The Mechanism Of Sulfiredoxin-1on Oxidative Injury In PC12Cells Via Nrf2/ARE Pathway

Cerebral ischemic diseases，such as stroke have high morbidity and,place high socioeconomic costs, on society. Oxidative stress occurs due toan imbalance between production of reactive oxygen species （ROS） and thecell’s capacity to neutralize them through its intrinsic antioxidant defenses.It has been suggested that small redox protein, sulfiredoxin1, mayhave neuroprotective function. Over-expression of Srxn1can protect thecortical neurons, and, astrocytes from oxidative damage caused by peroxide.Srxn1expression is lower in the lungs of patients with COPD than in thosewith nonemphysematous lungs.The present study was designed to explore the protective tendencies ofSrxn1toward H₂O₂-insulted rat pheochromocytoma cells （PC12） and themechanisms involved.Methods:1. Srxn1gene interference plasmid and a negative control plasmidwere transiently transfected into PC12cells by lipofectin2000, thentransfection efficiency of the plasmid vector to PC12cells was observed using a fluorescent microscope, the knock-down efficiency of threedifferent sh-Srxn1plasmid vectors in PC12cells were evaluated usingRT-PCR and Western blot analysis while β-actin as a reference.2. In our study, PC12cells were exposed to DMEM containing0,180μmol/L H₂O₂for24hours. Then cell viability was measured by andMTS assay.3. Three groups of cells were cultured by serum-free DMEMcontaining180μmol/L H₂O₂for24hours. Then samples were preparedusing ultrasonication, the maleicdialdehyde （MDA） level were thenassayed.4. Nuclear and cytoplasmic protein extracts wereprepared from each group PC12cells after H₂O₂injured, and theexpression of Nrf2and actin were determined by Western blotrespectively.5. Total RNA and protein were extracted after three groups of wereinduced by H₂O₂, and the expression of HO-1and NQO1in these groupswere determined by semi-quantitative RT-PCR assay.Results1. The Srxn1gene interference plasmid and negative control plasmidwere successfully constructed.Srxn1mRNA and protein expression in theSrxn1knockdown group （PLVT-575） were obviously lower than those innon-transfected group and negative vector transfected group, as examined using RT-PCR and Western blot. But there was no differences betweennon-transfected group and negative vector transfected group （P>0.05）.2. The cell viabilities were decreased in PC12cells after induced byH₂O₂at200μM for24hours markedly, while Srxn1knockdown increasedthe damage.3. MDA content（nmol/mgpro）of the three groups of cells wereincreased with the200μM H₂O₂，MDAcontent in Srxn1knockdown group（5.507±0.29） were higher than in non-transfected group and negativevector group. There was no differences between non-transfected group andnegative vector group （P>0.05）.4. After H₂O₂treatment, We found that Srxn1knockdown couldreduce the protein levels of Nrf2in the nucleus and decrease them in thecytoplasm of H₂O₂-injured PC12cells relative to non-transfected groupand negative control group （P<0.05）.5. After three groups of PC12cells induced by H₂O₂, the protein levelof actin in cytoplasm were increased while decreased in nucleus, Srxn1knockdown could reduce the protein levels of actin in the nucleus anddecrease them in the cytoplasm of H₂O₂-injured PC12cells relative tonon-transfected group and negative control group（P<0.05）.6. After H₂O₂treatment, HO-1mRNA expression were all increasedin non-transfected group （0.729vs.0.458）, negative control group （0.625vs.0.463）and Srxn1knockdown group （0.53vs.0.478）, compared with before H₂O₂treatment，HO-1mRNA expression in Srxn1knockdowngroup was much lower than that in other two groups. There was nodifference between non-transfected group and negative group（P>0.05）.Compared with before H₂O₂injuring, HO-1protein expression were allincreased in non-transfected group （1.107vs.0.63）, negative control group（1.019vs.0.581） and Srxn1low-expression group （0.754vs.0.644）, HO-1protein expression in Srxn1knockdown group was much lower than thatin other two groups （P<0.05）. There was no difference betweennon-transfected group and negative group（P>0.05）.7. After H₂O₂treatment, NQO1mRNA expression were all increasedin Srxn1knockdown group （0.827vs.0.557）, non-transfected group（0.868vs.0.547） and negative control group （0.915vs.0.566）,compared withbefore H₂O₂treatment，NQO1mRNA expression in Srxn1knockdowngroup was much lower than that in other two groups （P<0.05）. There wasno difference between non-transfected group and negative group （P>0.05）.compared with before H₂O₂treatment，NQO1protein expression in Srxn1knockdown group was much lower than that in other two groups （P<0.05）.There was no difference between non-transfected group and negativegroup （P>0.05）.Conclusion:1. Srxn1can protect PC12cells from H₂O₂injuring. Srxn1knockdown can obviously strengthen the oxidative damage in H₂O₂-induced PC12cells, increase the production of MDA. Thus Srxn1knockdown may enhance the injury of cells.2. It is certificated that Srxn1gene protect PC12cells from H₂O₂injuring. This neuroprotection of Srxn1may have been attributable to itsparticipation in the nuclear translocation of Nrf2and activation of theNrf2/ARE pathway nuclear translocation of Nrf2.