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

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的分子机制，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中起着重要作用，细胞凋亡可能在脓毒症急性肺损伤（ALI）中具有潜在的有害作用。此外，血管内皮细胞凋亡可能在脓毒症的发病机制中起作用。目的：我们验证了一种假设，即全身给药靶向fas相关死亡结构域（FADD）的小干扰RNA （siRNA）可能对脓毒性ALI和死亡率具有保护作用，该结构域将procaspase-8招募到诱导死亡的信号复合体中。我们还在小鼠多微生物内毒素休克模型中评估了caspase-8/caspase-3 siRNA的治疗效果。方法：采用盲肠结扎穿刺法（CLP）诱导BALB/c小鼠多微生物脓毒症。在CLP后10小时，使用转染试剂（Lipofectamine^(TM) RNAiMAX）进行siRN的体内递送。作为阴性对照，动物接受无意义（打乱）siRNA。结果：clp诱导的脓毒症小鼠表面死亡受体表达上调，FADD高表达和活化。DNA片段阶梯和TUNEL（转移酶介导的dUTP缺口末端标记）检测显示，sirna治疗可抑制脓毒症后肺和血管组织的凋亡诱导。此外，这些siRNA治疗阻止了CLP小鼠ALI的发展，这表明血气紊乱、组织学肺损伤和肺部炎症细胞的增加得到了极大的改善。此外，苏木精/伊红染色的脓毒主动脉切片显示内皮细胞部分脱离基膜。通过FADD siRNA或caspase-8/-3 siRNA的全身治疗，内皮细胞的这些组织病理学变化被显著阻止。最后，给药FADD siRNA或caspase-8/-3显著提高CLP小鼠的存活率。结论：这些结果表明包括FADD在内的死亡受体凋亡通路在脓毒性ALI中的病理生理学意义，以及FADD siRNA在脓毒性综合征基因治疗中的潜在用途。此外，sirna对caspase-8和caspase-3的基因沉默对多微生物内毒素休克具有深远的保护作用。预防血管内皮细胞凋亡似乎是，至少在一定程度上，负责其在内源性休克中的有益作用。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