Center for Translation Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Taiwan, Republic of China.
PLoS One. 2011;6(11):e27404. doi: 10.1371/journal.pone.0027404. Epub 2011 Nov 14.
Brain stem cardiovascular regulatory dysfunction during brain death is underpinned by an upregulation of nitric oxide synthase II (NOS II) in rostral ventrolateral medulla (RVLM), the origin of a life-and-death signal detected from blood pressure of comatose patients that disappears before brain death ensues. Furthermore, the ubiquitin-proteasome system (UPS) may be involved in the synthesis and degradation of NOS II. We assessed the hypothesis that the UPS participates in brain stem cardiovascular regulation during brain death by engaging in both synthesis and degradation of NOS II in RVLM.
METHODOLOGY/PRINCIPAL FINDINGS: In a clinically relevant experimental model of brain death using Sprague-Dawley rats, pretreatment by microinjection into the bilateral RVLM of proteasome inhibitors (lactacystin or proteasome inhibitor II) antagonized the hypotension and reduction in the life-and-death signal elicited by intravenous administration of Escherichia coli lipopolysaccharide (LPS). On the other hand, pretreatment with an inhibitor of ubiquitin-recycling (ubiquitin aldehyde) or ubiquitin C-terminal hydrolase isozyme L1 (UCH-L1) potentiated the elicited hypotension and blunted the prevalence of the life-and-death signal. Real-time polymerase chain reaction, Western blot, electrophoresis mobility shift assay, chromatin immunoprecipitation and co-immunoprecipitation experiments further showed that the proteasome inhibitors antagonized the augmented nuclear presence of NF-κB or binding between NF-κB and nos II promoter and blunted the reduced cytosolic presence of phosphorylated IκB. The already impeded NOS II protein expression by proteasome inhibitor II was further reduced after gene-knockdown of NF-κB in RVLM. In animals pretreated with UCH-L1 inhibitor and died before significant increase in nos II mRNA occurred, NOS II protein expression in RVLM was considerably elevated.
CONCLUSIONS/SIGNIFICANCE: We conclude that UPS participates in the defunct and maintained brain stem cardiovascular regulation during experimental brain death by engaging in both synthesis and degradation of NOS II at RVLM. Our results provide information on new therapeutic initiatives against this fatal eventuality.
在脑死亡期间，脑干心血管调节功能障碍的基础是延髓头端腹外侧区（RVLM）中一氧化氮合酶 II（NOS II）的上调，这是从昏迷患者的血压中检测到的生死信号的起源，该信号在脑死亡发生之前消失。此外，泛素-蛋白酶体系统（UPS）可能参与 NOS II 的合成和降解。我们评估了 UPS 通过参与 RVLM 中 NOS II 的合成和降解参与脑死亡期间脑干心血管调节的假设。
方法/主要发现：在使用 Sprague-Dawley 大鼠的临床相关脑死亡实验模型中，通过双侧 RVLM 微注射蛋白酶体抑制剂（乳胞素或蛋白酶体抑制剂 II）预处理，拮抗了静脉内给予大肠杆菌脂多糖（LPS）引起的低血压和生死信号的减少。另一方面，用泛素再循环抑制剂（泛素醛）或泛素 C 末端水解酶同工酶 L1（UCH-L1）预处理增强了所引发的低血压并削弱了生死信号的出现。实时聚合酶链反应、Western blot、电泳迁移率变动分析、染色质免疫沉淀和共免疫沉淀实验进一步表明，蛋白酶体抑制剂拮抗了增强的核 NF-κB 存在或 NF-κB 与 nos II 启动子之间的结合，并减弱了减少的细胞质中磷酸化 IκB 的存在。在 RVLM 中基因敲低 NF-κB 后，蛋白酶体抑制剂 II 已经阻碍了 NOS II 蛋白表达，进一步降低。在用 UCH-L1 抑制剂预处理并在 nos II mRNA 显著增加之前死亡的动物中，RVLM 中的 NOS II 蛋白表达显著升高。
我们得出结论，UPS 通过参与 RVLM 中 NOS II 的合成和降解，参与实验性脑死亡期间失效和维持的脑干心血管调节。我们的结果提供了针对这种致命事件的新治疗策略的信息。
