| 摘要: |
| 目的 研究制备一种pH响应性的钙-琥珀酸纳米颗粒(PCS NPs)，探讨其在肝细胞癌中通过代谢干扰与离子稳态失衡协同作用诱导肝细胞癌发生焦亡样细胞死亡的作用机制。方法 使用微乳液法构建一种PEG修饰的钙-琥珀酸纳米颗粒(PEGylated calcium succinate nanoparticles， PCS NPs)以Hepa1-6肝癌细胞为模型，并通过1H-NMR、FT-IR进行表征，采用CCK-8法与Calcein-AM/PI活死细胞双染检测其细胞毒性。进一步通过Fluo-8 AM、JC-1、DCFH-DA及BCECF-AM荧光探针，分别检测细胞存活率、胞内Ca2+水平、线粒体膜电位、活性氧（ROS）及pH的动态变化，再通过Western Blot检测NLRP3、Cleaved Caspase-1（p10亚基）、GSDMD-N等焦亡相关蛋白的表达，分析PCS NPs诱导肝细胞癌死亡的可能机制。结果 表征结果显示PCS NPs成功制备，该纳米颗粒在酸性条件下Ca2+释放明显增加，并可被 Hepa1-6 细胞有效摄取；CCK-8实验表明PCS NPs能显著抑制细胞活力。Calcein-AM/PI染色观察到膜完整性破坏等焦亡样特征；PCS NPs进入细胞后可引起胞内Ca2+的释放及胞内酸化，继而诱导线粒体去极化、ROS 水平升高。同时，Western Blot检测到NLRP3炎性小体、Caspase-1活化后产生的p10裂解亚基（Cleaved Caspase-1、p10）及GSDMD的N端剪切体（GSDMD-N）的蛋白表达。 结论 本研究构建了一种可在酸性条件下响应释放Ca2＋与琥珀酸的纳米颗粒PCS NPs。可通过琥珀酸介导的代谢干扰与Ca2+引起的离子稳态失衡产生协同作用，并触发焦亡相关信号蛋白表达变化。本研究结果表明，基于代谢干扰与离子稳态失衡的联合作用，可能在肝癌细胞死亡中发挥重要作用，为后续深入的分子机制研究与体内验证提供了实验基础。 |
| 关键词: 钙-琥珀酸纳米颗粒 肝细胞癌 焦亡 代谢紊乱 离子干扰 |
| DOI： |
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| Calcium–Succinate Nanoparticles Induce Pyroptosis-Like Cell Death in Hepatocellular Carcinoma via Metabolic–Ion Regulation |
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ZhouNiqiang
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Guangxi Medical University
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| Abstract: |
| Objective To prepare a pH-responsive calcium–succinate nanoparticle system (PCS NPs) and to investigate its potential mechanism in inducing pyroptosis in hepatocellular carcinoma through combined metabolic interference and disruption of ionic homeostasis.Methods PEGylated calcium succinate nanoparticles (PCS NPs) were constructed by the microemulsion method, with Hepa1-6 hepatocellular carcinoma cells employed as the in vitro model. The physicochemical properties of PCS NPs were characterized by 1H-NMR and FT-IR spectroscopy. Cellular cytotoxicity was evaluated using the CCK-8 assay and Calcein-AM/propidium iodide (PI) live/dead staining. Intracellular Ca2+ levels, mitochondrial membrane potential, reactive oxygen species (ROS) generation, and intracellular pH variations were dynamically monitored using Fluo-8 AM, JC-1, DCFH-DA, and BCECF-AM fluorescent probes, respectively, to elucidate the potential mechanisms underlying PCS NPs-induced hepatocellular carcinoma cell death.The expression of pyroptosis-associated marker proteins NLRP3, Cleaved Caspase-1 （p10 subunit), GSDMD-N was detected by Western blot to evaluate the involvement of related signaling pathways at the protein level. Results Characterization results confirmed the successful preparation of PCS NPs, which exhibited a significant increase in Ca2+ release under acidic conditions and could be efficiently internalized by Hepa1-6 cells. The CCK-8 assay demonstrated that PCS NPs markedly inhibited cell viability. Calcein-AM/PI staining revealed characteristic cellular swelling and membrane rupture, indicative of pyroptosis.Western blot analysis revealed upregulated expression of NLRP3 inflammasome, the p10 cleavage subunit of activated Caspase-1 (Cleaved Caspase-1 p10), and the N-terminal fragment of GSDMD (GSDMD-N), confirming the involvement of the canonical pyroptosis pathway. Following cellular uptake, PCS NPs induced intracellular Ca2+ release, which was accompanied by mitochondrial depolarization,elevated ROS production,and cytoplasmic acidification.Conclusion In this study, a pH-responsive PCS NPs system capable of releasing Ca2＋and succinate under acidic conditions was successfully constructed.The PCS NPs cantrigger pyroptosis characterized by Caspase-1 activation (p10 subunit) generation and GSDMD-N cleavage through the synergistic effects of succinate-mediated metabolic interference and Ca2+-inducedionic homeostasis imbalance. Collectively, these findings indicate that the interplay between metabolic perturbation and ionic imbalance may play a critical role in hepatocellular carcinoma cell death, providing an experimental basis for further mechanistic investigations and in vivo validation.
【Keywords】Calcium-succinate nanoparticles; Hepatocellular carcinoma; Pyroptosis; Metabolic disruption; Ion homeostasis |
| Key words: Calcium–succinate nanoparticles hepatocellular carcinoma pyroptosis metabolic dysregulation ionic disturbance |
