Peroxiredoxin 3 Inhibits Acetaminophen-Induced Liver Pyroptosis Through the Regulation of Mitochondrial ROS.
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ABSTRACT:
SUBMITTER: Wang Y
PROVIDER: S-EPMC8155593 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Acetaminophen (APAP) is one of the most frequently used drugs; however, its overdose leads to acute liver injury. Recently, studies have reported that the adduction of peroxiredoxin 6 (PRDX6), a member of the PRDX family of antioxidant enzymes, is associated with liver diseases. However, the role of PRDX6 in APAP-induced liver injury remains unclear. Here, we assessed both age-matched (about 12 weeks) PRDX6-overexpressing transgenic mice (PRDX6 mice) and wild type (WT) mice presenting acute liver injury induced by the intraperitoneal injection of APAP (500 mg/kg). Although PRDX6 is known as an antioxidant enzyme, PRDX6 mice unexpectedly demonstrated severe liver injury following APAP injection compared with WT mice. We observed that PRDX6 was hyperoxidized after APAP administration. Additionally, calcium-independent phospholipase A2 (iPLA2) activity and lysophosphatidylcholine (LPC) levels were markedly elevated in PRDX6 mice following APAP administration. Moreover, APAP-induced JNK phosphorylation was considerably increased in the liver of PRDX6 mice. MJ33, an inhibitor of PRDX6, attenuated APAP-induced liver injury both in WT and PRDX6 mice. Notably, MJ33 reduced the APAP-induced increase in JNK activation, iPLA2 activity, and LPC levels. Although SP600125, a JNK inhibitor, abolished APAP-induced liver injury, it failed to affect the APAP-induced hyperoxidation of PRDX6, iPLA2 activity, and LPC levels. These results suggested that PRDX6 was converted to the hyperoxidized form by the APAP-induced high concentration of hydrogen peroxides. In the liver, hyperoxidized PRDX6 induced cellular toxicity via JNK activation by enhancing iPLA2 activity and LPC levels; this mechanism appears to be a one-way cascade.
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Project description:Brevilin A (BA), a sesquiterpene lactone isolated from Centipeda minima herb, has been identified to exhibit potent anticancer activity. However, the potential pharmacological effect and mechanism of BA in regulating endothelial cell (EC) angiogenesis, a key event in tumor growth, is poorly understood. In this study, BA was shown to significantly prevent vascular endothelial growth factor (VEGF) induced EC angiogenic capacities in vitro, ex vivo, and in vivo. Subsequent functional assays revealed that BA dose dependently inhibited VEGF-stimulated survival, proliferation, migration, and triggered apoptosis activity in human umbilical vein endothelial cells (HUVECs), as well as suppressed the expression of antiapoptotic protein Bcl-2, increased the expression of proapoptotic protein caspase-3 and Bax, and suppressed PI3K/AKT pathway. Meanwhile, BA was also able to depolarize mitochondrial membranal permeability (MMP), accelerate mitochondrial superoxide accumulation, induce intracellular reactive oxygen species (ROS) production, and decreased intracellular glutathione (GSH) in HUVECs. Furthermore, both mitochondria-specific superoxide scavenger Mito-TEMPOL and broad-spectrum antioxidant N-acetyl-cysteine (NAC) dramatically abolished BA-induced mitochondrial dysfunction and mitochondrial ROS production, causing the reversion of PI3K/AKT pathway and repression of apoptosis, eventually correcting the impaired endothelial behavior in survival, growth, migration, and angiogenesis. Collectively, our data for the first time identified a new mechanism for antiangiogenic effect of BA in vascular EC, one that is based on the regulation of mitochondrial-dependent ROS overproduction.
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Project description:Acetaminophen (APAP) overdose is the primary cause of drug‑induced acute liver failure in numerous Western countries. NLR family pyrin domain containing 3 (NLRP3) inflammasome activation serves a pivotal role in the pathogenesis of various forms of acute liver injury. However, the cellular source for NLRP3 induction and its involvement during APAP‑induced hepatotoxicity have not been thoroughly investigated. In the present study, hematoxylin and eosin staining was performed to assess histopathological changes of liver tissue. Immunohistochemistry staining(NLRP3, Caspase‑1, IL‑1β, GSDMD and Caspase‑3), western blotting (NLRP3, Caspase‑1, IL‑1β, GSDMD and Caspase‑3) and RT‑qPCR (NLRP3, Caspase‑1 and IL‑1β) were performed to assess the expression of NLRP3/GSDMD signaling pathway. TUNEL staining was performed to assess apoptosis of liver tissue. The serum expression levels of inflammatory factors (IL‑6, IL‑18, IL‑1β and TNF‑α) were assessed using ELISA and inflammation of liver tissue was assessed using immunohistochemistry (Ly6G and CD68) and RT‑qPCR (TNF‑α, Il‑6, Mcp‑1, Cxcl‑1, Cxcl‑2). A Cell Counting Kit‑8 was performed to assess cell viability and apoptosis. Protein and gene expression were analyzed by western blotting (PCNA, CCND1) and RT‑qPCR (CyclinA2, CyclinD1 and CyclinE1). Through investigation of an APAP‑induced acute liver injury model (AILI), the present study demonstrated that APAP overdose induced activation of NLRP3 and cleavage of gasdermin D (GSDMD) in hepatocytes, both in vivo and in vitro. Additionally, mice with hepatocyte‑specific knockout of Nlrp3 exhibited reduced liver injury and lower mortality following APAP intervention, accompanied by decreased infiltration of inflammatory cells and attenuated inflammatory response. Furthermore, pharmacological blockade of NLRP3/GSDMD signaling using MCC950 or disulfiram significantly ameliorated liver injury and reduced hepatocyte death. Notably, hepatocyte Nlrp3 deficiency promoted liver recovery by enhancing hepatocyte proliferation. Collectively, the present study demonstrated that inhibition of the NLRP3 inflammasome protects against APAP‑induced acute liver injury by reducing hepatocyte pyroptosis and suggests that targeting NLRP3 may hold therapeutic potential for treating AILI.
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Project description:The mechanism by which acetaminophen (APAP) causes liver damage evokes many aspects drug metabolism, oxidative chemistry, and genetic-predisposition. In this study, we leverage the relative resistance of female C57BL/6 mice to APAP-induced liver damage (AILD) compared to male C57BL/6 mice in order to identify the cause(s) of sensitivity. Furthermore, we use mice that are either heterozygous (HZ) or null (KO) for glutamate cysteine ligase modifier subunit (Gclm), in order to titrate the toxicity relative to wild-type (WT) mice. Gclm is important for efficient de novo synthesis of glutathione (GSH). APAP (300 mg/kg, ip) or saline was administered and mice were collected at 0, 0.5, 1, 2, 6, 12, and 24 h. Male mice showed marked elevation in serum alanine aminotransferase by 6 h. In contrast, female WT and HZ mice showed minimal toxicity at all time points. Female KO mice, however, showed AILD comparable to male mice. Genotype-matched male and female mice showed comparable APAP-protein adducts, with Gclm KO mice sustaining significantly greater adducts. ATP was depleted in mice showing toxicity, suggesting impaired mitochondria function. Indeed, peroxiredoxin-6, a GSH-dependent peroxiredoxin, was preferentially adducted by APAP in mitochondria of male mice but rarely adducted in female mice. These results support parallel mechanisms of toxicity where APAP adduction of peroxiredoxin-6 and sustained GSH depletion results in the collapse of mitochondria function and hepatocyte death. We conclude that adduction of peroxiredoxin-6 sensitizes male C57BL/6 mice to toxicity by acetaminophen.
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Project description:The signaling pathways governing acetaminophen (APAP)-induced liver injury have been extensively studied. However, little is known about the ubiquitin-modifying enzymes needed for the regulation of APAP-induced liver injury. Here, we examined whether the Pellino3 protein, which has E3 ligase activity, is needed for APAP-induced liver injury and subsequently explored its molecular mechanism. Whole-body Peli3-/- knockout (KO) and adenovirus-mediated Peli3 knockdown (KD) mice showed reduced levels of centrilobular cell death, infiltration of immune cells, and biomarkers of liver injury, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), upon APAP treatment compared to wild-type (WT) mice. Peli3 deficiency in primary hepatocytes decreased mitochondrial and lysosomal damage and reduced the mitochondrial reactive oxygen species (ROS) levels. In addition, the levels of phosphorylation at serine 9 in the cytoplasm and mitochondrial translocation of GSK3β were decreased in primary hepatocytes obtained from Peli3-/- KO mice, and these reductions were accompanied by decreases in JNK phosphorylation and mitochondrial translocation. Pellino3 bound more strongly to GSK3β compared with JNK1 and JNK2 and induced the lysine 63 (K63)-mediated polyubiquitination of GSK3β. In rescue experiments, the ectopic expression of wild-type Pellino3 in Peli3-/- KO hepatocytes restored the mitochondrial translocation of GSK3β, but this restoration was not obtained with expression of a catalytically inactive mutant of Pellino3. These findings are the first to suggest a mechanistic link between Pellino3 and APAP-induced liver injury through the modulation of GSK3β polyubiquitination.
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Project description:Recent studies have demonstrated that ovarian granular cells (OGCs) pyroptosis is present in the ovaries of polycystic ovary syndrome (PCOS) mice and that NLRP3 activation destroys follicular functions. Metformin has been shown to protect against PCOS by reducing insulin resistance in women, whereas its role in OGC pyroptosis is unknown. This study aimed to investigate the impact of metformin on OGC pyroptosis and the underlying mechanisms. The results showed that treating a human granulosa-like tumor cell line (KGN) with metformin significantly decreased LPS-induced expression of miR-670-3p, NOX2, NLRP3, ASC, cleaved caspase-1, and GSDMD-N. Cellular caspase-1 activity; ROS production; oxidative stress; and the secretion of IL-1β, IL-6, IL-18, and TNF-α were also diminished. These effects were amplified by adding N-acetyl-L-cysteine (NAC), a pharmacological inhibitor of ROS. In contrast, metformin's anti-pyroptosis and anti-inflammatory effects were robustly ameliorated by NOX2 overexpression in KGN cells. Moreover, bioinformatic analyses, RT-PCR, and Western blotting showed that miR-670-3p could directly bind to the NOX2 (encoded by the CYBB gene in humans) 3'UTR and decrease NOX2 expression. Metformin-induced suppression of NOX2 expression, ROS production, oxidative stress, and pyroptosis was significantly alleviated by transfection with the miR-670-3p inhibitor. These findings suggest that metformin inhibits KGN cell pyroptosis via the miR-670-3p/NOX2/ROS pathway.
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Project description:Liver injury caused by acetaminophen (AP) overdose is a leading public health problem. Although AP-induced liver injury is well recognized as the formation of N-acetyl-p-benzoquinone (NAPQI), a toxic metabolite of AP, resulting in cell damage, emerging evidence indicates that AP-induced liver injury is also associated with gut microbiota. However, the gut microbiota-involved mechanism remains largely unknown. In our study, we found that vancomycin (Vac) pretreatment (100?mg/kg, twice a day for 4 days) attenuated AP-induced liver injury, altered the composition of gut microbiota, and changed serum metabolic profile. Moreover, we identified Vac pretreatment elevated cecum and serum 2-hydroxybutyric acid (2-HB), which ameliorated AP-induced cell damage and liver injury in mice by reducing AP bioavailability and elevating GSH levels. Our current results revealed the novel role of 2-HB in protecting AP-induced liver injury and add new evidence for gut microbiota in affecting AP toxicity. Graphical abstract Image 1 Highlights • Vac pretreatment attenuated AP-induced liver injury in rats.• Vac pretreatment elevated metabolite 2-HB both in cecum and serum.• 2-HB attenuated the AP-induced hepatotoxicity both in vitro and in vivo.
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Project description:Acetaminophen (APAP), a widely used analgesic/antipyretic agent, can cause liver injury through increased nitrative stress, leading to protein nitration. However, the identities of nitrated proteins and their roles in hepatotoxicity are poorly understood. Thus, we aimed at studying the mechanism of APAP-induced hepatotoxicity by systematic identification and characterization of nitrated proteins in the absence or presence of an antioxidant, N-acetylcysteine (NAC). The levels of nitrated proteins markedly increased at 2h in mice exposed to a single APAP dose (350mg/kg ip), which caused severe liver necrosis at 24h. Protein nitration and liver necrosis were minimal in mice exposed to nontoxic 3-hydroxyacetanilide or animals co-treated with APAP and NAC. Mass-spectral analysis of the affinity-purified nitrated proteins identified numerous mitochondrial and cytosolic proteins, including mitochondrial aldehyde dehydrogenase, Mn-superoxide dismutase, glutathione peroxidase, ATP synthase, and 3-ketoacyl-CoA thiolase, involved in antioxidant defense, energy supply, or fatty acid metabolism. Immunoprecipitation followed by immunoblot with anti-3-nitrotyrosine antibody confirmed that the aforementioned proteins were nitrated in APAP-exposed mice but not in NAC-cotreated mice. Consistently, NAC cotreatment significantly restored the suppressed activity of these enzymes. Thus, we demonstrate a new mechanism by which many nitrated proteins with concomitantly suppressed activity promotes APAP-induced mitochondrial dysfunction and hepatotoxicity.
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Project description:The flavonoid quercitrin has a strong antioxidant property. It is also reported to have a protective effect on the liver. However, the mechanism by which it exerts a protective effect on the liver is not fully understood. The objective of this article is to confirm the protective effect of quercitrin extracted from Albiziae flos on acetaminophen (APAP)-induced liver injury and to explain its mechanism. In the in vivo study, quercitrin was administered orally to BALB/c mice at a dose of 50, 100, and 200 mg/kg for seven consecutive days. APAP (300 mg/kg) was injected intraperitoneally after a last dose of quercitrin was administered. Determination of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), reactive oxygen species (ROS), superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GSH-Px), catalase (CAT), and malondialdehyde (MDA) levels showed that quercitrin effectively attenuated APAP-induced acute liver injury in mice. Results of the in vitro study showed that quercitrin reduced the levels of ROS, protected mitochondria from damage, and restored the activity of mitochondrial complex I in APAP-treated L-02 cells. The addition of rotenone which is an inhibitor of complex I blocked the protective effect of quercitrin. The expression of mitochondrial complex I was also maintained by quercitrin. Our results suggest that quercitrin can maintain the level of mitochondrial complex I in injured cells and restore its activity, which reduces the production of ROS, protects the mitochondria from oxidative stress, and has a protective effect on the liver.
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