Overexpression Of Mitofusin2 Decreased The Reactive Astrocytes Proliferation In Vitro Induced By Oxygen-glucose Deprivation/reoxygenation

Objective: Glia scar is a hallmark of late-stage brain stroke disease and a noteworthy risk factor for axonal regeneration and neuronal repair. Mitofusin2(Mfn2), known as hyperplasia suppressor gene, suppresses cell proliferation both in vitro and in vivo. This study is to elucidate the role of Mfn2 in reactive astrocytes induced by an in vitro oxygen-glucose deprivation/reoxygenation(OGD/R) model.Methods: The experiment was divided into OGD6h/R 0h group, OGD6h/R 6h group, OGD6h/R 12 h group and OGD6h/R 24 h group. At OGD6h/R 12 h the experiment was divided into Adv-GFP group, Adv-Mfn2 group and control group. Western blot analysis was applied to detect the expression of phosphorylation of Ras-Raf-ERK1/2、GFAP、PCNA、Cyclin D1 and Mfn2 in astrocytes subjected to OGD/R. The effect of mfn2 on proliferation of astrocytes was observed by Edu staining, the cell cycle was tested by flow cytometry. The changes of mfn2 gene were monitored by RT-PCR.Results: The expression of GFAP was consistently increased after the astrocytes subjected to OGD/R. The proteins of Ras-Raf-ERK1/2, CyclinD1 and PCNA increased consistently and reached a plateau at OGD/R12 h. The quiescent cells changed into reactive mitotic cells and the cell-cycle was transited from the G0/G1 phases to S and G2/M phases, with a peak at 12 h. Meanwhile, the expression of Mfn2 both in gene and protein levels was down-regulated. Compared with control groups and Adv-GFP groups subjected to reperfusion 12 h after OGD 6h, most of the astrocytes were blocked in the stage of G0/G1 and few entered into the S phase in Adv-mfn2 groups with Ras-Raf1-ERK1/2 signaling and biomarkers down-regulation.Conclusions: After subjected to OGD/R stimulation, the quiescent astrocytes changed into reactive mitotic cells, with the proteins of Ras-Raf-ERK1/2 up-regulation and the cell-cycle transition and both of mfn2 gene and protein down-regulation. Overexpression of Mfn2 can actually inhibit the proliferation of reactive astrogliosis via down-regulation the Ras-Raf-ERK1/2 pathway and cell-cycle arrest, lest the glia scar formation. These observations indicate that Mfn2 might be a potential novel hyperplasia suppressor gene for reactive gliosis and possibly an important therapeutic target for the treatment of glia scar.