The Role Of HMGB1 Succinylation Modification In NLRP3 Inflammatory Vesicle Activation-induced Changes In BSCB Function

ObjectiveSpinal Cord Injury(SCI)is one of the serious diseases of the central nervous In this study,an in vitro blood-spinal cord barrier model was established to study the effect of NLRP3 inflammatory vesicles on the succinylation of HMGB1 and the function of blood-spinal cord barrier by activating NLRP3 inflammatory vesicles,and then the effect of HMGB1 succinylation modification on the function of BSCB by inhibiting HMGB1 succinylation modification.To provide new targets and ideas for the treatment of SCI.Methods1.An in vitro BSCB model was constructed using immortalized human brain microvascular endothelium(hCMEC/D3)with spinal cord astrocytes(Ha-sc)in Transwells plates.After ROS treatment,Western-Blot was performed to detect Occludin,ZO-1,Claudin in NLRP3 inflammatory vesicles and BSCB tight junction protein(TJ)The relative expression of-5,and the in vitro BSCB permeability between the groups was detected by FITC-dextran.2.pcDNA3.1-NLRP3,pcDNA3.1-NLRP3 NC,NLRP3-homo-3449,si RNA NC were transfected into hCMEC/D3.hCMEC/D3 was used to construct an in vitro BSCB model with spinal astrocytes(Ha-sc)in Transwells plates,and after ROS treatment,the in vitro BSCB model was measured by RT-qPCR to detect the difference in succinylation expression of HMGB1 between groups,Western-Blot,RT-qPCR to detect the protein and gene expression of Occludin,ZO-1 and Claudin-5 between groups,and FITC-dextran to detect the in vitro BSCB permeability between groups.3.pcDNA3.1-NLRP3 was transfected into succinylation-modified mutant stable-transformed cell lines LV-HMGB1-K30A/K59A/K114 A,LV NC,and human spinal cord astrocytes(Ha-sc)in Transwells plates to construct in vitro BSCB models,and after ROS treatment,Western-Blot,RT-qPCR was performed to detect the expression of Occludin,ZO-1 and Claudin-5 proteins and genes between different groups.The in vitro BSCB permeability between the groups was detected by FITC-dextran.Results1.The results by Western-Blot,FITC-dextran assay showed that NLRP3 inflammatory vesicles expression was significantly increased,Occludin,ZO-1,Claudin-5 protein and gene expression was significantly decreased and BSCB permeability was significantly increased in vitro under the effect of ROS compared to the control group.2.The results by Western-Blot,RT-qPCR and FITC-dextran showed that after NLRP3 inflammatory vesicles were overexpressed,the level of succinylation modification of HMGB1 was significantly increased,the expression levels of Occludin,ZO-1,Claudin-5 proteins and genes were significantly decreased,and in vitro BSCB permeability was significantly increased.In contrast,when NLRP3 inflammatory vesicles were inhibited,the level of HMGB1 succinylation modification significantly decreased,the expression levels of Occludin,ZO-1,Claudin-5 proteins and genes significantly increased,and in vitro BSCB permeability significantly decreased.3.The results of Western-Blot,RT-qPCR and FITC-dextran assays showed that the expression levels of Occludin,ZO-1 and Claudin-5 proteins and genes were significantly increased and in vitro BSCB permeability was significantly reduced by inhibition of HMGB1 succinylation modification.ConclusionHMGB1 succinylation increases BSCB permeability in response to NLRP3 inflammatory vesicle induction.