N-acetylcysteine protects hepatocytes from hypoxia-related cell injury

Abstract

Aim of the study: Hepatocyte transplantation has been discussed as an alternative to liver transplantation in selected cases of acute and chronic liver failure and metabolic diseases. Immediately after infusion of hepatocytes, hypoxia-related cell injury is inevitable. N-acetylcysteine (NAC) has been suggested to attenuate hypoxic damage. This study's objective was to evaluate NAC's protective effect in a model of hypoxia-related hepatocyte injury.

Material and methods: HepG2 cells were used as a model for hepatocytes and were cultured under standardized hypoxia or normoxia for 24 hours with or without NAC. Growth kinetics were monitored using trypan blue staining. The activation of apoptotic pathways was measured using quantitative real-time PCR for Bcl-2/Bax and p53. The proportions of vital, apoptotic and necrotic cells were verified by fluorescence activated cell sorting using annexin V-labelling. The expression of hypoxia inducible factor 1 (HIF-1) was measured indirectly using its downstream target vascular endothelial growth factor A (VEGF-A).

Results: After NAC, cell proliferation increased under both hypoxia and normoxia by 528% and 320% (p < 0.05), while VEGF-A expression decreased under normoxia by 67% and 37% (p < 0.05). Compared to cells treated without NAC under hypoxia, the Bcl-2/Bax ratio increased significantly in cells treated with NAC. This finding was confirmed by an increased number of vital cells in FACS analysis.

Conclusions: NAC protects hepatocytes from hypoxic injury and ultimately activates anti-apoptotic pathways.

Keywords: HepG2; N-acetylcysteine; hepatocyte transplantation; hypoxia.

Fig. 1

Cell proliferation. Number of cells after 72 hours. Norm: 256 × 10³ ±24 249. NAC/norm: 696 × 10³ ±100 936 (p = 0.01). Hypo: 260 × 10³ ±77 382. NAC/hypo: 442 × 10³ ±6928 (p = 0.05)

Fig. 2

FACS. A) Proportion of necrotic, apoptotic and vital cells measured using annexin V labelling. Norm: vital cells: 82%; apoptotic cells: 3%; necrotic cells: 15%. NAC/norm: vital cells: 94%; apoptotic cells: 1%; necrotic cells: 5%. Hypo: vital cells: 68%; apoptotic cells: 5%; necrotic cells: 27%. NAC/hypo: vital cells: 82%; apoptotic cells: 3%; necrotic cells: 15%. B) Representative images of FACS analysis using annexin V (FL1-H) and PI (FL3-H) labelling for cells treated with or without NAC under normoxic and hypoxic conditions

Fig. 3

Activation of pro- and anti-apoptotic markers by PCR. A) Expression of Bax, Bcl-2 and p53. Norm: Expression under normoxic conditions as reference. NAC/norm: Bax: 0.34 (p = 0.0017); Bcl-2: 0.31 (p = 0.0031); p53: 0.58 (p = 0.0094). Hypo: Bax: 2.25 (p = 0.0034); Bcl-2: 1.95 (p = 0.0095); p53: 1.56 (p = 0.02). NAC/hypo: Bax: 4.06 (p = 0.0003); Bcl-2: 4.34 (p = 0.0007); p53: 2.57 (p = 0.005). B: Bcl-2/Bax ratio. Norm: Bcl-2/Bax ratio under normoxic conditions as reference. NAC/norm: 0.999 (p = 0.83). Hypo: 0.982 (p = 0.01). NAC/hypo: 0.996 (p = 0.0025)

Fig. 4

A) VEGF-A expression. Norm: VEGF-A expression under normoxic conditions as reference. Hypo: 7.26 (p = 0.0002). NAC/hypo: 6.93 (p = 0.6). B) VEGF-A expression measured by PCR under normoxic conditions. Norm: VEGF-A expression under normoxic conditions. NAC/norm: VEGF-A expression after 24 hours in normoxic conditions + NAC: 0.67 (p = 0.04). NAC/norm*: VEGF-A expression after 48 hours in normoxic conditions + NAC: 0.37 (p = 0.0017).