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Redox biology; Oxidative stress; Reactive oxygen species (ROS); Reactive nitrogen species (RNS); Cellular signaling

Introduction

Redox biology is a field that examines the roles of redox reactions in biological systems. These reactions are essential for maintaining cellular homeostasis, regulating signaling pathways, and protecting cells from oxidative stress. Redox reactions involve the transfer of electrons between molecules, influencing the oxidation state of various biomolecules [1,2]. This balance is critical for numerous cellular processes, including energy production, detoxification, and immune response. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are key players in redox biology. While they can be beneficial in small quantities, excessive ROS and RNS levels can lead to oxidative stress, damaging cellular components such as DNA, proteins, and lipids. This oxidative damage is implicated in the pathogenesis of many diseases, including cancer, neurodegenerative disorders, and cardiovascular diseases [3-6].

Methods

Cell culture and treatment

Human cell lines (e.g., HEK293, SH-SY5Y) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were maintained at 37°C in a humidified atmosphere with 5% CO₂. For treatment, cells were exposed to varying concentrations of hydrogen peroxide (H₂O₂) or nitric oxide donors to induce oxidative stress.

Measurement of ROS and RNS levels

Intracellular ROS levels were measured using the fluorescent dye 2',7'-dichlorofluorescin diacetate (DCFDA). Cells were incubated with 10µM DCFDA for 30 minutes at 37°C, followed by fluorescence measurement using a microplate reader. RNS levels were determined using the Griess reagent, which detects nitrite, a stable end-product of nitric oxide metabolism.

Western blotting

Protein expression levels of key redox-related enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx), were analyzed by Western blotting. Cells were lysed, and protein extracts were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with specific antibodies. Bands were visualized using chemiluminescence detection.

Statistical analysis

Data were analyzed using GraphPad Prism software. Differences between groups were assessed by one-way ANOVA followed by Tukey's post hoc test. A p-value of <0.05 was considered statistically significant.

Results

Induction of oxidative stress in cell lines

Treatment with H₂O₂ resulted in a dose-dependent increase in ROS levels in HEK293 cells Similarly, exposure to nitric oxide donors elevated RNS levels in SH-SY5Y cells These findings confirm the successful induction of oxidative stress in the cell models.

Modulation of redox enzyme expression

Western blot analysis revealed that oxidative stress significantly upregulated the expression of SOD and catalase in HEK293 cells. In contrast, GPx levels remained unchanged [7,8]. These results suggest a compensatory response to increased ROS levels, aimed at mitigating oxidative damage.

Impact on cellular viability

Cell viability assays demonstrated that prolonged exposure to high concentrations of H₂O₂ or nitric oxide donors significantly reduced cell viability. This effect was more pronounced in SH-SY5Y cells, indicating differential sensitivity to oxidative stress between cell types [9].

Discussion

Our findings highlight the crucial role of redox balance in maintaining cellular homeostasis. The upregulation of SOD and catalase in response to oxidative stress suggests an adaptive mechanism to counteract elevated ROS levels. However, the unchanged GPx expression indicates that different redox enzymes may be selectively regulated under stress conditions. The differential sensitivity of cell lines to oxidative stress underscores the complexity of redox biology [10]. The heightened vulnerability of SH-SY5Y cells to RNS suggests a potential link between redox dysregulation and neurodegenerative diseases. Further research is needed to elucidate the molecular mechanisms underlying this sensitivity and to explore potential therapeutic interventions.

Conclusion

This study provides insights into the dynamic interplay between redox reactions and cellular function. By understanding the mechanisms of redox regulation and the impact of oxidative stress, we can better appreciate the role of redox biology in health and disease. Future research should focus on identifying novel redox-related therapeutic targets and developing strategies to mitigate oxidative damage in disease contexts.

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