Poloxamer 188 Attenuates in vitro Traumatic Brain Injury-Induced Mitochondrial and Lysosomal Membrane Permeabilization Damage in Cultured Primary Neurons
Poloxamer 188 Attenuates in vitro Traumatic Brain Injury-Induced Mitochondrial and Lysosomal Membrane Permeabilization Damage in Cultured Primary Neurons
复制标题
泊洛沙姆 188 可减轻体外培养的原代神经元中创伤性脑损伤引起的线粒体和溶酶体膜通透性损伤
DOI：
10.1089/neu.2012.2425
复制
发表时间：
2013-04-01
影响因子：
4.2
通讯作者：
Zhao, Zi-Qin
中科院分区：
文献类型：
--
作者：
Luo, Cheng-Liang;Chen, Xi-Ping;Zhao, Zi-Qin
创伤性脑损伤（TBI）引起的急性膜损伤是一种严重的突发事件。然而，机械创伤的后续影响，包括线粒体和溶酶体膜通透性（MOMP和LMP）仍然难以捉摸。本研究的主要目的是评估假定的膜再密封剂泊洛沙姆188（P188）在MOMP和LMP中对明确定义的机械损伤的反应。使用体外细胞剪切装置（VCSD），机械性损伤导致培养的原代神经元的膜完整性的立即破坏，和神经元处理与P188或组织蛋白酶B抑制剂（CBI）VCSD后10分钟。P188对培养的原代神经元的保护作用，首先用光学显微镜观察，并通过MTT法和LDH法测定。JC-1染色法检测线粒体膜电位（Delta Psim）变化，Western blot法检测纯化线粒体中细胞色素c和截短的Bid（tBid）。此外，通过对纯化的溶酶体中的组织蛋白酶B和tBid进行印迹来检测溶酶体完整性。我们的结果表明，损伤后P188处理缓和了线粒体中Delta Psi m的耗散，并抑制了VCSD诱导的线粒体细胞色素c和溶酶体组织蛋白酶B的释放。组织蛋白酶B抑制剂（CBI）也可增加细胞活力，维持线粒体膜电位，并抑制VCSD诱导的细胞色素c从线粒体释放到胞浆。P188和CBI处理均降低了纯化的溶酶体上清液中tBid在胞浆中的积累，并降低了线粒体定位的tBid的量。这些数据表明，受损的神经元已经经历了线粒体和溶酶体膜通透性损伤，并且可以利用药理学干预来开发该机制。P188的神经保护作用似乎涉及组织蛋白酶B和TBID介导的线粒体启动细胞死亡之间的关系。
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 (Delta Psi m) 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 Delta Psi m 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.