| Background and objectiveCerebrovascular disease is one of the most common diseases worldwide. It is seriously threatening human life and health because of its high morbidity, disability rate and mortality. Ischemic cerebrovascular disease (ICVD) refers to occurrences of stenosis and occlusion of main arteries which resulted in the sudden loss of blood and oxygen to the cerebral tissue, especially in middle cerebral artery region. Local brain tissue is ischemic and necrotic ultimately, and appears corresponding clinical symptoms. Restoration of blood flow following ischemic stroke can be achieved by means of thrombolysis or mechanical recanalization. However, for some patients, reperfusion may exacerbate the injury initially caused by ischemia, producing a so-called "cerebral reperfusion injury". Cerebral ischemia-reperfusion injury is a complex pathophysiological process, there are many links in this cascade reaction, including energy shortage, cellular acidosis, increased releasing of excitatory amino acid, destabilization of intracellular Ca2+, production of free radicle, releasing of inflammatory cytokines and activation of apoptosis gene. These closely linked to each other cause and effect, overlapping each other, form a vicious cycle leading to brain cell apoptosis or necrosis. Despite great advances in the mechanism of the pathophysiology of cerebral ischemia, therapeutic options remain limited. One successful treatment strategy for salvaging ischemic tissue is reperfusion. Only recombinant tissue-plasminogen activator (rt-PA) for thrombolysis is currently approved for use in the treatment of this devastating disease. However, its use is limited by its short therapeutic window (3h-4.5h), complications derived essentially from the risk of hemorrhage, and the potential damage from reperfusion/ischemic injury. The other potential approach is to try to impede the ischemic cascade by targeting various components of the cascade that are deemed to be of importance. This latter approach is called the neuroprotection strategy. Despite many successful treatment experiments in animals regarding both infarct size reduction and improved functional outcome, no neuroprotective drug demonstrated unequivocal efficacy in clinical trials.The neuroprotective role of naturally occurring antioxidants has recently been a focus of attention. Mangiferin (MAG) is one such naturally occurring polyphenol that is widely found in many herbs such as Mangifera indica L and Anemarrhena asphodeloides Bunge. In the mangiferin molecule, four aromatic hydroxyl groups determine its strong antiradical and antioxidant properties. Numerous published in vitro and in vivo studies about mangiferin, demonstrated many pharmacological activities on inflammation, oxidative stress, tumor growth, microorganism infections, metabolic regulations, immune regulations, and radioprotection.The aim of this research is to set up the model of cerebral ischemia-reperfusion injury in vivo and in vitro and investigate the protective effects and mechanisms of mangiferin on the ischemic cerebral injury. It can provide a theoretical basis for safer and more reasonable uses of this therapy in clinic.MethodsIn vivo,(1) We set up the cerebral ischemia-reperfusion injury model by blocking middle cerebral artery with filament and observed effects of MAG on the neurological deficit score. Neurological deficits in the animals were assessed of the groups after2h of ischemia and24h of reperfusion. At24h after reperfusion, the behavioral characteristics were examined using a standard scale for a five-point neurological assessment (Longa et al.,1989). Briefly, rats were scored as follows:0=no deficit;1=failure to extend contralateral paw fully;2=circling to the contralateral side;3=falling to the contralateral side; and4=no spontaneous walking with a depressed level of consciousness. In the dose-dependence study, rats were randomly divided into6groups:model group, sham-operated group, positive control (edaravone) group, and MAG-treated groups (5mg/kg,10mg/kg and20mg/kg). In the sham-operated rats, the external carotid artery was surgically prepared for insertion of the filament but the filament was not inserted. All groups were injected, besides sham group and model group were gave isotonic Na chloride. HE staining was used to detect the histomorphological changes of brain tissue. The size of cerebral infarction and the water content were measured in the rats.(2) In the long-term experiment, the rats were randomly divided into6groups:model group, sham-operated group, positive control group, and MAG-treated groups of high, medium and low doses. Postural reflex test and limb use asymmetry test were performed as behavioral indexes at2th,4th,6th and8th day after the rats were subjected to focal cerebral ischemia-reperfusion injury for evaluating the recovery of motor function.(3) Biochemical evaluation of oxidative stress:The experiment designs the groups as above. The content of malonaldehyde (MDA), glutathione(GSH) and activity of superoxide dismutase(SOD) in brain tissue after cerebral ischemia for2h followed reperfusion for24h was determined by colorimetry.(4) Biochemical evaluation of inflammation:The experiment designs the groups as above. mRNA expression of cytokines TNF-a and IL-1β in brain tissue was measured by the RT-PCR method. ELISA experiments were next performed at protein levels. In vitro,(5) By establishing oxygen-glucose deprivation (OGD) model of PC12cells, we observed the morphological change of the cells. At the same time, MTT assay was employed to detect the cellular survival rate and biochemical method was used to determine LDH activity.Result1. Effect of MAG on neurological deficits in rats after focal cerebral ischemia-reperfusion injuryThe rats in sham-operated group have no deficit and were scored0; The rats in model group fail to extend contralateral paw fully and circle or fall to the contralateral side. The neurological score of ischemic model group was significantly high as compared with sham-operated group (P=0.000), indicating induction of ischemia. Treatment with MAG (10,20mg/kg) significantly reduced the neurological deficit (,P=0.023,P=0.030). Edaravone-treated animals exhibited a significantly improved neurological function (P=0.005) compared with the ischemic model group. However, we did not find a significant difference in neurobehavioral improvement in the5mg/kg MAG-treated groups (P=0.818).2. Effect of MAG on brain histopathology in rats after focal cerebral ischemia-reperfusion injuryHistomorphological changes:In model group, the ischemic cortex showed necrosis of neuron, swelling of cell, cytoplasm rarefaction, light dyeing and vacuolization, while in the sham group it showed no such pathological changes:the nucleolus was clear, round with intact karyolemma.Treatment groups to a certain extent were ameliorated. MAG of high-dose significantly ameliorated the above-mentioned pathological changes, compared with the model group, and necrosis cells obviously eliminated.3. Effect of MAG on cerebral infarct size, edema in rats after cerebral ischemia-reperfusion injuryImage analysis of TTC-stained coronal sections showed reproducible and readily detectable volume of infarction (31.51±9.78%) in MCAO group as compared to sham. In sham operation group, the infarct areas were unconspicuous. No significant difference was found in5mg/kg MAG-treated groups (P=0.093) in comparison to model group. The cerebral infarct size was significantly smaller in both10mg/kg and20mg/kg MAG-treated group than in MCAO model group(P=0.001,P=0.000). Edaravone-treated group has reduced the infarct volume significantly as compared with the MCAO ischemia group. Ischemia-reperfusion injury led to a significant increase of the brain water content as compared to sham operation group, suggesting the presence of brain edema. Treatment with MAG (5mg/kg,10mg/kg and20mg/kg) significantly limited the increase of brain water content after MCAO (P=0.006, P=0.000, P=0.000). Positive control (edaravone) group reduced the brain water content, the differences was statistically significant compared with the modle group (P=0.000). 4. Long-term motor functional assessment and body weight of MAG after cerebral ischemia-reperfusion injury①the weight variation ratio:Due to the surgical procedures, all the rats showed a loss of body weight after their operation, except rats in the sham-operated group. The weight of rats in the model group was significantly decreased. Compared with sham-operated group at each tested time point (2th、4th、6th and8th day), the weight variation ratio after MCAO in the model group was significantly decreased (-10.3612.55,-13.33±6.05,-12.97±7.61,-12.39±8.23) and the data presented highly significantly difference(all P (0.01). Compared with model group at each tested time point, the weight variation ratio of MAG in high dose (20mg/kg) group were just significantly increased (-5.34±10.30,-5.06±8.34) at4th and6th day (all P (0.05). Although statistically not significant, there was a general uptrend to body weight in the low and middle groups(5-.1Omg/kg), and the positive control group (6mg/kg) at each tested time point(2th、4th、6th and8th day) after surgery.②Postural reflex test:The limb function of rats in the sham-operated group was normal and scores0. Compared with sham-operated group(0±0) at each tested time point (2th,4th、6th and8th day), the model group were significantly increased (1.90+0.13,1.71±0.32,1.74±0.22,1.41±0.43) on the postural reflex test neurological score, and the data presented highly significantly difference (all P<0.01). Compared with model group, the rats treated with MAG (5mg/kg) showed no improvement on the limb abnormality after MCAO (all P>0.05). The rats treated with MAG (10、20mg/kg) showed significant improvement on the limb abnormality after MCAO (all P<0.01) and positive group treated with edaravone (6mg/kg) improved only at2th and6th day.③limb use asymmetry test:On limb use asymmetry test score, compared with sham-operated group(18.08±17.50,0±23.98,5±27.76,13.46+21.25) at each tested time point (2th、4th、6th and8th day), the model group were significantly increased (66.43±32.78,69.29±23.69,50.36±17.81,51.79±40.03) on the asymmetry test score, and the data presented highly significantly difference(P=0.000). Compared with the model group at each tested time point (2th、4tth、6th and8th day), only MAG high dose (20mg/kg) group were significantly decreased(37.22±10.34,25130.52,8.33±35.27,14.44±29.84)? and the data presented highly significantly difference(all P<0.01). The rats treated with MAG (5、10mg/kg) and edaravone (6mg/kg) showed improvement on the asymmetry test score after MCAO, but still significant difference at2th and8th day (all P<0.01).5. Effect of MAG on brain SOD, GSH and MDA levels after cerebral ischemia-reperfusionThe SOD activities in the ischemic hemisphere were significantly decreased (P=0.007) in ischemic model group as compared with the sham-operated group. Treatment with MAG (5mg/kg,10mg/kg and20mg/kg) significantly increased the SOD levels (P=0.044, P=0.009, f=0.001) compared with ischemic control group. Treatment with edaravone (6mg/kg/i.v.) significantly reversed the decreased SOD levels (P=0.012) as compared with the ischemic group.The GSH levels in the ischemic hemisphere were significantly decreased (P=0.000) in ischemic model group as compared with the sham-operated group. Treatment with MAG (20mg/kg) significantly increased the GSH content (P=0.002) compared with ischemic control group, but middle and low group (5mg/kg and10mg/kg)show no significant difference (P=0.994, P=0.171). Treatment with edaravone (6mg/kg) significantly reversed the decreased GSH levels (P=0.001) as compared with the ischemic group.Treatment with MAG (5mg/kg,10mg/kg and20mg/kg) significantly increased the GSH content (P=0.000, p=0.000) compared with ischemic control group. Treatment with edaravone (6mg/kg/i.v.) significantly reversed the decreased GSH levels (P=0.000) as compared with the ischemic group.The content of brain MDA in the ischemic hemisphere was significantly increased (P=0.000) in ischemic model group as compared with the sham-operated group. Treatment with MAG (5mg/kg, lOmg/kg and20mg/kg) significantly decrease the MDA content(P=0.000, P=0.000, P=0.000) compared with ischemic control group, indicating inhibition of lipid peroxidation after cerebral ischemia reperfusion. Treatment with edaravone (6mg/kg/i.v.) significantly decreased MDA content (P-0.000) as compared with the ischemic group.6. Effects of MAG on expression of TNF-a mRNA, IL-1β mRNA in cerebral ischemia/reperfusion injury rat brain tissueRT-PCR analysis showed that the expressions of IL-1β, TNF-a mRNA were seldom in brain of sham groups and all increased in brain of cerebral ischemia/reperfusion rat. Compared to the sham operation group, expression of TNF-a and IL-1β mRNA in ischemic brain tissue homogenate all increased after2h cerebral ischemia followed by24h reperfusion(P=0.004, P=0.000). Significantly, treatment with MGA (5mg/kg,10mg/kg and20mg/kg) dose-dependently decreased expression of IL-1β mRNA in tissue homogenate in comparison to the ischemic model grouptP=0.000, P=0.000, P=0.000). Meanwhile, treatment with MGA (5mg/kg,10mg/kg and20mg/kg) significantly decreased the mRNA expressions of TNF-a. Treatment with edaravone (6mg/kg/i.v.) significantly decreased the expressions of IL-1β and TNF-a mRNA as compared with the ischemic group (P=0.001, P=0.000). ELISA experiments were next conformed the results at protein levels.7. Effect of MAG on the morphology of PC12cell exposed to OGD injuryWhen cultured after3days, rat pheochromocytoma (PC12) cells in normal medium group, showed fine growing, well wall-adherented with lucent endochylema. The hallmarks of differentiation into neuronal-like cells are triangular or spindle-shaped cell body followed by outgrowth of multiple neurites from the cell body with multiple branches. The photographs of rat PC12cell that were exposed to oxygen-glucose deprivation showed obvious cell body damage, shrinkage, and cellular debris. In contrast, the cells treated with MAG (5μM,10μM and20μM) or edaravone (10μM) were much better preserved and did not show damage like that of model group. Thus, MAG appears to have a protective effect against oxygen-glucose deprivation-induced injury as well as edaravone. 8. Effect of MAG on the Cellular survival rate and LDH efflux of PC12cell exposed to OGD injuryThe three dosage groups of MAG (5μM,10μM,20μM) and positive control group (edaravone,10μM) showed a clear neuroprotective effect against oxygen-glucose deprivation induced ischemic injury. Compared with the normal control group, cellular survival rate in the model group significantly decreased (P=0.000). Moreover, we observed that, compared with model group, The three dosage groups of MAG increased cell survival rate (P<0.01), and more the MAG concentration, higher the cell survival rate. Compared with the normal control group, the level of LDH in the model group significantly increased (P<0.01). MAG (5μM,10μM,20μM) significantly decreased the LDH efflux (P<0.01), and more the MAG concentration, lower the level of LDH. Edaravone-treated group significantly significantly enhanced the cell viability (P<0.01) and reduced the level of LDH (P<0.01) as compared with the model group.ConclusionIn conclusion, MAG could increase the level of SOD and the content of GSH, decrease the content of MDA in brain tissue, which demonstrated its activity of scavenging free-radical and ability of attenuating lipid peroxidation damage; MAG could decrease the expression of IL-1β mRNA and TNF-a mRNA in brain tissue, which suppressed cerebral ischemia-reperfusion-induced inflammation.These results were next confirmed by ELISA at protein levels; meanwhile, MAG treatment significantly enhanced the cell viability and reduced the levels of LDH. Thus, MAG could ameliorate morphological changes, decrease the cerebral infarction size, attenuate tissue edema and improve neurological recovery, which could be of the important mechanisms of effective therapy effect after focal cerebral ischemia. |
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