medicine, Changsha, Hunan 410208, China) |
| Abstract: |
| Objective To investigate the effects and mechanism of action of Yishen Jiannao Formula (YSJNF) on neurona apoptosis in rats with vascular dementia (VD). Methods Sixty SPF-grade rats were randomized into a model preparation group (n=50) and a blank group (n=10). The model preparation group underwent staged ligation of the bilateral common carotid arteries, while the blank group had only separation of the bilateral common carotid arteries without ligation. Four weeks after surgery, 28 rats with successful model preparation, as confirmed by the Morris water maze test, were randomized into model group, donepezil group (0.45 mg·kg-1·d-1), as well as high-dose (24.3 g·kg-1·d-1) and low-dose (12.15 g·kg-1·d-1) YSJNF groups, with seven rats in each group, coupled with the blank group (n=10), totaling five groups. Four weeks after administration, Morris water maze was used to assess the learning and memory abilities of the rats. HE staining was employed to observe the histopathological changes in the cerebral cortex and hippocampal CA1 area, and TUNEL staining to evaluate the neuronal apoptosis in these areas. Immunohistochemistry was used to check the protein expression levels of cysteinyl aspartate-specific proteinase-3 (Caspase-3) and cysteinyl aspartate-specific proteinase-7 (Caspase-7) in the cerebral cortex and hippocampal CA1 area, and Western blot to examine the protein expression levels of nuclear factor-E2-related factor 2 (Nrf2), quinone oxidoreductase 1 (NQO1), and glutamate-cysteine ligase (GCL) in the hippocampal tissue. Results Compared with the blank group, the model group showed a significantly increased escape latency on the 5th day of the place navigation test, a significantly decreased number of the platform crossings in the spatial probe test, and a significantly shorter time spent in the target quadrant (P<0.01); it also exhibited significant pathological damage of the brain tissue and a markedly increased neuronal apoptosis rate (P<0.01); the positive expressions of Caspase-3 and Caspase-7 in the cerebral cortex and hippocampal CA1 area were significantly elevated (P<0.01), while the protein expressions of Nrf2, NQO1, and GCL in the hippocampal tissue were notably reduced in this group (P<0.01). Compared with the model group, the donepezil group and high- and low-dose YSJNF groups showed a significant reduction in the escape latency on the 5th day of the place navigation test, an increase in the number of platform crossings, and an obviously longer time spent in the target quadrant in the spatial probe test (P<0.05, P<0.01); these groups also demonstrated reduced pathological damage in the brain tissue and a significant decrease in neuronal apoptosis rate (P<0.01); furthermore, the positive expressions of Caspase-3 and Caspase-7 were significantly reduced (P<0.01), while the protein expressions of Nrf2, NQO1, and GCL in hippocampal tissue were significantly elevated in these groups (P<0.01). Conclusion YSJNF may improve the learning and memory abilities in VD rats, inhibit the protein expressions of Caspase-3 and Caspase-7, and reduce neuronal apoptosis, the mechanism of which may be related to the activation of the Nrf2/NQO1/GCL signaling pathway. |
| Key words: Yishen Jiannao Formula vascular dementia oxidative stress apoptosis nuclear factor-E2-related factor 2 quinone oxidoreductase 1 glutamate-cysteine ligase |
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