Moniaux Nicolas, Darnaud Marion, Garbin Kévin, Dos Santos Alexandre, Guettier Catherine, Samuel Didier, Amouyal Gilles, Amouyal Paul, Bréchot Christian, Faivre Jamila
INSERM, U1193, Centre Hépatobiliaire, Villejuif, F-94800, France; Université Paris-Sud, Faculté de Médecine, Villejuif, F-94800, France.
INSERM, U1193, Centre Hépatobiliaire, Villejuif, F-94800, France; Université Paris-Sud, Faculté de Médecine, Villejuif, F-94800, France; Assistance Publique-Hôpitaux de Paris (APHP), Hôpital Universitaire Paul Brousse, Villejuif, F-94800, France.
PLoS One. 2015 May 4;10(5):e0125584. doi: 10.1371/journal.pone.0125584. eCollection 2015.
Acute liver failure (ALF) is a rapidly progressive heterogeneous illness with high mortality rate and no widely accessible cure. A promising drug candidate according to previous preclinical studies is the Reg3α (or HIP/PAP) lectin, which alleviates ALF through its free-radical scavenging activity. Here we study the therapeutic targets of Reg3α in order to gain information on the nature of the oxidative stress associated with ALF.
Primary hepatocytes stressed with the reactive oxygen species (ROS) inducers TNFα and H2O2 were incubated with a recombinant Reg3α protein. ALF was induced in C57BL/6J mice by an anti-CD95 antibody. Livers and primary hepatocytes were harvested for deoxycholate separation of cellular and extracellular fractions, immunostaining, immunoprecipitation and malondialdehyde assays. Fibrin deposition was studied by immunofluorescence in frozen liver explants from patients with ALF.
Fibrin deposition occurs during experimental and clinical acute liver injuries. Reg3α bound the resulting transient fibrin network, accumulated in the inflammatory extracellular matrix (ECM), greatly reduced extracellular ROS levels, and improved cell viability. Hepatocyte treatment with ligands of death receptors, e.g. TNFα and Fas, resulted in a twofold increase of malondialdehyde (MDA) level in the deoxycholate-insoluble fractions. Reg3α treatment maintained MDA at a level similar to control cells and thereby increased hepatocyte survival by 35%. No antioxidant effect of Reg3α was noted in the deoxycholate-soluble fractions. Preventing fibrin network formation with heparin suppressed the prosurvival effect of Reg3α.
Reg3α is an ECM-targeted ROS scavenger that binds the fibrin scaffold resulting from hepatocyte death during ALF. ECM alteration is an important pathogenic factor of ALF and a relevant target for pharmacotherapy.
急性肝衰竭（ALF）是一种快速进展的异质性疾病，死亡率高且尚无广泛可用的治愈方法。根据先前的临床前研究，一种有前景的候选药物是Reg3α（或HIP/PAP）凝集素，它通过其自由基清除活性减轻急性肝衰竭。在此，我们研究Reg3α的治疗靶点，以获取与急性肝衰竭相关的氧化应激性质的信息。
用活性氧（ROS）诱导剂TNFα和H2O2应激的原代肝细胞与重组Reg3α蛋白一起孵育。通过抗CD95抗体在C57BL/6J小鼠中诱导急性肝衰竭。收获肝脏和原代肝细胞用于细胞和细胞外组分的脱氧胆酸盐分离、免疫染色、免疫沉淀和丙二醛测定。通过免疫荧光研究急性肝衰竭患者冷冻肝外植体中的纤维蛋白沉积。
在实验性和临床急性肝损伤期间会发生纤维蛋白沉积。Reg3α结合产生的瞬时纤维蛋白网络，积聚在炎性细胞外基质（ECM）中，大大降低细胞外ROS水平，并提高细胞活力。用死亡受体配体（如TNFα和Fas）处理肝细胞导致脱氧胆酸盐不溶性组分中的丙二醛（MDA）水平增加两倍。Reg3α处理使MDA维持在与对照细胞相似的水平，从而使肝细胞存活率提高35%。在脱氧胆酸盐可溶性组分中未观察到Reg3α的抗氧化作用。用肝素阻止纤维蛋白网络形成可抑制Reg3α的促生存作用。
Reg3α是一种靶向细胞外基质的ROS清除剂，它结合急性肝衰竭期间肝细胞死亡产生的纤维蛋白支架。细胞外基质改变是急性肝衰竭的重要致病因素和药物治疗相关靶点。
