Department of Urology, Zhongshan Hospital, Fudan University; Shanghai Key Laboratory of Organ Transplantation, 180 Fenglin Road, Shanghai 200032, PR China.
J Transl Med. 2013 Sep 13;11:210. doi: 10.1186/1479-5876-11-210.
The naked caspase-3 small interfering RNA (siRNA) infused into the renal artery during cold preservation was effective, but did not protect auto-transplant porcine kidneys with increased inflammation and apoptosis in our previous study. The mechanisms involved, in particular, whether siRNA or complementary systemic feedback eliciting innate immune responses are worthy to be further investigated.
The protein and mRNA expression of innate immunity-related molecules were detected by western blotting and quantitative PCR in the tissues previously collected from 48 h auto-transplant kidneys. The donor kidneys were retrieved from mini pigs and cold preserved by University of Wisconsin solution with/without 0.3 mg caspase-3 siRNA for 24 h.
The protein level of Toll like receptor (TLR) 3, TLR7, and their main adapters, TRIF and MyD88, was up-regulated in the siRNA preserved auto-transplant kidneys. The mRNA level of NF-κB and c-Jun was increased, as well as pro-inflammatory cytokines, including IL-1β, IL-6, TNF-α and interferon (IFN)-α, β and γ. In addition, the non-TLR RNA sensor PKR protein, but not RIG1, was also increased in the siRNA preserved auto-transplant kidneys.
The activation of innate immunity with amplified inflammatory responses in the caspase-3 siRNA preserved auto-transplant kidneys are associated with increased TLR3, TLR7 and PKR, which might be due to complementary systemic feedback, although persistent actions initiated by short-acting caspase-3 siRNA cannot be completely ruled out. These results provided valuable evidence to guide future siRNA design and pre-clinic studies.
在冷保存过程中向肾动脉注入裸 Caspase-3 小干扰 RNA（siRNA）在我们之前的研究中是有效的，但不能保护伴有炎症和细胞凋亡增加的自体移植猪肾。所涉及的机制，特别是 siRNA 或引发固有免疫反应的互补系统反馈是否值得进一步研究。
通过蛋白质印迹法和定量 PCR 检测先前从 48 小时自体移植肾脏组织中收集的与固有免疫相关分子的蛋白和 mRNA 表达。从迷你猪中取出供肾，并用含有/不含有 0.3mg Caspase-3 siRNA 的 University of Wisconsin 溶液冷保存 24 小时。
siRNA 保存的自体移植肾脏中 Toll 样受体（TLR）3、TLR7 及其主要衔接蛋白 TRIF 和 MyD88 的蛋白水平上调。NF-κB 和 c-Jun 的 mRNA 水平增加，以及促炎细胞因子，包括 IL-1β、IL-6、TNF-α 和干扰素（IFN）-α、β 和 γ。此外，siRNA 保存的自体移植肾脏中 PKR 蛋白而非 RIG1 的非 TLR RNA 传感器也增加。
在 Caspase-3 siRNA 保存的自体移植肾脏中固有免疫的激活和放大的炎症反应与 TLR3、TLR7 和 PKR 的增加有关，这可能是由于互补的系统反馈，尽管不能完全排除由短寿命的 Caspase-3 siRNA 引发的持续作用。这些结果为指导未来的 siRNA 设计和临床前研究提供了有价值的证据。
