Peroxiredoxin 3 Inhibits Acetaminophen-Induced Liver Pyroptosis Through the Regulation of Mitochondrial ROS

Abstract

Pyroptosis is a newly discovered form of cell death. Peroxiredoxin 3 (PRX3) plays a crucial role in scavenging reactive oxygen species (ROS), but its hepatoprotective capacity in acetaminophen (APAP)-induced liver disease remains unclear. The aim of this study was to assess the role of PRX3 in the regulation of pyroptosis during APAP-mediated hepatotoxicity. We demonstrated that pyroptosis occurs in APAP-induced liver injury accompanied by intense oxidative stress and inflammation, and liver specific PRX3 silencing aggravated the initiation of pyroptosis and liver injury after APAP intervention. Notably, excessive mitochondrial ROS (mtROS) was observed to trigger pyroptosis by activating the NLRP3 inflammasome, which was ameliorated by Mito-TEMPO treatment, indicating that the anti-pyroptotic role of PRX3 relies on its powerful ability to regulate mtROS. Overall, PRX3 regulates NLRP3-dependent pyroptosis in APAP-induced liver injury by targeting mitochondrial oxidative stress.

Keywords: APAP; NLRP3 inflammasome; PRX3; mitochondrial ROS (mtROS); pyroptosis.

Figure 1

Pyroptosis occurs in mice with APAP-induced liver injury. Liver and serum samples were obtained from mice treated with APAP (300 mg/kg) for 12 h. (A) Serum levels of ALT and AST were assessed, n=8. (B) Liver sections were stained with H&E (50 μm), and the damaged area are indicated with arrows. (C) Serum levels of LDH were assessed, n=8. (D) Pro-GSDMD/GSDMD-N, pro-caspase-1/caspase-1 p20, pro-IL-1β/IL-1β, and pro-IL-18/IL-18 protein levels in mice liver were determined, n=3. (E) Serum levels of IL-1β. (F) Serum levels of IL-18, n=8. **P<0.01.

Figure 2

Primary KCs and hepatocytes undergo pyroptosis upon APAP treatment. Primary cells were plated on rat tail collagen-coated plates, and APAP (10 mM/well) was administered after stable adherence. (A) Kupffer cellular supernatant levels of LDH were assessed, n=6. (B) GSDMD-N, caspase-1 p20, IL-1β, and IL-18 protein levels in primary KCs were determined, n=3. (C) Pro-GSDMD/GSDMD-N, pro-caspase-1/caspase-1 p20, pro-IL-1β/IL-1β, and pro-IL-18/IL-18 protein levels in primary hepatocytes were determined, n=3. (D) Hepatocellular supernatant levels of LDH were assessed, n=6. (E) Primary hepatocytes were obtained from untreated C57BL/6 mice and reseeded in a monolayer or the lower chamber of a transwell system. For coculture, primary KCs from untreated C57BL/6 mice were reseeded in the upper chamber of the transwell system. The levels of the indicated proteins (GSDMD-N, caspase-1 p20, IL-1β, IL-18) in the primary hepatocytes from the monolayer or transwell cultures were detected by western blotting, n=3. **P<0.01.

Figure 3

PRX3 silencing accelerates pyroptosis in APAP-induced hepatotoxicity in mice. (A) PRX3 protein levels in mice liver were determined, n=3. (B) Liver PRX3 mRNA expression, n=6. (C) Schematic representation of AAV9-TBG-shRNA injection through tail vein in mice. (D) Liver sections were analyzed by immunohistochemical staining for PRX3 (50μm). (E) Liver sections were stained with H&E (50μm). The damaged areas are indicated with arrows. (F) Serum levels of ALT and AST were assessed, n=8. (G) Serum levels of LDH were assessed, n=8. (H, I) Serum levels of IL-1β and IL-18, n=8. (J) PRX3, caspase-1 p20 and GSDMD-Nin mice liver were determined, n=3. (K) H₂O₂ level, n = 8.**P<0.01.

Figure 4

PRX3 regulates NLRP3-mediated pyroptosis in APAP-induced hepatotoxicity in vitro. Primary KCs and hepatocytes were transfected with NLRP3-specific siRNA and then exposed to APAP (10 mM). (A) Kupffer cellular supernatant levels of LDH were assessed, n=6. (B) NLRP3, GSDMD-N, caspase-1 p20, IL-1β, and IL-18 protein levels in primary KCs, n=3. (C) Hepatocellular supernatant levels of LDH were assessed, n=6. (D) NLRP3, GSDMD-N, caspase-1 p20, IL-1β, and IL-18 protein levels in primary hepatocytes, respectively, n=3. (E) Primary hepatocytes were transfected with pcDNA-PRX3 and then exposed to APAP (10 mM). PRX3, GSDMD-N, caspase-1 p20, IL-1β, and IL-18 protein levels in primary hepatocytes were determined, n=3. (F) Hepatocellular supernatant levels of LDH were assessed, n=6. (G) NLRP3 protein levels in primary hepatocytes, n=3. (H) Double immunofluorescence staining for PRX3 and NLRP3 in primary hepatocytes, 50 μm. (I) Hepatocellular supernatant levels of LDH were assessed, n=6. (J) Primary hepatocytes were transfected with si-PRX3 and then exposed to APAP (10 mM). PRX3, GSDMD-N, caspase-1 p20, IL-1β and IL-18 protein levels in primary hepatocytes were determined, n=3. (K) NLRP3 protein levels in primary hepatocytes, n=3. **P<0.01, *P<0.05.

Figure 5

PRX3 regulates the NLRP3 inflammasome through mitochondrial ROS after APAP overdose. Primary KCs and hepatocytes were cotransfected with PRX3- and NLRP3-specific siRNAs following APAP challenge. (A) Kupffer cellular supernatant levels of LDH were assessed, n=6. (B) NLRP3, GSDMD-N, caspase-1 p20, IL-1β and IL-18 protein levels in primary KCs n=3. **P<0.01. (C) Hepatocellular supernatant levels of LDH were assessed, n=6. (D) NLRP3, GSDMD-N, caspase-1 p20, IL-1β and IL-18 protein levels in primary hepatocytes, n=3. **P<0.01. PRX3 suppression was applied in primary hepatocytes via PRX3-specific siRNA transfection and subsequent Mito-TEMPO (0.2 mM) treatment before APAP intervention. (E) Representative fluorescence images of MitoSOX, 50 μm. (F) hepatocellular supernatant levels of LDH were assessed, n=6. (G) NLRP3, GSDMD-N, caspase-1 p20, IL-1β, and IL-18 protein levels in primary hepatocytes, n=3. **P<0.01.

Figure 6

Illustration of the mechanism by which PRX3 alleviates APAP-induced pyroptosis. Excessive APAP promotes NLRP3-mediated pyroptosis in mice. The crosstalk between KCs and hepatocytes amplifies the pyroptotic signal. PRX3 overexpression alleviates the above process by disrupting NLRP3 inflammasome activation. Moreover, PRX3 regulates NLRP3 inflammasome activation by targeting mitochondrial ROS.