Green Synthesis of Cerium Oxide Nanoparticles, Characterization, and Their Neuroprotective Effect on Hydrogen Peroxide-Induced Oxidative Injury in Human Neuroblastoma (SH-SY5Y) Cell Line

Abstract

Cerium oxide nanoparticles (CeO₂NPs) have a broad scale of applications in the biomedical field due to their excellent physicochemical and catalytic properties. The present study aims to synthesize the CeO₂NPs from Centella asiatica (C. asiatica) leaf extract, which has been used in Indian traditional medicine for its neuroprotective properties. The CeO₂NPs were characterized by ultraviolet-visible, X-ray diffraction, Fourier transform infrared, Raman spectroscopy, scanning electron microscopy- energy dispersive X-ray spectroscopy, and high-resolution transmission electron microscopy. The antioxidant property was evaluated by 2,2-di (4-tert-octyl phenyl)-1-picrylhydrazyl and OH radical assays. The neuroprotective potential was assessed against the oxidative stress (OS) induced by H₂O₂ in the human neuroblastoma (SH-SY5Y) cell line. CeO₂NPs exhibited significant DPPH and OH radical scavenging activity. Our results revealed that CeO₂NPs significantly increased H₂O₂-induced cell viability, decreased lactate dehydrogenase, protein carbonyls, reactive oxygen species generation, apoptosis, and upregulated antioxidant enzyme activity. Our findings suggest that the CeO₂NPs protect the SH-SY5Y cells from OS and apoptosis, which could potentially counter OS-related neurodegenerative disorders.

Figure 1

Characterization of CeO₂NPs synthesized using different volumes of C. asiatica extract. (a) UV–vis spectroscopy, (b) FTIR, (c) XRD pattern, and (d) Raman spectroscopy.

Figure 2

Morphology of CeO₂NPs synthesized using different volumes of C. asiatica extract. (a) SEM images, (b) EDX analysis, (c) HR-TEM images, and (d) SAED patterns.

Figure 3

Effects of CeO₂NPs on H₂O₂-induced cytotoxicity in SH-SY5Y cells. (a) Cells treated with varying concentration of H₂O₂ (3.125–400 μg/mL) for 24 h. (b) Cells pretreated with H₂O₂ followed by incubation with CeO₂NPs for 24 h, and cell viability was determined by MTT assay. (c) Cells pretreated with H₂O₂ followed by incubation with CeO₂NPs for 24 h, and cell cytotoxicity was measured by LDH assay. (d) Morphology of SH-SY5Y cells observed under phase contrast microscope. The data is expressed as mean ± SE from three independent experiments and analyzed by one-way ANOVA followed by Tukey’s test. *p < 0.05 significance compared with control; #p < 0.05 significance compared with H₂O₂-treated cells.

Figure 4

Cellular uptake of CeO₂NPs in SH-SY5Y cells. (a) Control, (b) CeO₂NPs (20 μg/mL), and (c) CeO₂NPs (40 μg/mL).

Figure 5

Effect of CeO₂NPs on antioxidant enzyme activities in H₂O₂-treated cells: (a) SOD, (b) CAT, and (c) GPx. The data is analyzed from three independent experiments by one-way ANOVA followed by Tukey’s test. *p < 0.05 significance compared with control; #p < 0.05 significance compared with H₂O₂-treated cells.

Figure 6

Effect of CeO₂NPs on PC levels in H₂O₂-treated SH-SY5Y cells. The data is analyzed from three independent experiments by one-way ANOVA followed by Tukey’s test. *p < 0.05 significance compared with control; #p < 0.05 significance compared with H₂O₂-treated cells.

Figure 7

Effect of CeO₂NPs on ROS generation in H₂O₂-treated SH-SY5Y cells. (a) ROS determination by flow cytometry. (b) Quantitative analysis of the ROS fluorescence intensity. The data is analyzed from three independent experiments by one-way ANOVA followed by Tukey’s test. *p < 0.05 significance compared with control; #p < 0.05 significance compared with H₂O₂-treated cells.

Figure 8

Effect of CeO₂NPs on H₂O₂-induced apoptosis in SH-SY5Y cells. (a) Morphology of cell nucleus using DAPI staining. (b) Flow cytometry. (c) Percentage of apoptotic cells. Data were expressed as mean ± SE (n = 3). Statistical analysis was performed by t-test. *p < 0.05 significance compared with control; #p < 0.05 compared with H₂O₂-treated cells.