Graphene oxide (GO) nanoparticles (NPs) have been widely applied in various fields, especially in biomedical applications. Extensive studies have suggested that GO can pass through the blood–brain barrier (BBB) and induce abnormal autophagy and cytotoxicity in the central nervous system (CNS). However, the effect and specific mechanism of GO on astrocytes, the most abundant cells in the brain still has not been extensively investigated. In this study, we systematically explored the toxicity and mechanism of GO exposure in the rat astroglioma-derived F98 cell line using molecular biological techniques (immunofluorescence staining, flow cytometry and Western blot) at the subcellular level and the signaling pathway level. Cells exposed to GO exhibited decreased cell viability and increased lactate dehydrogenase (LDH) release in a concentration- and time-dependent manner. GO-induced autophagy was evidenced by transmission electron microscopy (TEM) and immunofluorescence staining. Western blots showed that LC3II/I and p62 were upregulated and PI3K/Akt/mTOR was downregulated. Detection of lysosomal acidity and cathepsin B activity assay indicated the impairment of lysosomal function. Annexin V-FITC-PI detection showed the occurrence of apoptosis after GO exposure. The decrease in mitochondrial membrane potential (MMP) with an accompanying upregulation of cleaved caspase-3 and Bax/Bcl-2 further suggested that endogenous signaling pathways were involved in GO-induced apoptosis. The exposure of F98 cells to GO can elicit concentration- and time-dependent toxicological effects. Additionally, increased autophagic response can be triggered after GO treatment and that the blocking of autophagy flux plays a vital role in GO cytotoxicity, which was determined to be related to dysfunction of lysosomal degradation. Importantly, the abnormal accumulation of autophagic substrate p62 protein can induce capase-3-mediated apoptosis. Inhibition of abnormal accumulation of autophagic cargo could alleviate the occurrence of GO-induced apoptosis in F98 cells.

中文翻译：

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氧化石墨烯诱导的自噬通量和溶酶体功能的中断对F98细胞p62依赖性凋亡的影响

氧化石墨烯（GO）纳米颗粒（NPs）已广泛应用于各个领域，尤其是在生物医学应用中。大量研究表明，GO可以穿过血脑屏障（BBB）并在中枢神经系统（CNS）中诱导异常的自噬和细胞毒性。然而，GO对星形胶质细胞（脑中最丰富的细胞）的作用和特异性机制尚未得到广泛研究。在这项研究中，我们使用分子生物学技术（免疫荧光染色，流式细胞仪和蛋白质印迹）在亚细胞水平和信号通路水平上系统地探索了大鼠星形胶质瘤来源的F98细胞系中GO暴露的毒性和机制。暴露于GO的细胞以浓度和时间依赖性方式显示出降低的细胞活力和增加的乳酸脱氢酶（LDH）释放。GO诱导的自噬通过透射电子显微镜（TEM）和免疫荧光染色证明。蛋白质印迹显示，LC3II / I和p62被上调，而PI3K / Akt / mTOR被下调。溶酶体酸度检测和组织蛋白酶B活性测定表明溶酶体功能受损。Annexin V-FITC-PI检测显示GO暴露后发生凋亡。线粒体膜电位（MMP）的降低，伴随着裂解的caspase-3和Bax / Bcl-2的上调，进一步表明内源性信号通路与GO诱导的细胞凋亡有关。F98细胞暴露于GO可引起浓度和时间依赖性毒理学作用。此外，GO处理后可引发自噬反应增强，并且自噬通量的阻断在GO细胞毒性中起着至关重要的作用，这被确定与溶酶体降解的功能障碍有关。重要的是，自噬底物p62蛋白的异常积累可以诱导capase-3介导的细胞凋亡。抑制自噬货物的异常积累可以减轻GO诱导的F98细胞凋亡的发生。自噬底物p62蛋白的异常积累可诱导capase-3介导的细胞凋亡。抑制自噬货物的异常积累可以减轻GO诱导的F98细胞凋亡的发生。自噬底物p62蛋白的异常积累可诱导capase-3介导的细胞凋亡。抑制自噬货物的异常积累可以减轻GO诱导的F98细胞凋亡的发生。