Effect And Mechanism Of Nrf2 On The Glial Scar Formation After Spinal Cord Injury In Mice

BackgroundSpinal cord injury is a type of severe injury with complications of general sensation and motor function disorder.With the development of traffic and building industry,its incidence is increasing.Owing to the severe sequelae of nervous dysfunction,spinal cord injury(SCI)patients have poor self-care ability,which brings heavy psychical and economic burden.Current medical technology has limited effect on recovery of nervous function after SCI.Glial scar formation is an important factor influencing axon regeneration and functional recovery after SCI.Currently,the commonly used methods of inhibiting glial scar formation are as follows: inhibiting division growth of astrocyte after SCI;degrading chondroitin sulfate proteoglycan(CSPG)by medicine;inhibiting the expression of CSPG;gene blocking of glial fibrillary acidic protein(GFAP)and Vimentin(Vim);inhibiting glial scar formation by X ray;inhibiting inflammatory reaction after SCI;surgically resecting glial scar.Transcription factor nuclear factor E2-related factor 2(Nrf2)can inhibit inflammation in many diseases,such as autoimmune diseases,asthma,emphysema,rheumatic arthritis,colonitis,atherosclerosis.Nuclear factor-?b(NF-?b)is the main transcription factor regulating inflammation reaction.Nrf2 inhibits inflammation mainly by inhibiting the activation of NF-?b and attenuating expression of inflammatory factor such as matrix metalloproteinases,chemotactic factor and cell adhesion molecule.Nrf2 has neuroprotective effect in central nervous system(CNS),which can have neuroprotective effect on nerve degenerative disease,cerebral infarction,cerebral hemorrhage and craniocerebral trauma after activation.Thus we suppose that Nrf2 can inhibit glial scar information by inhibiting inflammation by regulating NF-?b signaling pathway,and hence we design this experiment.ObjectiveInstitute of Cancer Research(ICR)mice(Nrf2+/+,Nrf2-/-)were used to establish SCI models.Nrf2 reactivator sulforaphane(SFN)and gene knockout were used to regulate Nrf2 positively and negatively,the effect of Nrf2 on NF-?b signaling pathway,glial scar information,recovery of nervous function was observed,and the effect and mechanism of Nrf2 on gliar scar formation after SCI were also investigated.MethodsICR mice(Nrf2+/+,Nrf2-/-)were used to establish SCI models,which were interfered with Nrf2 activator SFN.SFN was diluted by corn oil and then was peritoneally injected after SCI at dose of 5mg/kg/d.It was injected for 7 times.Equal volume corn oil was peritoneally injected in the solvent control group.The experiment was divided into six groups: wild type sham operation group,wild type single injured group,wild type solvent control group,wild type SFN treatment group,Nrf2 knockout sham operation group,Nrf2 knockout injured group.The Basso Mouse Score(BMS)locomotor rating scale was used to evaluate mice's behavioral consequences at 1d,7d,14 d,21d,28 d after injury.Then the mice were sacrificed and the spinal cord tissue samples were harvested to observe the expression of Nrf2,P65,GFAP,VIM and CSPG by immunofluorescence assay at 7d after injury.The protein expression of Nrf2,P65,p-I?B?,GFAP,VIM and CSPG was determined by western blotting at 7d after injury,and GFAP,CSPG and GAP-43 were detected at 28 d after injury.The protein expression of TNF-? and IL-1? was determined by ELISA at 7d after injury.The m RNA expression of TNF-? and IL-1? was determined by PCR at 7d after injury.ResultsThe BMS score in the sham operation group was 9.After SCI,the hind limbs motor function in injured groups showed varied degrees of recovery.After 28 d,the BMS score in the single injured group was 2.17�0.41,which was 2.33�0.52 in the solvent control group,3.33�0.52 in the SFN treatment group,and 1.50�0.54 in the knockout injured group.SFN treatment group improved more remarkably than others(P <0.05).There was significant difference between each group except that between single injured group and solvent control group.Knockout injured group was the lowest than others(P <0.05).At 7d after SCI,astrocyte activation was observed in each SCI group by immunofluorescence,indicating that the expression of Nrf2 was most,P65,GFAP,CSPG and VIM were lowest in SFN treatment group,and the expressions of P65,GFAP,CSPG and VIM were most in knockout injured group.There was no remarkable difference between the single injured group and the solvent control group.At 7d after SCI,western blotting showed that the expressions of p-I?B?,P65,GFAP,VIM and CSPG in the SFN treatment group were decreased compared with other injured groups,but the expression of Nrf2 was increased(P<0.05).The expressions of p-I?B?,P65,GFAP,VIM and CSPG in knockout injured group were increased compared with other injured groups(P<0.05).The expression tendency of GFAP and CSPG at 28 d after SCI was the same as the 7d.At 28 d after SCI,GAP-43 in SFN treatment group was increased(P<0.05),while was decreased in knockout injured group(P<0.05).There was no remarkable difference between single injured group and solvent control group.At 7d after SCI,ELISA showed that the protein expressions of TNF-? and IL-1? were lowest in the SFN treatment group(P<0.05),while was most in the knockout injured group(P<0.05).There was no remarkable difference between the single injured group and the solvent control group.At 7d after SCI,PCR showed that the m RNA expressions of TNF-? and IL-1? were lowest in the SFN treatment group(P<0.05),while most in the knockout injured group(P<0.05).There was no remarkable difference between the single injured group and the solvent control group.ConclusionNrf2 could decrease release of inflammatory factors by inhibiting NF-?b signaling pathway after SCI,alleviate inflammatory reaction,inhibit the expressions of GFAP,VIM and CSPG,inhibit information of glial scar,increase the expression of GAP-43,promote axonal regeneration,and promote recovery of nerve function.