Nos2 deficiency enhances carbon tetrachloride-induced liver injury in aged mice

Document Type: Original Article


1 State Key Laboratory Cell Differentiation and Regulation

2 Henan International Joint Laboratory of Pulmonary Fibrosis

3 Henan center for outstanding overseas scientists of pulmonary fibrosis

4 College of Life Science

5 Institute of Biomedical Science

6 Overseas Expertise Introduction Center for Discipline Innovation of Pulmonary Fibrosis (111 Project), Henan Normal University, Xinxiang, Henan, China



Objective(s): As a multifunctional molecule, NO has different effects on liver injury. The present work aimed to investigate the effects of Nos2 knockout (KO) on acute liver injury in aged mice treated with carbon tetrachloride (CCl4).
Materials and Methods: The acute liver injury model was produced by CCl4 at 10 ml/kg body weight in 24-month-old Nos2 KO mice and wild type (WT) mice groups. The histological changes, transaminase and glutathione (GSH) contents, and the expressions of liver function genes superoxide dismutase (SOD2) and butyrylcholinesterase (BCHE), as well as apoptosis- and inflammation-associated genes were detected at 0, 6, 16, 20, 28, and 48 hr, respectively.
Results: Compared with WT aged mice, there are more fat droplets in liver tissues of Nos2 KO aged mice, and the serum levels of ALT and AST were elevated in the KO group; in addition, there was a decrease in the expression of SOD2 and BCHE and GSH content at multiple time-points. Furthermore, the expression of apoptosis protein CASPASE-3 was elevated from 20 to 48 hr, the same as CASPASE-9 at 28 and 48 hr and pro-apoptotic protein BAX at 6 and 28 hr, while the expression of apoptosis inhibitory protein BCL2 declined at 6 and 28 hr; at the same time the mRNA expressions of genes related to inflammation were increased at different extents in liver extracts of Nos2 KO aged mice.
Conclusion: Nos2 KO exacerbated liver injury probably by elevated oxidative stress, apoptosis and inflammation response in CCl4-induced aged mice liver intoxication model.


1. Wang T, Sun NL, Zhang WD, Li HL, Lu GC, Yuan BJ, et al. Protective effects of dehydrocavidine on carbon tetrachloride-induced acute hepatotoxicity in rats. J Ethnopharmacol 2008; 117:300-308.
2. Bhardwaj A, Khatri P, Soni M, Ali D. Potent herbal hepatoprotective drugs-A review. J Adv Sci Res 2011; 1:15-20.
3. Williams R. Global challenges in liver disease. Hepatology 2006; 44:521-526.
4. Sun H, Chen L, Zhou W, Hu L, Li L, Tu Q, et al. The protective role of hydrogen-rich saline in experimental liver injury in mice. J Hepatol 2011; 54:471-480.
5. Domitrovic R, Skoda M, Vasiljev Marchesi V, Cvijanovic O, Pernjak Pugel E, Stefan MB. Rosmarinic acid ameliorates acute liver damage and fibrogenesis in carbon tetrachloride-intoxicated mice. Food Chem Toxicol 2013; 51:370-378.
6. Yen FL, Wu TH, Lin LT, Cham TM, Lin CC. Naringenin-loaded nanoparticles improve the physicochemical properties and the hepatoprotective effects of naringenin in orally-administered rats with CCl4-induced acute liver failure. Pharm Res 2009; 26:893-902.
7. Lin X, Huang R, Zhang S, Zheng L, Wei L, He M, et al. Methyl helicterate protects against CCl4-induced liver injury in rats by inhibiting oxidative stress, NF-kappaB activation, Fas/FasL pathway and cytochrome P4502E1 level. Food Chem Toxicol 2012; 50:3413-3420.
8. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007; 39:44-84.
9. Bansal AK, Bansal M, Soni G, Bhatnagar D. Protective role of Vitamin E pre-treatment on N-nitrosodiethylamine induced oxidative stress in rat liver. Chem Biol Interact 2005; 156:101-111.
10. Pibiri M, Sulas P, Leoni VP, Perra A, Kowalik MA, Cordella A, et al. Global gene expression profile of normal and regenerating liver in young and old mice. Age 2015; 37:1-14.
11. Iakova P, Awad SS, Timchenko NA. Aging reduces proliferative capacities of liver by switching pathways of C/EBPalpha growth arrest. Cell 2003; 113:495-506.
12. Anantharaju A, Feller A, Chedid A. Aging Liver. A review. Gerontology 2002; 48:343-353.
13. Zeeh J, Platt D. The aging liver: structural and functional changes and their consequences for drug treatment in old age. Gerontology 2002; 48:121-127.
14. Forstermann U, Sessa WC. Nitric oxide synthases: regulation and function. Eur Heart J 2012; 33:829-837, 837a-837d.
15. Tabernero A, Nadaud S, Corman B, Atkinson J, Capdeville-Atkinson C. Effects of chronic and acute aminoguanidine treatment on tail artery vasomotion in ageing rats. Br J Pharmacol 2000; 131:1227-1235.
16. Chou TC, Yen MH, Li CY, Ding YA. Alterations of nitric oxide synthase expression with aging and hypertension in rats. Hypertension 1998; 31:643-648.
17. Li J, Billiar TR. Nitric Oxide. IV. Determinants of nitric oxide protection and toxicity in liver. Am J Physiol 1999; 276:G1069-1073.
18. Chen T, Zamora R, Zuckerbraun B, Billiar TR. Role of nitric oxide in liver injury. Curr Mol Med 2003; 3:519-526.
19. Diesen DL, Kuo PC. Nitric oxide and redox regulation in the liver: Part I. General considerations and redox biology in hepatitis. J Surg Res 2010; 162:95-109.
20. Laubach VE, Shesely EG, Smithies O, Sherman PA. Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death. Proc Natl Acad Sci U S A 1995; 92:10688-10692.
21. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25:402-408.
22. Hu L, Chen L, Yang G, Li L, Sun H, Chang Y, et al. HBx sensitizes cells to oxidative stress-induced apoptosis by accelerating the loss of Mcl-1 protein via caspase-3 cascade. Mol Cancer 2011; 10:43.
23. Tanaka N, Tanaka K, Nagashima Y, Kondo M, Sekihara H. Nitric oxide increases hepatic arterial blood flow in rats with carbon tetrachloride-induced acute hepatic injury. Gastroenterology 1999; 117:173-180.
24. Morio LA, Chiu H, Sprowles KA, Zhou P, Heck DE, Gordon MK, et al. Distinct roles of tumor necrosis factor-alpha and nitric oxide in acute liver injury induced by carbon tetrachloride in mice. Toxicol Appl Pharmacol 2001; 172:44-51.
25. Vulimiri SV, Berger A, Sonawane B. The potential of metabolomic approaches for investigating mode(s) of action of xenobiotics: case study with carbon tetrachloride. Mutat Res 2011; 722:147-153.
26. Szymonik-Lesiuk S, Czechowska G, Stryjecka-Zimmer M, Slomka M, Madro A, Celinski K, et al. Catalase, superoxide dismutase, and glutathione peroxidase activities in various rat tissues after carbon tetrachloride intoxication. J Hepatobiliary Pancreat Surg 2003; 10:309-315.
27. Yu H, Zheng L, Yin L, Xu L, Qi Y, Han X, et al. Protective effects of the total saponins from Dioscorea nipponica Makino against carbon tetrachloride-induced liver injury in mice through suppression of apoptosis and inflammation. Int Immunopharmacol 2014; 19:233-244.
28. Sun F, Hamagawa E, Tsutsui C, Ono Y, Ogiri Y, Kojo S. Evaluation of oxidative stress during apoptosis and necrosis caused by carbon tetrachloride in rat liver. Biochim Biophys Acta 2001; 1535:186-191.
29. Kuida K, Zheng TS, Na S, Kuan C, Yang D, Karasuyama H, et al. Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature 1996; 384:368-372.
30. Kim YM, Talanian RV, Billiar TR. Nitric oxide inhibits apoptosis by preventing increases in caspase-3-like activity via two distinct mechanisms. J Biol Chem 1997; 272:31138-31148.
31. Recknagel RO, Glende EA, Jr., Dolak JA, Waller RL. Mechanisms of carbon tetrachloride toxicity. Pharmacol Ther 1989; 43:139-154.
32. Franceschi C, Bonafe M, Valensin S, Olivieri F, De Luca M, Ottaviani E, et al. Inflamm-aging. An evolutionary perspective on immunosenescence. Ann N Y Acad Sci 2000; 908:244-254.
33. Gee JR, Ding Q, Keller JN. Modulation of apolipoprotein E and interleukin-1beta in the aging liver. Exp Gerontol 2005; 40:409-415.
34. Bohlinger I, Leist M, Barsig J, Uhlig S, Tiegs G, Wendel A. Interleukin-1 and nitric oxide protect against tumor necrosis factor alpha-induced liver injury through distinct pathways. Hepatology 1995; 22:1829-1837.
35. Taylor BS, Alarcon LH, Billiar TR. Inducible nitric oxide synthase in the liver: regulation and function. Biochemistry (Mosc) 1998; 63:766-781.
36. Mojena M, Hortelano S, Castrillo A, Diaz-Guerra MJ, Garcia-Barchino MJ, Saez GT, et al. Protection by nitric oxide against liver inflammatory injury in animals carrying a nitric oxide synthase-2 transgene. FASEB J 2001; 15:583-585.
37. Muriel P. Nitric oxide protection of rat liver from lipid peroxidation, collagen accumulation, and liver damage induced by carbon tetrachloride. Biochem Pharmacol 1998; 56:773-779.
38. Farrokhi M, Gashti MZ, Hoormand M, Bakhtiarian A, Habibi R. Combination therapy profoundly improved skin flap survival by modulating KATP channels and nitric oxide. Adv Med Sci 2019; 64:117-123.
39. Koner D, Banerjee B, Hasan R, Saha N. Antioxidant activity of endogenously produced nitric oxide against the zinc oxide nanoparticle-induced oxidative stress in primary hepatocytes of air-breathing catfish, Clarias magur. Nitric Oxide 2019; 84:7-15.
40. Aono K, Isobe K, Kiuchi K, Fan ZH, Ito M, Takeuchi A, et al. In vitro and in vivo expression of inducible nitric oxide synthase during experimental endotoxemia: involvement of other cytokines. J Cell Biochem 1997; 65:349-358.
41. Wang GS, Liu GT. Role of nitric oxide in immunological liver damage in mice. Biochem Pharmacol 1995; 49:1277-1281.
42. O’Donnell VB, Chumley PH, Hogg N, Bloodsworth A, Darley-Usmar VM, Freeman BA. Nitric oxide inhibition of lipid peroxidation: kinetics of reaction with lipid peroxyl radicals and comparison with alpha-tocopherol. Biochemistry 1997; 36:15216-15223.
43. Sansbury BE, Hill BG. Regulation of obesity and insulin resistance by nitric oxide. Free Radic Biol Med 2014; 73:383-399.
44. Sass G, Koerber K, Bang R, Guehring H, Tiegs G. Inducible nitric oxide synthase is critical for immune-mediated liver injury in mice. J Clin Invest 2001; 107:439-447.
45. Al-Shabanah OA, Alam K, Nagi MN, Al-Rikabi AC, Al-Bekairi AM. Protective effect of aminoguanidine, a nitric oxide synthase inhibitor, against carbon tetrachloride induced hepatotoxicity in mice. Life Sci 2000; 66:265-270.
46. Okuyama T, Nakatake R, Kaibori M, Okumura T, Kon M, Nishizawa M. A sense oligonucleotide to inducible nitric oxide synthase mRNA increases the survival rate of rats in septic shock. Nitric Oxide 2018; 72:32-40.
47. Fathy M, Khalifa E, Fawzy MA. Modulation of inducible nitric oxide synthase pathway by eugenol and telmisartan in carbon tetrachloride-induced liver injury in rats. Life Sci 2019; 216:207-214.