The Neuroprotective Mechanism Of IGF-2 In Neuromyelitis Optica Spectrum Disorder

Research background:Neuromyelitis optica spectrum disorder(NMOSD)is an immune system mediated central nervous system(CNS)disease.The discovery of aquaporin 4(AQP4)antibody has classified NMOSD from an autoimmune demyelinating disease to an autoimmune astrocyte disease.AQP4 is a kind of aquaporin driving two-way permeability on cell membrane,which is highly expressed in perivascular,astrocyte foot process and ependymal cells.Neuropathology,animal experiments,and clinical studies have provided a wealth of evidence to prove that AQP4 antibodies(AQP4 Ig G)are involved in the pathogenesis of NMOSD,and more in-depth mechanisms still need to be explored.High throughput proteomics sequencing is an objective and effective technical means to quantify proteins.It can deeply understand the disease development process from the downstream small molecule level,and has research significance in protein localization,post-translational modification,protein protein interaction(PPI),and individual specific physiological or pathological changes.Proteomics can identify all the proteins of an organism in a certain period of time,which may be most of the genetic information of cells,tissues or organs or even a kind of organism.It can not only provide hypotheses for clinical research,but also explain the biological changes of related diseases.Its function is not only to discover proteins,but also to describe the molecular mechanisms involved in the occurrence and development of diseases based on the disease background and the effective driving proteins involved.Insulin like growth factor 2(IGF-2)is a key protein that regulates growth during embryonic development,with a large and complex structure and regulatory mechanism.After the birth of mice,the expression of IGF-2 in all tissues decreased rapidly,but it was expressed sustainably in the brain,especially in the hippocampus.Studies have shown that IGF-2 is involved in brain memory,learning processes,and neuronal plasticity regulation,regulating neuronal survival by binding to related receptors to inhibit cell apoptosis.It plays a neuroprotective role in many nervous system disease,such as multiple sclerosis(MS),Parkinson’s disease(PD)and Alzheimer’s disease(AD).In this study,proteomics sequencing of NMOSD patients and normal controls to screen significant differential proteins,constructed in vivo and in vitro experimental models to verify the function of differential protein IGF-2,and explored the utility mechanism of IGF-2 for NMOSD.It provides a theoretical basis and research basis for the exploration of the pathogenesis and treatment means of NMOSD.Research methods:1.By collecting serum samples from clinical patients,data independent acquisition(DIA)quantitative technology combined with high performance liquid chromatography tandem mass spectrometry(LC-MS/MS)was used to screen the differential proteins between groups.Western blot(WB)was used to verify the differential proteins after screening and analysis.2.Establish the model of astrocytes injured by AQP4-Ig G,and detect the expression of astrocyte survival and apoptosis-related factors by immunofluorescence,real-time quantitative polymerase chain reaction(RT-q PCR)and WB.Overexpression of IGF-2 in astrocytes and administration of insulin-like growth factor 1 receptor(IGF-1R)inhibitor podophyllotoxin(PPP)were verified again to explore the effect of IGF-2 on the downstream pathway.3.The NMO animal model was constructed by intracerebral injection of AQP4-Ig G and human complement(h C),and the changes of body weight and XVI motor score of mice were observed.The brain and spinal cord tissues of mice were extracted to make sections for immunofluorescence detection of related protein expression.The total RNA and total protein in the brain and spinal cord were extracted and the expression of related factors was verified by RT-q PCR and WB.4.The changes of Glu content in the brain and spinal cord of NMO mice were detected by A340 nm spectrophotometry.Cellular immunofluorescence assay was used to detect the expression of excitatory amino acid transporter 2(EAAT2)in cells and explore the effect of IGF-2 on Glu homeostasis.Results:1.The differential proteins IGF-2,IGFBP-3 and IGFBP-6 were obtained by serum proteomics analysis.Compared with the normal control group,the expression of IGF-2 in NMOSD patients decreased significantly,and the expression of IGFBP-3 and IGFBP-6 increased(p<0.05),the fold change of IGF-2 was positively correlated with the patient EDSS score;After RTX treatment,the expression of IGF-2 increased compared with that of NMOSD patients,and the expression of IGFBP-3 and IGFBP-6 decreased(p<0.05).2.AQP4-Ig G/Con-Ig G was successfully extracted from the serum of NMOSD patients and normal control group,and the model of AQP4-Ig G astrocyte injury in vitro was constructed.After AQP4-Ig G intervention,the expression of IGF-2 and IGF-1R in astrocytes decreased,PI3K/AKT signal pathway was inhibited,and astrocyte apoptosis and the release of inflammatory factors were promoted(p<0.05).However,after overexpression of IGF-2 in astrocytes,the expression of IGF-1R increased,PI3K/AKT signal pathway was activated,and the expression of apoptotic protein and the release of inflammatory related factors were decreased.The above changes were inhibited after administration of IGF-1R inhibitor PPP.3.The NMO mouse model in vivo was established by injecting AQP4-Ig G and h C into the brain of mice.During the experiment,the weight of mice decreased significantly and the motor function was impaired(p<0.05).After the mice were injected with adenovirus containing IGF-2 into the brain before modeling,and then injected with AQP4-Ig G and h C,the changes of body weight and motor function were not significantly different from those of the control group(p>0.05),while the body weight of the positive control mice treated with RTX after modeling was not significantly changed from that before treatment,but the motor function was recovered from that before treatment(p>0.05).4.AQP4 Ig G can damage the Glu homeostasis of astrocyte,and the content of Glu in cell supernatant and cell increased significantly(p<0.01).After overexpression of IGF-2,AQP4 Ig G reduced the Glu content in the supernatant of astrocyte,and the ability of cells to ingest Glu recovered compared with the model group(p<0.05).The content of Glu in astrocyte overexpressing IGF-2 and adding PPP was higher than that in IGF-2 group.IGF-2 reduces Glu content in the brain and spinal cord of NMO mice(p<0.01).Conclusions:1.The serum IGF-2 levels in NMOSD patients were significantly reduced,which is related to the severity of clinical symptoms.2.IGF-2 activates the IGF-1R/PI3K/AKT signaling pathway,inhibits the release of pro-inflammatory factors,reduces central nervous system inflammation and myelin loss,and prevents motor function damage in NMO mice.3.IGF-2 reduces the accumulation of excessive glutamate in the central nervous system,and has protective effects such as improving cell activity and reducing neurotoxicity.