Institute for Experimental Surgery, Rostock University Medical Center, Schillingallee 69a, 18057, Rostock, Germany.
Silence Therapeutics GmbH, Berlin, Germany.
Apoptosis. 2019 Dec;24(11-12):934-945. doi: 10.1007/s10495-019-01571-7.
Acetaminophen (APAP)-induced acute liver failure (ALF) is a life-threatening disease with only a few treatment options available. Though extensive research has been conducted for more than 40 years, the underlying pathomechanisms are not completely understood. Here, we studied as to whether APAP-induced ALF can be prevented in mice by silencing the BH3-interacting domain death agonist (Bid) as a potential key player in APAP pathology. For silencing Bid expression in mice, siRNA was formulated with the liver-specific siRNA delivery system DBTC and administered 48 h prior to APAP exposure. Mice which were pre-treated with HEPES (vehicle) and siRNA served as siRNA controls. Hepatic pathology was assessed by in vivo fluorescence microscopy, molecular biology, histology and laboratory analysis 6 h after APAP or PBS exposure. Application of siRNA caused a significant decrease of mRNA and protein expression of Bid in APAP-exposed mice. Off-targets, such as cytochrome P450 2E1 and glutathione, which are known to be consumed under APAP intoxication, were comparably reduced in all APAP-exposed mice, underlining the specificity of Bid silencing. In APAP-exposed mice non-sterile inflammation with leukocyte infiltration and perfusion failure remained almost unaffected by Bid silencing. However, the Bid silencing reduced hepatocellular damage, evident by a remarkable decrease of DNA fragmented cells in APAP-exposed mice. In these mice, the expression of the pro-apoptotic protein Bax, which recently gained importance in the cell death pathway of regulated necrosis, was also significantly reduced, in line with a decrease in both, necrotic liver tissue and plasma transaminase activities. In addition, plasma levels of HMGB1, a marker of sterile inflammation, were significantly diminished. In conclusion, the liver-specific silencing of Bid expression did not protect APAP-exposed mice from microcirculatory dysfunction, but markedly protected the liver from necrotic cell death and in consequence from sterile inflammation. The study contributes to the understanding of the molecular mechanism of the APAP-induced pathogenic pathway by strengthening the importance of Bid and Bid silencing associated effects.
对乙酰氨基酚（APAP）诱导的急性肝衰竭（ALF）是一种危及生命的疾病，目前仅有少数治疗方法。尽管已经进行了 40 多年的广泛研究，但潜在的发病机制仍不完全清楚。在这里，我们研究了通过沉默 BH3 相互作用结构域死亡激动剂（Bid）是否可以预防 APAP 诱导的 ALF，Bid 是 APAP 病理中的一个潜在关键因素。为了在小鼠中沉默 Bid 表达，我们使用具有肝特异性 siRNA 递送系统 DBTC 的 siRNA 进行配方，并在 APAP 暴露前 48 小时给药。用 HEPES（载体）和 siRNA 预处理的小鼠作为 siRNA 对照。APAP 或 PBS 暴露后 6 小时通过体内荧光显微镜、分子生物学、组织学和实验室分析评估肝病理。APAP 暴露的小鼠中 siRNA 的应用导致 Bid 的 mRNA 和蛋白表达显著降低。众所周知，在 APAP 中毒的情况下，细胞色素 P450 2E1 和谷胱甘肽等靶点在所有 APAP 暴露的小鼠中都被类似地减少，这突出了 Bid 沉默的特异性。在 APAP 暴露的小鼠中，非无菌性炎症伴白细胞浸润和灌注衰竭几乎不受 Bid 沉默的影响。然而，Bid 沉默减少了肝细胞损伤，这在 APAP 暴露的小鼠中表现为 DNA 碎片化细胞的显著减少。在这些小鼠中，促凋亡蛋白 Bax 的表达也显著降低，最近 Bax 在调控性坏死的细胞死亡途径中变得重要，这与坏死性肝组织和血浆转氨酶活性的降低一致。此外，HMGB1 的血浆水平，一种无菌性炎症的标志物，显著降低。总之，Bid 表达的肝特异性沉默不能保护 APAP 暴露的小鼠免受微循环功能障碍，但显著保护肝脏免受坏死性细胞死亡，并因此免受无菌性炎症。该研究通过加强 Bid 的重要性及其相关沉默作用，有助于理解 APAP 诱导的致病途径的分子机制。
