Caffeine prevents experimental liver fibrosis by blocking the expression of TGF-b Jonathan Arauz a , Natanael Zarco b , Jose ´ Segovia b , Mineko Shibayama c , Victor Tsutsumi c and Pablo Muriel a Background There is a growing body of evidence that caffeine exerts beneficial effects on the liver; however, the molecular mechanisms by which caffeine exerts beneficial effects on the liver are poorly defined. Aims The aim of the present study was to examine the efficacy of caffeine in preventing thioacetamide (TAA)-induced cirrhosis in rats. Materials and methods Cirrhosis was induced by chronic TAA administration and the effects of coadministration of caffeine for 8 weeks were evaluated, including control groups. Results The administration of TAA induced liver cirrhosis, which was inhibited by caffeine. Caffeine prevents elevation of liver enzymes. Liver histopathology and hydroxyproline levels were significantly lower in the rats treated with TAA plus caffeine compared with TAA only. Caffeine shows antioxidant properties by restoring the redox equilibrium [lipid peroxidation and glutathione peroxidase (GPx) levels]. Western blot assays showed blockade of the expression of transforming growth factor-b and its downstream inductor connective tissue growth factor. Similarly, caffeine decreases messenger RNA levels of these profibrogenic proteins. In addition, caffeine inhibits hepatic stellate cells because of blockade of the expression of a-smooth muscle actin; in the western blot assay, we also found low levels of mRNA of collagen a1. Zymography assays showed that caffeine had an effect on the activity of matrix metalloproteinases 2 and 9, but no effect on the expression of tissue inhibitor of metalloproteinases-1, using RT-PCR. Conclusion Our results show that caffeine prevents experimental cirrhosis; the mechanisms of action are associated with its antioxidant properties and mainly by its ability to block the elevation of the profibrogenic cytokine transforming growth factor-b, which may be associated with attenuation of the inflammatory and fibrotic processes. Eur J Gastroenterol Hepatol 26:164–173  c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. European Journal of Gastroenterology & Hepatology 2014, 26:164–173 Keywords: caffeine, connective tissue growth factor, fibrosis, metalloproteinase, TGF-b, thioacetamide Departments of a Pharmacology, b Physiology, Biophysics and Neuroscience and c Infectomics and Molecular Pathogenesis, Cinvestav-IPN., Mexico, D.F. Mexico Correspondence to Pablo Muriel, PhD, Department of Pharmacology, Cinvestav-I.P.N. Apdo. Postal 14-740, Mexico 07000, D.F., Mexico Tel: +52 55 5747 3303; fax: +52 55 5747 3394; e-mail: pamuriel@cinvestav.mx Received 9 April 2013 Accepted 17 June 2013 Introduction Inflammation of the liver results from a variety of causes. The most prevalent cause of hepatic inflammatory dis- eases is hepatitis virus infection [1,2]. After the acute phase, different degrees of severity of inflammation may persist as chronic hepatitis in a considerable number of viral, autoimmune, and drug-induced hepatitis patients. The repeated liver tissue remodeling caused by frequent inflammation often leads to the development of fibrosis and cirrhosis [3,4]. Furthermore, progressive hepatic fibrosis is the final common pathway for most chronic liver injuries, leading to cirrhosis with a risk of liver failure and hepatocellular carcinoma (HCC) [5]. Hepatic chronic diseases are characterized by the gradual destruction of the parenchyma and accumulation of the extracellular matrix (ECM) (including collagens I, III, and IV) because of the increased synthesis and the inability to break down these proteins, leading to distor- tion of the hepatic architecture, resulting in liver fibrosis and then in cirrhosis [6]. Hepatic stellate cells (HSCs) play a crucial role in liver fibrosis. After liver injury, HSCs are activated and increase the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). MMPs and TIMPs are involved in matrix remodeling in both physiological and pathological processes [7]. Liver fibrosis is initiated by mechanisms leading to inflammation, which in turn activates a wound-healing response as a result of the production of the fibrogenic cytokine transforming growth factor-b (TGF-b). It is firmly established that the liver fibrogenic response is highly regulated by TGF-b. In addition, HSCs are induced by TGF-b to differentiate into myofibroblasts and to produce increased amounts of ECM proteins [8]. However, connective tissue growth factor (CTGF) has been considered as an important downstream modulator of TGF-b that is thus capable of amplifying the profibrogenic action of this cytokine in the liver and in other tissues [9]. Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in many popular beverages, including cocoa, tea, 164 Original article 0954-691X  c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins DOI: 10.1097/MEG.0b013e3283644e26 Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.