Ma Kai, Tian Tiantian, Li Xinyue, Pang Huan, Ning Xiaofan, Li Meng, Li Jiali, Luo Zhixuan, Liu Tianxiang, Liu Mengyue, Wang Mingqian, Zhao Chao, Song Xiuling, Du Haiying, Jin Minghua
School of Public, Health Jilin University, Changchun, Jilin, 130021, People's Republic of China.
Mol Neurobiol. 2025 Jun;62(6):7506-7524. doi: 10.1007/s12035-025-04724-9. Epub 2025 Feb 5.
A growing stream of research indicates that exposure to Silica nanoparticles (SiNPs) can cause nervous system damage, leading to the occurrence of neurodegenerative diseases such as Alzheimer's disease. However, the specific mechanism by which SiNPs cause neuroblast injury remains unclear and requires further research. This study established an in vitro experimental model of SH-SY5Y cells exposed to SiNPs and observed cell growth through an inverted fluorescence microscope. Cell viability was measured using an MTT assay. The intracellular ROS and Ca levels were detected by flow cytometry. Cell apoptosis was observed using both Hoechst33342 staining and TUNEL staining. The activities of SOD and ATPase and the content of ATP in the cells were tested by biochemical methods. The genes including parp-1, aif, par, ucp2, vdac and prdx3 were explored using quantitative real-time PCR. The expressions of PARP, AIF, PAR, Caspase-3, Caspase-9 and Cyt C proteins were evaluated by Western Blot. The immunofluorescence technique was used to observe the distribution of Parthanatos-related proteins induced by SiNPs. The results showed that SiNPs reduced cell survival rate, induced excessive ROS and Ca overload, decreased SOD activity, ATPase activity, intracellular and mitochondrial ATP content, increased the expression of mitochondrial function and PARP pathway related genes, as well as PARP and Caspase pathway protein expression, ultimately inducing cell apoptosis. As a further test of the roles of PARP and Caspase pathways in SiNPs induced SH-SY5Y cells death, we selected the PARP inhibitor Olaparib and Caspase inhibitor Z-VAD, and the above effects were significantly improved after treatment with the inhibitors. Conclusively, this study confirmed that SiNPs can generate excessive ROS production in SH-SY5Y cells, alter mitochondrial function, and induce cell death through Parthanatos and caspase dependent apoptotic pathways, which can coexist and interact with each other.
越来越多的研究表明，接触二氧化硅纳米颗粒（SiNPs）会导致神经系统损伤，进而引发神经退行性疾病，如阿尔茨海默病。然而，SiNPs导致神经母细胞损伤的具体机制仍不清楚，需要进一步研究。本研究建立了SH-SY5Y细胞暴露于SiNPs的体外实验模型，并通过倒置荧光显微镜观察细胞生长情况。使用MTT法检测细胞活力。通过流式细胞术检测细胞内活性氧（ROS）和钙（Ca）水平。使用Hoechst33342染色和TUNEL染色观察细胞凋亡情况。通过生化方法检测细胞中超氧化物歧化酶（SOD）和ATP酶的活性以及ATP的含量。使用定量实时PCR检测包括聚（ADP-核糖）聚合酶-1（parp-1）、凋亡诱导因子（aif）、聚（ADP-核糖）（par）、解偶联蛋白2（ucp2）、电压依赖性阴离子通道（vdac）和过氧化物还原酶3（prdx3）等基因。通过蛋白质免疫印迹法评估聚（ADP-核糖）聚合酶（PARP）、凋亡诱导因子（AIF）、聚（ADP-核糖）（PAR）、半胱天冬酶-3（Caspase-3）、半胱天冬酶-9（Caspase-9）和细胞色素C（Cyt C）蛋白的表达。使用免疫荧光技术观察SiNPs诱导的Parthanatos相关蛋白的分布情况。结果表明，SiNPs降低了细胞存活率，诱导了过量的ROS和Ca超载，降低了SOD活性、ATP酶活性、细胞内和线粒体ATP含量，增加了线粒体功能和PARP途径相关基因的表达，以及PARP和Caspase途径蛋白的表达，最终诱导细胞凋亡。作为对PARP和Caspase途径在SiNPs诱导的SH-SY5Y细胞死亡中作用的进一步测试，我们选择了PARP抑制剂奥拉帕尼（Olaparib）和Caspase抑制剂Z-VAD，用抑制剂处理后上述效应得到了显著改善。总之，本研究证实SiNPs可在SH-SY5Y细胞中产生过量的ROS，改变线粒体功能，并通过Parthanatos和Caspase依赖性凋亡途径诱导细胞死亡，这两种途径可以共存并相互作用。
