Investigating the Role of JNK Activation and Circulating BNP in Septic Hypotension

项目总结/摘要 脓毒症是由于对感染的压倒性免疫反应导致组织灌注不足， 器官衰竭最终死亡严重脓毒症引起的心肌抑制与高血压相关 住院死亡率。循环B型利钠肽（BNP）升高先前已被确定为 脓毒症心功能不全的标志物，与脓毒症患者预后不良相关。BNP编码 由Nppb基因产生，由心室肌细胞响应心肌应激而产生。BNP降低 通过促进血管舒张以减少后负荷和增加静脉容量， 促进尿钠排泄以减少血容量和前负荷。BNP是否对 心血管系统在脓毒症期间是活跃的，尚未进行评估，将在本申请中进行讨论。 先前的研究表明，药理学JNK抑制剂可保护LPS相关的心脏 通过恢复参与脂肪酸氧化的基因的表达来治疗功能障碍。JNK抑制剂治疗 完全消除LPS诱导的BNP mRNA上调。为了确定JNK的机制， 抑制可以阻止BNP的产生，我们的应用重点是cJun，一种已充分描述的JNK底物， 活化蛋白（AP）-1复合物的成员。启动子分析，然后进行人类基因组的Clustal比对。 而小鼠Nppb启动子揭示了两种启动子内遗传学上保守的AP-1基序。初步 数据显示在人心肌细胞AC 16细胞系中腺病毒递送活性cJun促进BNP 通过直接结合Nppb启动子进行转录。我们的建议将调查因果关系 BNP上调和心脏cJun激活之间的关系，这将使用LPS和CLP在体内进行评估。 诱导的鼠脓毒症，以评估JNK抑制防止BNP上调的潜力。我们的初步 数据提示，败血症患者循环BNP水平与疾病严重程度和心肌功能障碍相关， JNK抑制可减少BNP的产生，并可防止脓毒症诱导的 低血压对于具体目标2，我们的建议将从机制上确定BNP抑制是否参与了 JNK抑制剂的作用机制以及BNP抑制是否会防止低血压、前负荷降低和 在这些参数受损的脓毒症动物模型中降低心输出量。我们将翻译这些 通过比较脓毒症和脓毒性休克患者的循环BNP水平与 对照受试者，并通过确定BNP升高和心输出量减少之间的相关性， 心肌收缩功能拟议的研究将测试cJun负责的中心假设 BNP在脓毒症中上调，阻止BNP或JNK信号传导将通过增加血 压力和组织灌注。这些研究的结果可以作为未来治疗的基础， 抑制循环BNP升高难治性低血压患者的血压。

PROJECT SUMMARY/ABSTRACT Sepsis results from the overwhelming immune response to infection leading to tissue hypoperfusion, organ failure, and eventually death. Myocardial depression resulting from severe sepsis is associated with high in-hospital mortality. Elevated circulating B-type natriuretic peptide (BNP) has previously been identified as a marker of cardiac dysfunction in sepsis which correlates with poor prognosis in septic patients. BNP is encoded by the Nppb gene and is produced by the ventricular myocytes in response to myocardial stress. BNP reduces blood pressure peripherally by promoting vasodilation to reduce afterload, and by increasing venous capacitance and promoting natriuresis to reduce blood volume and preload. Whether or not these effects of BNP on the cardiovascular system are active during sepsis has not been evaluated and will be addressed in this application. Previous studies have shown that pharmacological JNK inhibition protects against LPS associated cardiac dysfunction by restoring expression of genes involved in fatty acid oxidation. Treatment with JNK inhibitor completely ablates the LPS-induced BNP mRNA upregulation. To identify the mechanisms by which JNK inhibition prevents BNP production, our application focuses on cJun, a well described substrate of JNK and member of the activating protein (AP)-1 complex. Promoter analysis followed by Clustal alignment of the human and mouse Nppb promoters reveals a phylogenetically conserved AP-1 motif within both promoters. Preliminary data shows that adenoviral delivery of active cJun in the human cardiomyocyte AC16 cell line promotes BNP transcription via direct binding to the Nppb promoter. Our proposal will investigate the causative association between BNP upregulation and cardiac cJun activation, which will be evaluated in vivo using LPS and CLP induced murine sepsis to evaluate the potential of JNK inhibition to prevent BNP upregulation. Our preliminary data suggests that circulating BNP levels correlate with disease severity and myocardial dysfunction in sepsis animal models, and that JNK inhibition reduces BNP production and protects against sepsis-induced hypotension. For specific aim 2, our proposal will identify mechanistically if BNP suppression is involved in the JNK inhibitor’s mechanism of action and if BNP inhibition will protect against hypotension, reduced preload, and reduced cardiac output in sepsis animal models where these parameters are impaired. We will translate these findings in human patients by comparing circulating BNP levels in patients with sepsis and septic shock with control subjects and by identifying the correlation between elevated BNP and reduced cardiac output and myocardial systolic function. The proposed studies will test the central hypothesis that cJun is responsible for BNP upregulation in sepsis, and that preventing BNP or JNK signaling will improve survival by increasing blood pressure and tissue perfusion. Results from these studies may serve as the basis for future therapies aimed at inhibiting circulating BNP to increase blood pressure in patients with refractory hypotension.