| Neuromyelitis Optica Spectrum Disease(NMOSD)is a rare autoimmune-mediated inflammatory demyelinating disease of the central nervous system involving the optic nerve and spinal cord,clinically characterized by female prevalence,high recurrence rate and high disability rate.It is also known as astrocyte disease because of its specific pathogenic antibody aqua-porin 4 immunoglobulin G(AQP4-IgG)targeting AQP4 on the astrocyte foot secondary astrocyte damage.Studies have shown that blood brain barrier(BBB)destruction and the activation of immune inflammation play a key role in the pathogenesis of NMOSD,and they directly affect the clinical severity and prognosis of NMOSD patients.The cumulative neurologic disability cau-sed by recurrent recurrence seriously impacts the quality of life.At present,drug options for the treatment of NMOSD are limited,and a considerable number of patients have poor drug response.There are large side effects in long-term application,so it is crucial to develop alternative or complementary treatment strategies.Aminoacyl-tRNA synthetase-interacting multi-functional protein 1,(AIMP1)is a novel pro-inflammatory factor with anti-angiogenic properties.Numerous studies have shown that AIMP1 functions as a multifunctional secreted protein in the activation of various immune cells,the release of proinflammatory factors,and neuronal death.Meanwhile,AIMP1,as a tumor suppressor,can increase the BBB and blood tumor barrier permeability by targeting the endothelium cell(EC)and tight junction(TJ)proteins.miRNA,as one of the most widely studied non-coding RNA,has become a major regulator of inflammation-induced gene expression changes in brain microvascular endothelial cells(BMECs)that constitute BBB.The main pathway through which it performs functions is to inhibit protein expression by inducing mRNA degradation or translation repression of the mRNA by complementary binding to the messenger RNA(mRNA)in the 3’untranslated region(3’UTR).We found that miRNA belonging to the let-7 family was significantly downregulated during inflammation,while overexpression of let7 reduced the secretion of proinflammatory factors and the expression of adhesion molecules in activated endothelial cells,and weakened monocyte adhesion/migration on BBB and ameliorated BBB dysfunction.Based on the above literature background,we found three unresolved questions:1)AIMP1,as a new proinflammatory factor,is significantly increased in the peripheral blood of various autoimmune diseases(such as rheumatoid arthritis,systemic lupus erythematosus,etc.),and inhibiting AIMP1 has potential therapeutic value.However,there are no relevant reports of AIMP1 levels in the peripheral blood of NMOSD patients yet.2)BBB destruction is closely related to the pathogenesis of NMOSD,and the repair of BBB can prevent NMOSD onset.Studies have shown that NMOSD serum can increase BBB permeability in vitro,while the specific molecular mechanism of BBB destruction in NMOSD is still unclear,and the role of AIMP1 with antiangiogenic properties in the regulation of BBB permeability in NMOSD needs to be further explored.3)Studies have shown that miRlet-7f-5p has antiinflammatory,BBB regulation functions,and we found a targeted binding site between the 3’UTR region of AIMP1 and miRlet-7f-5p through starbase software analysis.However,it is unclear that the changes of miRlet-7f-5p expression in BMECs induced by plasma from patient with NMOSD and whether miRlet-7 has a targeted regulatory relationship with AIMP1.Thus,for the above problems and combined with the relevant literature background,we have made three assumptions:1)Secreted AIMP1 protein may be elevated in the peripheral blood of NMOSD and may have some predictive value for the clinical severity of NMOSD;2)The NMOSD circula-ting factors may be involved in BBB permeability regulation in vitro by increasing AIMP1 expression in brain microvascular endothelial cells;3)brain endothelial miRlet7f-5p can target the 3’UTR region of AIMP1 to inhibit the generation of AIMP1 protein,and then alleviate NMOSD-induced BBB dysfunction.Our study aims to explore new therapeutic targets for the destruction of BBB in NMOSD,so as to protect BBB integrity and accelerate the recovery of damaged BBB,and provide new diagnosis and treatment ideas for NMOSD.Part One Clinical correlation analysis of secretory AIMP1 protein and neuromyelitis optica spectrum diseaseObjective:To explore the plasma AIMP1 level and AQP4-IgG+NMOSD patients and their clinical predictive value for disease severity.Methods:1.AQP4-IgG+NMOSD patients who met the inclusion criteria and ageand gender-matched concurrent health examinations were selected as the study subjects.2.General clinical data and laboratory and imaging data were collected from all study subjects,and the disability function of NMOSD patients was assessed by EDSS score.Meanwhile,2-3ml of peripheral venous blood was extracted,centrifuged(3000rpm/min,10-15min),the plasma was separated,frozen at-80℃.3.Plasma AIMP1 protein content was measured using enzyme-linked immunosorbent assay(ELISA)kits.Statistical analysis of the correlation between plasma AIMP1 level and the clinical severity,clinical characteristics,and laboratory indicators in NMOSD patients.Results:1.Plasma AIMP1 level were significantly higher in all NMOSD patients than in HCs(P<0.001).2.In patients with acute AQP4-IgG+NMOSD,the plasma AIMP1 level was significantly higher in the group before IVMP therapy than in the group after IVMP therapy(P<0.001).3.The plasma AIMP1 level in patients with AQP4-IgG+NMOSD in the acute phase was significantly higher than that in the remission period,P=0.021.4.In patients with acute AQP4-IgG+NMOSD,plasma AIMP1 level were significantly higher in EDSS≥4 than EDSS<4 groups(P=0.001).5.Plasma AIMP1 levels were positively correlated with EDSS score(r=0.485,P<0.001)in acute AQP4-IgG+NMOSD patients and negatively correlated with complement C3 concentration in blood(r=0.452,P=0.001).6.ROC analysis showed the result that 49.55pg/ml is the best intermediate concentration for predicting AQP4-IgG+NMOSD patients with EDSS≥4(AUROC 0.790,95%CI 0.653-0.926,P=0.0006).7.In both univariate and multivariate analyses,plasma AIMP1≥49.55 pg/ml was an independent predictor of moderate to severe AQP4-IgG+NMOSD(OR 0.032,95%CI 0.001-0.687,P=0.028).Conclusions:Plasma AIMP1 levels were higher in acute AQP4-IgG+NMOSD than in the remission NMOSD group and were positively correlated with the severity of NMOSD.IVMP therapy significantly reduced AIMP1 levels.Plasma AIMP1 levels appear to be a possible promising predictor of the moderate-severe AQP4-IgG+NMOSD disease.AIMP1 may be involved in the pathogenesis of NMOSD and predict the disease activity,severity,or effect of treatment in patients with NMOSD.Further studies should be performed to reveal the precise mechanisms of NMOSD.Part Two Silencing AIMP1 can ameliorate BBB dysfunction in neuromyelitis optica spectrum disorders by increasing the expression of the tight junction-associated protein ZO1Objective:This study aimed to explore whether AIMP1 is involved in the regulation of blood-brain barrier permeability in vitro and its specific molecular mechanism in NMOSD.Methods:1.The study subjects included 7 of acute AQP4-IgG+NMOSD patients,who had not received IVMP therapy at the time of sampling,and age-sex matched healthy controls(HCs),peripheral blood was extracted,centrifuged,plasma was isolated,and frozen at-80℃ for follow-up intervention experiments.2.An in vitro BBB model based on a single layer of hCMEC/D3 cells was established,and BBB model integrity was evaluated by a 4-h liquid level leakage assay.3.Experimental group and intervention:The experiment was divided into blank control group(Control group),healthy control group(Normal group)and NMOSD group.The in vitro BBB model and hCMEC/D3 cell line were intervened with 20%Fetal bovine serum(FBS),10%healthy control plasma+10%FBS and 10%NMOSD plasma+10%FBS,respectively.4.Endothelial cell viability was measured by CCK8,fluorescein sodium(Na-F)permeability coefficient was calculated to evaluate in vitro BBB model permeability,and tight junction microstructure was observed by transmission electron microscopy.5.The expression of ZO-1,AIMP1 mRNA in hCMEC/D3 cells after plasm intervention were quantitative detected by qPCR,ZO-1 and AIMP 1 protein were measured by Western blot.6.The ZO-1 protein was detected by immunofluorescence.7.siRNA AIMP1 Plasmids were transfected into hCMEC/D3 cells:1)Evaluation of the transfection efficiency:The experiments were divided into Control group,siRNA-NC group and siRNA-AIMP 1 group.Plasmid transfection carrying CY3 fluorescence were observed under an inverted fluores-cence microscope at 6h after transfection.AIMP1 mRNA level measured by qPCR after 24h and AIMP 1 protein expression by Western blot after 48h.2)Effect of AIMP1 silence on ZO1 protein in hCMEC/D3 cells induced by NMOSD plasma:Plasma from three patients with acute AQP4-IgG+NMOSD were randomly selected for intervention experiment,which was divided into NMOSD group,NMOSD+siRNA-NC group and NMOSD+siRNA-AIMP 1 group.The changes of ZO-1 protein in each group in hCMEC/D3 cells were determined by Western blot,and the Na-F permeability was calculated in each group.Results:1.The CCK 8 assay showed that the hCMEC/D3 cell viability was significantly decreased in the NMOSD group compared with the Normal group(P=0.006).2.Compared with the Normal group,the Na-F permeability coefficient of NMOSD group increased significantly,P=0.001,But,there was no significant difference between the normal group and the Control group,P=0.688.3.TEM showed that the TJ between the Control and Normal groups were linear and dense without significant loosening,but the NMOSD group was loose and the line continuity was damaged.4.ZO-1 expression in hCMEC/D3 cells induced by NMOSD plasma:ZO1 expression:the expression of ZO-1 mRNA in the NMOSD group was significantly lower than that in the Normal group(P=0.003),and there was no significant difference between the Normal group and the Control group(P=0.178).Compared with the Normal group,the ZO-1 protein in the NMOSD group was significantly reduced(P<0.001),while the content of ZO-1 protein in the Normal group and the Control group was not statistically significant(P=0.695).The immunofluorescence intensity of ZO1 in the NMOSD group was significantly weaker than that in the Control group and the Normal group.5.AIMP1 expression in hCMEC/D3 cells induced by NMOSD plasma:The expression of AIMP1 mRNA in NMOSD group was significantly upregulated compared with the Normal group(P=0.001),while there was no significant difference between the Control group and the Normal group(P=0.443).The content of AIMP1 protein in the NMOSD group was significantly higher than that in the Normal group(P<0.001),and there was no significant statistical difference between the Normal group and the Control group(P=0.740).6.siRNA-AIMP1 plasmid was more than 90%,and the AIMP1mRNA(P=0.012)and protein expression(P=0.004)in siRNA-AIMP1 group were significantly lower than that in siRNA-NC group.7.The expression content of ZO-1 protein in the NMOSD+siRNAAIMP1 group was significantly increased compared with the NMOSD group,P=0.011,while,compared with the NMOSD+siRNA-NC group,the ZO-1 protein expression was slightly higher,which was not statistically significant differences(P=0.447).The Na-F permeability was reduced in the NMOSD+siRNA-AIMP 1 group compared with the NMOSD group,P=0.018.but,the Na-F permeability of the NMOSD+siRNA-NC group was not significantly different from the NMOSD group,P=0.400.Conclusions:Plasma from patients with NMOSD can reduce the viability of hCMEC/D3 cells,destroy the structure of TJs and reduce the expression of ZO1 to increase the permeability of BBB in vitro,and AIMP1 expression were significantly up-regulated in hCMEC/D3 cells induced by plasma from patients with NMOSD.Silencing AIMP1 can ameliorate BBB dysfunction in neuromyelitis optica spectrum disorders by increasing ZO1 expression.Part Three AIMP1 is negatively regulated by miRlet-7f-5p in human cerebral vascular endothelial cellsObjective:This study was designed to explore the expression changes of miRlet-7f-5p in hCMEC/D3 cells induced by plasma from patients with NMOSD and whether the 3’UTR region of AIMP1 mRNA can be targeted by miRlet-7f-5p to inhibit its protein synthesis.Methods:1.The miRlet-7f-5p expression changes in hCMEC/D3 cells induced by plasma form patients with NMOSD were detected by qPCR:Plasma for the inter-vention hCMEC/D3 cells was obtained from seven acute phase AQP4IgG+NMOSD patients who did not receive IVMP therapy,and seven age-sex matched HCs.The experiment was divided into Control,Normal and NMOSD groups.The hCMEC/D3 cells was intervened with 20%FBS,10%HCs plasma+10%FBS,and 10%NMOSD plasma+10%FBS,Separately.The expression level of miRlet-7f-5p in hCMEC/D3 cells was detected by qPCR 24h after the intervention.2.Dual-luciferase reporter gene assay(target gene validation)experiment:Wild-type(wt)and mutant(mut)plasmid vectors for miRlet-7f-5p corresponding to the target sites of AIMP1 3’UTR were constructed:pmir-GLOAIMP1-wt and pmirGLO-AIMP1-mut.and then Wild-type and mutant plasmid vectors were co-transfected into 239T cells with the miRlet-7f-5p plasmid,firefly luciferase,and sea Renilla luciferase.Luciferase activity was then measured.Cells are grouped as follows:1)mimics NC+pmirGLO-AIMP1-wt2)miRlet-7f-5p mimics+pmirGLO-AIMP1-wt3)inhibitor NC+pmirGLO-AIMP1-wt4)miRlet-7f-5p inhibitor+pmirGLO-AIMP1-wt5)mimics NC+pmirGLO-AIMP1-mut6)miRlet-7f-5p mimics+pmirGLO-AIMP1-mut7)inhibitor NC+pmirGLO-AIMP1-mut8)miRlet-7f-5p inhibitor+pmirGLO-AIMP1-mut3.The miRlet-7f-5p plasmid was transfected into hCMEC/D3 cells:1)Evaluation of the plasmid transfection efficiency:The experiments were divided into Control,mimics-NC,mimics,inhibitor-NC and inhibitor groups,and the expression level of mirlet-7f-5p was determined by qPCR.Transfection efficiency observed under fluorescence microscopy.2)Effects of overexpression of miRlet-7f-5p on AIMP1 protein:The experiments were divided into mimics-NC,mimics,inhibitor NC and inhibitor groups,and the protein expression of AIMP1 was measured in hCMEC/D3 cells after plasmid transfection by Western blot.Results:1.The expression of miRlet-7f-5p in NMOSD group was significantly lower than that in Control group(P=0.017)and Normal group(P=0.013),while,there was no obvious difference between Control and Normal groups(P=0.210).2.The results of dual luciferase reporter gene assay showed that the relative luciferase activity of miRlet-7f-5p mimics+pmirGLO-AIMP1-wt decreased by 41%compared with mimics NC+pmirGLO-AIMP1-wt group(P=0.001).The relative luciferase activity of miRlet-7f-5p inhibitor+pmirGLO-AIMP1-wt was increased by 35%as compared to the inhibitor NC+pmirGLO-AIMP1-wt group(P<0.001).However,there was no significant difference in the relative luciferase activity between mimics NC+pmirGLOAIMP1-mut group and miRlet-7f-5p mimics+pmirGLO-AIMP1-mut group(P=0.673).Similarly,there was no statistically significant difference between the inhibitor NC+pmirGLO-AIMP1-mut group and the miRlet-7f-5p inhibitor+pmirGLO-AIMPl-mut group(P=0.525).3.The miRlet-7f-5p plasmid was efficiently transfected into hCMEC/D3 cells.Inverted fluorescence microscope(4×)field of view:the transfection efficiency of miRlet-7f-5p plasmids under CY3 fluorescent staining can reach about 85%.The result of qPCR test showed that the expression of miRlet-7f-5p in miRlet-7f-5p mimics group was significantly increased compared with Control group and miRlet-7f-5p mimics-NC group,averaging about 350-fold,P=0.019.The expression of miRlet-7f-5p in inhibitor group was significantly reduced compared with Control group and mimics-NC group,about 0.093 times that of Control group,P<0.001.There was no statistical difference between the Control,mimics NC and inhibitor NC groups,with P>0.05.4.The AIMP1 protein expression was significantly reduced in the mi-Rlet7f-5p mimics group compared with the mimics-NC group,P=0.024,while compared with the inhibitor NC group,the expression of AIMP1 protein in the miRlet-7f-5p inhibitor group was not significant difference,P=0.541.Conclusions:miRlet-7f-5p expression was decreased in hCMEC/D3 cells induced by plasma from patient with NMOSD.In hCMEC/D3 cells,miRlet-7f5p inhibited AIMP1 protein expression by specifically binding to the 3’UTR region of AIMP1. |
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