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 7β-acetoxy-8,13-epoxy-1α,6β,9α-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.

中文翻译：

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升高的 cAMP 可防止原代大鼠肝细胞中双氯芬酸诱导的毒性：由 cAMP/cAMP 调节的鸟嘌呤核苷酸交换因子直接激活的交换蛋白介导的保护作用


长期服用非甾体类抗炎药双氯芬酸可能会诱发药物性肝损伤（DILI）。双氯芬酸引起的肝损伤的机制已部分阐明，涉及线粒体损伤。升高的 cAMP 可保护肝细胞免受胆汁酸引起的损伤。然而，目前尚不清楚 cAMP 是否可以预防 DILI，如果可以，cAMP 的哪些下游靶标参与保护机制，包括经典蛋白激酶 A (PKA) 途径或替代途径，例如由 cAMP 直接激活的交换蛋白 (EPAC) ）。本研究的目的是调查 cAMP 和/或其下游靶标是否可以防止双氯芬酸诱导的肝细胞损伤。大鼠肝细胞暴露于 400 µmol/l 双氯芬酸。分别通过caspase-3活性测定和Sytox绿染色测量细胞凋亡和坏死。通过 JC-10 染色测量线粒体膜电位 (MMP)。分别通过定量聚合酶链反应（qPCR）和蛋白质印迹评估mRNA和蛋白质表达。 cAMP 升高剂 7 β -acetoxy-8,13-epoxy-1 α ,6 β ,9 α -triHydroxylabd-14-en-11-one (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)-喹啉甲醛 (CE3F4 ₎和 ESI-O5 用于评估 cAMP 及其效应器、PKA 或 EPAC 的作用。双氯芬酸暴露诱导细胞凋亡和肝细胞中 MMP 的损失。毛喉素和 IBMX 均可阻止双氯芬酸诱导的细胞凋亡。 EPAC 抑制而非 PKA 抑制消除了毛喉素和 IBMX 的保护作用。 Forskolin 和 IBMX 保留了 MMP，而两种 EPAC 抑制剂则减弱了这种作用。 EPAC1和EPAC2均在肝细胞中表达并定位于线粒体。 cAMP 升高可保护肝细胞免受双氯芬酸诱导的细胞死亡，这一过程主要涉及 EPAC。 cAMP/EPAC 通路可能是治疗 DILI 的新靶点。