JNK Signaling Pathway Mediates Acetaminophen-Induced Hepatotoxicity Accompanied by Changes of Glutathione S-Transferase A1 Content and Expression

Abstract

Acetaminophen (APAP) is an analgesic-antipyretic drug and widely used in clinics. Its overdose may cause serious liver damage. Here, we examined the mechanistic role of c-Jun N-terminal kinase (JNK) signaling pathway in liver injury induced by different doses of APAP. Male mice were treated with APAP (150 and 175 mg·kg^-1), and meanwhile JNK inhibitor SP600125 was used to interfere APAP-induced liver damage. The results showed that JNK signaling pathway was activated by APAP in a dose-dependent manner. C-Jun N-terminal kinase inhibitor decreased JNK and c-Jun activation significantly (P < 0.01) at 175 mg·kg^-1 APAP dose, and phosphorylation levels of upstream proteins of JNK were also decreased markedly (P < 0.05). In addition, serum aminotransferases activities and hepatic oxidative stress increased in a dose-dependent manner with APAP treatment, but the levels of aminotransferases and oxidative stress decreased in mice treated with JNK inhibitor, which implied that JNK inhibition ameliorated APAP-induced liver damage. It was observed that apoptosis was increased in APAP-induced liver injury, and SP600125 can attenuate apoptosis through the inhibition of JNK phosphorylation. Meanwhile, glutathione S-transferases A1 (GSTA1) content in serum was enhanced, while GSTA1 content and expression in liver reduced significantly with administration of APAP (150 and 175 mg·kg^-1). After inhibiting JNK, GSTA1 content in serum decreased significantly (P < 0.01); meanwhile, GSTA1 content and expression in liver enhanced. These findings suggested that JNK signaling pathway mediated APAP-induced hepatic injury, which was accompanied by varying GSTA1 content and expression in liver and serum.

Keywords: acetaminophen; c-Jun N-terminal kinase; glutathione S-transferases A1; hepatotoxicity; liver injury.

Figure 1

Dosage selection of APAP and SP600125. (A, B) Changes of ALT and AST activities with different dosages of APAP in serum of mice. (C, D) Changes of ALT and AST activities with 175 mg·kg⁻¹ APAP and different dosages of SP600125 in serum of mice. Values represented as means ± SD, n = 6. *Represents statistical differences caused by APAP. ^#Represents statistical differences caused by SP600125 under the same dose of APAP. 0.05 > P > 0.01 (*, ^#). P < 0.01 (**, ^##).

Figure 2

Activation of JNK signaling pathway under different dosages of APAP. (A) Western blot analyses of total tissue lysate for p-JNK, JNK, p-c-Jun, c-Jun, p-c-Fos, c-Fos, and β-actin (loading control). (B–D, F, G) Change of JNK activation (fold of p-JNK/JNK), c-Jun activation (fold of p-c-Jun/c-Jun), c-Fos activation (fold of p-c-Fos/c-Fos), ASK1 activation (fold of p-ASK1/ASK1), and MKK4 activation (fold of p-MKK4/MKK4) under different dosages of APAP in liver of mice with or without SP600125. (E) Western blot analyses of total tissue lysate for p-ASK1, ASK1, p-MKK4, MKK4, and β-actin (loading control). Values represented as means ± SD, n = 3. *Represents statistical differences caused by APAP. ^#Represents statistical differences caused by SP600125 under 150 mg·kg⁻¹ APAP; §Represents statistical differences caused by SP600125 under 175 mg·kg⁻¹ APAP. 0.05 > P > 0.01 (*, ^#, ^§). P < 0.01 (**, ^§§).

Figure 3

Inhibiting JNK ameliorated APAP-induced liver toxicity respect to ALT and AST. (A) Change of ALT activity under different dosages of APAP in serum of mice with or without SP600125. (B) Change of AST activity under different dosages of APAP in serum of mice with or without SP600125. Values represented as means ± SD, n = 6. *Represents statistical differences caused by APAP. ^§Represents statistical differences caused by SP600125 under the same dose of APAP. 0.05 > P > 0.01 (*). P < 0.01 (**, ^§§).

Figure 4

Changes of oxidative stress under different dosages of APAP in liver of mice with or without SP600125. (A) Change of MDA content under different dosages of APAP in liver of mice with or without SP600125. (B) Change of SOD activity under different dosages of APAP in liver of mice with or without SP600125. (C) Change of GSH content under different dosages of APAP in liver of mice with or without SP600125. (D) Change of GSH-Px activity under different dosages of APAP in liver of mice with or without SP600125. Values represented as means ± SD, n = 6. *Represents statistical differences caused by APAP. ^#Represents statistical differences caused by SP600125 under 150 mg·kg⁻¹ APAP. ^§Represents statistical differences caused by SP600125 under 175 mg·kg⁻¹ APAP. 0.05 > P > 0.01 (^#, ^§). P < 0.01 (**, ^##, ^§§).

Figure 5

Mouse hepatic pathology. Liver sections were stained by HE, scanned by slide scanner, and the images are displayed at 200× magnification. (A) Mice from control group. (B) Mice treated with 150 mg·kg⁻¹ APAP. (C) Mice treated with 150 mg·kg⁻¹ APAP and SP600125. (D) Mice treated with 175 mg·kg⁻¹ APAP. (E) Mice treated with 175 mg·kg⁻¹ APAP and SP600125.

Figure 6

Inhibition of JNK ameliorated APAP induced apoptosis. (A) Liver sections were stained by Hoechst 33258, examined by fluorescence microscope and the images are displayed at 200× magnification. (a) Mice from control group. (b) Mice treated with 150 mg·kg⁻¹ APAP. (c) Mice treated with 150 mg·kg⁻¹ APAP and SP600125. (d) Mice treated with 175 mg·kg⁻¹ APAP. (e) Mice treated with 175 mg·kg⁻¹ APAP and SP600125. (B) Western blot analyses of total tissue lysate for caspase-3, Bax and β-actin (loading control), n = 3. (a) Immunoblot graph of caspase-3 Bax and β-actin. (b, c) Changes of caspase-3 expression (fold of GSTA1/β-actin) and Bax expression (fold of Bax/β-actin) under different dosages of APAP in liver of mice with or without SP600125, n = 3. Values represented as means ± SD. *Represents statistical differences caused by APAP. ^#Represents statistical differences caused by SP600125 under 150 mg·kg⁻¹ APAP. ^§Represents statistical differences caused by SP600125 under 175 mg·kg⁻¹ APAP. 0.05 > P > 0.01 (^#). P < 0.01 (**, ^§§).

Figure 7

Changes of GSTA1 caused by different dosages of APAP in mice with or without SP600125. (A) Change of GSTA1 content under different dosages of APAP in serum of mice with or without SP600125, n = 6. (B) Change of GSTA1 content under different dosages of APAP in liver of mice with or without SP600125, n = 6. (C) Western blot analyses of total tissue lysate for GSTA1 and β-actin (loading control), n = 3. (D) Change of GSTA1 expression (fold of GSTA1/β-actin) under different dosages of APAP in liver of mice with or without SP600125, n = 3. Values represented as means ± SD. *Represents statistical differences caused by APAP. ^#Represents statistical differences caused by SP600125 under 150 mg·kg⁻¹ APAP. ^§Represents statistical differences caused by SP600125 under 175 mg·kg⁻¹ APAP. 0.05 > P > 0.01 (^#). P < 0.01 (**, ^##, ^§§).