Dept. Gastroenterology and Hepatology (F.A.A.M., M.B.-H., H.M.), Dept. Molecular Pharmacology, Groningen Research Institute of Pharmacy, Groningen Research Institute for Asthma and COPD, GRIAC (N.M., A.O., M.S.), Dept. Laboratory Medicine (M.B.-H., H.M.), University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; Inserm UMR-1048, Institut des Maladies Métaboliques et Cardiovasculaires, Univ Toulouse Paul Sabatier, Toulouse, France (F.L.); and Department of Integrative Biology and Pharmacology, Texas Therapeutics Institute, University of Texas Health Science Center at Houston, Houston, Texas (X.C.).
Dept. Gastroenterology and Hepatology (F.A.A.M., M.B.-H., H.M.), Dept. Molecular Pharmacology, Groningen Research Institute of Pharmacy, Groningen Research Institute for Asthma and COPD, GRIAC (N.M., A.O., M.S.), Dept. Laboratory Medicine (M.B.-H., H.M.), University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; Inserm UMR-1048, Institut des Maladies Métaboliques et Cardiovasculaires, Univ Toulouse Paul Sabatier, Toulouse, France (F.L.); and Department of Integrative Biology and Pharmacology, Texas Therapeutics Institute, University of Texas Health Science Center at Houston, Houston, Texas (X.C.)
Mol Pharmacol. 2021 Apr;99(4):294-307. doi: 10.1124/molpharm.120.000217. Epub 2021 Feb 11.
Chronic consumption of the nonsteroidal anti-inflammatory drug diclofenac may induce drug-induced liver injury (DILI). The mechanism of diclofenac-induced liver injury is partially elucidated and involves mitochondrial damage. Elevated cAMP protects hepatocytes against bile acid-induced injury. However, it is unknown whether cAMP protects against DILI and, if so, which downstream targets of cAMP are implicated in the protective mechanism, including the classic protein kinase A (PKA) pathway or alternative pathways like the exchange protein directly activated by cAMP (EPAC). The aim of this study was to investigate whether cAMP and/or its downstream targets protect against diclofenac-induced injury in hepatocytes. Rat hepatocytes were exposed to 400 µmol/l diclofenac. Apoptosis and necrosis were measured by caspase-3 activity assay and Sytox green staining, respectively. Mitochondrial membrane potential (MMP) was measured by JC-10 staining. mRNA and protein expression were assessed by quantitative polymerase chain reaction (qPCR) and Western blot, respectively. The cAMP-elevating agent 7-acetoxy-8,13-epoxy-1,6,9-trihydroxylabd-14-en-11-one (forskolin), the pan-phosphodiesterase inhibitor IBMX, and EPAC inhibitors 5,7-dibromo-6-fluoro-3,4-dihydro-2-methyl-1(2H)-quinoline carboxaldehyde (CE3F4 and ESI-O5 were used to assess the role of cAMP and its effectors, PKA or EPAC. Diclofenac exposure induced apoptotic cell death and loss of MMP in hepatocytes. Both forskolin and IBMX prevented diclofenac-induced apoptosis. EPAC inhibition but not PKA inhibition abolished the protective effect of forskolin and IBMX. Forskolin and IBMX preserved the MMP, whereas both EPAC inhibitors diminished this effect. Both EPAC1 and EPAC2 were expressed in hepatocytes and localized in mitochondria. cAMP elevation protects hepatocytes against diclofenac-induced cell death, a process primarily involving EPACs. The cAMP/EPAC pathway may be a novel target for treatment of DILI. SIGNIFICANCE STATEMENT: This study shows two main highlights. First, elevated cAMP levels protect against diclofenac-induced apoptosis in primary hepatocytes via maintenance of mitochondrial integrity. In addition, this study proposes the existence of mitochondrial cAMP-EPAC microdomains in rat hepatocytes, opening new avenues for targeted therapy in drug-induced liver injury (DILI). Both EPAC1 and EPAC2, but not protein kinase A, are responsible for this protective effect. Our findings present cAMP-EPAC as a potential target for the treatment of DILI and liver injury involving mitochondrial dysfunction.
慢性使用非甾体类抗炎药双氯芬酸可能会导致药物性肝损伤（DILI）。双氯芬酸诱导肝损伤的机制部分阐明，涉及线粒体损伤。升高的 cAMP 可保护肝细胞免受胆汁酸诱导的损伤。然而，目前尚不清楚 cAMP 是否能预防 DILI，如果能，cAMP 的哪些下游靶点参与了保护机制，包括经典的蛋白激酶 A（PKA）途径或 EPAC 等替代途径。本研究旨在探讨 cAMP 及其下游靶点是否能预防肝细胞中双氯芬酸诱导的损伤。用 400μmol/L 的双氯芬酸处理大鼠肝细胞。通过 caspase-3 活性测定和 Sytox 绿染色分别测定细胞凋亡和坏死。通过 JC-10 染色测定线粒体膜电位（MMP）。通过定量聚合酶链反应（qPCR）和 Western blot 分别评估 mRNA 和蛋白质表达。用 cAMP 升高剂 7-乙酰氧基-8,13-环氧-1,6,9-三羟基-14-烯-11-酮（forskolin）、泛磷酸二酯酶抑制剂 IBMX 和 EPAC 抑制剂 5,7-二溴-6-氟-3,4-二氢-2-甲基-1(2H)-喹啉羧酸醛（CE3F4 和 ESI-O5 来评估 cAMP 及其效应物 PKA 或 EPAC 的作用。双氯芬酸暴露诱导肝细胞凋亡和 MMP 丧失。forskolin 和 IBMX 均可预防双氯芬酸诱导的细胞凋亡。EPAC 抑制而非 PKA 抑制消除了 forskolin 和 IBMX 的保护作用。forskolin 和 IBMX 可维持 MMP，而两种 EPAC 抑制剂均减弱了这一作用。EPAC1 和 EPAC2 均在肝细胞中表达并定位于线粒体。cAMP 升高可防止肝细胞发生双氯芬酸诱导的细胞死亡，这一过程主要涉及 EPAC。cAMP/EPAC 途径可能是治疗 DILI 的新靶点。本研究有两个主要发现。首先，升高的 cAMP 水平通过维持线粒体完整性来防止原代肝细胞中双氯芬酸诱导的细胞凋亡。此外，该研究提出了大鼠肝细胞中线粒体 cAMP-EPAC 微区的存在，为药物性肝损伤（DILI）的靶向治疗开辟了新途径。EPAC1 和 EPAC2 而非蛋白激酶 A 负责这种保护作用。我们的研究结果表明 cAMP-EPAC 可能成为治疗 DILI 和涉及线粒体功能障碍的肝损伤的潜在靶点。
