Caffeine protects against alcohol-induced liver fibrosis by dampening the cAMP/PKA/CREB pathway in rat hepatic stellate cells
Introduction
Alcohol abuse and chronic alcohol consumption remain as a global public health problem. Excessive alcohol consumption is one of the critical causative factors leading to alcoholic liver disease (ALD) [1]. ALD is characterized by a wide spectrum of liver damage ranging from steatosis and steatohepatitis to fibrosis and cirrhosis [2]. Alcoholic liver fibrosis (ALF) is regarded as a turning point in ALD because it can lead to cirrhosis [3]. Accumulating evidence suggests that liver fibrosis is reversible and that recovery from cirrhosis may be possible [4], but the molecular determinants of fibrosis regression in animals and humans need to be more comprehensively defined.
The development of liver fibrosis in alcoholics has been linked to the oxidation of ethanol to the highly reactive compound acetaldehyde, which is the principal metabolite of alcohol and known to stimulate the production of several extracellular matrix (ECM) components, including type I collagen, by activating the hepatic stellate cell (HSC) [5], [6]. HSC activation represents a critical event in alcohol-induced fibrosis because these cells become the primary source of ECM in liver upon injury [7]. As response to acetaldehyde-induced stimulation, HSCs change from quiescent vitamin A-storing cells to activated myofibroblast-like cells, which proliferate and become fibrogenic [8].
Coffee is one of the most frequently consumed beverages worldwide [9]. Epidemiological studies have shown that coffee intake attenuates the progression of chronic liver disease, including alcoholic liver cirrhosis [10], [11], [12], although there are controversies on whether these beneficial effects are related to caffeine or other specific components in this popular beverage. In recent years, limited experimental data further suggest that caffeine intake exert beneficial effects in experimental liver fibrosis [13], [14], [15], [16]. Previous work from our laboratory has also indicated that caffeine could alleviate chronic alcohol-induced liver injury via inhibition of oxidative stress and inflammation [17]. However, to date, few studies have investigated the effects of caffeine directly on the HSCs and ALF.
Caffeine, a widely consumed psychoactive substance and an active ingredient in coffee [18], acts as a nonselective antagonist of adenosine A2A receptor (A2AR) [19]. During the past few years, a growing body of evidence indicates that A2AR is expressed on rat and human HSCs, and its activation plays a crucial role in the pathogenesis of hepatic fibrosis [20], [21], [22]. A2AR was originally identified by virtue of its ability to elevate intracellular levels of cAMP via receptor interaction with the heterotrimeric (alpha beta gamma) stimulatory G protein (Gs) and subsequent activation of adenylyl cyclase [23]. Using an in vitro cell culture model of acetaldehyde-induced HSC-T6 cells, we have previously demonstrated that caffeine inhibits the activation of acetaldehyde-induced HSC-T6 cells via A2AR mediated cAMP signal pathway [24]. However, the molecular mechanisms involved in the regulation of the cAMP-dependent pathway by which caffeine exerts beneficial effects in a rat model of ALF remain largely unclear.
In the present study, the rat model of ALF was induced by intragastric infusion of increasing concentration of ethanol 2 times per day for 12 weeks, and the primary HSCs were isolated from Sprague Dawley (SD) rats by in situ perfusion and density gradient centrifugation. Furthermore, we evaluated the protective effects of caffeine on rat model of ALF and assessed whether caffeine regulate the activation of HSCs via cAMP/PKA/CREB signaling pathway.
Section snippets
Materials and reagents
Caffeine and dimethyl sulfoxide were obtained from Sigma Chemical Co. (St. Louis, Mo, USA). Ethanol was obtained from Shanghai Suyi Chemical Reagent (Shanghai, China). DMEM and FCS were purchased from GIBCO (CA, USA). Reagent kits for ALT and AST were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). HA, LN, PIIINP and CIV radioimmunoassay kits were obtained from Beijing Huaying Bioengineering Research Company (Beijing, China). TRIzol reagent was obtained from
Animal vital signs and index of liver
In the study, a total of fifteen rats died: no rat died in the normal group; in model group, four rats died, including two deaths caused by gastric dilatation; in caffeine groups, eight rats died; in colchicine group, 3 rats died. The survival rate of the model was 85%. After 8-week and 12-week ethanol feeding, the weight of rats in the model group was less than that of the rats in normal group (P < 0.01). The weight of rats in caffeine groups and colchicine group was higher than that of rats in
Discussion
ALF is now widely recognized as an important public health problem, and the relevant studies on pathogenesis and therapeutic targets of ALF has increasingly become the hotspot of global attention [26]. Because of the complicated mechanism of ALF, it is difficult to develop a pathophysiology-based treatment. The understanding of the etiology and pathogenesis of ALF in depth may contribute to seeking effective therapeutic measures.
Resent epidemiological studies have indicated that consumption of
Acknowledgments
This project was supported by the National Science Foundation of China (No. 81270498), Anhui Provincial Natural Science Foundation (No. 11040606M194) and Anhui Provincial Key Projects of Scientific Research in Universities (No. KJ2012A148).
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2022, Toxicology LettersCitation Excerpt :It is involved in the pathophysiological regulation of a variety of diseases, including liver disease (Velazquez-Miranda et al., 2019). Previous research by our group has also explored the role of adenosine receptors in a variety of liver diseases (Yang et al., 2015; Liu et al., 2019; Wang et al., 2015). Peng et al. demonstrated that exposure to alcohol causes increased adenosine receptor expression.