Thonningianin A ameliorates acetaminophen-induced liver injury by activating GPX4 and modulating endoplasmic reticulum stress

Abstract

Introduction: Acetaminophen (APAP) is widely used as an analgesic and antipyretic. However overdose APAP can lead to acute liver injury (ALI), representing a significant challenge for public health due to limited treatment options. Current research highlights the need for safer and more effective therapies for APAP-induced liver injury, especially those that target oxidative and endoplasmic reticulum (ER) stress pathways. This study investigates the protective effects of Thonningianin A (TA), a flavonoid compound derived from Penthorum chinense Pursh, in mitigating APAP-induced hepatotoxicity.

Methods: The experimental design involved administering TA at doses of 20 mg/kg and 40 mg/kg to C57BL/6 mice prior to inducing hepatotoxicity with APAP.

Results and discussion: TA treatment significantly lowered plasma ALT and AST levels, inhibited the production of inflammatory cytokines, and reduced oxidative stress markers in liver tissues. Furthermore, TA modulated apoptosis-related proteins by increasing BCL-2 expression while decreasing CHOP and BAX levels. It alleviated endoplasmic reticulum (ER) stress by downregulating GRP78, p-PERK, and ATF4. Notably, liver-specific GPX4 knockdown, achieved through AAV-8-mediated shRNA delivery, abolished the hepatoprotective effects of TA, underscoring GPX4's essential role in mediating TA-induced hepatoprotection. These findings suggest TA as a promising therapeutic agent in managing APAP-induced liver injury, with its unique action on both oxidative and ER stress pathways contributing to its hepatoprotective efficacy.

Keywords: Acetaminophen; GPx4; acute liver injury; endoplasmic reticulum stress; hepatotoxicity; thonningianin A.

FIGURE 1

TA ameliorates APAP-induced liver injury in mice. (A) Schematic Diagram of the Animal Experiment. Fasted for 16 h, mice were administered TA [20 or 40 mg/kg, i.g.] 2 h prior to saline or APAP (300 mg/kg, i.p.) for 12 h. (B) Plasma levels of ALT. (C) Plasma levels of AST. (D) Liver tissues were subjected to H&E staining for histological examination. (E) Concentrations of hepatic IL-1β, IL-6, TNF-α, and MCP-1 were quantified using commercially available ELISA kits. All data were presented as mean ± SD, n = 5–8 per group. *P < 0.05 vs. corresponding control.

FIGURE 2

TA inhibits APAP-induced hepatocellular apoptosis. (A) Western blotting assessed hepatic BCL2 and BAX expression, with β-actin as a loading control. Band intensities were quantified using ImageJ. (B) q-PCR was conducted to evaluate the transcript levels of BCL2 and BAX-related genes. (C) Hepatic caspase 3 activity was quantified using a commercially available kit (fold of control). (D) Western blotting assessed hepatic CHOP expression, with β-actin as a loading control. Band intensities were quantified using ImageJ. (E) q-PCR was conducted to evaluate the transcript levels of the Chop gene. All data were presented as mean ± SD, n = 3 for Western blotting, n = 5–8 for others. *P < 0.05 vs. corresponding control.

FIGURE 3

TA mitigates APAP-induced hepatotoxicity through the alleviation of ER stress. (A) Western blotting assessed hepatic GRP78 expression, with β-actin as a loading control. Band intensities were quantified using ImageJ. (B) q-PCR was conducted to evaluate the transcript levels of the Grp78 gene. (C) Western blotting assessed hepatic phosphorylated-PERK, phosphorylated-eIF2α, PERK, and eIF2α protein expressions. Band intensities were quantified using ImageJ. (D) Western blotting assessed hepatic ATF4 expression, with β-actin as a loading control. Band intensities were quantified using ImageJ. (E) q-PCR was conducted to evaluate the transcript levels of the Atf4 gene. All data were presented as mean ± SD, n = 3 for Western blotting, n = 6 for others. *P < 0.05 vs. corresponding control.

FIGURE 4

TA mediates the enhancement of hepatic antioxidant function by GPX4. (A) Hepatic concentrations of MDA, alongside the enzymatic activities of SOD and CAT. (B) Hepatic concentrations of GSH-Px, GSH, and the GSH redox ratio (GSH/GSSG) were determined. (C) Western blotting assessed hepatic GPX4 expression, with β-actin as a loading control. Band intensities were quantified using ImageJ. (D) q-PCR was conducted to evaluate the transcript levels of the Gpx4 gene. All data were presented as mean ± SD, n = 3 for Western blotting, n = 6–8 for others. *P < 0.05 vs. corresponding control.

FIGURE 5

Liver-specific GPX4 knockdown abrogates the hepatoprotective effects of TA against APAP-induced hepatotoxicity. (A) Western blotting assessed the detected hepatic GPX4 knockdown efficiency in protein levels. Hepatocyte-specific GPX4 knockdown mice were created by AAV8-mediated delivery of a TBG promoter-driven shRNA targeting GPX4. Null-vector-injected mice served as control. (B) Plasma levels of ALT and AST. (C) Liver tissues were subjected to H&E staining for histological examination. (D) Concentrations of hepatic IL-1β, IL-6, TNF-α, and MCP-1 were quantified using commercially available ELISA kits. All data were presented as mean ± SD, n = 3 for Western blotting, n = 5 for others. *P < 0.05 vs. corresponding control.

FIGURE 6

Liver-specific GPX4 knockdown abrogates the protective effect of TA against APAP-induced hepatocyte apoptosis. (A) Western blotting assessed hepatic BCL2 and BAX expression, with β-actin as a loading control. Band intensities were quantified using ImageJ. (B) q-PCR was conducted to evaluate the transcript levels of BCL2 and BAX-related genes. (C) Hepatic caspase 3 activity was quantified using a commercially available kit (fold of control). (D) Western blotting assessed hepatic CHOP expression, with β-actin as a loading control. Band intensities were quantified using ImageJ. (E) q-PCR was conducted to evaluate the transcript levels of the Chop gene. All data were presented as mean ± SD, n = 3 for Western blotting, n = 5 for others. *P < 0.05 vs. corresponding control.

FIGURE 7

Liver-specific GPX4 knockdown inhibits TA-regulated ER stress in APAP-induced hepatotoxicity. (A) Western blotting assessed hepatic GRP78 expression, with β-actin as a loading control. Band intensities were quantified using ImageJ. (B) q-PCR was conducted to evaluate the transcript levels of the Grp78 gene. (C) Western blotting assessed hepatic phosphorylated-PERK, phosphorylated-eIF2α, PERK, and eIF2α protein expressions. Band intensities were quantified using ImageJ. (D) Western blotting assessed hepatic ATF4 expression, with β-actin as a loading control. Band intensities were quantified using ImageJ. (E) q-PCR was conducted to evaluate the transcript levels of the Atf4 gene. All data were presented as mean ± SD, n = 3 for Western blotting, n = 5 for others. *P < 0.05 vs. corresponding control.

FIGURE 8

Schematic illustration of the protective effects and mechanisms of TA APAP-induced liver injury.