Fty720 Induced Rat Spinal Cord Astrocytes Apoptosis Mechanism Research

Part I Optimized protocols for identification and primary culturesof spinal cord astrocyteObjective: This research was to establish optimized protocols for isolation andprimary cultures of spinal cord astrocytes.METHODS：Primary astrocytes cultures were prepared from1–2-day-old SD ratpups. Spinal cords were dissected under sterile conditions and the meninges wereremoved. Spinal cords were dissociated in0.25%trypsin for5to10min at37°C. Thecell suspension was centrifuged at1500rpm for10minutes at4℃.Cells were filteredthrough a200μm mesh cell dissociation sieve, and plated at a density of5.0×106cellsper25cm flask in Dulbecco’s modified Eagle’s medium supplemented with10%fetal bovine serum,100IU/ml penicillin, and100μg/ml streptomycin. Culturemedium was replaced48hours later and then changed every2～3days.Results: Before experiments, Immunocytochemistry showed that over95%of thecells stained positively for the astrocytic marker glial fibrillary acid protein.Conclusions: The method of culture spinal cord astrocytes is simple and feasible.These highly purified cells can be used for the study of spinal cord cell biology. Part Ⅱ FTY720induces apoptosis in rat spinal cord astrocytes viaJNK and Mitochondria pathwayObjective: To observe the effect of FTY720on spinal cord astrocytes cell viability,apoptosis and its possible mechanism.Methods: After rat spinal cord astrocytes were cultured and treated with increasingconcentrations of FTY720, a Cell Counting Kit-8(CCK-8) assay was used to evaluatecellular viability. Hoechest33342staining was used to observe the morphology ofastrocytes nuclei after FTY720treatment. Astrocyte apoptosis was observed by flowcytometry and the expression of Cleaved caspase3、caspase-9、P-JNK、Cyt c、Bax and Bcl-2were detected by Western blot.Results:(1) The CCK-8assay indicated that FTY720could inhibit the viability ofastrocytes，the mean effective dose (ED50) was10μM. Astrocyte nuclei werefragmented after FTY720treatment. Flow cytometry showed that the apoptosis rateincreased in a dose-dependent manner.(2) Pro-apoptotic proteins, such as cleavedcaspase3、P-JNK、Cyt c、Bax were up-regulated after FTY720treatment. Incontrast, anti-apoptotic protein Bcl-2was down-regulated after FTY720treatment.(3)Pretreatment with SP600125could significantly prevent FY720-induced cell viabilityloss and apoptosis.(4). Pretreatment with SP600125could significantly decreaseFY720-induced Cleaved-caspase-3protein levels.(5) Pretreatment with SP600125could significantly decrease FY720-induced P-JNK，Cyt c and Bax protein levelsand inhibit Cyt c release from the mitochondria.Conclusion: FTY720cloud inhibits spinal astrocytes cell viability via apoptosis.These results suggest that the JNK and mitochondria pathway may be involved in theFTY720-induced apoptosis of astrocytes.