Ski Regulates Proliferation And Migration Of Reactive Astrocytes Induced By Lipopolysaccharide （LPS） Through PI3K/AKt Pathway

Purpose: Our research group has previously confirmed that Ski is upregulated in astrocytes after spinal cord injury（SCI）,and knocked out Ski gene with small interfering si RNA.It was found that the physiological activity of reactive astrocytes decreased after Ski knockout.Therefore,we believe that Ski may be a regulatory factor of reactive glial scar formation after SCI.However,the function and exact mechanism of its overexpression in reactive astrocyte proliferation and migration after SCI remain unclear.In this study,we examined the effects of Ski overexpression on proliferation and migration of reactive astrocytes to explore the exact molecular mechanism.Methods: Astrocytes were extracted from cerebral cortex of 1-3 days old Sprague Dawley（SD）Suckling rats and cultured in vitro.The model of reactive astrocytes after spinal cord injury was established after lipopolysaccharide（LPS）induced astrocyte activation.Western bolt and RT-q PCR were used to detect the protein expression and m RNA expression of Ski and proliferation-related proteins PCNA,CDK4 and Cyclin D1 in activated astrocytes from normal and LPS-induced spinal cord injury cell models.The expression of Ski and astrocyte proliferation were detected by immunofluorescence dual staining and Ed U cell proliferation technique.Astrocytes were transfected with Ski-specific lentivirus,and the changes of cell cycle and proliferation activity were detected by flow cytometry,Ed U staining and CCK-8after Ski gene was overexpressed.The effect of Ski overexpression on the migration ability of reactive astrocytes was detected by Ski-specific lentivirus and Transwell assay.Meanwhile,the expression changes of related proteins in PI3K/Akt signaling pathway after Ski overexpression were detected by Western bolt and RT-q PCR,and PI3 K signaling pathway inhibitor LY294002 was further used to inhibit the activation of this signaling pathway.To explore the exact mechanism of Ski overexpression enhancing proliferation and migration of reactive astrocytes.Results: In LPS-induced in vitro astrocyte injury model,the m RNA and protein expression of Ski increased in a time-dependent manner,peaking at 4d.The expression patterns of PCNA,CDK4 and Cyclin D1 proliferative proteins in normal and activated astrocytes were consistent with that of Ski.Overexpression of Ski significantly enhanced proliferation and migration of reactive astrocytes,while inhibition of PI3K/Akt pathway activity significantly reduced the expression levels of Ski and proliferation-related proteins.Conclusion: This suggests that Ski is a novel molecule that regulates astrocyte biological characteristics after SCI and plays an important role in regulating astrocyte proliferation.Ski overexpression can effectively promote proliferation and migration of reactive astrocytes by activating the PI3K/Akt signaling pathway.These results suggest that Ski may be an important therapeutic target for preventing glial scar formation and promoting neurological recovery after SCI.