Regulation Of HMGB1 On P-gp In Ischemicized RBMECs

Objective:To investigate the change of HMGB1 in astrocytes after ischemic injury and its effection on P-glycoprotein and related mechanisms in rat brain microvascular endothelial cells in co-culture system.Method:1.Primary cultured astrocytes and brain microvascular endothelial cells were obtained from newborn SD rats within one week after birth.The morphologies of the cells were observed by an inverted microscope.Immunofluorescence staining was used to identify astrocytes.2.Primary cultured astrocytes were injuried with different concentrations of Na₂S₂O₄ solution,and the survival rate of the astrocytes after6 h was detected by MTT assay.3.Transwell co-culture system was established with transwell technology.Chopstick electrode was used to determine the change of transendothelial electrical resistance（TEER）within 10-11 days of the co-culture system.After TEER was stabilized,it was used in subsequent experiments.After astrocytes were damaged by different concentrations of Na₂S₂O₄ solution,the accumulation of fluorescein sodium injection（NaF）in the outer chamber of transwell and the apparent permeability（Papp）coefficient of it in the inner chamber were detected by a full-wavelength microplate reader to determine the optimal damage concentration at 6 h.Besides,Rh123 was added to the inner and outer chambers of the co-culture system,and the Papp value of Rh123 was measured to study the effect of Na₂S₂O₄ on the function of P-gp in rBMECs.4.Immunofluorescence double standard method was adopted to observe the nuclear translocation of HMGB1 in astrocytes after ischemic injury.Transwell technique was used to study the effect of HMGB1 on the funtion of P-gp in rBMECs after Na₂S₂O₄ induced ischemic.6 h of ischemic injury on astrocytes later,the conditioned medium was added with HMGB1,TLR4 and NF-κB inhibitors—the pyruvate ethyl ester,TAK-242 and PDTC respectively.The expression of each protein was determined by Western Blot to determine the relations of upstream and downstream in P-gp regulation.Results:1.The rat brain microvascular endothelial cells and astrocytes were successfully extracted,identified,purified and cultured.The rBMECs and glials transwell co-culture system was established.The system was not stable until the TEER value reached about 160?·cm.2.Astrocytes were induced to infiltrate with different concentrations of Na₂S₂O₄ solution for 6 h.The cell viability,accumulation of NaF,apparent permeability coefficient of NaF（Papp）and accumulation of interior and exterior chambers of Rh123 and apparent permeability coefficient（Papp）were measured.That indicated the tight junction structure of the in vitro BBB model remained intact after the astrocytes were induced to damage with a final concentration of 3.75 mM for 6 h.3.After the astrocytes were injured by Na₂S₂O₄ solution with a final concentration of 3.75 mM for 6 h the pro-inflammatory cytokine HMGB1 was translocated from the nucleus to the cytoplasm.The expression of HMGB1 was significantly up-regulated in the cytoplasm while it was significantly down-regulated in the nucleus.4.Astrocytes in the co-culture system of astrocytes and rBMECs were damaged by 3.75 mM Na₂S₂O₄ solution for 6 h,the expression of P-gp increased.5.The function and expression of P-gp were down-regulated after adding HMGB1,TLR4 and NF-κB inhibitors.And initially determine the upstream and downstream relationships of each factor:HMGB1→TLR4→NF-κB→P-gp.Conclusion:The vitro experiments demonstrate that nuclear translocation of nuclear HMGB1 occurs6 h after ischemic injury in rat astrocytes.Besides,it can regulate the function and expression of P-gp in rBMECs in BBB by HMGB1→TLR4→NF-κB→P-gp pathway.That provides experimental basis for further study of the brain permeability of the medicine after stroke.