Opuntia Milpa Alta Polysaccharides Extraction And Structure Identification And Protection Activity On Cerebral Ischemia

Opuntia Milpa Alta Polysaccharides Extraction and Structure identification and Protection Activity on Cerebral IschemiaAIMTo extract and isolate polysaccharides from Opuntia Milpa Alta (MAPs) and analysis the polysaccharides composition, further to investigate the effects and mechanisms of MAPs in vivo and in vitro model of cerebral ischemic injury demonstrated that the polysaccharides had potent neuroprotective. We tried to explore the protective effects of MAPs on cerebral ischemia in vivo with ischemia induced by cerebral hypoperfusion after the bilateral common carotid artery (2VO) and middle cerebral artery occlusion (MCAO). In vitro, rat cortical and hippocampal neurons have suffered from oxygen-glucose deprivation (OGD). To analysis the mechanisms of its action, we have systematically investigated the effect of MAPs on the expression of NMDAR2B, NMDAR1 and BDNF, the content of important neurotransmitter amino acid, the concentration of reactive oxygen species and intracellular calcium.METHODS AND RESULTSPartⅠMAPs extraction and structure identificationOpuntia Milpa Alta has been cut up and drying. We extracted polysaccharides from Opuntia Milpa Alta through water extraction and alcohol precipitation method and used DEAE-cellulose-52 column to purification the polysaccharides fragment. The contents of polysaccharides were determined by Phenol sulfuric Acid Method. MAPs composition was analyzed using Gas Chromatography-Mass Spectrometer (GC-MS) method. And the Molecular weight size through the Gel Permeation Chromatography (GPC) detection. Results:MAPs are extracted by our separation method contained and sugar to 84%. Sugar composition analysis revealed that MAPs consisted primarily of Mannose (6.37%), Rhamnose (14.94%), Xylose (1.99%), Arabinose (24.07%), Galactose (38.25%), Ribose (2.63%), Glucose (11.48%). The GPC analysis for Molecular weight MAPs were 5.40×105 and 2270.PartⅡProtective effects of MAPs on cerebral ischemia in vitroCultured rat cortical and hippocampal neurons suffered from oxygen-glucose deprivation (OGD) was served as an in vitro ischemia model. Primary cultures of neurons were exposed to 4 h OGD. MAPs were added 12 h before OGD initiated and maintained during OGD period. The effect of MAPs on cell activity was observed. Thiazoyl blue tetrazolium bromide (MTT) method and lactate dehydrogenase (LDH) release were detected to determined cell activity.2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA) staining was used to observe ROS in cortical neurons. Concentrations of extracellular amino acid (Glu), intracellular and calcium ([Ca2+]ⅰ) were detected by high performance liquid chromatography (HPLC) and fluorescent Ca2+ sensitive probe fura 2 acetoxylmethyl ester (Fura 2/AM) respectively in cortical neurons.Results:The extracted of MAPs could significantly inhibit the decrease of cell activity in cortical and hippocampal neurons induced by OGD. MAPs could decrease LDH level in cultures of cortical neurons suffered from OGD. At the same time, DCFH-DA staining showed that ROS in injured neurons was decreased after administration of MAPs. MAPs (0.5μg/mL 5μg/mL 50μg/mL effectively depressed of intracellular calcium ([Ca2+]ⅰ) and decrease the extracellular glutamate (Glu) level induced by OGD.PartⅢProtective effects of MAPs on cerebral ischemia in vivoChronic cerebral hypoperfusion in rats or mice was performed by permanent occlusion of the bilateral common carotid artery (2VO) and middle cerebral artery occlusion (MCAO). The MCAO model:Using mouse model of middle cerebral artery occlusion (MCAO) 2 h-reperfusion 24 h. Male mice were divided into three groups: sham-operated group, ischemic group, MAPs (200 mg/kg) treated group. In vivo cerebral infarct area was measured by tetrazolium staining, and neurological functional deficits were assessed at 4 and 24 h after reperfusion respectively. The 2VO model:Male mice were divided into five groups:sham-operated group, ischemic group, MAPs (100 mg/kg,200 mg/kg and 400 mg/kg) treated group. Male rats were divided into three groups:sham-operated group, ischemic group, MAPs (200 mg/kg) treated group. After ischemic impairment by 2VO, MAPs were administrated in treated groups and saline in sham-operated and ischemic groups. Morris water maze and Novel Object Recognition test were used to measure spatial learning and memory performance and animal behavior. Morphological change was examined by hematoxylin-eosin (HE) staining. Expressions of NMDAR1, NMDAR2B and BDNF were measured by real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) and expression of BDNF was measured by western blot analyses.Results:Chronic cerebral hypoperfusion resulted neurological functional deficits and increase cerebral infarct area, depress the cognitive ability, in spatial learning and memory impairments shown by longer escape latency and shorter time spent in the target quadrant. These behavioral dysfunctions were delayed degeneration of neurons, decreases in NMDAR2B and BDNF mRNA and BDNF protein levels. Intraperitoneally administration of MAPs (200 mg/kg) significantly reduced cerebral infarct area, and attenuated neurological functional deficits. Administration of MAPs markedly improved the spatial learning and memory dysfunction and spatial memory function, attenuated neuronal damage and enhanced the expression of NMDAR2B and BDNF.CONCLUSIONSMAPs are extracted by our separation method contained and sugar to 84%. Sugar composition analysis revealed that MAPs consisted primarily of Mannose (6.37%), Rhamnose (14.94%), Xylose (1.99%), Arabinose (24.07%), Galactose (38.25%), Ribose (2.63%), Glucose (11.48%). The GPC analysis for Molecular weight MAPs were 5.40×105 and 2270. In the pharmacological research, results show that MAPs have a significant neuroprotective effects in vivo and in vitro model of cerebral ischemic injury. The neuroprotective effects is partly due to its functions that are as follows:enhancement of NMDAR2B and BDNF expression; prevention of intracellular inhibition of excessive glutamate release, calcium overload and interrupt the cascade of events leading to neuronal injury and death in ischemia.