The Effect And Potential Mechanisms Of HMGB1 On Neural Stem Cells Migration And Regeneration After Spinal Cord Injury

BackgroundSpinal cord injury(SCI),which always occurs in young adults,is a devastating neurological disease and has becoming a cardinal public health concern.It not only results in physical trauma and emotional stress to patients,but also imposes huge economic burden to the family and society when SCI happens.Although the current treatments for SCI include early surgery to relieve pressure and stabilize spinal structure,dehydration,anti-inflammation,neurotrophic support,rehabilitation training,most of SCI patients still remain serious neurological dysfunction.The potential reasons for limited therapeutic effect might result from neural circuit damage,ischemia,hypoxia,inflammatory reaction,demyelination and glial scar formation,to introduce a hostile external environment around injured spinal cord,which are not conducive to nerve regeneration and rehabilitation,making it one of the most difficult diseases in modern spinal surgery field.Neural stem cells(NSCs),mainly lining in subventricular zone(SVZ)of the lateral ventricle and subgranular zone(SGZ)of dentate gyrus in adult mammal,are a subtype of multipotential cells with ability of self-renewal and capacity of differentiation into neurons,astrocytes and oligodentrocytes,playing an evident role in neurodevelopment and repair in adult mammals.NSCs could be activated,differentiated into functional neurons,and integrated into the damaged neural circuits to promote nerve function recovery after central nervous system(CNS)injury.Although previous studies have proven the beneficial effect of NSCs on neural repair and regeneration after CNS damage,the therapeutic effect is still limited due to the restricted ability of NSCs proliferation and migration towards the lesions.Therefore,exploring new methods combination with NSCs to enhance their proliferation,migration and preferred differentiation is a feasible approach to provide basic knowledge for cell replacement therapy and holds great importance for cell-based therapy in clinic.High mobility group box 1 protein(HMGB1)is one of the most widely expressed nucleoprotein member in the HMG family in mammals and HMGB1 is well known for its involvement in inflammatory response.Previous researches have found that it plays a significant role in cell migration,immune identification and inflammatory maintainment.Furthermore,previous investigation also reveals that HMGB1 could enhance differentiation of neuroblasts into neurons,thereby promoting functional recovery in CNS diseases.However,few researches focus on the relationship between HMGB1 and NSCs migration and differentiation,and potential mechanism.In addition,researches represent that transplanted NSCs participate in the repair of spinal cord after SCI through neuroprotection,re-myelinization and immunomodulation,etc.Hence,use of HMGB1 might serve as a new therapeutic target for SCI.However,only a small number of transplanted NSCs could survival for a long time resulting from unsatisfactory differentiation into neurons and limited migration potential owing to a hostile external environment around injured spinal cord.To overcome this obstacle,new methods are demanded to improve survival,migration potential and preferred differentiation of NSCs after transplantation.In order to verify the effect and underlying mechanism of HMGB1 on neural stem cells migration and regeneration after spinal cord injury,the NSCs were cultivated in vitro and SCI animal models were established in rats.Meanwhile,the underlying mechanism(s)was explored based on in vitro and in vivo studies.The details of the study were as follows:Part ? The role and mechanism of HMGB1 in migration of NSCs.ObjectiveThrough culturing NSCs in vitro,different concentration of HMGB1 was performed to evaluate the effect of HMGB1 on NSCs migration and proliferation.Different methods,such as western blotting and immunofluorescence,were carried out to investigate the possible mechanism of HMGB1 promoting NSCs migration.MethodsIn this part,cultured cells were isolated from E14.5 neocortex and they expressed the stem maker of nestin,one of NSCs maker,and held the capacity of differentiation into GFAP,Olig2 and DCX positive cells after incubation in differentiation medium,implying that the cultured cells were neural stem cells(NSCs).Then,cells were randomly divided into control group,0.01 ng/ml HMGB1 group,0.1 ng/ml HMGB1 group,1 ng/ml HMGB1 group and 10ng/ml HMGB1.Cell Counting Kit-8(CKK-8)method and neurosphere formation assays were conducted to assess the effect of different concentrations of HMGB1 on NSCs proliferation.Meanwhile,transwell assays were performed to determine the effect of HMGB1 on NSCs migration.Furthermore,the number of cells migrating from neurospheres and the migrating distance were calculated with HMGB1 addition by a phasecontrast microscope.Immunofluorescence and western blotting were applied to analyze the effects of HMGB1 on RAGE expression in NSCs.The effects and morphological changes of HMGB1 on filopodia formation of NSCs were observed by phalliodin detection and tubulin immunostaining.In addtion,FPS-ZM1,an antagonist of HMGB1/RAGE signaling pathway and RAGE si RNA were used to evaluate the role of HMGB1/RAGE signaling in HMGB1 potentiating NSCs migration.Results(1)Cells,isolated from neocortex,positively expressed nestin,one marker of NSCs,and held the capacity of differentiation into GFAP,Olig2 and DCX positive cells after incubation in differentiation medium using immunostaining assays,suggesting that the cultured cells were neural stem cells(NSCs).expression by immunofluorescence test.(2)The absorbance value at 450 nm was significantly increased after NSCs incubation in 1 ng/ml HMGB1 and 10 ng/ml HMGB1 for 3 days,compared to other groups.Meanwhile,the results indicated the diameter of neurospheres were much larger in 1 ng/ml and 10 ng/ml groups than that in other groups.Together,these data indicated that HMGB1 could promote NSCs proliferation in a dose-dependent manner.(3)The migration distance and cell number of NSCs after HMGB1 intervention were observed through transwell experiments and under phasecontrast microscope.Results showed that the distance and number of NSCs in 1 ng/ml HMGB1 migrating from neurospheres were greatly increased than that in vehicle.Next,transwell assays indicated that 1 ng/ml HMGB1 increased the mobility of NSCs than that in vehicle.Together,these data suggested that HMGB1 hold the capacity to enhance NSCs migration.(4)The results showed that NSCs treated with 1 ng/ml HMGB1 developed more filopodia formation.Meanwhile,the average number of processes of NSCs in 1 ng/ml HMGB1 significantly increased than that in vehicle,including leading processes and secondary branches.Furthermore,NSCs treated with 1 ng/ml HMGB1 upregulated RAGE expression.Together,these data suggest that NSCs treated with 1 ng/ml HMGB1 upregulates RAGE expression to promote filopodia formation.(5)To further uncover the role of RAGE playing in HMGB1 resulting in NSCs migration,50 n M FPS-ZM1,one of the RAGE antagonist,and RAGE-specific si RNA were used to detemine the role of RAGE playing in the NSCs migration indued by HMGB1.The data indicated that 50 n M FPS-ZM1 decreased the migrated distance and cell number after incubation for 24 hours.Furthmore,the downregulation of RAGE expression by si RNA were performed.The data indicated the same tendency as FPS-ZM1.In addition,the phalloidin staining and tubulin immunostaining were performed to assess the role of RAGE in filopodia formation and morphological structure changes.The results indicated the percent of filopodia formation decreased greatly to the vehicle level with the treatment of FPS-ZM1 and RAGE-specific si RNA.Meanwhile,the filopodia formation,primary processes and secondary branches were also decreased.Taken together,these data indicated that RAGE played an evident role in facilitating NSCs migration induced by HMGB1 and the underlying mechnism was promoting filopodia formation.ConclusionsThe NSCs migration ability could be potentiated by 1 ng/ml HMGB1 treatment through enhancing filopodia formation.Meanwhile,HMGB1/RAGE signaling pathway plays an evident role in NSCs migration induced by HMGB1.Part ? The role and mechanism of transplantation of NSCs preconditioned with HMGB1 in functional recovery after spinal cord injury in rats.ObjectiveThrough culturing NSCs in vitro and engraftment of NSCs preconditioned with HMGB1 in rats with SCI,the functional recovery was assessed.Moreover,different methods,such as western blotting and immunofluorescence assays,was performed to investigate the possible mechanism of engraftment of NSCs preconditioned with HMGB1 enhancing functional recovery after SCI.MethodsIn this part,NSCs were isolated from E14.5 neocortex of rats and the SCI model was established using Sprague Dawley rats.After rats were randomly assigned into various groups,Basso,Beattie and Bresnahan Locomotor Rating Scale(BBB),Mechanical allodynia assay and cold/hot plate heat sense assay were performed to evaluate the recovery of motion and sensory functional recovery.Then,the tissue structures in the damaged area in different groups was assessed using hematoxylin and eosin(H&E)stain and Nissl staining.Furthermore,the immunostaining were carried out to determine the number of neurons in the epicenter of injured spinal cord in different groups.In addition,the differentiation potential was determined using immunostaining and immunoblot assays to unravel the potential mechanism.Results(1)The results,collected from BBB scores,mechanical hypersensitivity and cold plate tests on days 1,7,14,21 and 28 after SCI in three groups: Control,NSCs and HMGB1+NSCs,indicated that rats in HMGB1+NSCs group showed the best improvement in locomotor recovery compared with the control and NSCs groups on days 14,21 and 28.Meanwhile,locomotor recovery in NSCs group presented better outcome compared with the control group on day 21 and 28.Next,the score for mechanical hypersensitivity was the highest in the HMGB1+NSCs group compared with the control and NSCs groups on days 14,21 and 28.Additionally,the mechanical stimulation score was higher in the NSCs group compared with the control group on days 14,21 and 28.In addition,the score using cold plate tests was highest in HMGB1+NSCs group compared with the control and NSCs groups on days 14,21 and 28.Furthermore,the score using cold plate tests was higher in NSCs group compared with the control group on days 21 and 28.These results suggested that Transplantation of HMGB1-preconditioned NSCs promoted functional recovery after SCI in rats.(2)The results obtained from H&E and Nissl staining indicated that the lesion area in morphologic characteristics in the HMGB1+NSCs group was significantly decreased compared with the NSCs and control groups.The lesion region in the NSCs group was smaller compared with the control group.Subsequently,HE staining showed the fewest number of basophilic nuclei and the smallest areas of spared tissue on day 21 post-SCI.Next,the number of functional Nissl bodies was the highest in the HMGB1+NSCs group,while the number of non-functional Nissl bodies was fewest,compared with the NSCs and control groups.It was suggested that transplantation of NSCs pre-treated by HMGB1 could promote the reshaping of nerve tissue structure in rats with SCI.(3)The expression level of ?III-tubulin,a neuron marker in the spinal cord injury area of SCI mice in the control group,NSCs group and HMGB1-NSCs group,was measured by western blotting and immunofluorescence assays.It was higher in HMGB1-NSCs group than that in the control group and NSCs group,suggesting that transplantation of NSCs pre-treated by HMGB1 could promote neuron survival in the epicenter of damaged spinal cord.(4)The expression level of ?III-tubulin and p-ERK in NSCs group,HMGB1-NSCs group and HMGB1-NSCs+U0126 were measured by western blotting and immunofluorescence assays.The results demonstrated that the expression level of ?III-tubulin and p-ERK was evidently increased in HMGB1-NSCs group.While,with addition of a specific ERK antagonist-U0126 could partially abrogate this enhanced differentiation potential into neurons induced by HMGB1,suggesting that ERK signaling pathway played an important role in HMGB1 resulting in NSCs differentiation into neurons.ConclusionsTransplantation of NSCs preconditioned with 1 ng/ml HMGB1 could significantly improve functional recovery via decreasing injured spinal cord atrophy and increasing the number of Tuj-1-positive cells at the epicenter of injured spinal cord.The increased number of neurons might derive from the survival of epibiotic neurons and the differentiation from engrafted NSCs preconditioned with 1 ng/ml HMGB1.Meanwhile,the potential mechanism might due to the activation of ERK signaling pathway.