The Influence Of Interference Of Small Molecular Protein Sulfiredoxin-1 To The Rat Cortical Astrocytes Oxidation Stress Damage

Background:The disease, disability and mortality rate of stroke and other brain pathogenesis of ischemic cerebrovascular is very high, which causing a serious burden to the society and family. Brain ischemia reperfusion produces oxidative stress, and once oxidative stress occurs, it will cause the scavenging capacity of imbalance of endogenous antioxidant defense system. Astrocytes injury plays an important role in ischemia disease. Sulfiredoxin-1 (Srxnl), a central endogenous antioxidant protein, has important neuroprotective functions in cerebral ischemia, which may be related to the antioxidant, anti-apoptosis and anti-inflammatory effects.The present study was designed to use lentiviral vector interference of Srxnl to preliminary explore the influence of interference of Srxnl to the rat cortical astrocytes oxidation stress, which reduced by oxygen-glucose deprivation (OGD) in vitro.Objective:To determine the influence of interference of Srxnl to the rat cortical astrocytes oxidation stress, which reduced by oxygen-glucose deprivation (OGD) in vitro.Methods:(1) Astrocytes were exposed to the different times of oxygen-glucose deprivation (OGD) (0h,2 h,4h,6 h,8 h), and then oxygen-glucose reoxygenation for 24 h. We chosen the optimal exposure time of OGD as determined by MTS and LDH assays.(2) Building three interfere lentiviral vector-encoding short hairpin RNAs (shRNAs) of Srxnl (shRNA-Srxnl-LV3 298, shRNA-Srxnl-LV3 385, shRNA-Srxnl-LV3 456) and one negative control (NC) shRNA. We transient transfected them to astrocytes separately. And then we observed the effiency of cell transfection under the inverted fluorescence microscope. And Srxnl shRNA knockdown efficiency was confirmed by Western blot (WB) and real-time quantitative PCR (QPCR).(3) We exposed transfected cells to the optimal exposure time of OGD to model oxidative stress injury. Then using MTS and LDH assays to detect cell viability and damage.(4) Buliding the complete oxidative stress model of Srxnl interference, and using WB and QPCR to measure the protein and the mRNA level of of Srxnl. Results: (1) The results of MTS and LDH showed that OGD exposure for 6 h, oxygen-glucose reoxygenation for 24 h is the optimal oxidative stress model.(2) The inverted fluorescence microscope showed that interference efficiency of Srxnl could reach about 85%.(3) Western blot analysis and real-time quantitative PCR showed that the interference efficiency of shRNA-Srxnl-LV3 456 was significant (p<0.01), compared with the control groups.(4) MTS and LDH assays showed that interference Srxnl attenuated cell viability and increased cell damage (p<0.05,p<0.01), compared with the control groups.Conclusion:These results suggested that Srxnl can protect primary rat cortical astrocytes from oxidative stress damage induced by OGD.