NS1619 Alleviated Brain Derived Extracellular Vesicles Induced Brain Injury by Regulating the BKCa channel and Nrf2/Ho-1/Nf-kB Pathway

Abstract Background Brain induced extracellular vesicle (BDEV) are increased after traumatic brain injury (TBI) but their role in secondary brain injury is unclear. The question whether and how BDEV is involved in secondary brain injury whether neuroprotective drugs BKCa channel openers NS1619 may attenuate BDEV-induced brain injury makes sense. Methods First, BDEV was extracted from enzymatically digested brains after TBI. Second, we injected BDEV and lactadherin to mimic the up- and down-regulation of BDEV respectively after TBI and determined the role of BDEV in vivo. In vitro, the membrane potential and calcium concentration of HT-22, bEnd3 and BV-2 were determined by DiBAC4 (3) staining and fluo4-AM staining respectively. The effects of BDEV and NS1619 on HT-22 were evaluated by CCK-8, LDH release assay, Na+/k+-ATPase activity, JC-1 staining, DHE staining, and 4-HNE staining respectively. The role of BDEV and NS1619 on the Nrf2/HO-1/p65 pathway was also evaluated in HT-22. Finally, we administration TBI mice with NS1619 to clarify the role of NS1619 against BDEV in vivo. Results BDEV injection aggravated and lactadherin mitigated TBI-induced EB leakage, brain edema, neuronal degeneration, apoptosis, ROS level, microgliosis, MMP-9 activity, and NF-kB activation. In vitro, BDEV-caused depolarized membrane potential and calcium overload were significantly attenuated by NS1619 in HT-22, bEnd3 and BV-2. BDEV markedly decreased cell viability, Na+/k+-ATPase activity and mitochondrial dysregulation, ROS, oxidative stress, NF-kB activation. NS1619 pretreatment alleviated above process and enhanced antioxidant system Nrf2/HO-1 in HT-22. NS1619 administration significantly improved TBI outcome. NS1619 facilitated microglial/macrophage phenotypic transformation and increased anti-inflammatory factor and decreased pro-inflammatory factors after TBI. Finally, NS1619 treatment reduced 4-HNE and NF-kB activation and enhanced Nrf2/HO-1 pathway. Conclusions BDEV aggravated brain injury after TBI by perturbing cell membrane potential, calcium homeostasis, oxidative stress and neuroinflammation. The BKCa channel opener NS1619 attenuated BDEV-induced pathological process in vitro and in vivo by modulating the BKCa channel and Nrf1/HO-1/p65 pathway.