目的 基于动力相关蛋白1（Drp1）与PTEN诱导激酶1（PINK1）/E3泛素-蛋白连接酶（PINK1/Parkin）协同调控线粒体质量控制（MQC），探讨地黄饮子（DHYZ）含药血清对β-淀粉样蛋白（Aβ）_25-35致HT22细胞氧化损伤的影响。方法 SD大鼠ig DHYZ（生药量90 g·kg^-1），每天2次，连续5 d，末次给药1 h，腹主动脉取血制备含药血清。DHYZ（5%含药血清）预给药30 min后，添加Aβ_25-35（10 μmol·L^-1）诱导HT22细胞建立阿尔茨海默病（AD）体外模型，继续培养24 h，DCFH-DA探针检测活性氧（ROS）含量，Calcein AM探针检测线粒体膜通透性转换孔（MPTP）开放情况，ELISA法检测细胞Aβ1-42含量，研究DHYZ对氧化损伤影响；透射电镜观察自噬小体； Western blotting检测Drp1、线粒体融合素1（Mfn1）、Mfn2、视神经萎缩1（Opa1）、PINK1、Parkin、LC3B、苄氯素1（Beclin1）的表达。预给药Drp1抑制剂Mdivi-1、DHYZ（5%含药血清）、Mdivi-1＋ DHYZ 30 min，添加Aβ25-35诱导HT22细胞建立AD体外模型，继续培养24 h，Western blotting检测总细胞、细胞质和线粒体中Drp1表达，免疫荧光检测线粒体Drp1含量，Western blotting检测线粒体自噬蛋白PINK1、Parkin表达。结果 与模型组比较，DHYZ组Aβ含量显著降低（P＜0.01），线粒体绿色荧光信号增强（P＜0.05），ROS荧光强度降低（P＜0.05）； DHYZ显著降低Drp1表达水平（P＜0.05），提高Mfn1、Mfn2、Opa1表达水平（P＜0.05），DHYZ组PINK1、Parkin、LC3B（P＜0.05）和Beclin1（P＜0.01）表达显著下降；电镜显示DHYZ组线粒体结构正常，偶见自噬体。与模型组比较，Mdivi-1组和DHYZ组细胞内Drp1（P＜0.05）和线粒体Drp1（P＜0.01）表达降低，胞质Drp1表达升高（P＜0.01），表明给药后能阻止线粒体聚集； DHYZ组PINK1（P＜0.05）、Parkin（P＜0.01）表达下调，DHYZ＋Mdivi-1组效果最明显（P＜0.01、0.001）。结论 DHYZ防治AD可能抑制Drp1介导的线粒体分裂，调节线粒体自噬PINK1/Parkin通路，进而调控MQC链。

Objective Based on the synergistic regulation of mitochondrial quality control (MQC) by dynamin related protein 1 (Drp1) and PTEN inducible putative kinase 1/E3 ubiquitin protein ligase (PINK1/Parkin), to explore the effect of Dihuang Yinzi (DHYZ) containing serum on the oxidative damage of HT22 cells induced by β- amyloid (Aβ)_25-35. Methods SD rats were ig administered with DHYZ (90 g·kg^-1 crude drug) twice a day for 5 consecutive days. One hour after the last administration, blood was collected from the abdominal aorta to prepare drug-containing serum. After pre-administration of DHYZ (5% drug-containing serum) for 30 min, Aβ_25-35 (10 μmol·L^-1) was added to induce an AD in vitro model in HT22 cells, and the cells were further cultured for 24 h. The content of reactive oxygen species (ROS) was detected by DCFH-DA probe, the opening of mitochondrial permeability transition pore (MPTP) was detected by Calcein AM probe, and the content of Aβ_1-42 in cells was detected by ELISA to study the effect of DHYZ on oxidative damage. Autophagosomes were observed by transmission electron microscopy, and the expressions of Drp1, Mfn1, Mfn2, Opa1, PINK1, Parkin, LC3B, and Beclin1 were detected by Western blotting. Drp1 inhibitor Mdivi-1, DHYZ (5% drug-containing serum), and Mdivi-1 + DHYZ were pre-administered for 30 min, and Aβ_25-35 was added to induce an AD in vitro model in HT22 cells. After 24 h of further culture, the expression of Drp1 in total cells, cytoplasm, and mitochondria was detected by Western blotting, the content of mitochondrial Drp1 was detected by immunofluorescence, and the expressions of mitochondrial autophagy proteins PINK1 and Parkin were detected by Western blotting. Results Compared with the model group, the Aβ content in the DHYZ group was significantly decreased (P<0.01), the green fluorescence signal of mitochondria was enhanced (P<0.05), and the fluorescence intensity of ROS was decreased (P<0.05). DHYZ significantly decreased the expression level of Drp1 (P<0.05), and increased the expression levels of Mfn1, Mfn2, and Opa1 (P<0.05). The expressions of PINK1, Parkin, and LC3B (P<0.05) and Beclin1 (P<0.01) in the DHYZ group were significantly decreased. Electron microscopy showed that the mitochondrial structure in the DHYZ group was normal, and autophagosomes were occasionally observed. Compared with the model group, the expressions of Drp1 in cells (P<0.05) and mitochondria (P<0.01) in the Mdivi-1 group and DHYZ group were decreased, and the expression of Drp1 in the cytoplasm was increased (P<0.01), indicating that administration could prevent mitochondrial aggregation. The expressions of PINK1 (P<0.05) and Parkin (P<0.01) in the DHYZ group were down-regulated, and the effect was most obvious in the DHYZ + Mdivi-1 group (P<0.01, 0.001). Conclusion DHYZ can prevent AD by inhibiting drp1-mediated mitochondrial division, regulating mitochondrial autophagy PINK1/parkin pathway, and then regulating the mitochondrial quality control chain.

R965

国家自然科学基金项目（81673860）；惠州学院自主创新能力提升计划“中医药认知障碍防治创新团队”（HZU202508）；国家级大学生创新创业训练计划项目（202510577030）