| ObjectiveTo investigate the protective effect and mechanism of monosialotetrahexosylgangliosideSodium （GM-1） on focal brain ischemia model produced by middle cerebral arteryocclusion. H2O2induced human umbilical vein endothelial cells（HUVEC）lesionswere also adapt to evaluate the protective effects and machnism of GM-1.MethodAnimal models and cultured cells methods were both used to complete thisresearch.1. Animal ModelsThe Wistar rats were divided into sham group, brain ischemia (model) group andGM-1treated group, in which group there are ten rats at least. The right middlecerebral artery of the rats in brain ischemia and GM-1treated group were oclusivedby nylon thread. The rats in GM-1treated group were treated with10mg/kg GM-1twice by intraperitoneal injection for7days while that in sham and brain ischemiagroups were treated only with1ml physiological salin. The rats in each group wereassessed by modified NSS standard everyday till being sacrificed. The brain wasobtained after eight days, and hematoxylin and eosin stain and immunohistochemistrywere used to evaluate the degree of ischemic lesions.2. Cell CultureThe HUVEC were divided into five groups, those are control group, H2O2treatedgroup, GM-1high, middle and low dose (10,5,1mg/l) group. The ability of cellproliferation was detected by CCK-8kit, and the cell cycle was analyzed by flowcytometry, total protein concentration were determined by Bradford method, theprotein expression of NF-κb was determined by immunofluorescence and western blotassay. Then the HUVEC were divided into nine groups, those are control group, H2O2treated group, CHIR-99021and LY294002treatment group with and without H2O2.The Western blot and realtime PCR were apllied to detect the protein and mRNAlevel of PI3K, Akt and GSK-3.Results1. Protect effects of GM-1on MCAO induced brain ischemia lesions in rats1.1Comparison of the general stateand nerve assessment of the rats invarious groupsThe rats in sham group had good spirits and normal activities while those in brainischemia group got poor mental state which had various degree activity reduceing andbehavioral disorder. Compared to that in brain ischemia group, the rats in GM-1treated group got similar symptoms at first three days and from the4thday, thesymptoms of cerebral ischemia significantly reduced (P<0.01) compared to the modelgroup.1.2HE staining of the rats’brain in various groupsThe shape and number of the nerve cells, glial cells and endoethial cells hhad noabnormal appearance. The main histological changes of the rats in brain ischemicgroup were neuronal degeneration, necrosis and glial cell reaction to different extent.The vascular of endothelial cells also increased in the brain ischemic group.1.3Immmunohistochemistry results of CD31stainingThe CD31positive signal were scattered or arranged into strips cords in the braintissue of the rats in the sham group. Compared with Sham group, the expression ofCD31increased significantly in ischemic area around the rats in the model group(P<0.01); and compared with the model group, the CD31signal in the GM-1groupincreased significantly (P<0.01).2. Effects of GM-1on the PI3K/GSK-3pathway2.1PI3K stainingCompared with the sham group, the PI3K positive signal decreaded significantly(P<0.05), which mainly expressed in vascular endothelial cells and glial cells.Compared with the brain ischemic group, the PI3K expression increased significantly(P<0.01). 2.2GSK-3stainingThe GSK-3positive signal mainly expressed in vascular endothelial cells andglial cells. In normal nerve cells, there were wake expression of the GSK-3.Compared with the sham group, the GSK-3positive signal increaded significantly(P<0.05); compared with the brain ischemic group, the GSK-3expression decreasedsignificantly (P<0.01).。3. H2O2and vascular endothelial cells of oxidized damage3.1The results of CCK-8The cell proliferation ability in the control group was higher than that in the H2O2treated group (P<0.001); the cell proliferation ability in the GM-1high, medium dosegroup was higher than that in the H2O2treated group, the difference was significant(P<0.001or P<0.05), and compared with the H2O2treated group, the difference of theproliferation ability in the GM-1low dose group is not significant.3.2The results of cytometryThe50.87%cells in the control group were in the G2/M phase and S phase,compared with that in the H2O2treated group (which is13.52%), the difference wassignificant (P<0.0001). The33.95%,30.70%and28.68%cells in the GM-1high,middle and low dose group were in G2/M phase and S phase, compared with the H2O2treated group, the difference was also significant (P<0.001or0.01).4. PI3K/GSK-3pathway and vascular endothelial cells of oxidized damage4.1The western blot results of PI3K and GSK-3The PI3K expression in the H2O2treated group was decreased significantlycompared with the centrol group (P<0.01); compared with the H2O2treated group,that in the high and low dose GM-1treated group increased significantly (P<0.05).Vsthe control group, the GSK-3expression were increased significantly (P<0.01). TheGSK-3expression in the GM-1group increased with the concentration of GM-1increasing, that in the high and low dose of GM-1treated group were increasedsignificantly (P<0.05) compared with the H2O2treated group.Vs the H2O2treated group, the PI3K, p-Akt and NF-κb in the nuclear expressionin the CHIR-99021and CHIR-99021+H2O2group were dencreased significantly(P<0.01) while the GSK-3expression was increased significantly (P<0.01); the PI3Kand p-GSK-3expression in the LY294002+H2O2group decresed significantly(P<0.01);the p-Akt expression in both groups above increased significantly (P<0.01). 4.2The results of real time PCRAfter the PI3K inhibitor (LY294002) was applied in the HUVEC damaged byH2O2, the expression of protein and mRNA of PI3K further decreased, thephosphorylation level of protein expression of Akt decreased, the protein expressionof GSK-3increased, the changes of the mRNA expression of Akt and GSK-3werenot obvious. After the GSK-3inhibitor (CHIR-99021) was applied in the HUVEC, theprotein and mRNA expression of GSK-3was decreased, and the changes of proteinand mRNA expression of PI3K and Akt were not obvious. After damaged by H2O2,the protein and mRNA expression of GSK-3decreased. While the changes proteinand mRNA expression of PI3K and Akt were not significant. After damaged by H2O2,the protein and mRNA expression of GSK-3were similar to those of CHIR-99021.5. NF-κb and vascular endothelial cells of oxidized damage5.1The western blot results of NF-κbThere was no significant difference to the NF-κb expression in the cytoplasm ofall cell culture groups while that in the nuclear showed difference significantly. In theH2O2treated group, the expression ratio of NF-κb in nuclear to cytoplasm is12.07±2.96，compared with the control group, the difference showed statisticalsignificance (P<0.05), while compared with H2O2treated group, the expression ratioof NF-κb in nuclear to cytoplasm in high and middle dose of GM-1treated groupwere increased significantly (P<0.01).5.2The double influoresence staing results of NF-κbThe immune-influoresence staing results showed that the NF-κb could expressedin both cytoplasm and nuclear, but the expression ratio of NF-κb in nuclear tocytoplasm showed statistical significance in various group when analyzed by ImagJ.The expression ratio of NF-κb in nuclear to cytoplasm in the high concentration ofGM-1treated group were increased significantly (P<0.05) compared with that ofH2O2treated group.Conclusion1. The GM-1has protective effects on brain ischemic lesion in which thevascular endoethial cells may play important roles.2. GM-1can delay the oxidative damage of intravascular cells, accelerate therepair, stimulate the proliferation, its mechanism may be related to PI3K-GSK signalpathway and NF-κb signal pathway by regulated with GM-1. Innovation point1. There were few reports about the protective effects of GM-1on vascularendothelial cells of oxidative damage.2. It is the first time to inform the mechanism of GM-1on the PI3K/GSK-3pathway and NF-κb. |
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