Traumatic brain injury (TBI) results in neuronal apoptosis, autophagic cell death and necroptosis. Necroptosis is a newly discovered caspases-independent programmed necrosis pathway which can be triggered by activation of death receptor. Previous works identified that necrostatin-1 (NEC-1), a specific necroptosis inhibitor, could reduce tissue damage and functional impairment through inhibiting of necroptosis process following TBI. However, the role of NEC-1 on apoptosis and autophagy after TBI is still not very clear. In this study, the amount of TBI-induced neural cell deaths were counted by PI labeling method as previously described. The expression of autophagic pathway associated proteins (Beclin-1, LC3-II, and P62) and apoptotic pathway associated proteins (Bcl-2 and caspase-3) were also respectively assessed by immunoblotting. The data showed that mice pretreated with NEC-1 reduced the amount of PI-positive cells from 12 to 48 h after TBI. Immunoblotting results showed that NEC-1 suppressed TBI-induced Beclin-1 and LC3-II activation which maintained p62 at high level. NEC-1 pretreatment also reversed TBI-induced Bcl-2 expression and caspase-3 activation, as well as the ratio of Beclin-1/Bcl-2. Both 3-MA and NEC-1 suppressed TBI-induced caspase-3 activation and LC3-II formation, Z-VAD only inhibited caspase-3 activation but increased LC3-II expression at 24 h post-TBI. All these results revealed that multiple cell death pathways participated in the development of TBI, and NEC-1 inhibited apoptosis and autophagy simultaneously. These coactions may further explain how can NEC-1 reduce TBI-induced tissue damage and functional deficits and reflect the interrelationship among necrosis, apoptosis and autophagy.
Acute membrane damage due to traumatic brain injury (TBI) is a critical precipitating event. However, the subsequent effects of the mechanical trauma, including mitochondrial and lysosomal membrane permeability (MOMP and LMP) remain elusive. The main objective of the current study was to assess the role of a putative membrane-resealing agent poloxamer 188 (P188) in MOMP and LMP in response to a well-defined mechanical insult. Using an in vitro cell shearing device (VCSD), mechanical injury resulted in immediate disruption of membrane integrity in cultured primary neurons, and neurons were treated with P188 or a cathepsin B inhibitor (CBI) after VCSD 10 min. The protective effect of P188 on cultured primary neurons was first detected visually with a light microscope, and measured by MTT assay and LDH assay. The validity of monitoring changes in mitochondrial membrane potential (ΔΨm) was measured by JC-1 staining, and Western blot for cytochrome c and truncated Bid (tBid) in purified mitochondria was also performed. In addition, lysosomal integrity was detected by blotting for cathepsin B and tBid in purified lysosomes. Our results showed post-injury P188 treatment moderated the dissipation of ΔΨm in mitochondria, and inhibited VCSD-induced cytochrome c release from mitochondria as well as cathepsin B from lysosomes. Cathepsin B inhibition (CBI) could also increase cell viability, maintain mitochondrial membrane potential, and repress VCSD-induced release of cytochrome c from mitochondria to cytosol. Both P188 and CBI treatment decreased the cytosolic accumulation of tBid in supernatant of purified lysosomes, and the amount of mitochondrial localized tBid. These data indicate injured neurons have undergone mitochondrial and lysosomal membrane permeability damage, and the mechanism can be exploited with pharmacological interventions. P188's neuroprotection appears to involve a relationship between cathepsin B and tBid-mediated mitochondrial initiation of cell death.
Plasmalemma permeability plays an important role in the secondary neuronal death induced by traumatic brain injury (TBI). Previous works showed that Poloxamer 188 (P188) could restore the intactness of the plasma membrane and play a cytoprotective action. However, the roles of P188 in blood-brain barrier (BBB) integrity and TBI-induced neural cell death are still not clear. In this study, mice were induced TBI by controlled cortical impact (CCI), and cerebral water content was measured to explore the profile of brain edema after CCI. Further, the regimen of P188 in mouse CCI models was optimized. The neurological test and BBB integrity assessment were performed, and the numbers of TBI-induced neural cell death were counted by propidium iodide (PI) labeling. The expression of apoptotic pathway associated proteins (Bax, cyt-c, caspase-8, caspase-9, caspase-3, P53) and aquaporin-4 (AQP4) was assessed by RT-PCR or immunoblotting. The data showed that the brain edema peaked at 24 h after TBI in untreated animals. Tail intravenous injection of P188 (4 mg/ml, 100 μl) 30 min before TBI or within 30 min after TBI could attenuate TBI-induced brain edema. P188 pre-treatment restored BBB integrity, suppressed TBI-induced neural cell death, and improved neurological function. TBI induced an up-regulation of Bax, cyt-c, caspase-8, caspase-9, caspase-3, and the expression of p53 was down-regulated by P188 pre-treatment. AQP4 mainly located on endothelial cells and astrocytes, and its expression was also regulated by P188 pretreatment. All these results revealed that P188 attenuates TBI-induced brain edema by resealing BBB and regulating AQP4 expression, and suppressed apoptosis through extrinsic or intrinsic pathway. Plasmalemma permeability may be a potential target for TBI treatment.
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