Potential usefulness of siRNAs for gene therapy ofsepsis-induced multiple organ failure

siRNA 在脓毒症引起的多器官衰竭基因治疗中的潜在用途

基本信息

批准号：
18590233
负责人：
HATTORI Yuichi
金额：
$ 2.46万
依托单位：
University of Toyama
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18590233/
关键词：
Sepsis RNA interference Apoptosis Endothelial cells Acute lung injury Caspase-8 -3 FADD Small interfering RNA(siRNA)RNA千渉短鎖干渉RNA (siRNA)内皮由来NO合成酵素 caspase-3

项目摘要

Rationale: Not only better understanding of the molecular mechanisms involved in the pathogenesis of sepsis and its resultant organ failure, but also new therapeutic approaches and targets are urgently needed. Accumulating evidence suggests that apoptosis plays an important role in the pathophysiology of sepsis, and apoptosis may be potentially detrimental in septic acute lung injury (ALI). Also, vascular endothelial cell apoptosis may play a role in the pathogenesis of the septic syndrome.Objectives: We tested the hypothesis that systemic administration of small interfering RNA (siRNA) targeting Fas-associated death domain (FADD), which recruits procaspase-8 into the death-inducing signaling complex, may be protective in septic ALI and mortality We also evaluated the therapeutic efficacy of caspase-8/caspase-3 siRNAs in a murine model of polymicrobial endotoxic shock.Methods: Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in BALB/c mice. In vivo delivery of siRN … More As was performed by using a transfection reagent (Lipofectamine^(TM) RNAiMAX) at 10 hours after CLP. As a negative control, animals received non-sense (scrambled) siRNA.Measurements and Main Results: In CLP-induced septic mice, surface expression of death receptors were up-regulated and FADD was highly expressed and activated. DNA fragmentation ladder and TUNEL (transferase-mediated dUTP nick end labeling) assays showed that treatment with siRNAs suppressed apoptosis induction in lungs and vascular tissues following sepsis. Moreover, these siRNA treatments prevented the development of ALI in CLP mice, as indicated by great improvements of blood-gas derangements, histologic lung damage, and increased pulmonary inflammatory cells. Furthermore, hematoxylin/eosin-stained sections of septic aorta displayed partial detachment of endothelial cells from the basal membrane. These histopathologic changes in endothelial cells were drastically prevented by systemic treatment with FADD siRNA or caspase-8/-3 siRNAs. Finally, administration of FADD siRNA or caspase-8/-3 dramatically improved the survival of CLP mice.Conclusions: These results indicate the pathophysiological significance of the death receptor apoptotic pathway, including FADD, in septic ALI and the potential usefulness of FADD siRNA for gene therapy of the septic syndrome. In addition, gene silencing of caspase-8 and caspase-3 with siRNAs provided profound protection against polymicrobial endotoxic shock. The prevention of vascular endothelial cell apoptosis appears to be, at least in part, responsible for their beneficial effects in endotoxic shock. Less

理由：不仅需要更好地了解脓毒症发病机制及其导致的器官衰竭，而且迫切需要新的治疗方法和靶点，越来越多的证据表明细胞凋亡在脓毒症的病理生理学中发挥着重要作用。脓毒症急性肺损伤 (ALI) 可能不利于健康。此外，血管内皮细胞凋亡可能在脓毒症综合征的发病机制中发挥作用。目的：我们测试了全身给药小剂量的假设。靶向 Fas 相关死亡结构域 (FADD) 的干扰 RNA (siRNA)，将 procaspase-8 募集到死亡诱导信号复合物中，可能对脓毒症 ALI 和死亡率具有保护作用。我们还评估了 caspase-8/caspase-3 的治疗效果多种微生物内毒素休克小鼠模型中的 siRNA。方法：通过 BALB/c 中的盲肠结扎和穿刺 (CLP) 诱导多种微生物败血症在 CLP 后 10 小时，使用转染试剂 (Lipofectamine^(TM) RNAiMAX) 进行 siRN 体内递送作为阴性对照，动物接受无义（乱序）siRNA。测量和主要结果：在 CLP 诱导的脓毒症小鼠中，死亡受体的表面表达上调，并且 FADD 高度表达并激活 DNA 片段化阶梯和 TUNEL（转移酶介导）。 dUTP 缺口末端标记）分析表明，siRNA 治疗可抑制脓毒症后肺和血管组织的细胞凋亡诱导，此外，这些 siRNA 治疗可防止 CLP 小鼠发生 ALI，血气紊乱、组织学肺损伤得到显着改善。此外，脓毒症主动脉的苏木精/伊红染色切片显示内皮细胞从基底膜部分脱离。 FADD siRNA 或 caspase-8/-3 siRNA 的全身治疗显着预防了内皮细胞的组织病理学变化。最后，给予 FADD siRNA 或 caspase-8/-3 显着改善了 CLP 小鼠的存活率。死亡受体凋亡途径（包括 FADD）在脓毒症 ALI 中的病理生理学意义以及 FADD siRNA 在基因治疗中的潜在用途此外，用 siRNA 对 caspase-8 和 caspase-3 进行基因沉默可提供针对多种微生物内毒素休克的深刻保护，预防血管内皮细胞凋亡似乎至少部分是其在内毒素中发挥有益作用的原因。震动较小