Sorbonne Université, UPMC, Univ Paris 06, 4 Place Jussieu, 75005 Paris, France; INSERM U1174, France; Univ. Paris Sud, Université Paris Saclay, 91405 Orsay, France.
INSERM U1174, France; Univ. Paris Sud, Université Paris Saclay, 91405 Orsay, France.
J Hepatol. 2018 Sep;69(3):644-653. doi: 10.1016/j.jhep.2018.05.020. Epub 2018 May 24.
BACKGROUND & AIMS: Liver fibrosis is characterized by the accumulation of extracellular matrix produced by hepatic myofibroblasts (hMF), the activation of which is critical to the fibrogenic process. Extracellular ATP, released by dying or stressed cells, and its purinergic receptors, constitute a powerful signaling network after injury. Although the purinergic receptor P2X4 (P2RX4) is highly expressed in the liver, its functions in hMF had never been investigated during liver fibrogenesis.
In vivo, bile duct ligation was performed and methionine- and choline-deficient diet administered in wild-type and P2x4 knock-out (P2x4-KO) mice. In vitro, hMF were isolated from mouse (wild-type and P2x4-KO) and human liver. P2X4 pharmacological inhibition (in vitro and in vivo) and P2X4 siRNAs (in vitro) were used. Histological, biochemical and cell culture analysis allowed us to study P2X4 expression and its involvement in the regulation of fibrogenic and fibrolytic factors, as well as of hMF activation markers and properties.
P2X4 genetic invalidation or pharmacological inhibition protected mice from liver fibrosis and hMF accumulation after bile duct ligation or methionine- and choline-deficient diet. Human and mouse hMFs expressed P2X4, mainly in lysosomes. Invalidation of P2X4 in human and mouse hMFs blunted their activation marker expression and their fibrogenic properties. Finally, we showed that P2X4 regulates calcium entry and lysosomal exocytosis in hMF, impacting on ATP release, profibrogenic secretory profile, and transcription factor activation.
P2X4 expression and activation is critical for hMF to sustain their activated and fibrogenic phenotype. Therefore, the inactivation of P2X4 may be of therapeutic interest during liver fibrotic diseases.
During chronic injury, the liver often repairs with fibrotic tissue, which impairs liver function, and for which there is currently no treatment. We found that a previously unexplored pathway involving the purinergic receptor P2X4, can modulate fibrotic liver repair. Therefore, this receptor could be of interest in the development of novel therapies for fibrotic liver diseases.
肝纤维化的特征是肝肌成纤维细胞（hMF）产生的细胞外基质积累，其激活对于纤维化过程至关重要。细胞外 ATP 由死亡或应激细胞释放，其嘌呤能受体构成损伤后的强大信号网络。尽管嘌呤能受体 P2X4（P2RX4）在肝脏中高度表达，但在肝纤维化过程中，其在 hMF 中的功能从未被研究过。
体内，在野生型和 P2x4 敲除（P2x4-KO）小鼠中进行胆管结扎和蛋氨酸-胆碱缺乏饮食。体外，从小鼠（野生型和 P2x4-KO）和人肝中分离 hMF。使用 P2X4 药理学抑制（体内和体外）和 P2X4 siRNAs（体外）。组织学、生化和细胞培养分析使我们能够研究 P2X4 表达及其在调节纤维生成和纤维溶解因子以及 hMF 激活标志物和特性中的作用。
P2X4 基因无效或药理学抑制可防止胆管结扎或蛋氨酸-胆碱缺乏饮食后小鼠肝纤维化和 hMF 积聚。人和小鼠 hMF 表达 P2X4，主要在溶酶体中。在人和小鼠 hMF 中无效 P2X4 会减弱其激活标志物的表达及其纤维生成特性。最后，我们表明 P2X4 调节 hMF 中的钙内流和溶酶体胞吐作用，影响 ATP 释放、促纤维化分泌谱和转录因子激活。
P2X4 的表达和激活对于 hMF 维持其激活和纤维生成表型至关重要。因此，在肝纤维化疾病中，抑制 P2X4 的失活可能具有治疗意义。
在慢性损伤期间，肝脏经常用纤维组织修复，这会损害肝功能，目前尚无治疗方法。我们发现，以前未被探索的涉及嘌呤能受体 P2X4 的途径可以调节纤维性肝修复。因此，该受体可能对纤维性肝病的新型治疗方法的开发具有重要意义。
