Different Levels Of Hydrogen Peroxide Regulate The Survive And Death On PC12 Cells Through Mitochondrial ROS

Historically, mitochondrial reactive oxygen species(m ROS) were thought to exclusively cause cellular damage and lack a physiological function. Accumulation of m ROS and oxidative damage have been linked to multiple pathologies. Thus, m ROS were originally envisioned as a necessary evil of oxidative metabolism, a product of an imperfect system. However, more and more evidences suggest that m ROS are critical for healthy cell function. m ROS is generally induced by cell stress, which include hypoxia, starvation, pathogen infection and growth factor stimulation. There have been numerous reports highlighting the importance of m ROS-dependent signaling in a variety of systems. Collectively, these data suggest that role of m ROS has evolved as a method of maintaining homeostasis and promote adaptation to stress.Studies have reported that oxidative stress can disrupt the function of endoplasmic reticulum and activative endoplasmic reticulum stress-autophagy pathway. Depending on the severity of oxidative stress, endoplasmic reticulum stress and the subsequent activation of autophagy can play the role of promoting cell survive or promoting cell death.Rat adrenal pheochromocytoma PC12 cell line is a common cell model for neurological research. When an animal serum medium is present, the body of PC12 cells were round, bright and the cell proliferate rapidly. Under these conditions, PC12 cells exhibit many characteristics of the immature nerve cells and can be converted into adrenal pheochromocytoma or the sympathetic nerve-like cells. Nerve growth factor can induce PC12 cells differentiate into sympathetic-like cells with morphology biochemical and physiological characteristics of neurons. These characteristics make PC12 cells a simple and convenient model in vivo for the the research of nervous system disease.PC12 cells were used for this study. Different concentrations of H2O2 were used to imitate different levels of oxidative stress. We investigated the role of mitochondrial ROS and endoplasmic reticulum stress-autophagy pathway in regulating the survive and death of PC12 cell on different levels of oxidative stress.Methods and results:1. After PC12 cells were treated with different concentrations of H2O2 for 6h, the cell viability was detected by the MTT assay. The results showed that low concentration of H2O2 had no effect on the survive of PC12 cells while high concentration of H2O2 promoted cell death, when compared with the control group.2. After PC12 cells were treated with a low concentration or high concentration of H2O2 for 6h, the morphology of cells were observed through an inverted optical microscope. The results showed that low concentrations of H2O2 had no effet on cell morphology while high concentrations of H2O2 changed cells’ morphology, when compared with the control group.3. After PC12 cells were treated with a low or high concentration of H2O2 for 6h, the expression of cleaved-caspase3 protein,a marker for cell apoptosis were detected by western blot. The results showed that low concentration of H2O2 did not induce apoptosis while high concentrations of H2O2 induced apoptosis when compared with the control group.4. After PC12 cells were treated with a low or high concentration of H2O2 for 6h, the change of mitochondrail membrane potential were detected using flow cytometry. The results showed that low concentration of H2O2 decreased the mitochondrail membrane potential slightly and high concentration of H2O2 decreased the mitochondrail membrane potential strongly when compared with the control group.5. After PC12 cells were treated with a low or high concentration of H2O2 for 6h, the level of mitochondrial ROS was stained with Mito SoxTM kit and viewed under fluorescent microscope. The results showed that low concentration of H2O2 decreased the level of the mitochondrail ROS slightly while high concentration of H2O2 increased the level of the mitochondrial ROS strongly when compared with the control group.6. After PC12 cells were treated with a low or high concentration of H2O2 for 6h, the expression of Bcl-2 protein, a antiapoptotic pathway associated protein, and the expression of caspase-9 protein, a mitochondrial apoptotic protein, was detected by western blot. The results showed that low concentration of H2O2 could upregulate the expression of antiapoptotic Bcl-2 protein, but has no effect on the expression of apoptotic caspase-9 protein; while high concentration of H2O2 decreased the expression of Bcl-2 but upregulated the expression of caspase-9.7. After PC12 cells were treated with a low or high concentration of H2O2 for 6h, the protein expressions related endoplasmic reticulum stress-autophagy pathway were detected by western blot. The results showed that low concentration of H2O2 upregulated the expression of GRP78, the endoplasmic reticulum stress protein, and had no effect on the expression of CHOP protein, the endoplasmic reticulum stress-mediated apoptosis protein, and on the expression of p62 and LC3,two autophagy-associated protein; while high concentration of H2O2 upregulated the protein expressions of GRP78, CHOP, p62 and LC3.Conclusion:When respond to different degrees of oxidation stress, cells mobilized different responses. Under mild oxidative stress, mitochondrial ROS mobilized the anti-apoptic signal to matain cellular homeostasis; under severe conditions of oxidative stress, mitochondrial ROS and endoplasmic reticulum stress-autophagy pathway triggered death signal simultaneously and induced the mitochondria-mediated apoptosis and the endoplasmic reticulum stress-mediated apoptosis.