N-acetylcysteine attenuates ischemia-reperfusion-induced apoptosis and autophagy in mouse liver via regulation of the ROS/JNK/Bcl-2 pathway

Abstract

Background: Hepatic ischemia-reperfusion injury (HIRI) remains a pivotal clinical problem after hemorrhagic shock, transplantation, and some types of toxic hepatic injury. Apoptosis and autophagy play important roles in cell death during HIRI. It is also known that N-acetylcysteine (NAC) has significant pharmacologic effects on HIRI including elimination of reactive oxygen species (ROS) and attenuation of hepatic apoptosis. However, the effects of NAC on HIRI-induced autophagy have not been reported. In this study, we evaluated the effects of NAC on autophagy and apoptosis in HIRI, and explored the possible mechanism involved.

Methods: A mouse model of segmental (70%) hepatic warm ischemia was adopted to determine hepatic injury. NAC (150 mg/kg), a hepatoprotection agent, was administered before surgery. We hypothesized that the mechanism of NAC may involve the ROS/JNK/Bcl-2 pathway. We evaluated the expression of JNK, P-JNK, Bcl-2, Beclin 1 and LC3 by western blotting and immunohistochemical staining. Autophagosomes were evaluated by transmission electron microscopy (TEM).

Results: We found that ALT, AST and pathological changes were significantly improved in the NAC group. Western blotting analysis showed that the expression levels of Beclin 1 and LC3 were significantly decreased in NAC-treated mice. In addition, JNK, p-JNK, Bax, TNF-α, NF-κB, IL2, IL6 and levels were also decreased in NAC-treated mice.

Conclusion: NAC can prevent HIRI-induced autophagy and apoptosis by influencing the JNK signal pathway. The mechanism is likely to involve attenuation of JNK and p-JNK via scavenged ROS, an indirect increase in Bcl-2 level, and finally an alteration in the balance of Beclin 1 and Bcl-2.

Figure 1. Effect of NAC on hepatic ischemia-reperfusion injury.

The ischemia-reperfusion and sham-operated mice were pretreated with NAC (150 mg/kg) or saline. (A) Serum AST and ALT levels were measured at 6, 12 and 24 h after reperfusion of the three groups. Data represent means (SD) (n = 6 mice per time point per group). *p<0.05 for saline VS saline + IR, # p<0.05 for saline +IR VS IR + NAC (150 mg/kg). (B) Photomicrographs of representative livers collected 6, 12 and 24 h after reperfusion, stained with hematoxylin and eosin (H&E), ×200 magnification. The necrotic areas were analyzed with Image-pro Plus 6.0, indicating there existed statistical significant among different groups [n = 6, *p<0.05 for saline VS saline + IR, # p<0.05 for saline +IR VS IR + NAC (150 mg/kg)].

Figure 2. Effect of NAC on regulation of apoptosis.

(A) The expression of Bax and Bcl-2 on cDNA levels were detected by real time PCR. ^* p<0.05 for saline VS saline + IR, ^# p<0.05 for saline +IR VS IR + NAC (150 mg/kg). (B) The expression of Bax and Bcl-2 on protein levels were detected with western blot. (C) The expression of Bax and Bcl-2 on protein levels were detected by immunohistochemistry staining in hepatic tissues at 12 h (×200 magnification). (D) TUNEL staining of hepatic tissues in three groups at 12 h to detect apoptotic cells (×400 magnification).

Figure 3. Effect of NAC on regulation of autophagy.

(A) The expression of Bclin 1 and LC3 on cDNA levels were detected by real time PCR. ^* p<0.05 for saline VS saline + IR, ^# p<0.05 for saline +IR VS IR + NAC (150 mg/kg). (B) The expression of Bclin 1 and LC3 on protein levels were detected with western blot. (C) The expression of Bclin 1 and LC3 on protein levels were detected by immunohistochemistry staining in hepatic tissues at 12 h (×200 magnification). (D) Morphology of autophagosomes in hepatocytes at 12 h detected by electron microscopy (×20000 magnification). Initial autophagic vacuole (AVi) containing a mitochondrion, endoplasmic reticulum membranes, and ribosomes (arrowheads). Degradative autophagic vacuole (AVd) is found to have degradation of contents (arrowheads).

Figure 4. Effect of NAC on regulation of JNK pathway, cytokines release and ROS generation.

(A) The expression of p-JNK and JNK on protein level was detected by western blot. The result of western blot were analyzed with quantity one [n = 3, *p<0.05 for saline VS saline + IR, # p<0.05 for saline +IR VS IR + NAC (150 mg/kg)]. The expression of p-JNK in hepatic tissue of different groups were shown by immunohistochemistry at 12 h (×200 magnification). The IOD of p-JNK in cytoplasm was analyzed by Image-Pro Plus 6.0. Date are showed as mean± SD [n = 3, *p<0.05 for saline VS saline + IR, # p<0.05 for saline +IR VS IR + NAC (150 mg/kg)]. The representative positive cells were indicated with red arrows. (B) The expression of NF-κB, IL-6, TNF-αon cDNA level were detected by real time PCR. *p<0.05 for saline VS saline + IR, # p<0.05 for saline +IR VS IR + NAC (150 mg/kg). And the expression of NF-κB, IL-6, TNF-α on protein level was detected by western blot. The result of the western blot were analyzed with quantity one [n = 3, *p<0.05 for saline VS saline + IR, # p<0.05 for saline +IR VS IR + NAC (150 mg/kg)]. (C) The generation of ROS was detected with ROS Fluorescent Probe-DHE. And ROS were measured and statistical analysis in three random vision fields by Image-Pro plus 6.0.