Differential Protein Study Of Protective Effect Of Hedysari Radix Polysaccharides On Brain Microvascular Endothelial Cells Of Oxidative Damage

Objective: To study the protective effect and protein expression spectrum impact of Hedysari Radix Polysaccharides(HRP) on Human brain microvascular endothelial cells(HBMEC) of oxidative damage induced by ox-LDL.Methods: Oxidative damage model of HBMEC were induced by 50 μg/mL ox –LDL. After the oxidative damage model of HBMEC was treated with different concentrations of HRP for 24 h, the cell survival rate was determined by MTT and morphologic changes were observed using phase-contrast microscopy. The total protein of HBMEC cells was extracted using cell lysate after HBMEC cells of oxidative damaged were treated by 20 μg /mL HRP for 24 h. After purification, the proteins were separated by using two-dimensional electrophoretogram(2-DE). After coomassie brilliant blue staining and image scanning, the electrophoretogram of the total protein of HBMEC cells was performed to analyze the differentially expressed proteins of drug group and model group using PD Quest 8.0 software. Mass spectrometry was applied to identify the differentially expressed proteins.Results: 1.The oxidative damage models were successfully estabished by 50μg/mL ox –LDL induced cause;Compared with model group, the cell survival rate and the cell number were significantly increased, and the structure of cells were obviously restore in HRP-treated group(2.5、5、10、20、40μg/mL). 2. The HBMEC cells proteins were purified using acetone precipitation method and separated in the optimal focusing condition, successfully established HBMEC cell protein two-dimensional electrophoresis conditions. 3. The outcomes of the 2-DE mappings with PD Quest 8.0 software are as follows: the protein expression profiling was significantly changed in HRP-treated group(20μg/mL). A total of 11 differentially-expressed protein spots were discovered and 7 protein spots among 11 protein spots were successfully identified by mass spectrometry as Heat shock 70 kDa protein, Glutathione synthetase, Calumenin, 26 S protease regulatory subunit 4, Gamma-enolase,40 S ribosomal protein SA and 78 kDa glucose-regulated protein, including 5 up regulated and 2 down regulated expressed proteins respectively.Conclusion: HRP has obvious protective effect on oxidative damage model of HBMEC induced by ox-LDL, the mechanism was related to endoplasmic reticulum stress, energy metabolism, scavenge free radicals, inhibition of cell apoptosis, and anti-oxidative stress.