| Objective: To evaluate the potential neuroprotective effect and the underlyingmechanism of Kaempferide-7-O-(4″-O-acetylrhamnosyl)-3-O-rutinoside (A-F-B)against cerebral I/R injury.Methods:150adult male Wistar rats were randomly divided into six groupswith20rats in each group:(1) Sham-operated group treated with equal volumes of0.5%sodium carboxymethyl cellulose;(2) Model group treated with equal volumesof0.5%sodium carboxymethyl cellulose;(3–5) A-F-B groups treated with A-F-B atdoses of12.5,25,50mg/kg;(6) Positive drug group treated with FlunarizineHydrochloride Tablets (FHT) at dose of2mg/kg. Each group was administratedintragastrically with its medicine once a day for3days.1h after the third dayadministration, rats were then subjected to2h of transient middle cerebral arteryocclusion (MCAO) followed by24h of reperfusion.Neurological deficit was investigated at6and24h of reperfusion. After24h ofreperfusion, rats were sacrificed and infarct volume, histopathological changes,oxidative stress-related biochemical parameters, neuronal apoptosis,apoptosis-related proteins and the expression of inflammatory cytokines were tested.Results:姓Compared with Sham group, Model group rats displayedsignificant neurological deficit at6h and24h of reperfusion (P<0.01). FHT (2mg/kg) or A-F-B (12.5,25and50mg/kg) treatment significantly improved theneurological score compared with the Model group (P <0.01).姓Compared withSham group, a significant infarction was observed at24h after reperfusion in Model group (P <0.01). Under the treatment with FHT (2mg/kg) or A-F-B (25and50mg/kg), the infarct volume was significantly smaller than that in Model group (P <0.01, P <0.05). Compared with Sham groups, most of the neurons in the Modelgroups of cortex neurons and hippocampal neurons had disappeared. Treatment withFHT (2mg/kg) or A-F-B (25and50mg/kg) significantly attenuated thepathophysiological changes of hippocampal neuronal cells. However, treatment withFHT or A-F-B had rarely effect on cortex neuronal cells.姓TUNEL-positiveapoptotic cells of cortex and hippocampus were significantly increased in the Modelgroups compared with Sham groups (P <0.01). FHT (2mg/kg) or A-F-B (25and50mg/kg) treatment effectively attenuated the cortex and hippocampus neuronal deathcaused by cerebral IR injury in rats, as indicated by significant increase of grayscalevalue, which indicated fewer apoptotic cells (P <0.01, P <0.05).姓Westernblotting showed that the expression of Bax, cleaved caspase-3and cleaved caspase-9markedly increased while Bcl-2dropped after I/R injury in Model group comparedwith Sham group (P <0.01). However, FHT (2mg/kg) or A-F-B (25and50mg/kg)treatment could remarkably attenuated Bax, cleaved caspase-3and cleavedcaspase-9(P <0.05, P <0.01). While the expression of Bcl-2rose by the treatmentof A-F-B (25and50mg/kg, P <0.05, P <0.01). The protein levels of P-NF-κB p65,ICAM-1, COX-2and iNOS were increased significantly after MCAO comparedwith Sham group (P <0.01). Treatment with FHT (2mg/kg) or A-F-B (12.5,25and50mg/kg) remarkably reduced the protein expression of P-NF-κB p65, ICAM-1andCOX-2(P <0.05, P <0.01). Meanwhile, A-F-B (25and50mg/kg) also inhibitedthe protein level of iNOS (P <0.05, P <0.01). QPCR test showed that after24h of reperfusion, all the values of inflammatory factors elevated significantly inModel groups compared with Sham groups (p <0.01). However, FHT (2mg/kg) orA-F-B (50mg/kg) could significantly inhibit the increase in the mRNA levels ofinterleukin-1β (IL-1β)(P <0.01), and A-F-B (25and50mg/kg) also markedly reduced interleukin-6(IL-6) and tumor necrosis factor-α TNF-α compared withModel group (P <0.05, P <0.01). In blood serum the activities of antioxidantenzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) weresignificantly decreased, while the content of malondiadehyde (MDA) was increasedsignificantly in the Model group compared with Sham group (P <0.05). FHT (2mg/kg) or A-F-B (25and50mg/kg) treatment induced significant elevation of SODand GSH-Px activities compared with the Model group (P <0.05). While A-F-B(12.5,25and50mg/kg) treatment significantly decreased the MDA contentcompared with Model group (P <0.05).Conclusion: The results indicated that A-F-B significantly protected brainagainst cerebral I/R injury in rats by its antioxidant action, anti-inflammatory actionand antiapoptosis. These results in vivo experiments suggest that A-F-B maybecome a promising neuroprotective natural product for the treatment of ischemiccerebrovascular diseases like stroke. |
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