NfκB Is Involved In The Cytotoxic Effect Of Anti-aquaporin-4 Antibody Positive Serum On Astrocytes

Objective: Neuromyelitis optica spectrum disorder(NMOSD)is an inflammatory demyelinating disorder of the central nervous system that primarily attacks the optic nerves and spinal cord.The majority of patients have clinical relapse and lead to blindness and paralysis.To date,no effective treatment for NMOSD has been proven,so it is important to understand the pathological mechanisms of NMOSD and find new therapeutic targets.Binding of NMOSD–specific serum autoantibody,water channel protein-4(AQP4)antibody to AQP4 on astrocytic foot processes,leads to primary astrocyte injury,inflammatory responses through complement dependent cytotoxicity.Complement-dependent cytotoxicity is thought to be the major initiating mechanism in NMOSD.Nuclear factor kappa B(NFκB)signal transducer pathway palys a critical role in the regulation of immune and inflammatory response and has been implicated in the pathogenesis of autoimmune demyelinating diseases such as multiple sclerosis.A recent research suggest that stimulation with AQP4 antibodies activates the canonical NFκB signaling pathway in astroglial cultures,the role of NFκB in the survival and death of astrocyte remains to be identified.This study investigates whether NFκB signal pathway involve the toxic effects of AQP4 antibody to astrocytes by using rat astrocyte cultures stimulated with AQP4-antibody-positive sera of NMOSD patients.Methods: The cerebral cortical purified astrocyte taken from newborn Sprague-Dawley rats were used for experiments.The cultured astrocytes were divided randomly into four groups.The control group,which treated with the healthy sera.The pyrrolidinedithiocarbamic acid(PDTC)group which preincubated with PDTC(10μmol/L)for 1h and then treated with healthy sera,The antibody positive group,which treated with the AQP4 antibody positive sera.The PDTC-pretreated group which exposed to AQP4 antibody positive sera after PDTC preincubating.After 12h’cultivation,astrocyte viability was tested by MTT assay.The nuclear translocation of NFκB p65 was detected by immunofluorescense staining.The protein expression of NFκB p65 and phosphorylated p65(p-p65)were assayed by western blot.Results:1 Identification of subcultured astrocytes The culture purity was assessed by immunofluorescence detection with mouse anti-gial fibrillary acidic protein(anti-GFAP)antibody.The result showed that 95% of cells were astrocytes;2 AQP4 antibody positive sera reduced the cellular viability of astrocytes,and NFκB inhibitor-PDTC protects astrocytes against treatment of AQP4 antibody positive sera To examine the effects of AQP4 antibody positive sera on astrocytes and confirm whether PDTC protected astrocytes from AQP4 antibody positive sera exposure,celluar viability was measured by MTS.The MTS results showed that the optical density(OD)value in the AQP4 antibody positive sera group(0.3897±0.045)was significantly decreased compared with that of the control group(0.5411±0.017)(P<0.05).The optical density value in the PDTC –pretreated group(0.4380±0.005)was obviously increased compared with that of the antibody positive group(P<0.05).There was not statistical difference between the control group and the PDTC group(0.5387±0.0140)(P>0.05);3 AQP4 antibody-positive sera causes nuclear translocation of NFκB To assess the effect of AQP4 antibody positive sera on astrocytic NFκB activity,we detected the nuclear translocation of NFκB p65 by immuofluorescence staining with antibodies to p65.No nuclear translocation of NFκB p65 were found in the control group and the PDTC group.The AQP4 antibody-positive sera could induce the translocation of NFκB p65 from the cytosol into the nucleus.,which was obviously inhibited by the pretreatment with PDTC;4 AQP4 antibody-positive sera induced the expression of NFκB p-p65 Western blot analysis revealed that no statistical difference of the expression levels of NFκB p65 among the control group(0.8029±0.045),the PDTC group(0.7880±0.047),the antibody positive group(0.7821±0.059)and the PDTC pretreated group(0.7093±0.044)(P>0.05).Howere,the p-p65 expression in antibody positive group(0.8197±0.027)strongly increased compared with control group(0.7439±0.032)(P<0.05).And PDTC pretreated group decreased the expression of p-p65(0.7027±0.020)compared with the antibody positive group(P<0.05),but had no statistical difference with the control group(P>0.05).Conclusions:1 AQP4 antibody-positive sera could induce astrocyte injury by activating NFκB signaling passway and inhibiting activity of NFκB protected astrocytes against the cytotoxic effect of AQP4 antibody positive serum.2 NFκB signal pathway may be involved in the pathogenesis of NMOSD.