Attenuation of Blood-Brain Barrier Disruption in Traumatic Brain Injury via Inhibition of NKCC1 Cotransporter: Insights into the NF-κB/NLRP3 Signaling Pathway

Following traumatic brain injury (TBI), inhibition of the Na⁺-K⁺-Cl^- cotransporter1 (NKCC1) has been observed to alleviate damage to the blood-brain barrier (BBB). However, the underlying mechanism for this effect remains unclear. This study aimed to investigate the mechanisms by which inhibiting the NKCC1 attenuates disruption of BBB integrity in TBI. The TBI model was induced in C57BL/6 mice through a controlled cortical impact device, and an in vitro BBB model was established using Transwell chambers. Western blot (WB) analysis was used to evaluate NOD-like Receptor family pyrin domain containing 3 (NLRP3) inflammasome and nuclear factor-kappaB (NF-κB) pathway proteins. Flow cytometry and transendothelial electrical resistance (TEER) were employed to assess endothelial cell Apoptosis levels and BBB integrity. ELISA was utilized to measure cytokines interleukin-1β (IL-1β) and matrix metalloproteinase-9 (MMP-9). Immunofluorescence techniques were used to evaluate protein levels and the nuclear translocation of the rela (p65) subunit. The Evans blue dye leakage assay and the brain wet-dry weight method were utilized to assess BBB integrity and brain swelling. Inhibition of NKCC1 reduced the level of NLRP3 inflammasome and the secretion of IL-1β and MMP-9 in microglia. Additionally, NKCC1 inhibition suppressed the activation of the NF-κB signaling pathway, which in turn decreased the level of NLRP3 inflammasome. The presence of NLRP3 inflammasome in BV2 cells led to compromised BBB integrity within an inflammatory milieu. Following TBI, an upregulation of NLRP3 inflammasome was observed in microglia, astrocytes, vascular endothelial cells, and neurons. Furthermore, inhibiting NKCC1 resulted in a decrease in the positive rate of NLRP3 inflammasome in microglia and the levels of inflammatory cytokines IL-1β and MMP-9 after TBI, which correlated with BBB damage and the development of cerebral edema. These findings demonstrate that the suppression of the NKCC1 cotransporter protein alleviates BBB disruption through the NF-κB/NLRP3 signaling pathway following TBI.

NKCC1; NLRP3; blood–brain barrier; inflammation; microglia; traumatic brain injury.