Kelleni MT, Abdelzaher WY, Adly M, Attya ME, Fawzy MA, Ibrahim MA. Targeting sirtuin 1/nuclear factor erythroid 2-related factor 2/tumor necrosis factor-α pathway to modulate hepatic ischemia reperfusion-induced injury. World J Hepatol 2025; 17(12): 110733 [DOI: 10.4254/wjh.v17.i12.110733]
Corresponding Author of This Article
Mina Thabet Kelleni, MD, PhD, Assistant Professor, Department of Medical Pharmacology, College of Medicine, Minia University, Main Road Shalaby Land, Minia 61519, Egypt. mina.kelleni@mu.edu.eg
Research Domain of This Article
Pharmacology & Pharmacy
Article-Type of This Article
Basic Study
Open-Access Policy of This Article
This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Mina Thabet Kelleni, Walaa Yehia Abdelzaher, Marly Adly, Mohamed Abdellah Ibrahim, Department of Medical Pharmacology, College of Medicine, Minia University, Minia 61519, Egypt
Walaa Yehia Abdelzaher, Department of Pharmacology, Faculty of Oral and Dental Medicine, Lotus University, Minia 61768, Egypt
Mina Ezzat Attya, Department of Pathology, Minia University, Minia 61519, Egypt
Michael A Fawzy, Department of Biochemistry-Faculty of Pharmacy, Minia University, Minia 61519, Egypt
Author contributions: Kelleni MT, Abdelzaher WY, Adly M, and Ibrahim MA were responsible for conceptualization of the concept, performing the experiments, writing, and revising the manuscript; Attya ME performed and wrote the histopathological and immunohistochemical examination; Fawzy MA performed and wrote the western blotting. All authors read, revised, and approved the manuscript.
Institutional review board statement: Permission was received from the Study Ethics Committee of the Faculty of Medicine, Minia University (No. 585/2023).
Institutional animal care and use committee statement: The National Institutes of Health’s guide for the care and use of laboratory animals was followed during the research. All methods were done in accordance with the relevant ethical guidelines and regulations.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Data sharing statement: All the necessary data are included in the manuscript. Any further data will be made available upon a reasonable request.
Open Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Mina Thabet Kelleni, MD, PhD, Assistant Professor, Department of Medical Pharmacology, College of Medicine, Minia University, Main Road Shalaby Land, Minia 61519, Egypt. mina.kelleni@mu.edu.eg
Received: June 13, 2025 Revised: July 24, 2025 Accepted: November 7, 2025 Published online: December 27, 2025 Processing time: 196 Days and 10.7 Hours
Abstract
BACKGROUND
Hepatic ischemia reperfusion (HIR) injury is a major complication affecting various major liver surgeries, including liver transplantation. Aprepitant (APRE), a neurokinin-1 receptor antagonist, is commonly used as an antiemetic to prevent chemotherapy-induced nausea and vomiting.
AIM
To assess the potential protective effect of APRE against HIR-induced liver injury via targeting the nucleotide-binding oligomerization domain-, leucine-rich repeat-, and pyrin domain-containing receptor 3/interleukin (IL)-1beta signaling pathway.
METHODS
Six groups of adult male Wistar albino rats were divided as follows: Sham group, Sham/APRE10 group (APRE 10 mg/kg), HIR group, HIR/APRE5 group (APRE 5 mg/kg), HIR/APRE10 group (APRE 10 mg/kg), and HIR/APRE20 group (APRE 20 mg/kg). Serum alanine transaminase, aspartate transaminase, liver malondialdehyde, total antioxidant capacity levels, as well as IL-6, sirtuin 1 (Sirt1), caspase-3, cleaved caspase-3, and tumor necrosis factor alpha biomarkers, were evaluated. Hepatic specimens were examined histopathologically and immunohistochemically for nuclear factor erythroid-2-related factor 2 (Nrf2) immunoexpression.
RESULTS
HIR resulted in hepatic damage, as evidenced by histopathological changes and a significant increase in serum alanine transaminase, aspartate transaminase, hepatic malondialdehyde, caspase-3, and tumor necrosis factor alpha levels. Additionally, there were significant increases in hepatic total antioxidant capacity and reductions in IL-6 and cleaved caspase-3 protein levels, as demonstrated by Western blot analysis, along with enhanced immunoexpression of Sirt1 and Nrf2. APRE has significantly reduced various parameters of oxidative stress, inflammation, and apoptosis, and a significant increase in liver Nrf2 immunoexpression, leading to a significant improvement in the histopathological changes.
CONCLUSION
In conclusion, targeting the Sirt1/Nrf2 signaling pathway, as demonstrated by APRE in our model, could present a promising therapeutic target to protect against HIR-induced liver injury during major liver surgeries.
Core Tip: This study demonstrates that aprepitant, a neurokinin-1 receptor antagonist, exerts hepatoprotective effects in a rat model of hepatic ischemia reperfusion by modulating the sirtuin 1/nuclear factor erythroid-2-related factor 2/tumor necrosis factor alpha signaling pathway. Aprepitant significantly reduced hepatic enzyme elevation, oxidative stress parameters, inflammatory cytokines, and apoptosis markers, while enhancing the pluripotent capacity of sirtuin 1/nuclear factor erythroid-2-related factor 2 expression, highlighting its potential as a therapeutic agent to mitigate HIR-induced liver injury during major liver surgeries, as well as highlighting the significant therapeutic pharmacological potential while targeting this pathway by other current and future drugs.
Citation: Kelleni MT, Abdelzaher WY, Adly M, Attya ME, Fawzy MA, Ibrahim MA. Targeting sirtuin 1/nuclear factor erythroid 2-related factor 2/tumor necrosis factor-α pathway to modulate hepatic ischemia reperfusion-induced injury. World J Hepatol 2025; 17(12): 110733
Hepatic ischemia reperfusion (HIR) induced liver injury is a common and almost inevitable complication that follows various liver surgeries such as liver resection and transplantation[1]. Due to its high prevalence, the development of preventive measures has become of great medical importance to reduce its detrimental impact on the overall survival of patients[2]. HIR-induced liver injury occurs in two consecutive phases: Ischemic and reperfusion phases. Both phases result in hepatic injury, necrosis, and apoptosis[3]. During the ischemic phase, both nutrient and oxygen supply are interrupted, resulting in adenosine triphosphate depletion and failure of adenosine triphosphate-dependent pumps. On the other hand, reperfusion causes reactive oxygen species (ROS) overproduction[4]. This hepatic injury triggers the recruitment of inflammatory cells and the subsequent release of inflammatory mediators such as interleukin-1 and tumor necrosis factor alpha (TNF-α), causing more liver damage and systemic inflammatory response syndrome[3,5]. Consequently, graft dysfunction or rejection, hepatic failure, and death may occur[6]. These detrimental effects can also extend to include several remote organs such as lungs, heart, and brain[5].
Aprepitant (APRE) is an antiemetic drug used for the treatment of vomiting induced by various chemotherapeutic agents. It exerts its action through antagonism of the neurokinin-1 receptor[7]. Different experimental studies have shown its antioxidant, anti-inflammatory, and anti-apoptotic role in various models such as nephrotoxicity and hepatotoxicity models[8], acute lung injury models[9], myocardial injury models[10], and epilepsy models[11]. We aimed to evaluate the possible protective and beneficial effect of APRE against HIR-induced liver injury by assessing its capability to modulate the sirtuin 1 (Sirt1)/nuclear factor erythroid-2-related factor 2 (Nrf2)/TNF-α signaling pathway.
MATERIALS AND METHODS
Chemicals and drugs
APRE was acquired from Merck Sharp and Dohme in the United Kingdom. We purchased xylazine from Adwia in Egypt and ketamine from Elice Pharma in Pakistan.
Animals and experimental design
48 adult male Wistar albino rats weighing 180-240 g were used. Rats were bought from El-Nahda University in Beni-Suef, Egypt. The rats were given a standard diet consisting of commercial rat chow and tap water ad libitum before being used in the study. They were also allowed to adapt to their environment for two weeks. The National Institutes of Health’s guide for the care and use of laboratory animals was followed during the research. Permission was received from the Study Ethics Committee of the Faculty of Medicine, Minia University (No. 585/2023) for the protocol of our experiment, and all methods were done in accordance with the relevant ethical guidelines and regulations, including regular assessment of animal behavior at least twice daily. The current study also complied with ARRIVE guidelines.
Six groups, each consisting of eight rats, were established with random distribution of rats as follows: (1) Sham group: Rats without HIR had an abdominal incision and were given carboxymethyl cellulose (CMC) orally[12]; (2) Sham/APRE10 group: APRE 10 mg/kg was suspended in CMC[13] and was given to rats orally every day for five days[12] prior to abdominal incision without HIR; (3) HIR group: Rats were given CMC then laparotomy was done and pringle maneuver was performed in the form of 30 minutes of ischemia and 1 hour of reperfusion[14]; (4) HIR/APRE5 group: APRE 5 mg/kg was suspended in CMC and was given to rats orally every day for five days in a row prior to HIR[10]; (5) HIR/APRE10 group: APRE 10 mg/kg was suspended in CMC and was given to rats orally every day for five days in a row prior to HIR[10]; and (6) HIR/APRE20 group: APRE 20 mg/kg was suspended in CMC and was given to rats orally every day for five days in a row prior to HIR[10]. The number of rats in our study was determined based on previous similar experimental studies in the literature evaluating HIR injury and pharmacological interventions[8,14].
Hepatic ischemia induction
The rats were fasted for 16 hours prior to the experiment. All surgical procedures were carried out under general anesthesia using intraperitoneal Xylazine (0.25 mg/kg) and ketamine injections (1 mg/kg)[15]. A midline incision was made in the rats’ abdomens. Pringle’s maneuver, which involved using bulldog clamps to totally obstruct the rats’ portal triad (portal vein, hepatic artery, and common bile duct) for 30 minutes, was used to produce hepatic ischemia[14]. A normal amount of saline at 37 °C was administered into the rat’s abdominal cavity while its abdomen was covered with plastic coverings to avoid visceral dehydration. By releasing the clamps, reperfusion was started when the rats’ temperature was adjusted to 37 °C using warming aids (a heater and lamps). Silk suturing was used to close the rat's abdominal cavity. An hour after reperfusion, the rats were sacrificed[14].
Blood and tissue sampling
Animal scarification was carried out at the final stage of the experiment, and rats’ serum was collected by centrifuging blood samples from the abdominal aorta for 10 minutes at 4000 × g using a centrifuge (Jantezki, T30, Germany). The serum was then stored at -80 °C for additional evaluation of different parameters. The liver was separated, with a portion stored at -80 °C for the biochemical assessment. Liver sections were preserved in 10% formalin for histological analysis. A homogenizer (Tri-R Stir-R homogenizer, Tri-R Instruments, Inc., Rockville Centre, NY, United States) was used to homogenize each gram of liver tissue in 5 milliliters of phosphate buffer saline, which was obtained by dissolving 0.2 g of KCl, 8.01 g of NaCl, 0.27 g of KH2PO4, and 1.78 g of Na2HPO4.2H2O in one litter of distilled water, adjusting the pH to 7.4, and centrifuging the resulting homogenate for 15 minutes at 4000 × g. For a further evaluation of several parameters, the supernatant was extracted and kept at -80 °C.
Biochemical analysis
Estimation of serum ALT, AST: A colorimetric assay based on the reaction reported by Gella et al[16] was used to measure the serum levels of alanine transaminase (ALT) and aspartate transaminase (AST) using Biosystems kits (No. 11533 and 11531, Barcelona, Spain), respectively.
Evaluation of total protein in hepatic tissue: A colorimetric commercial kit (Biodiagnostic Co., Egypt) was used to measure the total protein concentration in accordance with the manufacturer’s instructions, No. TP 20 20.
Evaluation of hepatic oxidative stress parameters: The malondialdehyde (MDA) kit (Biodiagnostic, Egypt, No. MD 25 29) was used to measure the amount of MDA, a biomarker of lipid peroxidation, in hepatic tissue. Total antioxidant capacity (TAC) was determined using a commercial colorimetric kit (Biodiagnostic, Egypt, No. TA 25 13) in accordance with the manufacturer’s instructions.
Evaluation of caspase-3 in lung tissue: The rat caspase-3 enzyme-linked immunosorbent assay kit was used to measure the amount of caspase-3 in the hepatic tissue homogenate in accordance with the manufacturer's instructions (Biovision, No. E4592-100).
Evaluation of TNF-α in hepatic tissue: Tumor necrosis factor alpha (TNF-α) level in liver tissue homogenate was determined by TNF-α enzyme-linked immunosorbent assay kit according to the manufacturer's instructions, No. RAB0480.
Western blotting analysis of IL-6, cleaved caspase-3 and Sirt1 in hepatic tissue
After boiling of tissue homogenates (50 μg of total proteins) for 5 minutes combining loading buffer containing 2-mercaptoethanol they were applied to 12% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) then running for 2 hours at 100 V. After electrophoresis, blotted proteins to polyvinylideneflouride (PVDF) membranes were blocked for 1 hour in a trisbuffered saline (TBS-T) blocking solution containing 5% (w/v) non-fat milk and 0.05% Tween-20. Incubation with primary antibodies Rabbit anti-interleukin (IL) 6 antibody (No. ab259341, Abcam, Cambridge, United Kingdom), anti-cleaved caspase-3 antibody (1:1000, No. ab214430, Abcam, Cambridge, United Kingdom) and Anti-Sirt1 antibody (No. ab189494, Abcam, Cambridge, United Kingdom) and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, United States) was allowed overnight at 4 °C. Goat anti-rabbit polyclonal immunoglobulin conjugated with horseradish peroxidase (1:5000) (Cell Signaling Technology Inc., MA, United States) in blocking buffer was used as a secondary antibody. Bands were visualized by chemiluminescence. Protein bands of all groups were quantified densitometrically as fold change relative to the normal control group after being normalized to β-actin using Image J Software.
Histopathological procedure and examination
The hepatic tissue specimens of the rats were dissected and preserved in a 10% formalin solution. These tissue specimens were dehydrated in ascending alcohol concentrations, processed, and embedded in paraffin. A 5 μm thick cross-section was cut using a microtome and placed on glass slides. Furthermore, these tissue sections were subjected to hematoxylin-eosin stain and examined and evaluated by a pathologist who was blinded to different research groups under an Olympus light microscope. The liver injury was scored as the following score: Score 0 representing normal liver tissue without any injury; score 1 representing slight damage involving less than 10% of centrilobular hepatocytes; score 2 representing moderate damage involving 10%-50% of centrilobular hepatocytes, and score 3 representing severe damage involving more than 50% of centrilobular hepatocytes. Liver injury was assessed by 3 parameters: Necrosis, degeneration, and inflammation[17].
Immunohistochemical staining of Nrf2 in hepatic tissue
Formalin-fixed paraffin-embedded hepatic tissue blocks were sectioned into 5 µm sections using a microtome. Then, these tissue sections were placed on positively charged glass slides and were immunohistochemically stained manually. Initially, these tissue sections were dewaxed, rehydrated, and endogenous peroxidase blocked by immersion in 3% H2O2 solution for 30 minutes. Immersion of the slides in sodium citrate puffer (pH = 6) for 2 times, 10 minutes each, in a microwave was used for antigen retrieval. Blocking of non-specific staining is performed by treating the slides with an ultraviolet block. Polyclonal rabbit anti-Nrf2 antibody (ABclonal Technology Co., Ltd., Wuhan, China) was added to each slide at 1:100 dilution and incubated overnight in the humidity chamber at 4 °C. After that, the biotinylated secondary antibody was added to each slide for 30 minutes at room temperature. One drop of diaminobenzidine substrate-chromogen was applied to each slide for 15 minutes or till the brown coloration was seen. Then, slides were dipped in Mayer’s hematoxylin to be counterstained. Regarding the evaluation and scoring of Nrf2 expression, the slides were examined under light microscope magnification × 200 or × 400. Nrf2 staining intensity was scored into: Score (0) representing no staining; score (1) weak, score (2) moderate, and score (3) strong staining[18].
Statistical analysis
Means ± SEM were used to express the results. One-way analysis of variance (ANOVA) and Tukey’s test were used to analyze the results. P-values less than 0.05 were regarded as significant differences. For statistical analysis, GraphPad Prism (version 7 for Windows, GraphPad software, San Diego, CA, United States; Available from: https://www.graphpad.com/) was utilized.
RESULTS
Effect of different doses of APRE on the rats’ survival rate after HIR injury
The survival of all groups was observed throughout the maneuver. Our findings revealed that 37.5% of rats in the HIR group died during ischemia and reperfusion phases. While HIR/APRE5, HIR/APRE10, and HIR/APRE20 groups had mortality 25%, 12.5% and zero, respectively. HIR/APRE5, HIR/APRE10, and HIR/APRE20 groups showed a significant improvement in the overall survival 75%, 87.5% and 100% compared to the HIR group (62.5%) (Table 1).
Table 1 Effect of different doses of aprepitant on survival rate of hepatic ischemia reperfusion injury in rats.
Effect of different doses of APRE on ALT and AST in HIR induced liver injury in rats
The results of the current study revealed that HIR produced a significant increase in ALT and AST levels as compared to the sham and Sham/APRE10 groups. Administration of HIR/APRE5, HIR/APRE10, and HIR/APRE20 showed a significant reduction in levels of serum ALT compared to HIR, with no significant difference in its levels between HIR/APRE10 and HIR/APRE20 groups. Meanwhile, administration of APRE5, APRE10, and APRE20 with HIR showed a significant reduction in levels of serum AST in a dose-dependent manner compared to HIR (Table 2).
Table 2 Effect of different doses of aprepitant on alanine transaminase and aspartate transaminase in hepatic ischemia reperfusion-induced liver injury in rats.
Effect of different doses of APRE on hepatic oxidative stress parameters in HIR induced liver injury in rats
The results of current study revealed that HIR produced a significant increase in levels of hepatic MDA with a significant reduction in hepatic TAC levels as compared to Sham and Sham/APRE10 groups while administration of APRE5, APRE10 and APRE20 with HIR showed a significant reduction in hepatic MDA and increase in hepatic TAC levels in a dose dependent manner compared to HIR group (Table 3).
Table 3 Effect of different doses of aprepitant on hepatic oxidative stress parameters in hepatic ischemia reperfusion-induced liver injury in rats.
Effect of different doses of APRE on hepatic anti-inflammatory parameters in HIR induced liver injury in rats
HIR injury revealed a significant increase in hepatic TNF-α level and IL-6 expression as compared to Sham and Sham/APRE10 groups. On the other hand, HIR/APRE5, HIR/APRE10, and HIR/APRE20 groups showed a significant decrease in the level of hepatic TNF-α and IL-6 expression in a dose-dependent manner compared to the HIR group (Figure 1).
Figure 1 Effect of different doses of aprepitant on hepatic tumor necrosis factor alpha in hepatic ischemia reperfusion-induced liver injury in rats.
Results are considered significantly different when P < 0.05. aP < 0.05 significantly different from Sham group; bP < 0.05 significantly different from Sham/aprepitant (APRE) 10; cP < 0.05 significantly different from hepatic ischemia reperfusion (HIR) group; dP < 0.05 significantly different from HIR/APRE5; eP < 0.05 significantly different from HIR/APRE10 group. TNF-α: Tumor necrosis factor alpha; APRE5: Aprepitant 5 mg/kg; APRE10: Aprepitant 10 mg/kg; APRE20: Aprepitant 20 mg/kg; HIR: Hepatic ischemia reperfusion; IL-6: Interleukin-6.
Effect of different doses of APRE on hepatic caspase-3 in HIR induced liver injury in rats
HIR group revealed a significant elevation in hepatic caspase-3 in comparison to the Sham and Sham/APRE10 groups, while giving APRE5, APRE10, and APRE20 with HIR showed a significant reduction in hepatic caspase-3 in a dose-dependent manner compared to the HIR group (Figure 2).
Figure 2 Effect of different doses of aprepitant on hepatic caspase-3 level in hepatic ischemia reperfusion-induced liver injury in rats.
Results are considered significantly different when P < 0.05. aP < 0.05 significantly different from Sham group; bP < 0.05 significantly different from Sham/aprepitant (APRE) 10; cP < 0.05 significantly different from hepatic ischemia reperfusion (HIR) group; dP < 0.05 significantly different from HIR/APRE5; eP < 0.05 significantly different from HIR/APRE10 group. APRE5: Aprepitant 5 mg/kg; APRE10: Aprepitant 10 mg/kg; APRE20: Aprepitant 20 mg/kg; HIR: Hepatic ischemia reperfusion.
Effect of different doses of APRE on hepatic Sirt1 expression in HIR induced liver injury in rats
Our study showed that Sirt1 decreased in the HIR group in comparison to the Sham and Sham/APRE10 groups. On the other hand, HIR/APRE5, HIR/APRE10, and HIR/APRE20 groups showed a significant increase in the level of hepatic Sirt1 compared to the HIR group, with no significant difference between different APRE doses (Figure 3).
Figure 3 Effect of different doses of aprepitant on hepatic sirtuin 1 western blotting in hepatic ischemia reperfusion-induced liver injury in rats.
Results are considered significantly different when P < 0.05. aP < 0.05 significantly different from Sham group; bP < 0.05 significantly different from Sham/aprepitant 10; cP < 0.05 significantly different from hepatic ischemia reperfusion group. APRE5: Aprepitant 5 mg/kg; APRE10: Aprepitant 10 mg/kg; APRE20: Aprepitant 20 mg/kg; HIR: Hepatic ischemia reperfusion; SIRT-1: Sirtuin1.
APRE on hepatic histopathology in HIR induced liver injury in rats
Slides of both Sham (Figure 4A) and Sham/APRE10 (Figure 4B) groups showed normal liver architecture composed of a normal central vein (brown arrows) lined by endothelial cells with normal polygonal hepatocytes (blue arrows) with eosinophilic cytoplasm arranged around it, with the absence of any inflammation. Slides of the HIR group (Figure 4C) showed marked centrilobular necrosis (black arrow), hepatocyte degeneration, and inflammatory cell infiltrate. Slides of the HIR/APRE5 group (Figure 4D) showed moderate hepatocyte necrosis (black arrow), degeneration, and inflammatory cell infiltration (yellow arrow). Slides of the HIR/APRE10 group (Figure 4E) showed mild hepatocyte necrosis (black arrow), degeneration, and inflammatory cell infiltrate (yellow arrow). HIR/APRE20 group (Figure 4F) showed mild degeneration and inflammatory cells infiltrate (blue arrows) with no necrosis (Figure 4).
Figure 4 Histopathology of hepatic tissues in hepatic ischemia reperfusion-induced liver injury in rats.
A and B: Slides of both Sham (A) and Sham/aprepitant (APRE) 10 group (B) groups showed a normal central vein (brown arrows) lined by endothelial cells with normal polygonal hepatocytes (blue arrows); C: Slides of the hepatic ischemia reperfusion (HIR) group showed marked centrilobular necrosis (black arrow); D: Showed moderate hepatocyte necrosis (black arrow), degeneration, and inflammatory cell infiltration (yellow arrow). E: Slides of the HIR/APRE10 group showed mild hepatocyte necrosis (black arrow), degeneration, and inflammatory cell infiltrate (yellow arrow); F: The HIR/APRE20 group. Results are considered significantly different when P < 0.05. aP < 0.05 significantly different from Sham group; bP < 0.05 significantly different from Sham/aprepitant (APRE) 10; cP < 0.05 significantly different from hepatic ischemia reperfusion (HIR) group; dP < 0.05 significantly different from HIR/APRE5; eP < 0.05 significantly different from HIR/APRE10 group. APRE5: Aprepitant 5 mg/kg; APRE10: Aprepitant 10 mg/kg; APRE20: Aprepitant 20 mg/kg; HIR: Hepatic ischemia reperfusion.
Effect of different doses of APRE on hepatic Nrf2 immunohistochemical expression in HIR induced liver injury in rats
The HIR/APRE5, HIR/APRE10, and HIR/APRE20 groups showed an increase in the immune staining of hepatocytes in a dose dependent manner in comparison to HIR group that showed a decrease in the number of immunoreactive hepatocytes with no significant difference between either HIR/APRE10 group and HIR/APRE20 group or sham and sham/APRE10 groups (Figure 5).
Figure 5 Immunohistochemical expression of nuclear factor erythroid-2-related factor 2 in hepatic tissues in hepatic ischemia reperfusion-induced liver injury.
A: Sham group; B: Sham/aprepitant (APRE) 10 group; C: Hepatic ischemia reperfusion (HIR) group; D: HIR/APRE5 group; E: The HIR/APRE10 group; F: The HIR/APRE20 group. The HIR/APRE5 (D), HIR/APRE10 (E), and HIR/APRE20 groups (F), showed an increase in the immune staining of hepatocytes in a dose dependent manner in comparison to HIR group (C), that showed a decrease in the number of immunoreactive hepatocytes with no significant difference between either HIR/APRE10 group and HIR/APRE20 group or sham (A), and sham/APRE10 (B), groups. Results are considered significantly different when P < 0.05. aP < 0.05 significantly different from Sham group; bP < 0.05 significantly different from Sham/aprepitant (APRE) 10; cP < 0.05 significantly different from hepatic ischemia reperfusion (HIR) group; dP < 0.05 significantly different from HIR/APRE5; eP < 0.05 significantly different from HIR/APRE10 group. APRE5: Aprepitant 5 mg/kg; APRE10: Aprepitant 10 mg/kg; APRE20: Aprepitant 20 mg/kg; HIR: Hepatic ischemia reperfusion.
DISCUSSION
HIR is an important etiology of hepatic damage that commonly complicates major liver surgeries as liver transplantation and partial hepatectomy. This results in numerous detrimental effects in the liver as well as in other remote organs[5]. The present study showed that the HIR rats’ group mortality rates were significantly high, and this could be explained by systemic inflammatory response syndrome and diffuse infiltration of both liver and different remote organs by inflammatory cells, platelet aggregation, and small blood vessels occlusion by thrombi[19]. Furthermore, there is a marked reduction in rats’ cardiac output and instability of the circulatory system due to the complete occlusion of hepatic blood vessels[20]. This agreed with previous studies that concluded similar findings[21,22]. APRE-treated rats’ groups showed a significant reduction in the mortality rates, which occurred in the reperfusion phase, in a dose-dependent manner. This mortality reduction was maximized in the APRE20 group, and this could be attributable to the mitigation of reperfusion-related injury mechanisms, including oxidative stress and inflammatory cytokine release, which is explained by APRE’s anti-inflammatory and antioxidant characteristics[23].
Similarly, liver aminotransferases, ALT and AST, are normally present inside hepatocytes and considered as sensors of hepatocyte damage that help early detection of liver injury and are used to evaluate the liver function[24,25]. In our study, HIR resulted in liver damage, evidenced by a significant increase in liver aminotransferases. Consistent with our results, several previous studies showed that HIR caused a significant increase in liver aminotransferases[26-28]. On the other hand, pretreatment with APRE in different doses resulted in a reduction of liver aminotransferases in a dose-dependent manner, and this agreed with Un and co-authors who have also concluded the potential hepatoprotective role of APRE[8].
Moreover, HIR caused a significant oxidative stress in liver tissue, as proved by an increase in liver MDA and a significant reduction in TAC levels. This was in line with several previous studies[26,27,29-32]. Tissue injury produces excess MDA, which modifies the structure of molecules through interacting with the free amino group of protein, resulting in the generation of MDA-modified protein adducts that turn them immunogenic[33]. Also, the TAC value indicates the antioxidant state of the organism, including the synergic and redox interactions among different chemicals in the biological fluids[34]. Furthermore, it was observed that APRE pretreatment in different doses with HIR has significantly improved oxidative stress parameters. This matched with several previous studies[8,35,36]. These studies have shown that neurokinin-1 receptor activation elevated the oxidative stress in tissues by ROS overproduction, while blocking APRE reduced the ROS production owing to its antioxidant characteristics.
Regarding the liver histopathological changes, our study demonstrated that HIR resulted in marked tissue damage in the form of marked hepatocyte degeneration and necrosis, and inflammatory cell infiltrate. This finding agreed with previous studies performed by Wang et al[37] and Xia et al[38]. Interestingly, co-administration of APRE resulted in dose-dependent amelioration of these detrimental effects through decreasing the degree of hepatocyte degeneration, necrosis, and inflammatory cell infiltrate. This is matching with Un and colleagues who found that administration of APRE in different doses caused amelioration of histopathological injuries of both liver and kidney caused by cisplatin in rats due to its anti-inflammatory effect[8].
In the same manner, Sirt1 is a sensor of cellular stresses and its activation maintains mitochondrial homeostasis. Previous studies showed that the increase of Sirt1 expression alleviated the HIR induced liver damage[39-41], and our study showed that APRE pretreatment with HIR resulted in an increase in Sirt1 western expression, which could explain its hepatoprotective effect. Notably, although the HIR/APRE20 group showed an apparent increase in Nrf2 expression compared to the Sham group, this difference did not reach statistical significance.
Correspondingly, Nrf2 is a transcriptional factor that has anti-inflammatory and cytoprotective effects via activation of certain antioxidant enzymes such as superoxide dismutase and glutathione peroxidase[42]. Previous studies showed the protective effect of increased Nrf2 in different HIR models[43-46]. Our study demonstrated that APRE pretreatment increased Nrf2 immunohistochemical expression. Equally, the decrease of Sirt1 and Nrf2 was previously demonstrated in renal ischemia reperfusion[47,48]. These studies were in accordance with our present study, which showed a significant decrease in Sirt1 western blotting and Nrf2 immunohistochemical expression caused by HIR.
Regarding the role of inflammation, cytokines, e.g., TNF-α and IL-6, liberated from Kupffer cells during HIR aggravate the inflammatory response and cause more liver damage[49]. Our study showed that HIR caused an increase in hepatic TNF-α level and IL-6 expression. While administration of APRE with HIR resulted in a statistically significant reduction in a dose-dependent manner and this came in accordance with previous studies[26,28,29,31]. Moreover, previous studies have shown that APRE can inhibit the expression of chemokines and cytokines in several inflammatory conditions such as rheumatoid arthritis, hepatotoxicity, and nephrotoxicity[8,50].
Likewise, in HIR, the release of cytochrome c in response to mitochondria swelling causes the activation of the caspase cascade, initiating the apoptosis of hepatocytes[51]. In agreement with several previous studies, our present study revealed that HIR resulted in an increase in hepatic tissue caspase 3[28,52,53]. Our results demonstrated that pretreatment with APRE led to a significant reduction in hepatic cleaved caspase-3, as confirmed by Western blot analysis, indicating an attenuation of apoptosis, and it resulted in attenuation of hepatic tissue caspase 3 in a dose-dependent manner that supports the concept that APRE confers hepatoprotective effects, at least in part, by suppressing apoptotic pathways activated during HIR. This finding was in line with Hafez and colleagues, who reported the anti-apoptotic role of APRE in rats’ renal toxicity model[12].
Importantly, a primary limitation of this study is the incomplete validation of the Nrf2 pathway mechanism. Due to our limited resources, we were unable to perform Western blot analyses for Kelch-like ECH-associated protein 1 - Nrf2’s classic upstream regulator - or for key downstream antioxidant targets such as heme oxygenase-1 and NAD(P)H quinone oxidoreductase1. Furthermore, we could not assess Nrf2’s nuclear translocation using immunofluorescence co-staining (Nrf2/4’,6-diamidino-2-phenylindole) or through nuclear/cytoplasmic fractionation followed by Western blot. The unavailability of immunofluorescence and fractionation methods in our laboratories in Egypt represents a recognized constraint in fully delineating the molecular events underpinning APRE’s modulation of the Nrf2 pathway. Future studies are recommended to address these mechanistic aspects more comprehensively.
CONCLUSION
In conclusion, our present study revealed that targeting the Sirt1/Nrf2/TNF-α signaling pathway via APRE reduced the degree of liver injury caused by HIR and had a hepatoprotective effect. APRE caused a statistically significant reduction in the levels of liver enzymes, MDA, TNF-α, and caspase-3 after the administration of APRE. Moreover, it caused a statistically significant increase in hepatic TAC, Sirt1, and Nrf2 expression. Our findings highlight the significant therapeutic pharmacological potential while targeting this pathway by other current and future drugs.
ACKNOWLEDGEMENTS
We are truly grateful for the thorough and highly constructive comments received during the anonymous peer-review process. These insightful suggestions have greatly contributed to improving the clarity, rigor, and overall quality of our manuscript.
Footnotes
Provenance and peer review: Invited article; Externally peer reviewed.
Peer-review model: Single blind
Specialty type: Gastroenterology and hepatology
Country of origin: Egypt
Peer-review report’s classification
Scientific Quality: Grade C
Novelty: Grade B
Creativity or Innovation: Grade C
Scientific Significance: Grade B
P-Reviewer: Li B, PhD, Full Professor, China S-Editor: Bai SR L-Editor: A P-Editor: Zhang YL
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