目的 探讨川芎嗪调控线粒体自噬抑制bEnd.3细胞衰老的分子机制。方法 bEnd.3细胞随机分为对照组、模型组和川芎嗪低、中、高浓度(25、50、100μmol·L^-1)组，除对照组外，使用H₂O₂(200μmol·L^-1)诱导bEnd.3细胞衰老，通过免疫荧光双染检测CD31/P53、CD31/P21蛋白的表达，Transwell实验检测细胞迁移能力，细胞流式实验检测细胞周期，血管形成实验检测细胞成管能力，β-半乳糖苷酶染色检测细胞衰老，Western blotting检测bEnd.3细胞P53、P21、P16、核纤层蛋白B（Lamin B）、过氧化物酶体增殖物激活受体γ（PPARγ）、FUN14结构域包含蛋白1（FUNDC1）表达，荧光探针检测线粒体与溶酶体共定位情况，用Discovery Studio软件进行川芎嗪与PPARγ的分子对接，用We Mol在线平台进行分子动力学模拟。结果 模型组中CD31/P53及CD31/P21双阳性细胞比例较对照组显著增加（P<0.01），证明模型构建成功；与对照组比较，模型组细胞的迁移能力、细胞成管能力、PPARγ和FUNDC1蛋白表达均明显减少，Lamin B蛋白显著减少，而P53、P21、P16蛋白表达显著增加，β-半乳糖苷酶染色阳性细胞数量明显增加，细胞显著阻滞在G₀/G₁期，差异均具有统计学意义（P<0.01）；线粒体和溶酶体共定位明显减少。与模型组比较，川芎嗪干预后，细胞的迁移能力、细胞成管能力、PPARγ和FUNDC1蛋白表达明显增加，Lamin B蛋白表达显著增加，而P53、P21、P16蛋白表达显著减少，β-半乳糖苷酶染色阳性细胞数量明显减少，显著改善细胞在G₀/G₁期阻滞，差异均具有统计学意义（P<0.01），线粒体和溶酶体共定位明显增加；分子对接与动力学模拟表明川芎嗪与PPARγ具有较好的靶向作用力。结论 川芎嗪通过调控PPARγ-FUNDC1信号轴增强线粒体自噬，清除受损线粒体，从而抑制bEnd.3细胞衰老。

Objective To explore the molecular mechanism by which tetramethylpyrazine regulates mitochondrial autophagy to inhibit senescence in bEnd.3 cells. Methods bEnd.3 cells were randomly divided into a control group, a model group, and tetramethylpyrazine low-, medium-, and high-concentration(25, 50, and 100 μmol·L^-1) groups. Except for the control group, bEnd.3 cells were induced to senescence with H₂O₂(200 μmol·L^-1). The expression of CD31/P53 and CD31/P21 proteins was detected by immunofluorescence double staining. Cell migration ability was detected by Transwell assay. Cell cycle was detected by flow cytometry. Cell tube formation ability was detected by vascular formation assay. Senescent cells were detected by β-galactosidase staining. The expression of P53, P21, P16, lamin B, peroxisome proliferator-activated receptor γ（PPARγ）, and FUN14 domaincontaining protein 1（FUNDC1） in bEnd.3 cells was detected by Western blotting. The co-localization of mitochondria and lysosomes was detected by fluorescence probe. Molecular docking and molecular dynamics simulation were performed using Discovery Studio software and WeMol online platform, respectively. Results The proportion of CD31/P53 and CD31/P21 double-positive cells in the model group was significantly increased compared with the control group（P ＜ 0.01）, indicating successful model construction. Compared with the control group, the cell migration ability, cell tube formation ability, and the expression of PPARγ and FUNDC1 proteins in the model group were significantly decreased, the expression of Lamin B protein was significantly decreased, while the expression of P53, P21, and P16 proteins was significantly increased, the number of β-galactosidase staining positive cells was significantly increased, and cells were significantly arrested in the G₀/G₁ phase, with statistically significant differences（P ＜ 0.01）. The co-localization of mitochondria and lysosomes was significantly decreased. Compared with the model group, after tetramethylpyrazine intervention, the cell migration ability, cell tube formation ability, and the expression of PPARγ and FUNDC1 proteins were significantly increased, the expression of Lamin B protein was significantly increased, while the expression of P53, P21, and P16 proteins was significantly decreased, the number of β-galactosidase staining positive cells was significantly decreased, and the cell arrest in the G₀/G₁ phase was significantly improved, with statistically significant differences（P ＜ 0.01）. The co-localization of mitochondria and lysosomes was significantly increased. Molecular docking and molecular dynamics simulation indicated that tetramethylpyrazine had a good targeting force with PPARγ. Conclusion Tetramethylpyrazine inhibits senescence in bEnd.3 cells by regulating the PPARγ-FUNDC1 signaling axis to enhance mitochondrial autophagy and remove damaged mitochondria.

R285.5

广西中医药大学赛恩斯新医药学院校级科研项目(2024ZZA001)