Regulation Of Blood-spinal Barrier Permeability By MiRNA-138-5p Induced By Succinylation Of HMGB1

ObjectiveThe effect of miRNA-138-5p on the permeability of BSCB induced by succinylation of HMGB1 was studied by establishing a Blood-spinal cord barrier(BSCB)model in vitro.Methods1.In vitro BSCB model was constructed with immortable human microvascular endothelial(hCMEC/D3)and spinal cord astrocytes(Ha-sc)in Transwells plate.Lv-hmgb1-k30 a /K59A/K114 A and LV NC were transfected into hCMEC/D3.hCMEC/D3 and spinal astrocytes(Ha-sc)were used to construct BSCB models in vitro in Transwells plates.Differences in HMGB1 succinylation and miR-138-5p expression among all groups were detected by RT-qPCR.The protein and gene expressions of occludin,ZO-1,claudin-5 and cavwolin-1 among all groups were detected by Western-Blot and RT-qPCR,and the in vitro BSCB permeability among all groups was detected by FITC-dextran.2.antagomiR-138-5p,antagomiR-138-5p NC,agomiR-138-5p,agomiR-138-5pNC were transfected into hCMEC/D3.hCMEC/D3 and spinal astrocytes(Ha-sc)were used to construct BSCB models in vitro in Transwells plates,and the expression difference of miR-138-5p among all groups was detected by RT-qPCR.The protein and gene expressions of occludin,ZO-1,claudin-5 and caveolin-1 among all groups were detected by Western-Blot and RT-qPCR,and the in vitro BSCB permeability among all groups was detected by FITC-dextran.3.antagomiR-138-5p,antagomiR-138-5p NC,agomiR-138-5p,agomiR-138-5pNC were transfected into succinylated mutant stable cell lines LV-HMGB1-K30A/K59A/K114 A and LV NC.The BSCB model was constructed in vitro with spinal astrocytes(Ha-sc)in Transwells plate,and the expression difference of miR-138-5p among all groups was detected by RT-qPCR.The protein and gene expressions of occludin,ZO-1,claudin-5 and caveolin-1 among all groups were detected by Western-Blot and RT-qPCR,and the in vitro BSCB permeability among all groups was detected by FITC-dextran.Resμlts1.The detection results of Western-Blot,RT-qPCR and FITC-dextran showed that: Inhibited succinylation,miR-138-5p expression decreased significantly,occludin,ZO-1,claudin-5 and caveolin-1 protein and gene expression increased significantly,and BSCB permeability decreased significantly in vitro.2.The detection results of Western-Blot,RT-qPCR and FITC-dextran showed that: After miR-138-5p overexpression,the protein and gene expression levels of occludin,ZO-1,claudin-5 and caveolin-1 decreased significantly,and the permeability of BSCB increased significantly in vitro.However,after inhibiting miR-138-5p expression,the protein and gene expression levels of occludin,ZO-1,claudin-5 and caveolin-1 increased significantly,and the permeability of BSCB decreased significantly in vitro.3.The detection results of Western-Blot,RT-qPCR and FITC-dextran showed that: Inhibiting the succinylation of HMGB1 can significantly increase the expression of Occludin,ZO-1 and Claudin-5 proteins and genes by inhibiting the expression of miR-138-5p,thus significantly reducing the permeability of BSCB in vitro.ConclusionsSuccinylation of HMGB1 increases the permeability of BSCB by up-regulating miRNA-138-5p and inhibiting the expression of ZO-1,Occludin,Claudin-5 and caveolin-1 proteins.