Liver diseases are of great importance for public health due to their socioeconomic impact, given that they are associated with high treatment costs, long periods of outpatient follow-up, and hospital admissions.1 Those diseases cause high morbidity and mortality, as a result of the progression to cirrhosis, a common final stage of chronic liver diseases.2 In the United States, cirrhosis caused approximately 49,500 deaths and was the eighth leading cause of death in 2010.3 According to the World Health Organization (WHO), liver cirrhosis is the 11th leading cause of death worldwide.4

There are numerous causes of liver disease, due to liver inflammation or cholestasis. It can end in cirrhosis, which is defined as a diffuse process of nodular formation and fibrosis after liver cell necrosis, the common outcome of chronic liver lesions.5

Regardless of the etiologic agent, cirrhosis can be asymptomatic in its compensated form. However, when decompensated, one or more liver failure complications appear, such as encephalopathy, which is one of the most prevalent.6,7

In patients with liver cirrhosis, hepatic encephalopathy is present in about 30 to 45% of cases and minimal hepatic encephalopathy is present in 80% of those individuals.8–10 It is a potentially reversible metabolic disorder that consists of the functional disorder of the central nervous system, associated with hepatocellular insufficiency. Hepatic encephalopathy results from acute or chronic liver diseases and the presence of portosystemic shunts, which can be spontaneous or surgical (portosystemic intrahepatic transjugular shunting). Ammonia is the main element related to its development, in addition to the interference of several neurotoxins and various factors, such as cerebral edema, GABAergic tone, and the microelements, zinc and manganese.11–13 Because a large part of ammonia is metabolized to urea inside the liver through the urea cycle, the serum ammonia level increases in cases of liver dysfunction, causing it to cross the blood-brain barrier and become toxic to the central nervous system.13,14 It is therefore understood that substances that decrease said concentration can be used in the management of said complication, and caffeine (CAF) is one of them.15

CAF is a pharmacologically active alkaloid that belongs to the group of methylxanthines.16 It is present in several types of food, such as chocolates, soft drinks, teas, energy drinks, and coffee, which is its main source.16,17 Coffee intake has recently been linked to a reduced risk of presenting with several chronic diseases, such as type 2 diabetes mellitus, Parkinson’s disease, inflammatory diseases, and liver diseases.18,19 Specifically regarding liver diseases, CAF consumption has been linked to a decrease in liver enzyme levels and a lower risk of fibrosis.20,21

CAF also increases the activity level of several enzymes that affect the urea cycle, including carbamoyl-phosphate synthetase 1, ornithine transcarbamylase, argininosuccinate synthetase, and argininosuccinate lyase.22,23 All of those enzymes work in favor of the cycle, i.e., they catalyze the urea formation reaction, then increase its synthesis and decrease serum ammonia concentration.24 In addition, one of the main effects of CAF is the stimulation of the central nervous system, by antagonizing adenosine receptors.25

Based on the above, the aim of the present study was to analyze the effects of CAF on the progression of cholestatic liver fibrosis induced by ligation of the common bile duct (LCBD) and correlate its use with the development of encephalopathy caused by hyperammonemia, resulting from liver disease.

The liver enzymes, ALT and AST, are markers of toxicity and severity of liver damage, and their release into the bloodstream demonstrates the presence of damage to the liver parenchyma.38 In our study, the LCBD-induced liver damage, which was evidenced by the significant increase of those enzymes in the blood, a fact already corroborated by other studies using the same model.27,39 As reported in similar studies, CAF treatment decreased the release of those enzymes into the bloodstream, showing a hepatoprotective effect and possible improvement in the liver architecture.30,40

With the LCBD, direct bilirubin from the liver is unable to complete its flow through the bile ducts to the intestine, regurgitating and accumulating in the liver, and is captured by the hepatic sinusoids, increasing its serum concentration.41 With the chronicity of biliary obstruction, the bile acids accumulated in the liver begin to damage the cells responsible for conjugating indirect bilirubin, derived from hemolysis, into direct bilirubin, which causes the serum level of indirect bilirubin to also increase.5,41 In the present study, both the direct bilirubin and indirect bilirubin levels in the ligated animals were increased. The ligated group that was untreated had the highest mean total bilirubin of the groups. On the other hand, the caffeine-treated group showed a decrease in indirect bilirubin, compared with the untreated ligated group, demonstrating that CAF administration resulted in less damage to the cells responsible for the conjugation of indirect bilirubin into direct bilirubin. According to the literature, total bilirubin levels greater than 2 or 3mg/dl are sufficient for the pigment to leak into the skin and mucous membranes and lead to the development of jaundice.5,41 In both LCBD groups, the amount of bilirubin was greater than 2mg/dl, which explains the intense yellowish color visible in the animals after day 7 of the experiment.

Hepatomegaly was also macroscopically evident. That condition is often associated with hepatobiliary diseases and is caused by the retention of bile content in the liver tissue and by the diffuse damage resulting from its accumulation.42,43 In the histopathologic assessment of the liver in the groups that underwent LCBD, both areas had only slight architectural distortion of the parenchyma and areas with intense fibrosis, similar to results from other studies using the same model.27,39,42 The parameters of architectural distortion, portal enlargement, ductal proliferation, and hepatic fibrosis were considered moderate/accentuated in all the untreated ligated animals.

Hepatic myofibroblasts, derived from stellate cells and portal fibroblasts, are the main cells involved in the formation of hepatic fibrosis.44 Those cells express adenosine receptors, which when activated (usually by liver injury), stimulate the formation of fibrosis through myofibroblasts. CAF is an antagonist of adenosine receptors, i.e., it prevents the activation of those receptors and consequently decreases the formation of fibrotic tissue.45 TGF-β is the main regulatory cytokine of the liver and one of its functions is to activate liver stellate cells, stimulating the formation of fibrosis.46 According to the literature, TGF-β levels are increased in experimental models of liver disease, but when CAF is administered, TGF-β levels are lower, meaning there is less activation of the stellate cells due to downregulation of that cytokine.47,48

In vitro studies have shown that CAF also prevents the activation of liver stellate cells by inhibiting the Snail-1 transcription factor, which is essential in the activation of those cells. CAF also activates the enzyme, Nrf2, which regulates antioxidant protein expression. Consequently, it acts by reducing oxidative stress, which is one of the causes of liver fibrosis.30 Through all those mechanisms, CAF exerts its hepatoprotective effect, resulting in less liver damage, thus releasing fewer transaminases into the bloodstream.

In the present study, the liver tissue of the ligated animals treated with CAF generally had a histologic pattern that was more similar to the physiologic one. The induced liver damage could be viewed under microscopy, but there was less damage than that seen in the untreated LCBD group.

As a result, a dose of 50mg/kg/day of CAF, in the presence of cholestatic liver fibrosis, exerted a hepatoprotective effect, especially with respect to the formation of fibrotic tissue, corroborating its effects on injured liver tissue, described in recent studies.30,45,47,48

In hepatic encephalopathy, the main microscopic changes in the encephalon are edema and Alzheimer type II astrocytes,49,50 which are modified cells that show swelling, enlarged nuclei, and nuclear inclusions.41 Laminar necrosis and a spongiform appearance can also be found in the deep layers of the cerebral cortex, subcortical white matter, basal nuclei, and the cerebellum.51

In the present study, the untreated ligated animals intensely presented with the characteristic parameters of encephalopathy, mainly edema and Alzheimer type II astrocytes. Those features were classified as accentuated in 100% of the animals, corroborating the findings of other studies in the literature.28,52

The result of the histologic analysis of the encephalon slides of the LCBD group treated with CAF was satisfactory, with the administration of CAF producing a significant decrease in edema, Alzheimer type II astrocytes, the spongiform aspect, and laminar necrosis. Edema and Alzheimer type II astrocytes, the main histologic changes observed in encephalopathy,49,50 were classified as accentuated in only 20% of the animals, whereas, in the LCBD + H2O group, they were accentuated in 100% of the animals.

In the last days of the experiment, the animals in the untreated LCBD group were clearly more lethargic, confused, disoriented, and apathetic than the animals in the treated group. During gavage, the animals did not struggle against the administration of H2O, unlike the other groups or at other times of the experiment. That reduced consciousness was demonstrated and confirmed by the open field test (OFT), during which the animals showed a decrease in two of the three parameters used to assess exploratory and locomotor activity.29 That state of consciousness is characterized as grade II or grade III of hepatic encephalopathy, according to the West Haven criteria (Table 1), which was expected for the encephalopathy model chosen.28,53,54 Those findings corroborate the results of other studies that used a similar methodology to induce hepatic encephalopathy.28,52,55,56

In the LCBD group supplemented with CAF, no signs of lethargy or prostration were observed. The mean values of the three parameters assessed in the OFT were similar to those in the control groups, showing that the administration of CAF prevented the animals from having the clinical signs characteristic of grade II and grade III of the West Haven criteria.29,53

CAF is known to have a modulatory effect on the state of consciousness due to its stimulating effect on the central nervous system, by antagonizing adenosine receptors.25,57 CAF also decreases the serum concentration of ammonia,57 by increasing the activity of several enzymes in the urea cycle, especially carbamoyl phosphate synthetase 1.22,23 It catalyzes the urea formation reaction, increasing the synthesis of the compound, using ammonia as a substrate, which, in excess, can lead to the development of hepatic encephalopathy.24,53 Through those mechanisms, CAF, at a dose of 50mg/kg/day, prevented the hepatic encephalopathy from progressing to the most severe degrees. In the human body, that dose is equivalent to 280mg/day for a 70kg adult, corresponding to two or three cups of coffee per day.33 That amount of CAF is extremely safe. Side effects, such as anxiety, bipolar disorders, insomnia, psychomotor agitation, and tachycardia, are caused only with larger doses (>400mg/day).58 Thus, CAF would be a viable alternative in the management of patients with liver disease and/or hepatic encephalopathy.

Based on the above, and with the results of the present study, we conclude that the effect of caffeine as a stimulant of the central nervous system, associated with its catalytic action in the urea cycle and its hepatoprotective effect of attenuating liver injury, prevented induced hepatic encephalopathy from progressing to the most severe degrees. It impeded the appearance of clinical signs and the decrease in exploratory and locomotor activity, characteristic of West Haven criteria grade II and grade III,29 and attenuated the appearance of histologic changes in the encephalon. Future studies that measure cerebral ammonia and determine the urea cycle enzymes are strongly recommended for a better understanding of the pathophysiology involved in the prevention of hepatic encephalopathy severity, with the use of caffeine.

The present work was supported by the Mackenzie Evangelical College of Paraná (grant number 03/2017,082017/072018). The funding source had no involvement in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The authors declare that there is no conflict of interest.