摘要

BACKGROUND & AIMS:Porphyrias result from anomalies of heme biosynthetic enzymes and can lead to cirrhosis and hepatocellular cancer. In mice, these diseases can be modeled by administration of a diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), which causes accumulation of porphyrin intermediates, resulting in hepatobiliary injury. Wnt/β-catenin signaling has been shown to be a modulatable target in models of biliary injury; thus, we investigated its role in DDC-driven injury.
METHODS:β-Catenin (Ctnnb1) knockout (KO) mice, Wnt co-receptor KO mice, and littermate controls were fed a DDC diet for 2 weeks. β-Catenin was exogenously inhibited in hepatocytes by administering β-catenin dicer-substrate RNA (DsiRNA), conjugated to a lipid nanoparticle, to mice after DDC diet and then weekly for 4 weeks. In all experiments, serum and livers were collected; livers were analyzed by histology, western blotting, and real-time PCR. Porphyrin was measured by fluorescence, quantification of polarized light images, and liquid chromatography-mass spectrometry.
RESULTS:DDC-fed mice lacking β-catenin or Wnt signaling had decreased liver injury compared to controls. Exogenous mice that underwent β-catenin suppression by DsiRNA during DDC feeding also showed less injury compared to control mice receiving lipid nanoparticles. Control livers contained extensive porphyrin deposits which were largely absent in mice lacking β-catenin signaling. Notably, we identified a network of key heme biosynthesis enzymes that are suppressed in the absence of β-catenin, preventing accumulation of toxic protoporphyrins. Additionally, mice lacking β-catenin exhibited fewer protein aggregates, improved proteasomal activity, and reduced induction of autophagy, all contributing to protection from injury.
CONCLUSIONS:β-Catenin inhibition, through its pleiotropic effects on metabolism, cell stress, and autophagy, represents a novel therapeutic approach for patients with porphyria.
LAY SUMMARY:Porphyrias are disorders resulting from abnormalities in the steps that lead to heme production, which cause build-up of toxic by-products called porphyrins. Liver is commonly either a source or a target of excess porphyrins, and complications can range from minor abnormalities to liver failure. In this report, we inhibited Wnt/β-catenin signaling in an experimental model of porphyria, which resulted in decreased liver injury. Targeting β-catenin affected multiple components of the heme biosynthesis pathway, thus preventing build-up of porphyrin intermediates. Our study suggests that drugs inhibiting β-catenin activity could reduce the amount of porphyrin accumulation and help alleviate symptoms in patients with porphyria.

译文

背景与目的: 由于血红素生物合成酶的异常，导致了肝硬化和肝癌。在小鼠中，这些疾病可以通过服用含有 3,5-diethoxycarbonyl-1，4-二氢 collidine (DDC) 的饮食来模拟，DDC 会导致卟啉中间体的积累，从而导致肝胆损伤。Wnt/β-连环蛋白信号已被证明是胆损伤模型中的一个可调节的靶点; 因此，我们研究了其在 DDC 驱动损伤中的作用。
方法: β-连环蛋白 (Ctnnb1) 基因敲除 (KO) 小鼠、 Wnt 共受体 KO 小鼠和同窝对照组被喂食 DDC 饮食 2 周。β-连环蛋白通过给予 β-连环蛋白 dicer-substrate RNA (DsiRNA)，结合脂质纳米粒，在 DDC 饮食后，然后每周 4 周后，在肝细胞中被外源性抑制。在所有实验中，收集血清和肝脏; 通过组织学、蛋白质印迹和实时聚合酶链反应分析肝脏。通过荧光、偏振光图像的定量和液相色谱-质谱联用法来测量卟啉。
结果: 与对照组相比，DDC 喂养的缺乏 β-连环蛋白或 Wnt 信号的小鼠肝损伤减少。与接受脂质纳米颗粒的对照组小鼠相比，在 DDC 喂养过程中受到 DsiRNA β-连环蛋白抑制的外源性小鼠也表现出较小的损伤。对照肝脏含有大量的卟啉沉积物，这些沉积物在缺乏 β-连环蛋白信号的小鼠中很大程度上是不存在的。值得注意的是，我们确定了一个关键血红素生物合成酶的网络，这些酶在 β-连环蛋白缺失的情况下受到抑制，防止了有毒原porphyrins 的积累。此外，缺乏 β-连环蛋白的小鼠表现出较少的蛋白质聚集体，提高蛋白酶体活性，减少自噬的诱导，所有这些都有助于保护免受损伤。
结论: β-连环蛋白抑制通过其对代谢、细胞应激和自噬的多效性，代表了一种新的治疗方法。
总结: 斑岩是由导致血红素产生的步骤中的异常导致的疾病，血红素会导致称为斑岩的有毒副产物的积累。肝脏通常是过量的斑岩的来源或目标，并发症范围从轻微异常到肝功能衰竭。在本报告中，我们在一个实验模型中抑制 Wnt/β-连环蛋白信号，导致肝损伤减少。靶向 β-连环蛋白影响血红素生物合成途径的多个组成部分，从而防止卟啉中间体的积累。我们的研究表明，抑制 β-连环蛋白活性的药物可以减少斑岩的蓄积量，并有助于缓解斑岩患者的症状。