Acetylsalicylic acid (Aspirin®) and liver regeneration: experimental study in rats

ABSTRACT Objective: to evaluate the influence of acetylsalicylic acid (ASA) on cell proliferation after partial hepatectomy in rats. Methods: 40 male Wistar rats were separated into four groups of ten rats each. Groups 1 and 2 (controls): undergoing 30% partial hepatectomy and, after one day (group 1) and seven days (group 2), to euthanasia; daily administration of 0.9% saline solution (1mL per 200g of body weight). Groups 3 and 4 (experimental): undergoing 30% partial hepatectomy and, after one day (group 3) and seven days (group 4), to euthanasia; daily administration of ASA (40mg/mL, 1mL per 200g of body weight). The absolute number of cells stained with PCNA was counted in photomicrographs, in five fields, and it was calculated the mean of positive cells per animal and per group. Results: the final mean of PCNA+ cells per group was: in group 1, 17.57 ± 6.77; in group 2, 19.31 ± 5.30; in group 3, 27.46 ± 11.55; and, in group 4, 12.40 ± 5.23. There was no significant difference at the two evaluation times in the control group (p=0.491), but there was in the experimental group (p=0.020), with a lower number of PCNA+ cells on the seventh day. The comparison between the two groups, on the first day, showed more PCNA+ cells in the livers of the animals that received ASA (p=0.047), and on the seventh day the number was lower in the experimental group (p=0.007). Conclusion: ASA induced greater hepatocyte proliferation.


INTRODUCTION
O ne of the main characteristics of the liver is its ability to regenerate.However, the primary response of the organ to different lesions is not always this, and it may concur with fibrosis and hepatocyte necrosis 1 .Hepatic fibrosis results from a sustained response to chronic liver injuries and thrombosis of intrahepatic vessels, which stimulate the exacerbated secretion of the extracellular matrix by stellate cells 2 .Necrosis occurs in contexts of severe hypoxemic or toxic lesions, situations in which liver cells, unable to maintain their basic homeostatic functions, lose their integrity 3 .
The liver has a unique ability to regenerate.
Even after removing 70% of its total mass, the remaining tissue is able to regenerate, recovering its original volume and function 4 .This process depends on a series of cytokines and growth factors, which stimulate angiogenesis and coordinate hepatocyte hypertrophy and hyperplasia.
Platelets seem to play a dual role in liver regeneration: they release pro and anti-fibrotic cytokines, which can either exacerbate parenchymal fibrosis and impair regeneration, or suppress this fibrogenesis and stimulate the regenerative process 5 .Furthermore, the formation of microthrombi in the hepatic venous system Biondo-Simões Acetylsalicylic acid (Aspirin®) and liver regeneration: experimental study in rats mediated by platelet aggregation is associated with liver atrophy and fibrosis in transplanted livers 6 .Therefore, it is suggested that anti-thrombogenic therapies may exert some influence on the reduction of liver fibrosis in these cases 7 .
Acetylsalicylic acid (ASA), like other antiplatelet drugs, seems to play an important role in the modulation of liver fibrosis 7 .ASA is an anti-inflammatory medication with antiplatelet aggregation properties widely used in clinical practice.Its main mechanism of action is the inhibition of the enzyme cyclooxygenase (COX), which converts arachidonic acid into prostaglandins 8 , substrates for the synthesis of thromboxane A2, a compound produced by platelets and responsible for platelet aggregation 9 .
The influence of ASA on the progression of liver fibrosis and its role on the progression of chronic liver diseases are widely discussed topics in the literature.
Studies show that treatment with ASA may be associated with a lower risk of progression of liver fibrosis, a reduction in the risk of developing hepatocarcinoma and mortality from chronic liver diseases [10][11][12][13] .
Most studies address the use of the medication on the progression of fibrosis in already cirrhotic livers [14][15][16][17] , and the role of ASA is poorly understood in liver regeneration in healthy livers.Thus, the present study aims to evaluate the influence of acetylsalicylic acid administration on liver regeneration in a model of partial hepatectomy in healthy male Wistar rats (non-cirrhotic), taking into account its anti-inflammatory and anti-platelet aggregation effects.The temperature was maintained at 20 ± 2 degrees centigrades, the air changes, the characteristic of the environment and the luminosity according to 12-hour light and dark cycles.The animals were kept in polypropylene boxes, appropriate for the species, containing white shavings (changed daily), in groups of five animals per box.They received water and standard commercial food, suitable for the species, ad libitum.

METHODS
The sample was randomly divided into four groups, with ten rats each.The animals in groups 1 and 2 constituted the controls and those in groups 3 and 4, the experiments.All underwent partial hepatectomy.
The animals in the control groups received daily sodium chloride solution, 0.9%, 1mL per 200g of body weight, by gavage, and those in the experimental groups received acetylsalicylic acid 40mg/mL, 1mL per 200g of body weight 20 .The animals in groups 1 and 3 were euthanized 24 hours after the intervention and those in groups 2 and 4 on the seventh day after the intervention.The medication was started one day before the intervention and was maintained until euthanasia.
Partial hepatectomy was performed according to the modified method by Higgins and Anderson (1931) 21 , with resection of approximately 30% of the liver.Anesthesia was performed by a veterinariananesthetist, with intramuscular injection of ketamine hydrochloride (50mg/kg) and xylazine hydrochloride (20mg/kg), complemented with induction via inhalation with isoflurane 1 to 1.5% under mask, associated with 100% of oxygen.Trichotomy of the ventral abdominal wall was performed, antisepsis was carried out with polyvinylpyrrolidone-iodine (PVP-I) and a median incision of four centimeters.The ligamentum teres hepatis was sectioned, isolating the left lateral lobe, which, after ligation with 4.0 cotton thread, was resected.After the review of hemostasis, laparorrhaphy was performed in two planes, the first, the peritoneum-muscle-aponeurotic plane and the second, the skin, with synthesis in continuous running with a 4.0 nylon monofilament thread.Intramuscular dipyrone (10mg/kg) was used for analgesia.After the period determined for each group, euthanasia was performed under anesthesia, according to the protocol described in the Guidelines for the Practice of Euthanasia of the National Council for the Control of Animal Experimentation (2013) 22 and the Brazilian Guide for Good Practices in Euthanasia in Animals of the Federal Council of Veterinary Medicine (2013).Anesthetic induction was performed with inhaled isoflurane and thiopental sodium solution (10mg/kg) was administered intravenously, followed by cardiac puncture for administration of 10% potassium chloride solution (5mg/kg).
Collection of the remaining liver started with trichotomy and antisepsis of the abdominal wall.This was followed by the opening of the cavity with a "U" incision, which, when folded cranially, allowed for the exposure and total resection of the organ.
The dried pieces were fixed in 10% buffered formalin, sending them for histological analysis.Fourmicrometer-thick sections were prepared for staining with immunohistochemistry with primary monoclonal proliferating cell nuclear antigen (PCNA) antibody using the strepto-avidin-biotin-peroxidase technique.
The analysis of cell proliferation was based on counting the absolute number of cells labeled by anti-PCNA (PCNA+ cells) in photomicrographs of histological sections and subsequent calculation of the mean number of positive cells per animal and per group (Figure 1).
The ZEN Blue software (Carl Zeiss Microimaging, Jena, Germany) was used for cell counting.For each animal, four slides were made and, on each slide, five fields with an area of 4.84cm², randomly marked, were analyzed at 400x magnification.The counting was carried out by two independent observers and, if there was an interobserver difference greater than 30% between the final means obtained for the group, the analysis was carried out by a third observer.
The collected data underwent statistical analysis using two-tailed non-parametric Mann-Whitney tests for comparison between groups 1 versus 3 and 2 versus 4; and Kruskal-Wallis for comparison between groups 1 versus 2 and 3 versus 4, adopting p<0.05 (5%) as the level of significance.

RESULTS
There were two deaths in groups 2 and 4 and three deaths in groups 1 and 3.One animal in group 1 and one in group 3 were excluded from the statistical analysis due to a technique artifact in the histological preparation.
The mean of PCNA+ cells between all groups ranged from 7.10 to 44.70, with a total mean of 18.60 ±

DISCUSSION
The partial hepatectomy model in rats is the classic for studying liver regeneration, and it has been used for decades 23 .The model described by Higgins and Anderson (1931) 21 proposes the surgical removal of two thirds of the liver, resulting in immediate activation of the regenerative process and complete recovery of the organ mass after seven to ten days [24][25][26] .The popularity of the model is based on two important aspects: the  The analysis of the first day showed a much higher number of replicating hepatocytes in the ASA group than in the control group (p=0.047).The sevenday evaluation showed a greater number of hepatocytes in replication in the control group (p=0.007).These data demonstrate that, at first, when the regenerative process was installed, animals treated with ASA had a more active replication rate.At seven days, the untreated group showed a greater number of replicating hepatocytes.This observation allows us to imply that the replication process took place earlier and with greater speed in the treated group.
Although Miyaoka et al. 33 have pointed out cell hypertrophy as the main initial mechanism for increasing liver mass after partial hepatectomy in rats, Marongiu et al. 34 demonstrated that hepatocyte hyperplasia is the key mechanism in the evolution of liver regeneration, with hypertrophy being only transitory as part of the process.Therefore, in the present study, mass gain and hepatocyte hypertrophy were not evaluated, the effects of ASA on liver regeneration are highlighted only by hepatocyte hyperplasia, with PCNA+ cell count.

Wang et al. demonstrated thrombocytopenia
and medication-mediated platelet dysfunction as determining factors in reducing the rate of liver regeneration in rats 35 .Platelets accumulate in the remaining liver tissue within 5 to 15 minutes after partial hepatectomy in rats 36,37 and disappear within an hour of the procedure, which suggests a platelet mitogenic stimulus in the very early stages of the postoperative period 36 .
In humans, there is evidence that postoperative platelet count influences the functionality and regeneration of transplanted liver tissue, as well resection of liver tissue is not associated with massive necrosis of the remaining tissue and the regeneration time can be precisely measured 27 .
The induction of regeneration occurs at a time when virtually all hepatocytes are quiescent in phase G028; DNA synthesis begins 12-16 hours after hepatectomy and peaks at 24-48 hours 23,29 .Thus, the first 24 hours after the procedure provide a single period for studying hepatocytes, which start the cell cycle, going from the G0 to G1 state and from G1 to the S phase in relative synchrony 30 .The regeneration rate then peaks between one and four days (when analyzed in terms of liver weight, liver regeneration rate and hepatocyte proliferation), being completed after eight days 31 .
In the present study, the impact of acetylsalicylic acid (ASA) on liver regeneration after partial hepatectomy in rats was analyzed.It was decided to modify the technique described by Higgins and Andersen (1931), adopting a single resection of the left hepatic lobe (approximately 30% of the organ volume), due to the high mortality when the pilot study was carried out, and 70% of the liver mass was ressected.Liver regeneration was evaluated one and seven days after the procedure, according to the literature indication that DNA synthesis reaches its peak 24 hours 29 after resection and that the regeneration process is complete after seven to ten days [24][25][26]31 .
There are several methods to quantify liver regeneration, including: liver mass, mitotic cell count, identification of DNA synthesis, and immunohistochemical methods.Among these, staining with proliferating cell nuclear antigen (PCNA) antibody is one of the most common 32 .PCNA is an auxiliary protein of the DNA polymerase delta enzyme, essential for DNA replication in prokaryotic cells.Its expression is cell cycle dependent, being initially detected in the G1 phase, with a peak in the S phase.In this regard, it acts as the morbidity and mortality of patients undergoing hepatectomy 35,38,39 .Conversely, thrombocytosis and platelet infusion in the portal vein seem to stimulate liver regeneration after hepatectomy 35,37 .
The exact mechanism by which platelets stimulate liver regeneration is still unclear.In vivo studies demonstrate that inflammatory cytokines and growth factors released by platelets may be crucial for the initiation of hepatocyte proliferation 35,36,40 , and direct contact between platelets and endothelial cells of the hepatic sinusoids seem to play an important role in stimulating to the release of these platelet factors 41 .In vitro studies demonstrate that serotonin, stored in platelets and released at sites of injury as part of the platelet hemostatic action, is a potent mitogenic agent and tissue remodeler 42,43 .Thus, the reduction in platelet number or activity after partial hepatectomy could negatively influence the liver regeneration process.
On the other hand, when evaluating the influence of antiplatelet agents in models of liver cirrhosis in rats, Assy et al. 20 demonstrated that ASA in low doses was able to prevent the progression of fibrosis and to stimulate liver regeneration in cirrhotic livers.Poujol-Robert et al. 14 observed an association between the use of ASA and a reduction in the progression of fibrosis rates in transplant patients with hepatitis C.

CONCLUSION
The results of this study demonstrate that the use of ASA stimulated hepatocyte proliferation.
Acetylsalicylic acid (Aspirin®) and liver regeneration: experimental study in rats In the intra-group comparison, there was no significant difference when comparing the control groups (1 and 2) (p=0.439),although a greater number of PCNA+ cells was found on the seventh day (1 day = 17.57± 6.77 and 7 days = 19.31± 5.30).In the experimental group (treated with ASA) there was a higher number of cells on the first day and a lower number after seven days (p=0.020)(1 day = 26.78± 12.08 and 7 days = 12.40 ± 5.23).In the inter-group comparison, it could be verified that there was a greater number of PCNA+ cells in the experimental group (ASA) in the one-day evaluation (Group 1 = 17.57± 6.77 and Group 3 = 27.46 ± 11 .55),this difference was significant (p=0.047).The comparison between the two groups, after seven days, showed that there was a greater number of PCNA+ cells in the control group (p=0.007),(Group 2 = 19.31± 5.30 and Group 4 = 12.40 ± 5.23).
Palavras-chave: Regeneração Hepática.Aspirina.Hepatectomia.R E S U M O R E S U M O