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

基本原理：不仅要更好地了解脓毒症及其导致的器官衰竭的发病机制，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过程中起着重要作用，细胞凋亡在脓毒症急性肺损伤（ALI）中可能是潜在的有害因素。此外，血管内皮细胞凋亡可能在脓毒症的发病机制中发挥作用。目的：我们测试了这样的假设，即系统性施用靶向Fas相关死亡结构域（FADD）的小干扰RNA（siRNA），其将半胱氨酸天冬氨酸蛋白酶原-8募集到死亡诱导信号复合物中，我们还评估了caspase-8/caspase-3 siRNA在多微生物内毒素休克小鼠模型中的治疗功效。采用盲肠结扎穿孔法（CLP）建立BALB/c小鼠多菌性脓毒症模型。siRN的体内递送 关于我们 在CLP后10小时，通过使用转染试剂（LipofectamineTM RNAiMAX）进行。作为阴性对照，动物接受无义（乱序）SiRNA.Measurements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被上调，并且FADD被高度表达和激活。DNA片段化梯和TUNEL（转移酶介导的dUTP缺口末端标记）分析显示，用siRNA治疗抑制脓毒症后肺和血管组织中的细胞凋亡诱导。此外，这些siRNA治疗预防了CLP小鼠中ALI的发展，如血气紊乱、组织学肺损伤和肺部炎性细胞增加的极大改善所示。此外，苏木精/伊红染色的脓毒症主动脉切片显示内皮细胞从基底膜部分脱离。内皮细胞中的这些组织病理学变化通过用FADD siRNA或半胱天冬酶-8/-3 siRNA的系统性治疗而被彻底阻止。最后，管理的FADD siRNA或caspase-8/-3显着改善生存的CLP mice.Conclusions：这些结果表明的病理生理意义的死亡受体凋亡途径，包括FADD，在脓毒症ALI和潜在的有用性FADD siRNA的基因治疗的脓毒症综合征。此外，用siRNA沉默半胱天冬酶-8和半胱天冬酶-3提供了对多微生物内毒素休克的深刻保护。血管内皮细胞凋亡的预防似乎是，至少部分地，负责他们的有益效果，内毒素休克。少

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