滋肾活血方通过Sirt1/PGC1a信号通路改善血管性痴呆模型大鼠海马线粒体功能的机制研究

Effect of Zishen Huoxue Formula on Mitochondrial Function of Vascular Dementia Model Rats Based on Sirt1/PGC1a Signal Pathway

关键词:  滋肾活血方  血管性痴呆  Sirt1/PGC-1α  线粒体功能

Key words:  zishen houxue formula  vascular dementia  Sirt1/PGC-1α  mitochondrial function

摘要:

目的：探讨滋肾活血方对2-VO大鼠神经元线粒体功能的改善作用与Sirt1/PGC-1α信号通路之间的关系。方法：选用SPF级SD大鼠，6-8周龄，采用改良双侧颈总动脉结扎法（2-VO）构建VD大鼠模型，随机分为假手术组、2-VO组、盐酸多奈哌齐组（0.00045g·kg-1）、滋肾活血方低、中、高剂量组（8.9g·kg-1、17.8g·kg-1、35.6g·kg-1）。干预4周后水迷宫实验检测学习记忆能力；苏木精-伊红染色（HE）观察海马CA1区病理形态变化；Fluoro-JadeB（FJB）荧光染色观察神经元变性与死亡情况；透射电子显微镜（TEM）观察线粒体超微结构；化学发光法检测海马组织三磷酸腺苷（ATP）含量；二氢乙锭（DHE）荧光染色检测活性氧（ROS）含量；免疫荧光检测过氧化物酶体增殖受体γ辅激活因子α（PGC-1α）表达；蛋白免疫印迹法（WB）检测沉默信息调节因子2同源物1（Sirt1）、PGC-1α、B淋巴细胞瘤-2基因（Bcl-2）以及Bcl-2相关X蛋白（Bax）蛋白表达。结果：与假手术组相比，2-VO组大鼠逃避潜伏期延长（P＜0.001），穿越平台次数减少（P＜0.001），目标象限停留时间缩短（P＜0.001），学习记忆能力明显受损；海马CA1区神经元数量减少，形态异常；FJB阳性神经元数量增多（P＜0.001），线粒体超微结构受损；海马组织中ATP含量下降（P＜0.001），海马CA1区ROS含量显著上升（P＜0.001），PGC-1α表达水平下降（P＜0.001）；海马组织中Sirt1和PGC-1α蛋白表达量下调（P＜0.001），Bcl-2/BAX比值下调（P＜0.001）；与2-VO组相比，滋肾活血方低、中、高组干预2-VO大鼠逃避潜伏期明显缩短（P＜0.01，P＜0.001），目标象限停留时间延长（P＜0.01，P＜0.001），中、高剂量组2-VO大鼠平台穿越次数显著增加（P＜0.01，P＜0.001）；海马CA1区神经元病理形态改善，FJB阳性细胞数量减少（P＜0.01，P＜0.001），线粒体超微结构损伤改善，海马组织中ATP含量增加（P＜0.001），海马CA1区ROS含量降低（P＜0.01，P＜0.001），PGC-1α表达水平上升（P＜0.01，P＜0.001）；海马组织中Sirt1和PGC-1α蛋白表达量上调（P＜0.01，P＜0.001），Bcl-2/BAX比值上升（P＜0.001）。结论：滋肾活血方可能通过激活Sirt1/PGC-1α信号通路，改善线粒体形态与功能，减少神经元的变形损伤与凋亡，进而促进认知功能的改善。

Abstract:

OBJECTIVE: To investigate the effect of Zishen Huoxue Formula on the function of damaged neurons and mitochondria in 2-VO rats based on Sirt1/PGC-1α signaling pathway. METHODS: SPF SD rats, 6-8 weeks old, were selected to construct vascular dementia rat model by 2-vessel occlusion(2-VO) and were randomly divided into sham-operated group, 2-VO group, donepezil group(0.00045g·kg-1), and low-dose(8.9g·kg-1), medium-dose(17.8g·kg-1) and high-dose(35.6g·kg-1) Chinese medicine groups. After 4 weeks of intervention, the water maze test detected learning and memory ability. Hematoxylin-eosin staining (HE) was used to observe the pathological changes in the CA1 region of the hippocampus. Fluoro-JadeB (FJB) fluorescence staining was used to observe neuronal degeneration. Transmission electron microscopy (TEM) was used to observe the ultrastructure of mitochondria. The kit was used to detect adenosine triphosphate (ATP) content in hippocampal tissue. Dihydroethidium (DHE) fluorescence staining was used to detect the content of reactive oxygen species (ROS). IF staining was used to detect the expression of peroxisome proliferation receptor γ coactivator α (PGC-1α). Western blot (WB) was used to detect the expressions of PGC-1α, silencing information regulator 2 homolog 1 (Sirt1), B lymphocytoma-2 gene (Bcl-2) and Bcl-2-related X protein (Bax). RESULTS: Compared with the Sham group, the 2-VO group had a longer escape latency（P＜0.001）, a decrease in the number of platform crossings（P＜0.001）, a shorter stay time in the target quadrant（P＜0.001）, and significantly impaired learning and memory ability. The number of neurons in the CA1 region of the hippocampus was reduced and the morphology was abnormal. The number of FJB-positive neurons increased（P＜0.001）, and the mitochondrial ultrastructure was damaged. The ATP content in the hippocampus decreased（P＜0.001）, and the ROS content in the CA1 region of the hippocampus increased significantly（P＜0.001），and the expression level of PGC-1α decreased（P＜0.001）. The protein expressions of Sirt1 and PGC-1α were down-regulated（P＜0.001）, and the Bcl-2/BAX ratio was down-regulated（P＜0.001）; Compared with the 2-VO group, the escape latency of the 2-VO rats in the low, medium and high-dose groups was significantly shortened（P＜0.01，P＜0.001）, and the residence time of the target quadrant was prolonged（P＜0.01，P＜0.001）, and the number of platform crossings of 2-VO rats in the medium and high-dose groups was significantly increased（P＜0.01，P＜0.001）. The pathological morphology of neurons in the hippocampus CA1 region was improved, the number of FJB-positive cells decreased, the mitochondrial ultrastructure damage was improved（P＜0.01，P＜0.001）, the ATP content in hippocampal tissues increased（P＜0.001）, and the ROS content in hippocampal CA1 region decreased（P＜0.01，P＜0.001），and the expression level of PGC-1α increased（P＜0.01，P＜0.001）. The protein expressions of Sirt1 and PGC-1α were up-regulated（P＜0.01，P＜0.001）, and the Bcl-2/BAX ratio increased（P＜0.001）. CONCLUSION: Zishen Huoxue Formula may reduce the level of hippocampal oxidative stress, improve mitochondrial morphology and function, reduce neuronal deformation damage and apoptosis by activating the Sirt1/PGC-1α signaling pathway, and promote the improvement of cognitive function.