Role Of Mitofusin-2 In Reactive Astrocytes Proliferation In Vitro And In Vivo

Objectives: Reactive astrocytes become activated in response to spinal cord injury,and reactive astrogliosis is considered an hindrance to axonal regeneration and functional recovery by forming glial scar.Mitofusin 2(Mfn2),which is known as a crucial gene of hyperplasia suppressor,has been reported to negatively regulate cell proliferation.The present study aimed at exploring the role of Mitofusin-2 in reactive astrocytes proliferation in vitro and in vivo.Methods: Part 1: The experiment was divided randomly into scratched 0h group,scratched 6h group,scratched 12 h group,scratched 24 h group and scratched 48 h group.At scratched 24 h,the experiment was divided into Adv-GFP group,Adv-GFP-Mfn2 group and control group.Cell immunofluorescence staining was made to observe the morphology change of GFAP.Western-blot analysis was applied to detect the expression of Ras-Raf-ERK1/2、GFAP、PCNA、Cyclin D1 and Mfn2 in astrocytes.Part 2: Adult famale SD rats(250–300 g)were divided into three groups(n = 10): sham-operated SCI group(n = 24)and two SCI group treated with either Adv-GFP(n = 24)or Adv-Mfn2-GFP(n = 24).The animals were killed at 3 days after SCI(n =10).Based on Western Blotting,immunocytochemical and immunofluorescene analyses,we will detect the tranfection of recombinant adenovirus and quantify the relationship of overexpression of Mfn2 and GFAP and CSPGs in spinal cord tissue in both experimental groups.Result:Part 1: The GFAP expression of astrocytes increasing after being scratched,while the expression of Mfn2 was down-regulated.The proteins of Ras-Raf-ERK1/2,Cyclin D1 and PCNA increased consistently and reached a plateau at 24 h.Adv-Mfn2-GFP group overexpressed Mfn2,compared with control groups and Adv-GFP,while the expression of GFAP,Ras-Raf-ERK1/2,Cyclin D1 and PCNA.Part 2: Western-blot analysis and immunofluorescene analyse showed that Mfn2 expression increased after Adv-Mfn2-GFP infection.Immunocytochemical and immunofluorescene analyses revealed that Ad-Mfn2-GFP transfection can increase Mfn2 expression,which may decrease the expression of GFAP and CSPGs.Conclusion:After scratched stimulation,Mfn2 expression was down-regulated.Overexpression of Mfn2 can actually inhibit the proliferation of reactive astrogliosis via down-regulation the Ras-Raf-ERK1/2 pathway and cell-cycle proliferation factort.Overexpressing Mfn2 may have an effect that suppressing reactive astrogliosis proliferation in rat.In a word,anti-proliferative effect of Mfn2 may serve as a promising therapeutic intervention in CNS diseases with excessive astrogliosis.