Neferine, a bisbenzylisoquinoline alkaloid, offers protection against cobalt chloride-mediated hypoxia-induced oxidative stress in muscle cells

摘要

Abstract BackgroundNeferine, a bisbenzylisoquinoline alkaloid, isolated from Nelumbo nucifera has a wide range of biological activities. Cobalt chloride (CoCl₂) was known to mimic hypoxic condition. In the present study, we assessed the cytoprotective effect of neferine against CoCl₂-induced oxidative stress in muscle cells.MethodsRhabdomyosarcoma cells were exposed to different concentrations of CoCl₂, and the IC₅₀ value was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Lactate dehydrogenase and NO assays were performed in order to determine the cytotoxic effect of CoCl₂. Reactive oxygen species generation and cellular antioxidant status were determined for evaluating oxidative stress. For analyzing the effect of neferine on CoCl₂-induced apoptosis, propidium iodide staining was performed.ResultsThe results of the present study indicate that CoCl₂ induces cell death in a dose-dependent manner. Neferine pretreatment at 700?nM concentration offers better cytoprotection in the cells exposed to CoCl₂. Lactate dehydrogenase and NO release in the culture medium were restored after neferine pretreatment. CoCl₂ triggers time-dependent reactive oxygen species generation in muscle cells. Further, results of propidium iodide staining, mitochondrial membrane potential, and intracellular calcium accumulation confirm that neferine offers protection against CoCl₂-induced hypoxic injury. Depleted activities of antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase due to CoCl₂ exposure were also reinstated in the group that received neferine pretreatment.ConclusionOur study suggests that neferine from N. nucifera offers protection to muscle cells by counteracting the oxidative stress induced by CoCl₂. Keywords cobalt chloride ; hypoxia ; neferine ; oxidative stress ; reactive oxygen species prs.rt("abs_end"); 1. Introduction Cobalt chloride (CoCl₂) has widely been used to mimic hypoxia in cell culture, and it is known to activate hypoxic signaling. Hypoxia is a pathophysiological condition characterized by an increase in reactive oxygen species (ROS) and a change in intracellular redox level. Oxygen flux in the mitochondria of skeletal muscle increases up to 100-fold at high altitude and during physical exercise, which in turn results in an increased generation of free radicals. Physical exercise results in oxidative stress, which is associated with increases in lipid peroxidation (LPO) and protein oxidation. 1 High-altitude physiology may be divided into studies of short-term changes that occur with exposure to hypobaric hypoxia (the acute response to hypoxia) and studies of longer-term acclimatization and adaptation. Acute exposure to the ambient atmosphere at extreme altitude (for example, above 8000 m) is rapidly fatal. 2 Acclimatization is the set of beneficial processes whereby lowland humans respond to a reduced inspired partial pressure of oxygen. These changes tend to reduce the gradient of oxygen partial pressure from ambient air to tissues (classical oxygen cascade), and are distinct from the pathological changes that lead to altitude illness. Adaptation to high altitude describes changes that have occurred over a number of generations as a result of natural selection in a hypobaric hypoxic environment, and this can be observed in some groups of high-altitude residents. Cellular response to hypoxia may provide important clues about impaired cellular function and neuronal cell death. ROS have been proposed to act as second messengers in redox-sensitive signal transduction pathways and can damage biomolecules. 3 Reactive oxygen intermediates, superoxide radicals (O₂ ? ), hydrogen peroxide (H₂O₂), and hydroxyl radicals (OH), are produced mainly in mitochondria. ROS act as physiological modulators of some mitochondrial functions, but may also damage mitochondria. Oxygen-derived radicals are implicated in LPO events and are critical in injuries after ischemia. 4 and 5 Physical exercise leads to temporary ischemia in muscles. During strenuous exercise, muscle oxygen consumption increases tremendously to as high as 100–200 times that in normal resting conditions. 6 The sudden increase and influx of oxygen cause a calcium overload in cells, leading to an influx of inflammatory cells into reperfused tissue. This will lead to the generation of ROS that are considered responsible for muscle fatigue during exercise. 7 Hence, oxidative stress plays a significant role in the initiation and progression of fatigue. Oxidative stress, more specifically LPO, is known to play an important role in the pathophysiology of exercise intolerance at high altitude. Exposure to high altitude appears