Beneficial effects of N-acetylcysteine on protease-antiprotease balance in attenuating bleomycin-induced pulmonary fibrosis in rats

Document Type : Original Article


1 Department of Pathology, V. P. Chest Institute, University of Delhi, Delhi-110007, India

2 Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala, Punjab, India

3 Department of Biochemistry, V. P. Chest Institute, University of Delhi, Delhi-110007, India


Objective(s): The role of N-acetylcysteine (NAC) as an anti-oxidant in attenuating bleomycin-induced pulmonary fibrosis has been reported. However, its effect on parenchymal remodeling via regulating the protease-antiprotease balance is not fully defined. Therefore, the present study was designed to explore the possible role of matrix metalloproteinases (MMP), tissue inhibitors of metalloproteinases (TIMP) and transforming growth factor-β1 (TGF-β1) pathway and their modulation by NAC in attenuating bleomycin-induced pulmonary fibrosis in rats.
Materials and Methods: Bleomycin sulphate (7 units/kg) was instilled inside the trachea to induce pulmonary fibrosis. The time course of TGF-β1, MMP-9, TIMP-1,3 mRNA and protein expression, TGF-β1 and hydroxyproline levels were evaluated on days 7, 14, and 28. NAC (0.3 mmol/kg and 3 mmol/kg) was administered in bleomycin-instilled animals.
Results: NAC treatment significantly attenuated bleomycin-induced histopathological changes by decreasing interstitial inflammation and reducing the deposition of extracellular matrix proteins such as collagen. Moreover, it increased the mRNA and protein expression of MMP-9 and decreased the expression of TIMP-1,3 in alveolar epithelial cells (AECs), interstitial macrophages and inflammatory cells. Indeed, there was decrease in the MMP-9/TIMP ratio in bleomycin-instilled rats, which increased with NAC treatment. Moreover, NAC attenuated bleomycin-induced increased expression of TGF-β1 and total lung collagen levels.
Conclusion: NAC attenuates bleomycin-induced pulmonary fibrosis by normalizing the protease-antiprotease balance and favoring the degradation of collegen to reduce fibrosis.


1. Shahidi S, Zargooshnia S, Asl SS, Komaki A, Sarihi A. Influence of N-acetyl cysteine on beta-amyloid-induced Alzheimer’s disease in a rat model: A behavioural and electrophysiological study. Brain Res Bull 2017;131:142-149.
2. Rapado-Castro M, Dodd S, Bush AI, Malhi GS, Skvarc DR, On ZX, et al. Cognitive effects of adjunctive N-acetyl cysteine in psychosis. Psychol Med 2017;47:866-76.
3. Yu WC, Tian LY, Cheng W. Efficacy study of edaravone and acetylcysteine towards bleomycin-induced rat pulmonary fibrosis. Int J Clin Exp Med 2015 15;8:8730-8739.
4. Li S, Yang X, Li W, Li J, Su X, Chen L, et al. N-acetylcysteine downregulation of lysyl oxidase activity alleviating bleomycin-induced pulmonary fibrosis in rats. Respiration 2012;84:509-517.
5. Wynn TA. Integrating mechanisms of pulmonary fibrosis. J Exp Med 2011; 208:1339-1506.
6. Lanchou J, Corbel M, Tanguy M, Germain N, Boichot E, Theret N, et al. Imbalance between matrix metalloproteinases (MMP-9 and MMP-2) and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in acute respiratory distress syndrome patients. Crit Care Med 2003; 31:536-542.
7. Talmi-Frank D, Altboum Z, Solomonov I, Udi Y, Jaitin DA, Klepfish M, et al. Extracellular matrix proteolysis by MT1-MMP contributes to influenza-related tissue damage and mortality. Cell Host Microbe 2016 ;20:458-470.
8. Wang Y, Yamamoto Y, Kuninaka Y, Kondo T, Furukawa F. Forensic potential of MMPs and CC chemokines for wound age determination. J Forensic Sci 2015; 60:1511-1515.
9. Hayakawa T, Yamashita K, Ohuchi E, Shinagawa A. Cell growth-promoting activity of tissue inhibitor of metalloproteinases-2 (TIMP-2). J Cell Sci 1994; 2373-2379.
10. García-Alvarez J, Ramirez R, Checa M, Nuttall RK, Sampieri CL, Edwards DR, et al. Tissue inhibitor of metalloproteinase-3 is up-regulated by transforming growth factor-beta1 in vitro and expressed in fibroblastic foci in vivo in idiopathic pulmonary fibrosis. Exp Lung Res 2006; 32:201-214.
11. Lu P, Takai K, Weaver VM, Werb Z. Extracellular matrix degradation and remodeling in development and disease. Cold Spring Harb. Perspect Biol 2011; 3:a005058–a005058.
12. Fukuda Y, Ishizaki M, Kudoh S, Kitaichi M, Yamanaka N. Localization of matrix metalloproteinases-1, -2, and -9 and tissue inhibitor of metalloproteinase-2 in interstitial lung diseases. Lab Invest 1998; 78, 687-698.
13. Hayashi T, Stetler-Stevenson WG, Fleming MV, Fishback N, Koss MN, Liotta LA, et al. Immunohistochemical study of metalloproteinases and their tissue inhibitors in the lungs of patients with diffuse alveolar damage and idiopathic pulmonary fibrosis. Am J Pathol 1996;149, 1241-1256.
14. Border WA, N.N., Transforming growth factor beta in tissue fibrosis. N Engl J Med 1994.
15. Feige, JJ, Quirin N, Souchelnitskiy S. TGF-beta, un peptide biologique sous contrôle: formes latentes et mécanismes d’activation. M/S Med Sci 1996; 12:929-939.
16. Hoyt, DG, Lazo, JS. Alterations in pulmonary mRNA encoding procollagens, fibronectin and transforming growth factor-beta precede bleomycin-induced pulmonary fibrosis in mice. J Pharmacol Exp Ther 1988; 246, 765-771.
17. Khalil N, Bereznay O, Sporn M., Greenberg AH. Macrophage production of transforming growth factor beta and fibroblast collagen synthesis in chronic pulmonary inflammation. J Exp Med 1989; 170:727-737.
18. Yu Q, Stamenkovic I. Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis. Genes Dev. 2000;14: 163-176.
19. Kang HR., Soo JC, Chun GL, Homer RJ, Elias JA. Transforming growth factor (TGF)-??1 stimulates pulmonary fibrosis and inflammation via a Bax-dependent, Bid-activated pathway that involves matrix metalloproteinase-12. J Biol Chem 2007;282: 7723-7732.
20. Gurujeyalakshmi G, Wang Y, Giri SN. Taurine and Niacin block lung injury and fibrosis bydown regulating bleomycin induced activation of transcription nuclear factor-kB in mice. JPET 2000; 293: 82-90.
21. Oury TD, Thakker K, Menache M, Chang LY, Crapo JD, Day BJ. Attenuation of bleomycin-induced pulmonary fibrosis by a catalytic antioxidant metalloporphyrin. Am J Respir Cell Mol Biol 2001; 25:164-169.
22. Ashcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol 1988; 41: 467-470.
23. Reddy GK, Enwemeka CS. A simplified method for the analysis of hydroxyproline in biological tissues. Clin. Biochem. 1996; 29: 225-229.
24. Nguyen DH, Zhou T, Shu J, and Mao JH. “Quantifying chromogen intensity in immunohistochemistry via reciprocal intensity.” Cancer InCytes 2013; 2:e.
25. Yang HK, Jeong KC, Kim YK, Jung ST. Role of matrix Metalloproteinase (MMP) 2 and MMP-9 in Soft tissue sarcoma. Clin Orthop Surg. 2014; 6: 443-454
26. Reinert T, Serodio C, Arthur F, Nunes P, Alves A, Scheliga DS. Bleomycin-induced lung injury. J Cancer Res. 2013; 1-9.
27. Mata M, Ruíz A, Cerdá M, Martinez-Losa M, Cortijo J, Santangelo F, Serrano-Mollar A, Llombart-Bosch A, Morcillo EJ. Oral N‐acetylcysteine reduces bleomycin-induced lung damage and mucin Muc5ac expression in rats. Eur Respir J 2003;22: 900-905
28. Liu T, De Los Santos FG, Phan SH. The bleomycin model of pulmonary fibrosis. Methods Mol Biol. 2017;1627:27-42.
29. Cabrera S, Gaxiola M, Arreola JL, et al. Overexpression of MMP9 in macrophages attenuates pulmonary fibrosis induced by bleomycin. Int J Biochem Cell Biol 2007; 39: 2324–2338.
30. Shapiro, S.D., Fliszar, C.J., Broekelmann, T.J., Mecham, R.P., Senior, R.M., Welgus, H.G. Activation of the 92-kDa gelatinase by stromelysin and 4-aminophenylmercuric acetate. Differential processing and stabilization of the carboxyl-terminal domain by tissue inhibitor of metalloproteinases (TIMP). J Biol Chem 1995; 270:6351-6356.
31. Watanabe, H., Nakanishi, I., Yamashita, K., Hayakawa, T., Okada, Y. Matrix metalloproteinase-9 (92 kDa gelatinase/type IV collagenase) from U937 monoblastoid cells: correlation with cellular invasion. J Cell Sci 1993;104: 991–9.
32. Reichenstein M, Reich R, LeHoux, JG, Hanukoglu I. ACTH induces TIMP-1 expression and inhibits collagenase in adrenal cortex cells. Mol. Cell. Endocrinol. 2004; 215, 109–114.
33. Selman M, Ruiz V, Cabrera S, Segura L, Barrios R, Pardo A. TIMP-1,-2,-3, and -4 in idiopathic pulmonary fibrosis. A prevailing nondegradative lung microenvironment ? Am J Physiol Lung Cell Mol Physiol 2000; 279, 562–574.
34. Zuo WL, Zhao JM, Huang JX, Zhou W, Lei ZH, Huang YM, Huang YF, Li HG. Effect of bosentan is correlated with MMP-9/TIMP-1 ratio in bleomycin-induced pulmonary fibrosis. Biomed Rep. 2017; 6:201-205.
35. Brew K, Nagase H. The tissue inhibitors of metalloproteinases (TIMPs): An ancient family with structural and functional diversity. Biochim. Biophys. Acta Mol Cell Res 2010;1803:55-71.
36. Gill SE, Huizar I, Bench EM, Sussman SW, Wang Y, Khokha R, Parks WC. Tissue inhibitor of metalloproteinases 3 regulates resolution of inflammation following acute lung injury. Am J Pathol 2010;176:64-73.
37. Anadón A, Castellano V, Rosa Martínez-Larrañaga M. Biomarkers of drug toxicity. Biomarkers Toxicol. 2014;593-607.
38. Bonnans C, Chou J, Werb Z. Remodelling the extracellular matrix in development and disease. Nat Rev Mol Cell Biol 2014;15:786-801.
39. Cu A, Ye Q, Sarria R, Nakamura S, Guzman J, Costabel U. N-acetylcysteine inhibits TNF-alpha, sTNFR, and TGF-beta1 release by alveolar macrophages in idiopathic pulmonary fibrosis in vitro. Sarcoidosis Vasc Diffus Lung Dis 2009; 26:147-154.