Department of Foot and Ankle Surgery, Chinese Medicine Hospital of Shizhong District of Leshan 12511002451599964Y, 614000 Leshan, Sichuan, China.
Department of Gynaecology, Leshan Hospital of Traditional Chinese Medicine, 614000 Leshan, Sichuan, China.
Discov Med. 2023 Oct;35(178):777-786. doi: 10.24976/Discov.Med.202335178.72.
Doxorubicin (Dox) is a clinical first-line broad-spectrum anticancer agent. A dose-dependent cardiotoxic and myelosuppressive response limits the clinical use of Dox. Recent research indicates that Dox-induced cardiotoxicity is associated with senescent cell accumulation and that antiaging therapy can alleviate aging-related disorders. Cepharanthine (Cep) is commonly used to treat various acute and chronic illnesses, including leukopenia, snakebites, dry mouth, and hair loss. Whether Cep alleviates Dox-induced senescence is unknown.
The expression of genes and proteins associated with aging was examined using NIH3T3 cell lines. The experiments were divided into a control group, a Dox group, and a Cep group on different days. NIH3T3 senescent cells were detected by senescence-β-galactosidase (SA-β-Gal) staining, and Western blotting was used to detect the protein levels of p16, p53, AMP-activated protein kinase (AMPK), mammalian target of the rapamycin (mTOR), p62, and Light Chain 3 (LC3). Fluorescence was used to detect the expression of monomeric red fluorescence protein-green fluorescence protein-Light Chain 3 (mRFP-GFP-LC3) and LC3 puncta in NIH3T3 cells. Real-time quantitative reverse transcription polymerase chain reaction (RT‒qPCR) was used to test the expression of senescence-associated secretory phenotypes (SASP: Interleukin 6 (IL-6), Interleukin 1 beta (IL-1β), and Interleukin 8 (IL-8)). Cell Counting Kit-8 (CCK-8) was used to assess NIH3T3 cell viability.
Here, we reported that Cep reversed the Dox-induced increase in the proportion of SA-β-Gal-positive cells and the high expression of aging-related proteins (p53, < 0.05; p16, < 0.05) and aging-related genes (, < 0.05; , < 0.05; , < 0.05) on the 3rd day. Mechanistically, Cep reduced the increase in the levels of phospho-mTOR ( < 0.05) on Days 1 and 3 and p62 protein ( < 0.05) caused by Dox on Day 1 and reversed the decline in LC3II/LC3I levels ( < 0.05) caused by Dox on Day 3, which is associated with the regulation of senescence. Additionally, the viability of NIH3T3 cells was significantly increased in the concentration range of 0.5-5 μM Cep ( < 0.05).
We first found that Cep could suppress SA-β-Gal activity ( < 0.05) and the development of SASP. Additionally, in Cep-treated cells, Cep could restore autophagy dysfunction and suppress the mTOR signaling pathway. This research provides a new view on the mechanics of aging and autophagy and aids in developing novel antiaging drugs.
多柔比星（Dox）是一种临床一线广谱抗癌药物。剂量依赖性的心脏毒性和骨髓抑制反应限制了 Dox 的临床应用。最近的研究表明，Dox 诱导的心脏毒性与衰老细胞的积累有关，而抗衰老治疗可以缓解与衰老相关的疾病。石蒜碱（Cep）常用于治疗各种急性和慢性疾病，包括白细胞减少症、蛇咬伤、口干和脱发。Cep 是否能减轻 Dox 诱导的衰老尚不清楚。
使用 NIH3T3 细胞系检测与衰老相关的基因和蛋白的表达。实验分为对照组、Dox 组和 Cep 组，在不同的天数进行。衰老β-半乳糖苷酶（SA-β-Gal）染色检测 NIH3T3 衰老细胞，Western blot 检测 p16、p53、AMP 激活蛋白激酶（AMPK）、哺乳动物雷帕霉素靶蛋白（mTOR）、p62 和 Light Chain 3（LC3）的蛋白水平。荧光法检测 NIH3T3 细胞中单体红色荧光蛋白-绿色荧光蛋白-LC3（mRFP-GFP-LC3）和 LC3 斑点的表达。实时定量逆转录聚合酶链反应（RT-qPCR）检测衰老相关分泌表型（SASP：白细胞介素 6（IL-6）、白细胞介素 1β（IL-1β）和白细胞介素 8（IL-8））的表达。细胞计数试剂盒-8（CCK-8）检测 NIH3T3 细胞活力。
我们发现 Cep 逆转了 Dox 诱导的 SA-β-Gal 阳性细胞比例和衰老相关蛋白（p53，<0.05；p16，<0.05）和衰老相关基因（，<0.05；，<0.05；，<0.05）在第 3 天的高表达。在第 1 天和第 3 天，Cep 降低了 Dox 引起的磷酸化 mTOR（<0.05）水平的升高，在第 1 天降低了 Dox 引起的 p62 蛋白（<0.05）水平的升高，并在第 3 天逆转了 Dox 引起的 LC3II/LC3I 水平的下降（<0.05），这与衰老的调节有关。此外，在 0.5-5 μM Cep 的浓度范围内，NIH3T3 细胞的活力显著增加（<0.05）。
我们首次发现 Cep 可抑制 SA-β-Gal 活性（<0.05）和 SASP 的发展。此外，在 Cep 处理的细胞中，Cep 可以恢复自噬功能障碍并抑制 mTOR 信号通路。这项研究为衰老和自噬的机制提供了新的视角，并有助于开发新的抗衰老药物。
