读书丸调控PI3K/Akt通路改善Aβ25~35诱导HT22细胞线粒体凋亡的机制研究*

作者：梅本圆1，宋 琳2，朴钟源3，李 莉1

单位：1.黑龙江中医药大学基础医学院，黑龙江 哈尔滨 150040； 2.惠州学院生命科学学院，广东 惠州 516007； 3.惠州市第三人民医院/广州医科大学附属惠州医院，广东 惠州 516002

引用：引用：梅本圆，宋琳，朴钟源，李莉.读书丸调控PI3K/Akt通路改善Aβ25~35诱导HT22细胞线粒体凋亡的机制研究[J].中医药导报,2026,32(1):21-28.

DOI：10.13862/j.cn43-1446/r.2026.01.004

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摘要：目的：研究古方读书丸通过线粒体凋亡途径对Aβ25~35诱导HT22细胞的保护作用及机制。方法：利用10 μmol/L的Aβ25~35诱导HT22细胞构建体外阿尔茨海默病（AD）模型，CCK-8法检测细胞活力筛选最佳培养时间及读书丸浓度，实验设置对照组、模型组、读书丸低剂量组（0.05 mg/mL）、读书丸中剂量组（0.10 mg/mL）、读书丸高剂量组（0.50 mg/mL）和盐酸多奈哌齐组。各组细胞给药预处理1 h加入Aβ25~35诱导48 h，利用试剂盒检测细胞内Aβ1~42、SOD、MDA含量，荧光探针检测线粒体总量、线粒体ROS、线粒体膜电位及通透性转换孔开放水平，透射电镜观察线粒体结构，qRT-PCR检测线粒体DNA拷贝数，蛋白质印迹法（Western blotting）检测PI3K、p-PI3K、Akt、p-Akt、Bax、Bcl-2、Caspase-3蛋白表达。结果：与对照组比较，模型组HT22细胞活力明显下降，细胞内Aβ1~42、MDA含量增加，SOD含量降低，线粒体总量减少，线粒体ROS增加，线粒体膜电位坍塌下降，线粒体通透性转换孔大量开放，线粒体肿胀、线粒体嵴断裂，线粒体DNA拷贝数降低（P＜0.05），细胞内p-PI3K/PI3K、p-Akt/Akt及Bcl-2蛋白表达减少（P＜0.05），Bax、Caspase-3蛋白表达增加（P＜0.05）。与模型组比较，经读书丸干预后，Aβ25~35诱导的HT22细胞活力明显增加，细胞内Aβ1~42、MDA含量减少，SOD活性升高，线粒体总量增加，线粒体ROS累积减少，线粒体膜电位上升，线粒体通透性转换孔关闭，被破坏的线粒体结构有所改善；读书丸高剂量组线粒体DNA拷贝数增加（P＜0.05），细胞内p-PI3K/PI3K、p-Akt/Akt及Bcl-2蛋白表达增加（P＜0.05），Bax、Caspase-3蛋白表达减少（P＜0.05）。结论：读书丸能够明显改善Aβ25~35诱导的HT22细胞线粒体氧化应激损伤和细胞凋亡，其潜在作用机制可能与调控PI3K/Akt信号通路有关。

关键词：阿尔茨海默病；读书丸；线粒体；氧化应激；凋亡；PI3K/Akt信号通路

Abstract：

Objective: To investigate the protective effect and mechanism of the ancient formula Dushu pill on Aβ25-35-induced HT22 cells through the mitochondrial apoptosis pathway. Methods: An in vitro Alzheimer's disease (AD) model was established by inducing HT22 cells with 10 μmol/L Aβ25-35. Cell viability was detected by the CCK-8 method to screen for the optimal culture time and concentration of Dushu pill. The experiment was set up with control group, model group, Dushu pill low-dose group (0.05 mg/mL), Dushu pill medium-dose group (0.10 mg/mL), Dushu pill high-dose group (0.50 mg/mL), and donepezil hydrochloride group. After the cells in each group were pretreated with the corresponding drugs for 1 h, Aβ25-35 was added for 48 h of induction. Commercial kits were used to detect intracellular contents of Aβ1-42, superoxide dismutase (SOD), and malondialdehyde (MDA). Fluorescent probes were used to detect mitochondrial mass, mitochondrial ROS, mitochondrial membrane potential, and the opening level of the mitochondrial permeability transition pore. Mitochondrial structure was observed by transmission electron microscopy. Mitochondrial DNA copy number was detected by qRT-PCR. Protein expression of PI3K, p-PI3K, Akt, p-Akt, Bax, Bcl-2, and Caspase-3 was detected by Western blotting. Results: Compared with the control group, the model group showed significantly decreased HT22 cell viability, increased intracellular contents of Aβ1-42 and MDA, decreased SOD content, reduced mitochondrial mass, increased mitochondrial ROS, collapse (decrease) of mitochondrial membrane potential, massive opening of the mitochondrial permeability transition pore, mitochondrial swelling and cristae breakage, and decreased mitochondrial DNA copy number (P<0.05). The expression levels of p-PI3K/PI3K, p-Akt/Akt, and Bcl-2 protein in the cells decreased (P<0.05), while the expression of Bax and Caspase-3 protein increased in model group (P<0.05). Compared with the model group, after intervention with Dushu pill, the viability of Aβ25-35-induced HT22 cells significantly increased, intracellular contents of Aβ1-42 and MDA decreased, SOD activity increased, mitochondrial mass increased, mitochondrial ROS accumulation decreased, mitochondrial membrane potential rose, the mitochondrial permeability transition pore closed, and the damaged mitochondrial structure was improved. In the Dushu pill high-dose group, the mitochondrial DNA copy number increased (P<0.05), the expression levels of p-PI3K/PI3K, p-Akt/Akt, and Bcl-2 protein in the cells increased (P<0.05), and the expression of Bax and Caspase-3 protein decreased (P<0.05). Conclusion: Dushu pill can significantly ameliorate Aβ25-35-induced mitochondrial oxidative stress damage and apoptosis in HT22 cells, and its potential mechanism may be related to the regulation of the PI3K/Akt signaling pathway.

Key words：Alzheimer's disease; Dushu pill; mitochondria; oxidative stress; apoptosis; PI3K/Akt signaling pathway

发布时间：2026-01-30

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