Abstract
p66Shc, a master regulator of mitochondrial reactive oxygen species (mtROS), is a crucial mediator of hepatocyte oxidative stress. However, its functional contribution to acetaminophen (APAP)-induced liver injury and the mechanism by which it is modulated remain unknown. Here, we aimed to assess the effect of p66Shc on APAP-induced liver injury and to evaluate if circular RNA (circRNA) functions as a competitive endogenous RNA (ceRNA) to mediate p66Shc in APAP-induced liver injury. p66Shc-, miR-185-5p-, and circ-CBFB-silenced mice were injected with APAP. AML12 cells were transfected with p66Shc, miR-185-5p, and circ-CBFB silencing or overexpression plasmids or siRNAs prior to APAP stimulation. p66Shc was upregulated in liver tissues in response to APAP, and p66Shc silencing in vivo protected mice from APAP-induced mitochondrial dynamics perturbation and liver injury. p66Shc knockdown in vitro attenuated mitochondrial dynamics and APAP-induced hepatocyte injury. Mechanically, p66Shc perturbs mitochondrial dynamics partially by inhibiting OMA1 ubiquitination. miR-185-5p, which directly suppressed p66Shc translation, was identified by microarray and bioinformatics analyses, and its overexpression attenuated mitochondrial dynamics and hepatocyte injury in vitro. Furthermore, luciferase, pull-down and RNA immunoprecipitation assays demonstrated that circ-CBFB acts as a miRNA sponge of miR-185-5p to mediate p66Shc in APAP-induced liver injury. circ-CBFB knockdown also alleviated APAP-induced mitochondrial dynamics perturbation and hepatocyte injury. More importantly, we found that the protective effects of circ-CBFB knockdown on p66Shc, mitochondrial dynamics and liver injury were abolished by miR-185-5p inhibition both in vivo and in vitro. In conclusion, p66Shc is a key regulator of APAP-induced liver injury that acts by triggering mitochondrial dynamics perturbation. circ-CBFB functions as a ceRNA to regulate p66Shc during APAP-induced liver injury, which may provide a potential therapeutic target.
Highlights
Acetaminophen (APAP) is a safe antipyretic and analgesic drug at therapeutic levels
We first addressed the crucial role of p66Shc in APAP-induced liver injury
We uncovered that p66Shc perturbed mitochondrial dynamics execution in the liver and hepatocytes
Summary
Acetaminophen (APAP) is a safe antipyretic and analgesic drug at therapeutic levels. APAP overdose is the most common cause of acute liver failure in the US and UK1. APAP is metabolized in hepatocytes and converted by cytochrome P-450 2E1 (CYP2E1) to. N-acetyl-p-benzoquinoneimine (NAPQI), which covalently binds to intracellular proteins, especially mitochondrial proteins, to form APAP adducts[2,3]. The formation of APAP adducts in the mitochondria results in mitochondrial dysfunction, releasing mitochondrial cell death factors and leading to nuclear DNA fragmentation and hepatocyte death[4,5,6]. Hepatocyte survival and the therapeutic outcome of APAP-induced liver injury may benefit from treatments that minimize mitochondrial damage and/or enhance mitochondrial function.
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