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Neuroprotective Effects of Deproteinized Calf Serum in Ischemic Stroke
Weiwei Li
Anchen Guo
Ming Sun6, 
Jiachuan Wang
Qun Wang- 1Department of Neurology, Beijing Tiantan Hospital, Capital Medical University, Beijing, China
- 2China National Clinical Research Center for Neurological Diseases, Beijing, China
- 3Department of Surgery, University of Cincinnati, Cincinnati, OH, United States
- 4Beijing Key Laboratory of Translational Medicine for Cerebrovascular Disease, Beijing, China
- 5Beijing Institute of Brain Disorders, Collaborative Innovation Center for Brain Disorders, Capital Medical University, Beijing, China
- 6Department of Neuropharmacology, Beijing Neurosurgical Institute, Beijing, China
- 7Department of Pathology, Shenzhen Traditional Chinese Medicine Hospital, The Fourth Clinical Medical College of Guangzhou University of Chinese Medicine, Shenzhen, China
Deproteinized calf serum (DCS) may have neuroprotective effects after ischemic stroke. The aim of this study is to investigate whether and how the DCS inhibits neuronal injury following cerebral ischemia. Rats were subjected to 2 h transient middle cerebral artery occlusion (MCAO). One dose of 0.125 mg/gbw DCS was given immediately after reperfusion. Neurological deficit and infarct volume at 24 h post-MCAO in DCS-treated rats were lower than those in vehicle-treated rats (p < 0.0005). In cultured neurons model, cell viability was decreased, and apoptosis was increased by oxygen-glucose deprivation/reperfusion (OGD/R) (p < 0.0005). These effects of OGD/R were attenuated by 0.4 μg/μl DCS (p < 0.05) that were validated by CCK8 cell viability assay, phycoerythrin–Annexin V Apoptosis Detection assay, and TUNEL assay. Furthermore, the increase of intracellular ROS level in cultured neurons was suppressed by DCS (p < 0.05). Compared with cells subjected to OGD/R, the expression level of Bax protein decreased, and bcl-2 protein increased after DSC treatment (p < 0.05). Overall, the neuroprotective effects of DCS following cerebral ischemia may in part be due to decreased ROS production and inhibition of apoptosis.
Introduction
Stroke is an acute disorder caused by abnormal blood supply to the brain and a major cause of morbidity and mortality worldwide [(1), p. 161–176]. Stroke is the fifth leading cause of death and main cause of disability in adults in the USA [(2), p. 165260]. Recent estimates suggest that nearly 795,000 individuals experience a stroke every year in the USA, corresponding to one victim every 40 s [(2), p. 165260]. Ischemic stroke accounts for 60–80% of all cases, and hemorrhagic stroke accounts for the remainder [(3), p. 7–32]. The main aim of therapy for acute ischemic stroke is to achieve rapid revascularization and thus restoration of blood flow [(4), p. 967–973, (5), p. 1066–1072]. Thrombolysis with recombinant tissue-type plasminogen activator (tPA) within a few hours of symptom onset is recognized as a potentially effective treatment for acute ischemic stroke and widely used in the clinical setting [(6), p. CD000213]. Surgical treatments have also been developed, including retrievable self-expanding stents for thrombectomy of acute proximal intracranial artery occlusions [(7), p. 12–20]. However, despite costing more than 70 billion dollars per year in the USA alone, the available treatments are limited in scope and predominantly supportive in nature [(2), p. 165260].
Ischemic stroke results from the blockade of an artery to the brain from in situ thrombosis or an embolus from another artery or the heart. Due to the brain's limited capacity to store glucose and utilize anaerobic metabolism, the blood flow deficit leads to rapid neurodegeneration and an expanding infarct core surrounded by potentially salvageable penumbral tissue that is the primary target for treatment strategies [(8), p. 975–980]. The development of novel therapies requires an understanding of the processes underlying the brain injury that follows stroke. These mechanisms include excitotoxicity, acidotoxicity, ionic imbalance, oxidative stress, nitrative stress, inflammation, and apoptosis [(9), p. 297–309, (10), p. 1167–1186]. Neuronal apoptosis, a major mechanism of cell death induced by cerebral ischemia/reperfusion (I/R) injury and the balance between the expressions of antiapoptotic Bcl-2 protein and proapoptotic Bax protein, is critical for regulating apoptotic cell death [(11), p. 1334–1340]. Bcl-2 exerts a survival function in response to a wide range of apoptotic stimuli through inhibition of mitochondrial cytochrome c release. Bax is a key component for apoptosis induced by mitochondrial stress. Upon stimulation of the apoptotic pathway, Bax forms oligomers and translocates from the cytosol to the mitochondrial membrane to interact with pore proteins in the mitochondrial membrane and increases the membrane's permeability. This leads to the release of cytochrome c from the mitochondria, activation of caspase-9, and initiation of the caspase-dependent apoptotic pathway.
Oxidative stress and mitochondrial dysfunction are widely recognized as making an important contribution to neuronal apoptosis following I/R injury. Dysfunction of the mitochondrial respiratory chain during ischemia leads to the generation of reactive oxygen species (ROS) [(12), p. 712–718]. Numerous in vivo and in vitro studies have provided evidence implicating important roles for ROS and mitochondrial-dependent apoptosis in the death of neuronal tissue following I/R injury [(13), p. 98–106, (14), p. 1491–1499].
Deproteinized calf serum (DCS, also known as Actovegin, AODEJIN® Avanc Pharmaceutical Co., Ltd., Jinzhou, China), a deproteinized ultrafiltrate of calf blood composed of more than 200 biological substances, has been used in clinical practice for a variety of indications including ischemic stroke and brain injury, peripheral arterial and venous perfusion disorders, diabetic polyneuropathy, and skin trauma [(15), p. 80–88]. DCS has been reported to have multiple metabolic effects, including improved oxygen utilization and uptake, enhanced cellular energy metabolism, and increased glucose uptake and oxidation [(16), p. 266–274]. These effects may contribute to the beneficial effects of DCS in patients with diabetic polyneuropathy [(17), p. 1181–1187]. Therefore, it is possible that DCS may exert a neuroprotective effect by enhancing energy metabolism in the brain after ischemia or injury. DCS has been reported to reduce focal neurologic deficits in patients with ischemic stroke, enhance cognitive function in patients with ischemic stroke [(18), p. 873–875] or vascular mild cognitive impairment, improve the functional rehabilitation of patients after ischemic and hemorrhagic stroke, correct immunometabolic disturbances in patients with chronic cerebral ischemia, and facilitate recovery after brain injury. In addition, DCS was found to enhance cell survival in the hippocampal CA1 region and improve spatial learning and memory in rats subjected to transient forebrain ischemia [(19), p. 1623–1630]. DCS has also been reported to reduce oxidative stress, inhibit apoptosis, and maintain excitatory synapses (in a concentration-dependent manner) in cultured primary rat neurons challenged with amyloid peptide Aβ [(16), p. 266–274]. DCS was also observed to attenuate H2O2-induced apoptosis of human neuroblastoma cells [(20), p. 215–217].
We hypothesized that the neuroprotective effects of DCS may be mediated, at least in part, by reduced generation of ROS. Therefore, the present study utilized a rat model of ischemic stroke and cultured neuronal cells to investigate whether DCS inhibited neuronal injury following ischemia by reducing ROS and suppressing apoptosis.
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