Mora Fabio Alejandro Aguilar, Musheshe Nshunge, Arroyave Ospina Johanna C, Geng Yana, Soto Juan M, Rodrigo José A, Alieva Tatiana, Buist-Homan Manon, Lezoualc'h Frank, Cheng Xiaodong, Schmidt Martina, Moshage Han
Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
Deptartment Molecular Pharmacology, Groningen Research Institute of Pharmacy, Groningen Research Institute for Asthma and COPD, GRIAC, University Medical Center Groningen University of Groningen, Groningen, The Netherlands.
Biomed Pharmacother. 2021 Nov;143:112072. doi: 10.1016/j.biopha.2021.112072. Epub 2021 Aug 28.
It has been shown that the antidiabetic drug metformin protects hepatocytes against toxicity by various stressors. Chronic or excessive consumption of diclofenac (DF) - a pain-relieving drug, leads to drug-induced liver injury via a mechanism involving mitochondrial damage and ultimately apoptotic death of hepatocytes. However, whether metformin protects against DF-induced toxicity is unknown. Recently, it was also shown that cAMP elevation is protective against DF-induced apoptotic death in hepatocytes, a protective effect primarily involving the downstream cAMP effector EPAC and preservation of mitochondrial function. This study therefore aimed at investigating whether metformin protects against DF-induced toxicity via cAMP-EPACs.
Primary rat hepatocytes were exposed to 400 µmol/L DF. CE3F4 or ESI-O5 were used as EPAC-1 or 2 inhibitors respectively. Apoptosis was measured by caspase-3 activity and necrosis by Sytox green staining. Seahorse X96 assay was used to determine mitochondrial function. Mitochondrial reactive oxygen species (ROS) production was measured using MitoSox, mitochondrial MnSOD expression was determined by immunostaining and mitochondrial morphology (fusion and fission ratio) by 3D refractive index imaging.
Metformin (1 mmol/L) was protective against DF-induced apoptosis in hepatocytes. This protective effect was EPAC-dependent (mainly EPAC-2). Metformin restored mitochondrial morphology in an EPAC-independent manner. DF-induced mitochondrial dysfunction which was demonstrated by decreased oxygen consumption rate, an increased ROS production and a reduced MnSOD level, were all reversed by metformin in an EPAC-dependent manner.
Metformin protects hepatocytes against DF-induced toxicity via cAMP-dependent EPAC-2.
已有研究表明，抗糖尿病药物二甲双胍可保护肝细胞免受多种应激源的毒性作用。慢性或过量服用双氯芬酸（DF）——一种止痛药物，会通过涉及线粒体损伤并最终导致肝细胞凋亡死亡的机制，引发药物性肝损伤。然而，二甲双胍是否能预防DF诱导的毒性尚不清楚。最近，研究还表明，cAMP升高可保护肝细胞免受DF诱导的凋亡死亡，这种保护作用主要涉及下游cAMP效应物EPAC以及线粒体功能的维持。因此，本研究旨在探讨二甲双胍是否通过cAMP-EPAC途径预防DF诱导的毒性。
原代大鼠肝细胞暴露于400 μmol/L的DF中。分别使用CE3F4或ESI-O5作为EPAC-1或EPAC-2抑制剂。通过半胱天冬酶-3活性检测细胞凋亡，用Sytox green染色检测细胞坏死。采用Seahorse X96分析仪测定线粒体功能。使用MitoSox检测线粒体活性氧（ROS）的产生，通过免疫染色测定线粒体锰超氧化物歧化酶（MnSOD）的表达，并通过三维折射率成像测定线粒体形态（融合与裂变比率）。
二甲双胍（1 mmol/L）可保护肝细胞免受DF诱导的凋亡。这种保护作用依赖于EPAC（主要是EPAC-2）。二甲双胍以不依赖于EPAC的方式恢复线粒体形态。DF诱导的线粒体功能障碍表现为氧消耗率降低、ROS产生增加和MnSOD水平降低，而二甲双胍以依赖于EPAC的方式将这些指标全部逆转。
二甲双胍通过依赖于cAMP的EPAC-2保护肝细胞免受DF诱导的毒性。
