Abstract
Oxidative stress and mitochondrial dysfunction are related to disease pathogenesis. Oligodeoxynucleotide containing CpG motifs (CpG ODN) demonstrate possibilities for immunotherapy applications. The aim of the present work is to explore the underlying mechanism of the cytoprotective function of CpG ODN by employing the oxidative stress modulation in immune cells. We used the imaging flow cytometry to demonstrate that tert-butyl hydroperoxide (t-BHP) induces mitochondrial-mediated apoptosis and ROS production in RAW264.7 cells. After pretreatment with CpG ODN, the percentage of apoptotic cells and ROS production was both markedly reduced. The decrease in mitochondrial membrane potential (MMP) induced by t-BHP was partially reversed by CpG ODN. The t-BHP induced upregulation of the expression of apoptosis-related proteins (cleaved-caspase 3, cleaved-caspase 9, cleaved-PARP, and bax) was notably decreased in the presence of CpG ODN. Furthermore, we found that CpG ODN enhanced phosphorylation of ERK1/2 and Akt to inhibit ROS production. In conclusion, the protective effect of CpG ODN in mitigation of t-BHP-induced apoptosis is dependent on the reduction of ROS.
Highlights
Oxidative stress and mitochondrial dysfunction are potentially related to the pathogenesis of various diseases, such as cardiovascular disease, ischemia/reperfusion injury, alcoholic hepatitis, diabetes, Parkinson’s disease, and age-related macular degeneration (AMD) [1]
We determined that apoptosis was the primary cell death form in the stimulation of tert-butyl hydroperoxide (t-BHP) using Annexin V and propidium iodide (PI) staining (Figures 1(b) and 1(c))
reactive oxygen species (ROS) production was analyzed by imaging flow cytometry; the results showed that significant increases in DCFHDA fluorescence indicated increases of intracellular ROS in RAW264.7 cells exposed to a high dose of t-BHP (Figures 3(c)-3(f))
Summary
Oxidative stress and mitochondrial dysfunction are potentially related to the pathogenesis of various diseases, such as cardiovascular disease, ischemia/reperfusion injury, alcoholic hepatitis, diabetes, Parkinson’s disease, and age-related macular degeneration (AMD) [1]. Overproduction of ROS is a harmful process that causes injury to cellular components, including DNA, proteins, lipids, mitochondria, and membrane structures. Excess ROS generation and release cause a series of oxidative stress responses, which serve to further aggravate mitochondrial dysfunction, leading to cell injury and death [4], subsequently accelerating disease progression [5]. It is Oxidative Medicine and Cellular Longevity well-known that oxidizing agents can induce cell apoptosis, including macrophages and hepatocytes [6]
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