Efefcts Of SARA On Oxygen-glucose Depirvation In PC12Cell Line

Objective To explore the effect of smad anchor for receptor activation (SARA)repression on the sensitivities of nerve growth factor (NGF)-induced differentiatedrattus PC12pheochromocytoma cells to oxygen-glucose deprivation (OGD) injury.And to provide the experimental basis for further understanding the neuronalprotective effect of ActivinA/Smads pathway in ischemic damage.Methods Nerve growth factor (NGF,50ng/ml) was used to induce thedifferentiation of rattus PC12pheochromocytoma cells to nerve-like cells. AfterMicrotubule-associated protein2immunostaining, nerve-like PC12cells wereobserved under the confocal microscopy to identify its neuronal characteristic.Oxygen-glucose deprivation (OGD) model was introduced to those nerve-like PC12cells by hyposulfite of soda for3,6,12and24h, to imitate the pathological processthat nerve cells suffered during the ischemic stroke. Through RNA interferencetechnology, chemically synthesized small interference RNA (siRNA), specificallytargeting SARA gene, was used to pre-repress the expression of SARA, formingexperimental group, negative control group and black control group. The transcriptionand translation of SARA, Smad2and the phosphorylation of Smad2in nerve-likePC12cells was detected by Real-Time RT-PCR and Western blot after24h of siRNAtransfection, before and after3h of OGD treatment. The sensitivities of transfectednerve-like PC12cells to OGD injury for3,6,12,24h were detected by methylthiazolyl tetrazolium（MTT）assay. Cell cycle and apoptosis were measured by flowcytometry after SARA transfection.Results After5days of NGF exposure, PC12cells were successfullydifferentiated into nerve-like cells with MAP2highly expression. After24h of siRNAtransient transfection, the mRNA and protein expressions of SARA were dramaticallydecreased by more than70%compared to that in the negative control group. Therepression of SARA before OGD exposure reduced the expressions of Smad2,3,4mRNA, total and phosphorylated Smad2protein, and increased the sensitivities of nerve-like cells to OGD damage (by80%, more than95%, more than95%,27.1%and40.6%repression respectively), but it did not affect the cell cycle distribution andmRNA expressions of Smad7. OGD injury down-regulated the mRNA and proteinexpressions of SARA in negative control group of nerve-like PC12cells by11%and14%，but increased the mRNA expressions of SARA in experimental group by329.8%, to a level that had no significant difference to that in negative control groupfollowed the same treatment. The expressions of Smad2mRNA, total andphosphorylated Smad2protein in negative control group were increased by15%,150%and20%respectively, after3h of OGD treatment. The expressions of Smad2mRNA were increased in experimental group, total and phosphorylated Smad2proteinwere also increased to the level that had no significant difference to the controls, dueto3h of OGD exposure.Conclusions ActivinA/Smads pathway was activated in nerve-like PC12cells byOGD treatment. The pre-repression of SARA decreased the protective effects ofActivinA/Smads pathway in nerve-like PC12cells after ischemic damage,down-regulating the expressions of Smad2mRNA, total and phosphorylated Smad2protein.