Protective effect of N-acetylcysteine against lipopolysaccharide injury in hepatocytes of neonatal mice

Abstract

The present study provides supportive evidence for the effective prevention and treatment of lipopolysaccharide (LPS)-induced hepatocyte injury in neonatal mice by N-acetylcysteine (NAC). Hepatocytes of neonatal mice were obtained through collagenase digestion of the liver. The hepatocytes were treated either with LPS (10 ?g/mL) alone or with NAC (5 mmol/L) for 1 h before the addition of LPS (10 ?g/mL). After LPS treatment, 12 wells of the cultured hepatocytes and supernatants were harvested at 0, 6, and 12 h, respectively. Levels of alanine aminotransferase (ALT) and nitric oxide (NO) in the supernatant were biochemically quantified and reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect the expression of inducible nitric oxide synthase (iNOS) mRNA after different treatments. At 0 h following the treatment of primary cultured hepatocytes with LPS, the levels of ALT, NO and iNOS mRNA in the supernatant were (21.1 ± 4.78) u/L, (1.6 ± 0.31) ?mol/L and 0.17 ± 0.023, respectively; at 6 h, (59.8 ± 8.59) u/L, (6.6 ± 0.81) ?mol/L, and 0.71 ± 0.091; and at 12 h, (89.6 ± 15.30) u/L, (7.8 ± 1.01) ?mol/L, and 0.71 ± 0.097. The levels of ALT, NO and iNOS mRNA at 6 and 12 h increased significantly, compared to those at 0 h (P < 0.01). In contrast to LPS treatment alone, pretreatment with NAC before LPS addition significantly reduced the levels of ALT, NO and iNOS mRNA in the supernatant at 6 h to (40.8 ± 7.30) u/L, (3.2 ± 0.71) ?mol/L, and 0.41 ± 0.060; and at 12 h to (55.4 ± 5.48) u/L, (4.0 ± 0.71) ?mol/L, and 0.40 ± 0.067, respectively (P < 0.01). However, the levels of ALT, NO and iNOS mRNA at 0 h did not change significantly with both treatment approaches. NAC has protective effects in hepatocytes of neonatal mice against LPS-induced injury as shown by the reduced levels of ALT, NO and iNOS mRNA when primary hepatocytes were treated with NAC prior to LPS stimulation. We postulate that NAC exhibits its protective function by inhibiting LPS-induced transcription of iNOS, resulting in decreased levels of NO.

References

1. Li J Quan N Bray T M Supplementation of N-acetylcysteine normalizes lipopolysaccharideinduced nuclear factor kappaB activation and proinflammatory cytokineproduction during early rehabilitation of protein malnourished miceJ Nutr 2002 132(11)32863292
2. Majano P L Medina J Zubía I Sunyer L Lara-Pezzi E Maldonado-Rodríguez A López-Cabrera M Moreno-Otero R N-Acetyl-cysteine modulatesinducible nitric oxide synthase gene expression in human hepatocytesJ Hepatol 2004 40(4)632637. doi:10.1016/j.jhep.2003.12.009
3. Weiskirchen R Gressner A M Isolation and culture of hepaticstellate cellsMethods Mol Med 2005 11799113
4. Arterburn L M Zurlo J Yager J D Overton R M Heifetz A H A morphological study of differentiatedhepatocytes in vitroHepatology 1995 22(1)175187
5. Beath S V Hepaticfunction and physiology in the newbornSeminNeonatol 2003 8(5)337346. doi:10.1016/S1084‐2756(03)00066‐6
6. New K J Eaton S Elliott K R Spitz L Quant P A Effect of lipopolysaccharide and cytokineson oxidative metabolism in neonatal rat hepatocytesJ Pediatr Surg 2001 36(2)338340. doi:10.1053/jpsu.2001.20710
7. Bergamini S Rota C Canali R Staffieri M Daneri F Bini A Giovannini F Tomasi A Iannone A N-acetylcysteine inhibits in vivo nitric oxide productionby inducible nitric oxide synthaseNitricOxide 2001 5(4)349360. doi:10.1006/niox.2001.0356
8. Zhang W Z Cui S C Wang J Q Lang Z W Zhu R P Distribution of inducible nitric oxidesynthase in the liver of severe hepatitisZhonghua Chuan Ran Bing Za Zhi 2005 23(1)3839(in Chinese)
9. Labib R Abdel-Rahman M S Turkall R N-acetylcysteine pretreatment decreases cocaine and endotoxin-inducedhepatotoxicityJ Toxicol Environ HealthA 2003 66(3)223239. doi:10.1080/15287390306370