| BACKGROUD AND OBJECTIVEMultiple sclerosis(MS)is an inflammatory autoimmune disease of the central nervous system,with clinical manifestations varying from sensory and visual disturbances and motor disturbances to cognitive impairments.MS,with repeated attacks eventually resulted in permanent physical disability,is the most common disabling neurological disease in young people from age 20 to 40,even in young women.Up to 2013,more than2.3 million patients worldwide have suffered from this disease,causing a heavy burden to society and family.The blood-brain barrier(BBB)is mainly composed of brain microvascular endothelial cells connected by tight junctions(TJs)and adhesion molecules.As the connecting structure of BBB endothelial cells,TJs like occludin,claudin-5,ZO-1,ZO-2,etc.,restrict the entry of substances and toxins from the peripheral environment into the brain parenchyma,thereby maintain the stability of the central nervous system(CNS).The destruction of the TJs structure leads to an increase in the permeability of the BBB,then peripheral toxic and harmful substances invade into the brain parenchyma,which eventually results in CNS dysfunction.As a typical pathological change of MS,the disruption of BBB is also a necessary condition for the pathogenesis in MS.Therapies on BBB have been one of the most promising therapeutic targets for MS.For example,MS classic drugs likes corticoid and interferon beta can improve the blood-brain barrier disruption and relieve clinical symptoms via different mechanisms.However,due to the complex pathological mechanism of blood-brain barrier disruption,currently there is no treatment strategy directly aiming at repairing BBB,which greatly limits the clinical application in targeting the “critical step” of MS patients.Therefore,it is urgent to explore the molecular mechanism of blood-brain barrier disruption in MS.Repulsive guidance molecule A(RGMa)is an axon-guiding molecule involved in the pathogenesis of various central nervous system diseases.And RGMa exhibits potent immunomodulatory effects in multiple sclerosis.Studies have found that the expression of RGMa was significantly increased in experimental autoimmune encephalomyelitis(EAE)mice.After RGMa-specific antibody application,the expression of INF-γ,IL-17 and IL-2 in the central nervous system was significantly reduced,and the demyelination and neurological function scores were also significantly improved.Besides,previous study found that RGMa enhanced the adhesion of activated CD4+ T cells to intracellular adhesion molecule-1 in the EAE mice,which suggests the relationship among blood-brain barrier,peripheral immune cells and RGMa.Our research group have previously found that RGMa was also expressed in brain vascular endothelial cells.Therefore,given that RGMa is involved in the pathogenesis and progression of multiple sclerosis,it is worth exploring whether RGMa acts on the blood-brain barrier or causes its disruption.Normally,RGMa binds to the type 1 membrane protein neogenin to exert biological functions such as axon guidance or neuronal survival.Except that,as a co-receptor of bone morphogenetic proteins(BMPs),RGMa binding to BMP and BMP receptor,activates the canonical Smad pathway or the non-canonical Smad pathway,and participates in cell proliferation,migration and survival processes.Although RGMa exerts function by combining its main receptor neogenin in the CNS,many studies have pointed out that the BMP/BMP receptor signaling pathway was involved in angiogenesis and endothelial cells regulation.Therefore,whether RGMa plays a biological function in the BBB through the BMP/BMP receptor signaling pathway,and how RGMa functions through this pathway remains to be further explored.Yes-associated protein(YAP)is one of the main effectors of the Hippo signaling pathway.As a transcriptional co-activator,YAP is involved in transcriptional regulation.Studies have pointed out that YAP is involved in regulating the stability of the BBB.YAP-knockout mice showed discontinuity and thinning of the basement membrane of BBB.In addition,after applying Erb B4 in subarachnoid hemorrhage mice,the expression of YAP was upregulated,then maintained the stability of the blood-brain barrier and improved neurological function.In a word,as a newly discovered important regulator of blood-brain barrier,it is urgent and necessary to further explore the role of YAP in MS.In addition,as downstream effectors of BMP2,Smad1/4 interacting with YAP,competes with TAED1,and inhibits the cooperative transcriptional activity of YAP.Therefore,it is worth exploring whether RGMa,BMP/BMP receptors,and YAP play roles in the disruption of the blood-brain barrier in MS.literature has pointed out that the existence of soluble protein form of RGMa promotes its extensive functions.Some studies have also confirmed that RGMa is expressed in both Th17 cells and bone marrow derived cells,and plays a role in neuronal death and adhesion.Therefore,whether soluble RGMa in serum exists a non-negligible role in diseases deserves further study.In this study,we aim to explore the target and possible mechanism of RGMa on BBB,and the relationship between the expression of serum RGMa in MS patients and the severity and activity of the disease,providing more options for the treatment of MS patients.PART Ⅰ: EFFECT OF RGMA ON BLOOD-BRAIN BARRIER IN EXPERIMENTALAUTOIMMUNE ENCEPHALOMYELITIS MICEObjective:To observe the expression of BBB TJs protein ZO-1 and RGMa,BMP2,type 2 BMP receptor(BMPR2),as well as YAP in brain and spinal cord of EAE mice,we constructed EAE mice model and detected the expression of these proteins.Methods:1.100 healthy female C57BL/6 mice were selected for EAE modeling and divided into control group,immunization post 0-day group,7-day group,14-day group and 21-day group.2.The 5-point scoring standard(Kono et al.)was used to evaluate the neurological function in EAE mice.3.HE staining of brain and spinal cord tissue was used to observe lymphocyte infiltration.4.EAE mice on day 7,day 14 and day 21,as well as control mice were enrolled,and were injected with 2% Evans blue Dye(EBD)through tail vein.2 hours later,mice were anesthesia and sacrificed.To observe the destruction of BBB,qualitative and quantitative analysis was used to detect the content of EBD in brain and spinal cord.5.Western blot was used to detect the expression of ZO-1,RGMa,BMP2,BMPR2 and YAP proteins in the brain and spinal cord of EAE mice in each group.6.The double-labeling of RGMa,BMP2,BMPR2,YAP with the vascular endothelial cell marker CD31 in the brain and spinal cord of EAE mice were detected by immunofluorescence.7.Knocking down RGMa by lentivirus in EAE mice,and the neurological function of EAE mice in the three groups was observed.The transfection efficiency of lentivirus was verified by immunofluorescence.8.Evans blue staining and quantitative detection was performed to evaluate the BBB breakdown of EAE mice in the three groups.9.Western blot was used to detect the expression changes of RGMa,BMP2,BMPR2,YAP and BBB tight junction proteins ZO-1 and claudin-5in EAE mice of three groups.Results:1.Neurological function score and HE staining showed that EAE model was successfully constructed.2.Evans blue detection indicated that the concentration of EBD in the brain and spinal cord of EAE mice in 14 days and 21 days groups were higher than that in the control group(P<0.05).3.Western blot results showed that the protein expression of RGMa,BMP2 and BMPR2 in the brain and spinal cord of EAE mice was increased in day 14 and 21 group(P<0.05),while the protein expression of YAP and ZO-1 was decreased(P<0.05).4.Immunofluorescence results showed that RGMa,BMP2,BMPR2 and YAP were co-labeled with vascular endothelial cell marker CD31 in brain and spinal cord.5.After RGMa knockdown,the neurological deficit score evaluation showed that the score was decreased in EAE+LV-sh RGMa group than that in EAE and EAE+LV-NC group.And EBD detection showed the concentration of EBD was reduced in brain and spinal cord of EAE+LV-sh RGMa group compared with EAE and EAE+LV-NC group(P< 0.05).Besides,Western blot indicated that the protein expression levels of ZO-1 and claudin-5 in brain and spinal cord of EAE+ LV-sh RGMa group were increased compared with those of EAE and EAE+LV-NC group(P< 0.05),and the YAP protein expression increased(P<0.05),while the protein expression levels of BMP2 and BMPR2 decreased(P<0.05).Conclusions:1.The BBB of EAE mice was significantly damaged on day 14 and 21 post immunization,and the protein expression levels of RGMa,BMP2 and BMPR2 increased,while the protein expression level of YAP decreased.2.After RGMa knockdown,the damage of BBB in EAE mice was reduced,and the expression levels of BMP2 and BMPR2 protein were decreased,while the expression level of YAP protein was increased,suggesting that RGMa regulates BBB breakdown in EAE mice may be related to BMP2,BMPR2 and YAP.PART II: THE ROLE AND MECHANISM OF RGMA ON THE TIHGT JUNCTION PROTEIN OF IN-VITRO BLOOD-BRAIN BARRIER MODELObjective:Cerebrovascular endothelial cells,one of the main structures of the blood-brain barrier,and the tight junctions between endothelial cells play an important role in CNS diseases.The destruction of the TJs structure leads to an increase in the permeability of the BBB,then peripheral toxic and harmful substances invade into the brain parenchyma,which eventually results in CNS dysfunction.In vitro culture of endothelial cells is often used to construct in-vitro BBB model to study the mechanism of BBB destruction.To explore the effect and mechanism of RGMa on the in-vitro BBB model,in this part,we cultured human brain microvascular endothelial cells(HBMECs),which was used as an in-vitro BBB model,by upregulating or knock-downing RGMa,and followed by interfered the expression of BMPR2 or YAP,the expression of BBB tight junction proteins was observed,to explore the effect of RGMa on tight junction protein in in-vitro BBB model and its possible mechanism.Methods:1.HBMECs were cultured in vitro,and immunofluorescence was used to detect the expression of RGMa,BMP2,BMPR2 and YAP in HBMECs.2.HBMECs were cultured in transwell chamber,and cells were transfected with lentivirus LV-RGMa and control LV-NC for 4 days.To evaluate the permeability of HBMECs transwell monolayer,4k Da FITC-dextran was added to the upper chamber of the transwell,and after30,60 and 90 minutes,the concentrations of FITC-dextran in the lower chamber of transwell in LV-RGMa group and LV-NC group were detected by multimodal microplate reader.And the evaluation of integrity of monolayer of HBMECs on the transwell membrane was by staining with0.5% crystal violet solution.3.HBMECs were cultured in vitro,overexpressed with LV-RGMa,4days later,cells were fixed with glutaraldehyde,and observed TJs by transmission electron microscope.4.HBMECs were cultured in vitro,overexpressed with LV-RGMa,4days later,the protein was extracted,and the protein expression of ZO-1and claudin-5,as well as RGMa,BMP2,BMPR2 and YAP were detected by Western blot.And immunofluorescence was used to detect the fluorescence expression of ZO-1 and claudin-5.5.HBMECs were cultured in vitro,overexpressed with LV-RGMa,48??hours later,cells were transfected with siBMPR2 to silence BMPR2,Cells were divided into four groups: LV-NC+siNC group,LV-RGMa+siNC group,LV-NC+siBMPR2 group and LV-RGMa+siBMPR2 group.Extracted cell proteins,the protein expression of ZO-1 and claudin-5,as well as BMPR2 and YAP were detected by Western blot.Meanwhile,immunofluorescence was used to detect the fluorescence expression of ZO-1 and claudin-5.6.HBMECs were cultured in vitro,overexpressed with LV-RGMa,and 20 u M LPA was used to activate YAP 2 hours before cell protein extraction.Cells were divided into four groups: LV-NC+ethonal group,LV-RGMa+ethonal group,LV-NC+LPA group and LV-RGMa+LPA group.The protein expression of ZO-1,claudin-5,and YAP were detected by Western blot.Immunofluorescence was used to detect the fluorescence expression of ZO-1 and claudin-5.7.siRGMa was used to silence RGMa gene in HBMECs,cell protein was extracted and detected the protein expression of ZO-1 and claudin-5,as well as RGMa,BMP2,BMPR2 and YAP by Western blot.Immunofluorescence was used to detect the fluorescence expression of ZO-1 and claudin-5.8.siRGMa was used to silence RGMa gene in HBMECs,100 u M mnTBAP was used to activate BMPR2 1 hour before cell protein extraction.Cells were divided into four groups: siNC+DMSO group,siRGMa+DMSO group,siNC+mnTBAP group and siRGMa+mnTBAP group.Western blot was used to detect the protein expression of ZO-1 and claudin-5,as well as BMPR2 and YAP.And immunofluorescence was used to detect the fluorescence expression of ZO-1 and claudin-5.9.siRGMa was used to silence RGMa gene in HBMECs,2u M verteporfin was used to inhibit YAP 24 hours before cell protein extraction.Cells were divided into four groups: siNC+DMSO group,siRGMa+DMSO group,siNC+VP group and siRGMa+VP group.Western blot was used to detect the protein expression of ZO-1 and claudin-5,as well as YAP.Immunofluorescence was used to detect the expression of ZO-1 and claudin-5.Results:1.immunofluorescence results showed that RGMa,BMP2,BMPR2 and YAP were expressed in HBMECs.2.Compared with the control group(LV-NC group),crystal violet staining of HBMECs cells showed that the integrity of the monolayer in RGMa overexpression group(LV-RGMa group)was damaged.Meanwhile,after 90 min,the permeability of LV-RGMa group was significantly higher than that of the control group(LV-NC group)(P<0.05).3.Transmission electron microscopy of HBMECs showed that the destruction of TJs in LV-RGMa group was heavier than that in the LV-NC control group.4.Compared with the control group(LV-NC group),Western blot results showed that the protein expression of BMP2 and BMPR2 was significantly increased(P<0.05)in LV-RGMa group,while the protein expression of ZO-1,claudin-5 and YAP was significantly decreased (P<0.05).immunofluorescence results showed that the fluorescence expression of ZO-1 and claudin-5 was decreased in LV-RGMa group as compared with LV-NC group.On the contrary,compared with the control group(siNC group),Western blot results showed that the protein expression of BMP2 and BMPR2 was significantly decreased(P<0.05),while protein expression of ZO-1,claudin-5 and YAP was significantly increased in siRGMa group(P<0.05).immunofluorescence results showed that the fluorescence expression of ZO-1 and claudin-5 was upregulated in siRGMa group as well.5.After silencing BMPR2 gene following overexpressing RGMa,Western blot results showed that the BMPR2 protein expression was decreased in the BMPR2 silencing group(LV-RGMa+siBMPR2 group)compared with the control group(LV-RGMa+siNC group),while the protein expression of ZO-1,claudin-5 and YAP was increased(P<0.05).Meanwhile,immunofluorescence results showed that the fluorescence expression of ZO-1 and claudin-5 was increased in LV-RGMa+siBMPR2 group.On the contrary,after BMPR2 activation following RGMa silencing,Western blot results showed that the protein expression of BMPR2 was increased in siRGMa+mnTBAP group,while the protein expression of ZO-1,claudin-5 and YAP was decreased(P<0.05).At the same time,immunofluorescence results showed that the fluorescence expression of ZO-1 and claudin-5 was decreased in siRGMa+mnTBAP group.6.After YAP activation following RGMa overexpression,Western blot detection showed that the protein expression of YAP,ZO-1and claudin-5 was increased in LV-RGMa+LPA group(P<0.05)compared with the control group(LV-RGMa+ethonal group).Besides that,immunofluorescence results showed that the fluorescence expression of ZO-1 and claudin-5 was increased in LV-RGMa+ LPA group.On the contrary,after inhibition of YAP following silencing RGMa gene,Western blot results showed that the protein expression of YAP,ZO-1 and claudin-5in the YAP inhibition group(siRGMa+VP group)was decreased(P<0.05),and immunofluorescence results showed that the fluorescence expression of ZO-1 and claudin-5 was decreases in siRGMa+VP group.Conclusions:1.When RGMa was overexpressed in in-vitro BBB model,BBB tight junction protein ZO-1 and claudin-5 expression was decreased,however,the expression of ZO-1 and claudin-5 increased when RGMa was silenced,suggesting that RGMa is a negative regulator of BBB dysfunction.2.In the in-vitro BBB model,up-regulation of RGMa increased the expression of BMP2 and BMPR2,while decreased the expression of YAP.Up-regulation of RGMa followed by BMPR2 knockdown or YAP activation alleviated BBB breakdown.on the contrary,down-regulation of RGMa decreased the expression of BMP2 and BMPR2,while increased the expression of YAP.Down-regulation of RGMa followed by BMPR2 activation or YAP inhibition induced BBB breakdown,suggesting RGMa regulate BBB dysfunction may be through the BMP2/BMPR2-YAP signaling pathway,and regulation of RGMa may serve as a potential therapeutic target for BBB disruption.PART III: STUDY ON THE EXPRESSION OF RGMA IN THE PERIPHERAL BLOOD IN RELAPSING-REMITTING MULTIPLE SCLEROSIS PATIENTSObjective:In vitro study has confirmed that RGMa regulated the permeability of the BBB,suggesting that RGMa may be a therapeutic target for CNS diseases(such as MS)manifested by BBB disruption.To explore the role of RGMa plays in MS patients,we detected the expression of serum RGMa in patients with relapsing-remitting multiple sclerosis(RRMS)in different phase.By analyzing serum RGMa combined with expanded disability status scale(EDSS),cerebrospinal fluid(CSF)test,blood lipid test,we explored the relationship between the expression of serum RGMa in RRMS patients and disease severity,as well as disease activity.Methods:1.Blood samples were collected from 86 patients with RRMS(62relapsing RRMS patients with Gd+ enhanced Magnetic Resonance Imaging(MRI)lesions and 24 remitting RRMS patients)within 48 hours of admission to the department of neurology,the first affiliated hospital of Chongqing Medical University and 115 remitting RRMS patients during follow-up from December 2017 to June 2021.And age and sex matched 63 healthy controls were collected.2.After the blood samples were naturally agglutinated in 4°C,the supernatant was centrifuged at low temperature and stored in-80°C.3.ELISA kit was used to detect the expression of serum RGMa in relapsing RRMS patients with Gd+ enhanced MRI lesions,remitting RRMS patients and healthy controls.4.The clinical features such as clinical manifestation,blood lipid test,CSF test and EDSS score at admission of the hospitalized patients were collected.5.The nonparametric test was used to compare the expression difference of serum RGMa in relapsing RRMS patients with Gd+ enhanced MRI lesions,remitting RRMS patients and healthy controls,t test was used to compare the expression difference of serum RGMa in same RRMS patients during two phases.Spearman test and point-biserial correlation test were used to analyze the correlation between serum RGMa and clinical features of hospitalized RRMS patients.Results:1.The expression level of serum RGMa in RRMS patients was significantly higher than that in healthy controls(P<0.01).2.The expression level of serum RGMa in relapsing RRMS patients with Gd+ enhanced MRI lesions was significantly higher than that in remitting RRMS patients(P<0.01).3.Serum RGMa in hospitalized RRMS patients was positively correlated with EDSS score at admission(P<0.01).4.The expression level of serum RGMa in hospitalized RRMS patients with CSF oligoclonal band(OCB)positive was higher than that in hospitalized RRMS patients with CSF OCB negative(P<0.01).5.The expression level of serum RGMa in male hospitalized RRMS patients was higher than that in female hospitalized RRMS patients(P<0.05).Conclusions:1.The expression level of serum RGMa in RRMS patients was significantly higher than that in the healthy control,and the expression level of serum RGMa in relapsing RRMS patients with Gd+ enhanced MRI lesions was also significantly higher than that in remitting RRMS patients.Meanwhile,the expression level of RGMa in RRMS patients with CSF OCB positive was higher than that in RRMS patients with CSF OCB negative,suggesting that serum RGMa may be a marker of disease activity in RRMS patients.2.The expression level of serum RGMa was positively correlated with EDSS score of hospitalized RRMS patients,suggesting that RGMa may be one of the biomarkers for predicting the severity of disease in RRMS patients.3.The expression level of serum RGMa in male RRMS patients was higher than that in female RRMS patients,suggesting that a gender difference in serum RGMa expression level. |
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