Protective effects of treprostinil and ischemic preconditioning on hepatic ischemia-reperfusion injury and biomarkers in experimental studies in rats

Abstract

BACKGROUND

Hepatic ischemia-reperfusion injury (HIRI) remains one of the major causes of postoperative liver dysfunction following extensive hepatectomy and liver transplantation. Owing to its progressive and dynamic nature, HIRI may lead to multiple organ failure and a worsened outcome. Treprostinil is a relatively new synthetic prostacyclin analog with a potential beneficial effect against HIRI. Ischemic preconditioning (IP) is a promising method to protect against HIRI.

AIM

To investigate HIRI biomarkers, their effects on liver and heart, and the effects of treprostinil and IP on these processes.

METHODS

Forty male Wistar albino rats aged 3-4 months were randomly assigned to four groups of ten, subjected to a 3-hour surgical intervention, and then sacrificed. Hepatic ischemia was induced by clamping the hepatoduodenal ligament for 30 minutes, followed by reperfusion for 120 minutes. Treprostinil (100 ng/kg/minute for 24 hours) or IP before HIRI, no protection, and a sham operation were applied accordingly in each group. Liver and heart histopathology and specific serum and hepatic tissue biomarkers were assessed.

RESULTS

HIRI deteriorated hepatocellular function and exacerbated liver and myocardial damage in the control group. Furthermore, HIRI triggered cytokine overexpression and protein carbonyl content (P < 0.001). Compared with those in the HIRI group, lower troponin I, tumor necrosis factor-α, endothelin-1, and interleukin-1β in serum and liver tissue were significantly correlated with reduced cellular necrosis and improved hepatocellular function in the treprostinil group (P < 0.001). Similar but less pronounced effects were observed in the IP group. Both treprostinil and IP had protective effects in hepatic and cardiac tissues. However, treprostinil showed slightly superior cardioprotective efficacy, as evidenced by a statistically significant difference in troponin I levels (P < 0.05) and histopathological scoring of myocardium samples, but there were no differences in the other parameters.

CONCLUSION

HIRI results in oxidative stress and cytokine overexpression, which deteriorate hepatic function and accelerates myocardial damage. Treprostinil and IP are promising strategies for preventing reperfusion-induced cellular and systemic damage.

Key Words: Hepatic ischemia-reperfusion injury; Liver surgery and transplantation; Oxidative stress; Prostaglandins; Prostacyclin; Treprostinil; Ischemic preconditioning

Core Tip: Hepatic ischemia-reperfusion injury is a serious condition that causes oxidative stress with a systemic inflammatory response. In addition to the liver, it may cause damage to remote organs, particularly the myocardium, with disastrous results. Consequently, its prevention is crucial, especially in liver surgery and transplantation. This experimental preclinical study in rats evaluated the cytoprotective effect of treprostinil compared with ischemic preconditioning on the liver and heart. Biomarkers were assessed for a better understanding of the pathophysiology of systemic manifestations, thus opening new future perspectives for the design of clinical trials assessing treprostinil efficacy.

INTRODUCTION

Hepatic ischemia-reperfusion injury (HIRI) is a major complication associated with liver surgery and other clinical conditions, such as trauma, hemorrhagic shock and resuscitation[1]. The management of patients with liver disease often requires complex surgical procedures, including major hepatectomy and liver transplantation. Warm HIRI remains the main cause of impaired hepatic function and acute liver failure following liver surgery. However, cold HIRI occurs during hypothermic graft preservation prior to liver transplantation[2,3]. Because the liver is a critical organ in humans and is responsible for supporting metabolism, immune responses, coagulation pathways, vitamin storage, macro- and micronutrient absorption and detoxification, and any restriction or obstruction of the blood supply can lead to hepatic hypoxia and liver damage. Restoration of the blood supply and reoxygenation of ischemic liver tissue often leads to further hepatocellular damage, which is primarily moderated by reactive oxygen species (ROS)[4]. Numerous cells and factors are involved in the complex mechanisms underlying HIRI, including neutrophils, Kupffer cells, platelets, cytokines, chemokines, vasoactive agents, adhesion molecules and various signaling mediators[5,6]. The development of ischemic hepatocellular damage is associated with anaerobic metabolism, metabolic acidosis, intracellular calcium overload, mitochondrial dysfunction, and oxidative stress. Inflammatory cascade activation is strongly linked to the induction of programmed cell death pathways[7]. Apoptosis, pyroptosis and ferroptosis are the main regulated cell death mechanisms involved in HIRI. Apoptosis involves both receptor-mediated and mitochondrial interactions with the transforming growth factor-β-activated kinase 1, c-Jun N-terminal kinase, p38 mitogen-activated protein kinase, apoptosis signal-regulating kinase 1, and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathways. Pyroptosis is another form of programmed cell death triggered by inflammasome activation (NLRP3, NACHT, LRR, and PYD domains-containing protein 3), caspase-1/11, and gasdermin D, contributing to inflammation. Ferroptosis, a form of programmed cell death characterized by iron-dependent lipid peroxidation, has also been identified as a key contributor to HIRI. Therapeutic strategies targeting these pathways using natural compounds, microRNAs, histone deacetylase inhibitors, and signaling modulators have shown promising results in preclinical models[8,9].

As HIRI progresses, it can trigger extensive local and systemic phenomena, resulting in liver failure, remote organ damage and multiple organ dysfunction syndrome[10]. ROS and cytokine release following hepatic ischemia-reperfusion leads to the inflammatory cell and platelet aggregation in distant organs[11]. Systemic inflammatory reactions, along with increased vascular permeability and a hypercoagulable state, are associated with dysfunction of other organs, including the lungs, kidneys, myocardium, gastrointestinal tract, pancreas and brain[10,12,13]. Myocardial damage has been described as a serious complication of HIRI and is mainly observed in patients who have undergone liver transplantation. According to current knowledge, cardiac injury following HIRI may cover a large spectrum of clinical manifestations[14,15]. Overall, HIRI may significantly increase morbidity and mortality rates in cases of extensive and irreversible organ damage. Although multiple pharmacological agents and surgical techniques have been explored to attenuate HIRI, there are currently no definitive and effective therapeutic methods[7,16].

Prostaglandins are a group of active lipid compounds with fundamental regulatory effects on human physiology and metabolism[17]. Many studies have demonstrated the hepatoprotective effect of prostaglandins through direct or indirect signaling pathways. Inhibiting ROS generation, preventing neutrophil aggregation, improving hepatic metabolism and regulating cytokine production constitute notable protective actions against HIRI[18,19]. Prostaglandin PGI₂ (prostacyclin) is produced from arachidonic acid in cell membranes by endothelial and vascular muscle cells using the cyclooxygenase pathway[20]. In the context of HIRI, PGI₂ has been demonstrated to exert cytoprotective effects through its potent vasodilative, anti-inflammatory, and antiapoptotic effects and its ability to prevent platelet aggregation[21,22].

Treprostinil is a chemically stable synthetic PGI₂ analog that has a relatively long half-life and strong potency; however, it is a high-cost medication. Treprostinil was approved by the United States Food and Drug Administration (FDA) in 2009 for the management of patients with pulmonary arterial hypertension (group 2 of the World Health Organization classification)[23,24]. Treprostinil directly induces vasodilation in both pulmonary and systematic vascular beds, thereby reducing arterial pressure and improving cardiac output[25]. Furthermore, experimental and clinical data have demonstrated the inhibition of platelet aggregation and smooth muscle cell proliferation, along with anti-inflammatory effects, in the setting of pulmonary arterial hypertension[26]. In addition to its approved use, treprostinil has been administered under several other conditions in an off-label context[27-30]. Currently, treprostinil is under investigation for HIRI, chronic limb ischemia, digital ischemia, pulmonary hypertension in interstitial lung disease (group 3) and chronic thromboembolic pulmonary hypertension (group 4)[31,32]. Recent published experimental data demonstrated a reduction in intracellular enzyme release and modulation of hepatic drug transporter expression and improved liver function in rat livers treated with treprostinil during preservation and reperfusion[33].

Ischemic preconditioning (IP) refers to brief (5-10 minutes) repeated episodes of ischemia caused by inflow vascular occlusion due to compression of the hepatoduodenal ligament (Pringle maneuver) followed by reperfusion, which renders the liver less susceptible to subsequent prolonged ischemia[34,35]. First reported by Murry et al[36] in 1986, IP has been established as a low-cost and effective technique to protect against HIRI, although an optimal protocol has not yet been defined. The underlying protective mechanisms and molecular processes of IP are still not fully understood. Several studies have reported a decrease in ROS generation, attenuation of oxidative stress and decreased proinflammatory cytokine and adhesion molecule levels, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1, and decreased neutrophil infiltration. Furthermore, IP reduces the apoptosis rate via the upregulation of antiapoptotic proteins and the inhibition of caspase-3 activation. Finally, IP improves hepatic microcirculation in the setting of cold and warm ischemia by inducing nitric oxide (NO) production, reducing endothelial dysfunction and enhancing sinusoidal perfusion[2,37-39]. The clinical effects of IP have been assessed in liver resection and transplantation, especially in deceased donor scenarios[40]. According to the results, IP improved postoperative liver function, particularly in those receiving grafts with steatosis or from extended-criteria donors, which are more susceptible to HIRI[41,42].

Numerous studies have evaluated the underlying mechanisms of HIRI, but the effects on remote organs have not been completely investigated. To our knowledge, myocardial damage in the context of HIRI and the protective effect of IP on the heart have not been adequately studied. Furthermore, there are few reports regarding the experimental and clinical use of treprostinil in liver graft preservation and transplantation cases, but no studies have evaluated the protective effects of treprostinil on systemic pathophysiological disorders associated with HIRI. Experimental studies of HIRI and its biomarkers will lead to a greater understanding of the underlying mechanisms and evaluation of local and systematic damage. Given that there have been some occasional off-label applications of treprostinil in liver surgery and transplantation, this experimental preclinical study in rats aimed to reveal reliable biomarkers and evaluate the assessment of liver and myocardial damage in the context of HIRI and the protective effects of treprostinil and IP.

MATERIALS AND METHODS

Animals

The animals were handled according to the Science-based Guidelines for Laboratory Animal Care published by the National Research Council Institute for Laboratory Animal Research in November 2003. A total of 40 male Wistar albino rats aged 3-4 months and weighing approximately 400-450 g were used. The animals were housed in special individually ventilated cages (one per cage), with each cage connected to a central ventilation circuit. The cages were maintained at a stable room temperature of 23°C, with humidity levels ranging from 50%-60%, noise levels below 50 dB and an artificial 12-hour light/dark cycle. The animals had free access to tap water and were fed a standard chow diet throughout the study. The experimental protocol was designed to prevent pain, discomfort and distress in the animals. The sample size was determined using G*Power software. The calculation was based on sample sizes derived from previous studies employing comparable experimental designs and methodologies.

Animal groups

Rats were randomly assigned to four groups of ten animals each according to the protocol. All animals in were subjected to a 3-hour surgical intervention. In group I (HIRI), hepatic ischemia was induced by encirclement and occlusion of the hepatoduodenal ligament with an atraumatic bulldog clamp for 30 minutes, followed by removal of the clamp and hepatic reperfusion for 120 minutes. In group II (HIRI + treprostinil), 100 ng/kg/minute treprostinil (Remodulin 1 mg/mL, Galenica AE, Greece, Chemical abstracts service registry number: 81846-19-7) was continuously administered for 24 hours via subcutaneous osmotic minipumps (Alzet Inc., Cupertino, CA, United States), which were implanted approximately 24 hours prior to the main surgical procedure. Prior to implantation, the pumps were primed for 18 hours according to the manufacturer’s instructions. For implantation, animals were anesthetized using intraperitoneal injection of ketamine hydrochloride (Ketamin-Actavis, Institute of Pharmaceutical Research and Technology, Athens, Greece) or dexmedetomidine (Dexmedetomidine, Ever Valinject, Oberburgau, Austria) at doses of 75 mg/kg and 0.5 mg/kg, respectively. For analgesia, tramadol (Tramal, Vianex, Athens, Greece) was administered at a dose of 25 mg/kg via intraperitoneal injection. A small area on the dorsum, slightly caudal to the scapulae, was shaved and prepared with 10% povidone iodine. A small 1.5-cm incision was made, and a pocket was created in the subcutaneous tissue using curved mosquito forceps. A minipump filled with treprostinil was inserted into the subcutaneous pocket, and the wound was closed in a single layer with simple interrupted sutures using 3-0 silk (Figure 1A). The procedure lasted approximately 10 minutes, followed by recovery and monitoring of the animals. Postoperatively, the animals had free access to water and food for 24 hours before the main operation. After 24 hours, the hepatic ischemia-reperfusion conditions were reproduced according to the protocol (30 minutes and 120 minutes, respectively).

Figure 1 Subcutaneous osmotic minipump implantation. A: Subcutaneous osmotic minipump implantation in rats pre-treated with treprostinil; B: An occlusion of hepatoduodenal ligament using atraumatic bulldog clamp.

In group III (HIRI + IP), a brief period of hepatic ischemia was induced by clamping the hepatoduodenal ligament using an atraumatic bulldog clamp for 10 minutes, followed by reperfusion for 15 minutes prior to subsequent prolonged ischemia for 30 minutes. After the clamp was removed, hepatic reperfusion was allowed for 120 minutes. In group IV (sham operation), the animals underwent a surgical procedure (anesthesia, laparotomy) without the induction of hepatic ischemia-reperfusion conditions and with no additional intervention (treprostinil administration, IP). In all the groups, blood, liver and heart tissue samples were collected after the end of the reperfusion time interval of 120 minutes, and the subjects were euthanized.

Operative procedure

All rats were anesthetized with an intraperitoneal injection of ketamine hydrochloride (Ketamin-Actavis, Institute of Pharmaceutical Research and Technology, Athens, Greece) and dexmedetomidine (Dexmedetomidine, Ever Valinject, Oberburgau, Austria) at doses of 75 mg/kg and 0.5 mg/kg, respectively, while analgesia was achieved with an intraperitoneal injection of tramadol (Tramal, Vianex, Athens, Greece) at a dose of 25 mg/kg. The animals were immobilized and weighed, the abdomen was shaved, and the skin was prepared with 10% povidone iodine. All surgical interventions were performed by the same surgeon on a warm, stable dissection table in a sterile environment using sterile surgical instruments. A laparotomy was performed with a midline incision of approximately 4-5 cm. After the control of the peritoneal cavity, the liver was located, and hepatic ischemia was induced by encirclement and occlusion of the hepatoduodenal ligament with an atraumatic bulldog clamp for 30 minutes (Figure 1B). The clamp was removed after 30 minutes of ischemia, and reperfusion was applied for 120 minutes. Between interventions, the abdominal wound was closed in a single layer with continuous 3-0 nylon. After 120 minutes of reperfusion, the laparotomy was reopened, and blood samples were drawn from the right ventricle. Blood, liver and heart tissue samples were collected for biochemical and histopathological examinations, and the subjects were euthanized.

A portion of each blood sample was placed in a tube with ethylenediaminetetraacetic acid (EDTA) and centrifuged at 5000 rpm for 15 minutes at 4°C. Serum samples were placed in Eppendorf tubes, immediately frozen in liquid nitrogen and stored at -80°C until analysis. The remaining blood samples were sent for biochemical analysis. Frozen liver samples (1 g each) were minced on ice and homogenized in 9 mL of phosphate-buffered saline (pH = 7.4). To lyse the cells, the samples were subjected to six freeze-thaw cycles. After homogenization, the samples were centrifuged at 5000 rpm for 5 minutes at 4°C. The supernatants were collected (200 μg each), placed in Eppendorf tubes and stored at -80°C until analysis. The livers and hearts of all experimental animals were placed in 10% neutral buffered formalin and sent for histopathological examination.

Biochemical analysis

Serum levels of glutamic oxaloacetic transaminase (SGOT) (Medicon Hellas, Athens, Greece), glutamic pyruvic transaminase (SGPT) (Medicon Hellas, Athens, Greece) and lactate dehydrogenase (LDH) (Medicon Hellas, Athens, Greece) were measured as markers of liver damage using a spectrophotometric method. To determine the presence of myocardial damage, serum levels of creatine phosphokinase (CPK) (Medicon Hellas, Athens, Greece) were measured using spectrophotometry, and analysis of high-sensitivity serum troponin I (Dxl 800-Access, Beckman Coulter, Brea, CA, United States) levels was performed using a chemiluminescence immunoassay. The serum concentration of endothelin-1 (ET-1), a potent vasoconstrictor peptide with chemotactic features, was determined as an inflammatory response marker by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (rat endothelin 1 ELISA kit, Antibodies.com Europe AB, Stockholm, Sweden).

Serum levels of the oxidative stress markers TNF-α (rat TNF-α ELISA kit, Antibodies.com Europe AB, Stockholm, Sweden) and IL-1β (rat IL-1β ELISA kit, Antibodies.com Europe AB, Stockholm, Sweden) were measured by ELISA. To further assess the inflammatory response during HIRI, IL-1β levels were also determined in liver tissue (rat IL-1β ELISA kit, Antibodies.com Europe AB, Stockholm, Sweden). Protein carbonyls are early indicators of the protein oxidative modification process, which is an indicator of oxidative stress. For the quantitative determination of the protein carbonyl concentrations in serum and liver tissue, a high-sensitivity ELISA method was used (Protein Carbonyl Assay Kit, Antibodies.com, Cambridge, United Kingdom).

Histopathological examination

Liver and heart tissue samples were fixed in 10% neutral buffered formalin and embedded in paraffin. Histological sections (3-4 μm thick) were obtained from paraffin blocks, stained with hematoxylin-eosin and prepared for histopathological examination. Sample evaluation was performed blindly by two pathologists using a light microscope (Nikon eclipse 50i, Nikon Corporation, Tokyo, Japan) and a digital camera (Nikon DS-5 ML1, Nikon Corporation, Tokyo, Japan). Regarding liver tissue sections, 10 fields of view were examined at 200 × magnification. The following histopathological features were evaluated in the liver: Hepatocyte necrosis, presence of inflammatory cells, edema and hemorrhage. Hepatocyte necrosis in the liver samples was scored from 0-3, where 0 = absence of necrosis; 1= < 2 foci per hepatic lobule; 2 = 3-5 foci per hepatic lobule; and 3 = > 5 foci per hepatic lobule (modified classification of Savvanis et al[43]). Regarding infiltration of portal spaces with inflammatory cells, a modification of the classification reported by Ibrahim[44] was applied, and the above parameters were scored from 0-3, where 0 = None; 1= Presence in single cells; 2 = Presence in < 30% of cells; and 3 = Presence in < 60% of cells. In addition, the presence of Mallory-like bodies (cytoplasmic eosinophilic inclusions) and hemorrhage was evaluated. These parameters were assigned scores of 0 or 1 for their absence or presence, respectively.

Approximately 25 fields of view in heart tissue samples were evaluated for the presence of cardiomyocyte necrosis, inflammatory cell infiltration and loss of striation at 400 × magnification, and edema (interstitial and cytoplasmic) was evaluated at 200 × magnification, according to a modified version of the classification described by Papoutsidakis et al[45]. The above parameters based on the severity level were scored from 0-3, where 0 indicated none; 1 indicated mild; 2 indicated moderate; and 3 indicated severe.

Statistical analysis

Analysis was performed using SPSS Statistics 20.0 software (IBM Corp, Armonk, NY, United States). Continuous and ordinal variables are presented as the mean ± SD. The Shapiro-Wilk test was used to separate parametric variables from nonparametric variables. To detect significant differences between the control group and each intervention group separately, independent samples t test and the Mann-Whitney U test were used for parametric and nonparametric variables, respectively. To detect significant differences among the three intervention groups, one-way ANOVA and the Kruskal-Wallis test were used for parametric and nonparametric variables, respectively, with post hoc analysis using the independent samples t test for parametric variables or the Mann-Whitney U test for nonparametric variables. Histopathological results were treated as ordinal variables. All tests were two-tailed, and significance was set at P < 0.05.

RESULTS

Transaminases

Serum SGOT (946.4 ± 59.1 U/L, P < 0.001) and SGPT (1210.6 ± 234.4 U/L, P < 0.001) levels were greater in the HIRI group than in the sham group (SGOT 144.3 ± 19.4 U/L, SGPT 67.2 ± 11.6 U/L), confirming hepatic injury (Figure 2A). Treprostinil treatment significantly reduced serum transaminase levels (SGOT, 616.3 ± 42.9 U/L, P < 0.001; SGPT, 734.4 ± 226.9 U/L, P = 0.001). Compared with those in the HIRI group, the serum transaminase levels in the IP pre-treated group also markedly differed (586.4 ± 32.8 U/L, P < 0.001; SGPT 768.1 ± 210.7 U/L, P = 0.03). We did not identify a significant difference between the two intervention groups (P = 0.053).

Figure 2 Effect of treprostinil and ischemic preconditioning in rats subjected to hepatic ischemia-reperfusion injury. A: Effect of treprostinil and ischemic preconditioning (IP) on serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), and lactate dehydrogenase (LDH) levels in rats subjected to hepatic ischemia-reperfusion injury (HIRI). Serum SGOT, SGPT, and LDH levels in the HIRI group were significantly different from sham group. In treprostinil and IP groups, serum SGOT, SGPT, and LDH levels were significantly decreased compared to HIRI; B: Effect of treprostinil and IP on serum creatine phosphokinase (CPK) values in rats subjected to HIRI. Serum CPK levels in the HIRI group were significantly different from the sham group, confirming the presence of myocardial injury. In treprostinil and IP groups, serum CPK levels were reduced compared to the HIRI group, but no significant difference was observed; C: Effect of treprostinil and IP on serum troponin I values in rats subjected to HIRI. Serum troponin I levels in the HIRI group were significantly different from the sham group, confirming the presence of cardiac damage. The treprostinil group significantly reduced serum troponin I levels. Rats pre-tretated with IP also had lower serum troponin I levels, but no statistically significant difference was observed; D: Effect of treprostinil and IP on serum tumor necrosis factor-α (TNF-α) and endothelin-1 (ET-1) values in rats subjected to HIRI. Serum TNF-α and ET-1 levels in the HIRI group were significantly elevated compared with the sham group, confirming the upregulated inflammatory response. In the treprostinil and IP group, serum TNF-α and ET-1 levels were significantly reduced compared to the HIRI group. Black lines on the top of the bars represent SD. HIRI: Hepatic ischemia-reperfusion injury; IP: Ischemic preconditioning; SGOT: Serum glutamic-oxaloacetic transaminase; SGPT: Serum glutamic pyruvic transaminase; LDH: Lactate dehydrogenase; CPK: Creatine phosphokinase; TNF-α: Tumor necrosis factor-α; ET-1: Endothelin-1. ^aP < 0.001, ^bP < 0.05, and ^cP > 0.05.

LDH

Liver ischemia-reperfusion induced hepatocellular injury, as evidenced by an increase in serum LDH levels (5896.0 ± 592.2 U/L, P < 0.001) in the HIRI group compared with the sham group (791.5 ± 101.9 U/L; Figure 2A). The treprostinil- and IP-treated groups presented markedly lower LDH levels (1439.7 ± 339.9 U/L and 2039.3 ± 904.9 U/L, respectively; P < 0.001), whereas there was no significant difference between the two treatment groups (P = 0.19).

CPK

CPK levels were significantly elevated in the HIRI group (5213.7 ± 1437.3 U/L, P < 0.001) compared with those in the sham group (1211.8 ± 216.1 U/L). Both treatment groups presented a reduction in CPK levels (treprostinil, 3767.4 ± 1245.7 U/L, P = 0.07; IP, 3801.8 ± 872.8 U/L, P = 0.10) compared with those in the HIRI group, although no statistically significant difference was observed (Figure 2B). There was no statistically significant difference between the two intervention groups (P = 1.00).

Troponin I

Compared with the sham group (253.9 ± 131.0 pg/mL), the hepatic ischemia-reperfusion group presented with cardiac damage, as evidenced by increased serum troponin I levels (3846.9 ± 2441.2 pg/mL, P = 0.008). Treprostinil treatment significantly reduced troponin I levels (186.3 ± 253.7 pg/mL, P < 0.001). Similarly, animals pretreated with IP had reduced serum troponin I levels (2609.3 ± 1146.6 pg/mL), but no significant difference was detected (P = 0.32; Figure 2C). The Treprostinil group had a marked reduction in troponin I levels compared with the IP group (P < 0.06).

Serum TNF-α

Hepatic ischemia-reperfusion induced an inflammatory response, as evidenced by increased serum TNF-α levels (8.43 ± 0.89 pg/mL, P < 0.001) compared with those in the sham group (3.69 ± 0.62 pg/mL). Compared with HIRI, both treprostinil and IP significantly reduced TNF-α levels (treprostinil, 6.52 ± 0.72 pg/mL, P < 0.001; IP, 5.64 ± 0.87 pg/mL, P < 0.001; Figure 2D). There was no statistically significant difference between the two interventions (P = 0.09).

Serum ET-1

Serum ET-1, a marker of the inflammatory response, was significantly elevated in the HIRI group (5.82 ± 0.27 pg/mL, P < 0.001) compared with the sham group (1.76 ± 0.41 pg/mL). Animals treated with treprostinil presented significantly lower serum ET-1 levels (4.48 ± 0.75 pg/mL, P < 0.001) than those in the HIRI group, and those in the IP group also had markedly lower levels (4.00 ± 0.68 pg/mL, P < 0.001; Figure 2D). The comparison between the two treatments revealed no statistically significant difference (P = 0.35).

IL-1β

In our study, an excessive inflammatory response was significantly associated with hepatic ischemia-reperfusion, as demonstrated by increased serum and liver tissue IL-1β levels (serum IL-1β, 118.57 ± 8.72 pg/mL; liver IL-1β, 165.34 ± 10.68 pg/mL; P < 0.001) in the HIRI group compared with the sham group (serum IL-1β, 58.15 ± 6.21 pg/mL; liver IL-1β, 19.42 ± 1.34 pg/mL). Treprostinil treatment significantly decreased HIRI-induced inflammation (serum IL-1β, 89.56 ± 5.00 pg/mL; liver IL-1β, 128.88 ± 5.05 pg/mL; P < 0.001). Similarly, IP treatment markedly reduced the HIRI-induced inflammatory response by decreasing serum and tissue IL-1β expression (serum IL-1β, 83.97 ± 9.36 pg/mL; liver IL-1β, 123.18 ± 4.97 pg/mL; P < 0.001). The comparison of the two protective methods revealed no significant difference (serum and tissue, P = 0.42 and P = 0.26, respectively; Figure 3A).

Figure 3 Effect of treprostinil and ischemic preconditioning in rats subjected to hepatic ischemia-reperfusion injury. A: Effect of treprostinil and ischemic preconditioning (IP) on serum and liver tissue interleukin-1β (IL-1β) concentrations in rats subjected to hepatic ischemia-reperfusion injury (HIRI). Serum and tissue IL-1β levels in the HIRI group were significantly increased compared to the sham group, confirming the induction of the inflammatory response. In the treprostinil and IP group, serum and liver tissue IL-1β concentrations were significantly reduced compared to the HIRI group; B: Effect of treprostinil and IP on serum and liver tissue protein carbonyl concentrations in rats subjected to HIRI. Serum and tissue protein carbonyl levels in the HIRI group were significantly increased compared to the sham group, confirming the induction of oxidative stress and inflammatory response. Treprostinil and IP reduced serum protein carbonyl concentration, but the difference was not statistically significant. In contrast, treprostinil and IP significantly reduced liver tissue protein carbonyl levels compared to the HIRI group; C: Effect of treprostinil and IP on hepatocyte necrosis and Mallory-like bodies formation in rats subjected to HIRI. Hepatocyte necrosis was present in the HIRI group, but no statistically significant was revealed when compared to the sham group, in contrast with Mallory-like bodies formation. Treprostinil significantly reduced the extent of cell necrosis compared to the HIRI group. Cell necrosis was also lower in the IP group, but there was no significant difference compared to the HIRI group. The presence of Mallory-like bodies was reduced in treprostinil group compared to the HIRI group, although with no statistically significant difference. However, the Mallory-like bodies formation was higher in the IP group than in the HIRI group; D: Effect of treprostinil and IP on portal inflammatory cell infiltrations and liver hemorrhage occurrence in rats subjected to HIRI. Portal inflammatory cell infiltration was present in the HIRI group compared to sham group, but no statistically significant difference was revealed, in contrast with liver hemorrhage. Both treatments significantly reduced the degree of inflammatory cell aggregation, but no statistically significant difference was observed for the occurrence of hemorrhagic lesions when compared to the HIRI group. Black lines on the top of the bars represent SD. HIRI: Hepatic ischemia-reperfusion injury; IP: Ischemic preconditioning; IL-1β: Interleukin-1β. ^aP < 0.001, ^bP < 0.05, and ^cP > 0.05.

Protein carbonyl concentration

In addition to cytokine levels, oxidative stress was also assessed with the measurement of protein carbonyl concentrations in serum and liver tissue. Hepatic ischemia-reperfusion induced oxidative stress in liver tissue, as evidenced by increases in the protein carbonyl concentrations in the serum (4.37 ± 1.69 nmol/mg, P = 0.037) and liver tissue (6.29 ± 0.67 nmol/mg, P < 0.001) compared with the sham group (serum, 2.64 ± 0.47 nmol/mg; tissue, 2.88 ± 0.13 nmol/mg). Compared with HIRI, treprostinil treatment reduced serum protein carbonyl levels (3.67 ± 0.86 nmol/mg, P = 0.95), although the difference was not statistically significant. A greater decrease in the serum protein carbonyl concentration was observed in the IP group, although the difference was not significant (2.81 ± 1.52 nmol/mg, P = 0.14). In contrast, both interventions significantly reduced the tissue protein carbonyl concentration compared with the HIRI group (treprostinil, 4.17 ± 0.40 nmol/mg; IP, 4.15 ± 0.19 nmol/mg; P < 0.001; Figure 3B). There was no statistically significant difference in the serum or tissue protein carbonyl levels between the two treatments (serum, P = 0.60; tissue, P = 1.00).

Histopathological examination

The predominant histopathological findings of the liver were hepatocyte necrosis, hemorrhage, inflammatory cell aggregates and the presence of Mallory-like bodies. Despite the presence of hepatocyte necrosis in the HIRI group (1.29 ± 0.49), it was not statistically significantly different than the sham group (1.00 ± 0.00, P = 0.17; Figure 3C). The sham group unexpectedly presented mild tissue damage, which may not reflect true pathology but rather natural variability, surgical stress or technical factors. Compared with HIRI, treprostinil treatment significantly reduced the extent of cell necrosis (0.00 ± 0.00, P < 0.001). Cell necrosis was also lower in the IP group than in the HIRI group, but the difference was not significant (0.67 ± 0.87, P = 0.11). Mallory-like bodies are irregular cytoplasmic eosinophilic inclusions in hepatocytes that are associated with cellular damage and inflammation. The formation of Mallory-like bodies is often linked to hepatocyte injury in the context of cirrhosis, alcoholic steatohepatitis and drug hepatotoxicity. Mallory-like body presence is not specific to a particular liver disease, but their occurrence can reflect the type and extent of liver damage[46]. The presence of Mallory-like bodies was significantly greater in the HIRI group (0.71 ± 0.49, P = 0.008) than in the sham group (0.00 ± 0.00), but no statistically significant difference was found in the treprostinil group (0.30 ± 0.48, P = 0.10; Figure 3C). The presence of Mallory-like bodies was greater in the IP group than in the HIRI group (1.11 ± 0.93, P = 0.32). Compared with that in the sham group, hepatic ischemia-reperfusion led to an inflammatory response, as evidenced by increased but not statistically significant portal inflammatory cell aggregation consisting of neutrophilic and/or mononuclear cells in the HIRI group (1.43 ± 0.54, P = 0.35; 1.17 ± 0.41; Figure 3D). Compared with HIRI, both treatment options significantly reduced the degree of inflammatory cell aggregation (treprostinil, 0.10 ± 0.32, P < 0.001; IP, 0.67 ± 0.50; P = 0.011). The occurrence of liver hemorrhage was significantly greater in the HIRI group (0.86 ± 0.69, P = 0.017) than in the sham group (0.00 ± 0.00). Both treatments resulted in lower hemorrhage intensity scores, but the difference was not statistically significant (treprostinil, 0.80 ± 0.42, P = 0.83; IP 0.67 ± 0.71, P = 0.60; Figure 3D). A comparison between the two treatments revealed statistically significant advantages of treprostinil in the attenuation of portal inflammatory cell infiltration (P = 0.012) and the formation of Mallory-like bodies (P = 0.027), whereas no significant differences were observed in the degree of hepatocyte necrosis (P = 0.05) or the presence of hemorrhagic lesions (P = 0.62). The histopathological assessment of liver tissue is summarized in Figure 4.

Figure 4 Histopathological findings of liver samples in sham group, hepatic ischemia-reperfusion injury group, treprostinil-treated group, and ischemic preconditioning group. A: Sham group; histological section from a rat of the sham group shows normal architecture. Hematoxylin and eosin (HE), original magnification 100 ×; B: Hepatic ischemia-reperfusion injury (HIRI) group; rat subjected to HIRI presents sinusoidal dilatation and portal spaces with inflammatory cell infiltration (mostly neutrophilic). HE, original magnification 100 ×; C: HIRI group; hepatic hemorrhages and sinusoidal hyperemia accompanied by hepatocyte necrosis, primarily characterized by pyknotic nuclei. Intracytoplasmic eosinophilic bodies are present in numerous hepatocytes while in lower center, there is vacuolar degeneration. Inset shows Mallory-like hyaline bodies. HE, original magnification 100 ×, inset 400 ×; D: HIRI group, hyperhemia and microhemorrhages and multiple necrotic hepatocytes characterized by homogenous eosinophilic cytoplasm and pyknotic or absent nucleus, are detectable. HE, original magnification 200 ×; E: Treprostinil treated group; treatment with treprostinil shows less disturbed/normal lobular architecture, mild sinusoidal hyperhemia and sparse inflammatory cells in portal spaces (upper right) and central vein dilatation (lower left), C × 100; F: Ischemic preconditioning group; rats pre-treated with IP preserve hepatic lobular architecture. However sparse hepatocyte necrosis and mild periportal neutrophilic infiltration are observed. HE, original magnification 100 ×.

Heart tissue samples were evaluated for the presence of cardiomyocyte necrosis, interstitial and/or intravascular edema, presence of wavy fibers, loss of striation and inflammatory cell infiltration. Specifically, animals subjected to hepatic ischemia-reperfusion presented with marked cardiomyocyte necrosis (1.86 ± 0.69, P = 0.003) compared with animals in the sham group (0.50 ± 0.55), but no statistically significant difference was found compared with those in the treprostinil group (1.90 ± 0.32, P = 0.86). Animals subjected to IP prior to HIRI presented higher cardiomyocyte necrosis scores (2.33 ± 0.71, P = 0.20) than animals in the HIRI group (Figure 5A). The extent of cell edema was greater in the HIRI group (2.57 ± 0.54, P = 0.010) than in the sham group (1.67 ± 0.52). Both treatment groups resulted in decreased edema, but the difference was not statistically significant (treprostinil, 2.40 ± 0.52, P = 0.52; IP, 2.33 ± 0.50, P = 0.38; Figure 5A). Myocardial inflammatory cell infiltrations, mostly mononuclear, were greater in the HIRI group (1.43 ± 0.54, P = 0.08) than in the sham group (1.00 ± 0.00), but the difference was not statistically significant. Both interventions reduced myocardial inflammatory cell infiltration (treprostinil, 0.90 ± 0.57, P = 0.26; IP, 1.22 ± 0.67, P = 1.00), but the differences were not statistically significant (Figure 5B). Wavy myocardial fibers and loss of striation are characteristic morphological signs of ischemic injury, especially when associated with focal edema. Wavy fibers were present in the HIRI group (1.71 ± 0.49, P = 0.20) but not in the sham group (1.33 ± 0.52), and no statistically significant differences were detected in the treatment groups (treprostinil, 1.70 ± 0.68, P = 0.96; IP, 1.78 ± 0.67, P = 0.84; Figure 5B). The degree of striation loss was significantly greater in the HIRI group (2.00 ± 0.58, P = 0.001) than in the sham group (0.67 ± 0.52). Both treatment options reduced striation loss, but the differences were not statistically significant (treprostinil, 1.50 ± 0.53, P = 0.08; IP, 1.56 ± 0.53, P = 0.13; Figure 5B). Additionally, comparison of the two protective methods revealed no difference in the degree of cardiomyocyte necrosis (P = 0.12), edema (P = 0.78), inflammatory cell infiltration (P = 0.75), presence of wavy fibers (P = 0.80), or loss of striation (P = 0.82). The histopathological assessment of the myocardial tissue samples is summarized in Figure 6.

Figure 5 Effect of treprostinil and ischemic preconditioning in rats subjected to hepatic ischemia-reperfusion injury. A: Effect of treprostinil and ischemia-reperfusion (IP) on cardiomyocyte necrosis and edema in rats subjected to hepatic ischemia-reperfusion injury (HIRI). The degree of cardiomyocyte necrosis and edema was significantly increased in the HIRI group compared to the sham group. Nonetheless, no statistically significant difference was found between either of the treatment groups and the HIRI group; B: Effect of treprostinil and ischemia-reperfusion on myocardium inflammatory cell infiltrations, presence of wavy fibers and loss of striation in rats subjected to HIRI. The degree of myocardial inflammatory cell infiltrations, presence of wavy fibers and loss of striation was increased in the HIRI group compared to the sham group, but a statistically significant difference was only detected for the loss of striation. There was no statistically significant difference between either treatment group and the HIRI group. Black lines on the top of the bars represent SD. HIRI: Hepatic ischemia-reperfusion injury; IP: Ischemic preconditioning. ^bP < 0.05, and ^cP > 0.05.

Figure 6 Representative histopathological images of heart tissue in sham group, hepatic ischemia-reperfusion injury group, treprostinil treated group, and ischemic preconditioning group. A: Sham group; the myocardium in a rat from the sham group shows well-preserved cardiomyocytes. Mild interstitial edema, sinusoidal hyperhemia and presence of wavy fibers located subepicardially are also observed. Hematoxylin and eosin (HE), original magnification, 100 ×; B: Hepatic ischemia-reperfusion injury group (HIRI); rat subjected to HIRI shows loss of striation with homogenously eosinophilic sarcoplasm, a large mononuclear cell aggregate and pyknotic nuclei (necrosis). HE, original magnification, 100 ×; C: Treprostinil-treated group; treprostinil-treated rat shows mild edema and cardiomyocyte necrosis. HE, original magnification, 200 ×; D: Ischemic preconditioning (IP) group; rats subjected to hepatic IP show mild interstitial edema, segmental loss of striation and eosinophilic sarcoplasm. HE, original magnification, 400 ×; E: IP group, rats subjected to hepatic IP show hyperhemia, hemorrhage and edema. HE, original magnification, 100 ×; F: IP group; rats subjected to hepatic IP show eosinophilic sarcoplasm, pyknotic nuclei necrosis and presence of sparse mononuclear cells. HE, original magnification, 200 ×.

DISCUSSION

For decades, HIRI has been the subject of intense research, as its pathophysiological mechanisms are highly responsible for hepatic dysfunction and liver failure during liver surgery and transplantation, as well as other clinical conditions. The underlying pathways are extremely complex and are not completely understood, although much progress has been made in the interpretation of this phenomenon[4,47]. According to current knowledge, reperfusion of previously ischemic hepatic parenchyma results in ROS production, oxidative stress, mitochondrial dysfunction, cytokine overexpression and inflammatory cell aggregation[48]. In addition to hepatic cell injury, the acute systemic inflammatory response may lead to damage to distant organs, resulting in multiple organ failure[7,10,49]. Research in the field of HIRI provides ongoing information for the assessment and management of liver and distant organ damage, although an ideal protective method has yet to be identified. Recent studies have shown that treprostinil may have hepatoprotective effects during liver preservation and transplantation, although its effect on distant organ damage in the context of HIRI has not been investigated[30,33,50]. Similarly, there are insufficient data on the protective effects of liver IP against myocardial damage during HIRI.

In our study, we used a common protocol of hepatic ischemia-reperfusion, consisting of 30 minutes of hepatic blood inflow occlusion followed by 120 minutes of reperfusion. This particular HIRI model is typically used for the experimental study of hepatic damage and the testing of therapeutic strategies or drug interventions. This approach appears to be an ideal model for assessing acute biochemical, histological and molecular liver injury[51,52]. We utilized intraperitoneal administration of anesthetic and analgesic drugs. Notably, the peritoneum has a high absorptive capacity and is a commonly used pathway for drug delivery in experimental studies involving rats. For many compounds, this method offers bioavailability similar to that of intravenous injection and has been extensively utilized across a range of pharmacological agents[53].

With respect to liver and myocardial damage in the context of HIRI, the present study revealed that 30 minutes of hepatic ischemia followed by 120 minutes of reperfusion resulted in deterioration of liver function and exacerbation of hepatocellular and myocardial damage, as evidenced by increases in the serum SGOT (P < 0.001), SGPT (P < 0.001), LDH (P < 0.001), CPK (P < 0.001), and troponin I (P = 0.008) levels in the HIRI group compared with those in the sham group. Moreover, HIRI led to an exaggerated inflammatory response and oxidative stress, as revealed by statistically significant increases in the serum TNF-α (P < 0.001), ET-1 (P < 0.001), and IL-1β levels (P < 0.001), liver tissue IL-1β (P < 0.001) level, and serum and liver tissue protein carbonyl levels (P = 0.037 and P < 0.001, respectively) in the HIRI group compared with the sham group. Histopathological examination revealed a significantly greater incidence of Mallory-like bodies (P = 0.008) and hemorrhagic lesions (P = 0.017) in liver tissue samples from the HIRI group than in those from the sham group. The presence of hepatocyte necrosis and portal inflammatory cell infiltration was also greater in the HIRI group than in the sham group, but the difference was not statistically significant (P = 0.17 and P = 0.35, respectively); this finding may be attributed to the short duration of our experiment, which was insufficient for necrosis development. Histopathological examination of the heart demonstrated myocardial injury in the context of HIRI, as evidenced by the significantly greater incidence of cardiomyocyte necrosis, edema and striation in the HIRI group (P = 0.003, P = 0.010, and P = 0.001, respectively) than in the sham group. The incidence of wavy fibers and myocardial inflammatory cell infiltration was also higher in the HIRI group than in the sham group, but the difference was not statistically significant (P = 0.20 and P = 0.08, respectively). Although troponin I significantly decreased after treprostinil treatment (P < 0.001), necrosis scores did not significantly improve (P = 0.86). This discrepancy may indicate a difference between reversible and irreversible myocardial damage. Troponin I is a highly sensitive biomarker that may be released not only in response to irreversible cell death but also during mild cell injury, such as transient membrane permeability alterations induced by oxidative stress. Accordingly, it can be assumed that treprostinil attenuated myocardial injury by stabilizing stressed but viable cardiomyocytes, thereby promoting functional recovery, a process reflected by the reduction in serum troponin I levels. However, the therapeutic intervention appeared insufficient to rescue cells that had already sustained irreversible injury, which may explain the absence of a statistically significant difference in histological necrosis compared with that in the control group. These findings support the hypothesis that treprostinil exerts protective effects during both the ischemic and reperfusion phases, ultimately contributing to the preservation of cells that might have otherwise undergone death during reperfusion. This may explain the observed reduction in troponin I, despite the absence of a statistically significant difference in total necrosis scores, which reflects cumulative irreversible damage from both the ischemic and reperfusion phases. Treprostinil administration was more effective in limiting injury progression during the reperfusion phase than in ameliorating irreversible cell injury during the initial ischemia phase[54].

Numerous pharmacological agents and techniques have been explored to attenuate HIRI during hepatectomy and liver transplantation[2,55]. Prostaglandins are members of the active lipid compound family called eicosanoids, which have been broadly evaluated for their hepatoprotective effects. Prostaglandins act as chemical messengers and exhibit various effects that may contribute to the minimization of HIRI, such as potent vasodilation, inhibition of platelet aggregation, and anti-inflammatory and antioxidative effects[56]. Prostaglandin E1 and PGI₂ analogs reduce the overexpression of inflammatory mediators and improve the hepatic circulatory microenvironment in HIRI and liver transplantation cases[21,22,57,58]. Treprostinil (remodulin) is the most recent FDA-approved synthetic PGI₂ analog used for the treatment of patients with pulmonary arterial hypertension. In contrast to other PGI₂ analogs, treprostinil has a stable structure, greater potency and longer half-life (2-4 hours), which allows manageable administration, accessible dose optimization and fewer adverse events[59]. Treprostinil is available in four possible formulations: Intravenous, subcutaneous, oral and inhaled. The subcutaneous route is preferred for treprostinil administration because it allows continuous drug infusion without further dilution and rapid and complete absorption[60]. Specially designed subcutaneous pumps promote treatment management and dose optimization. In our study, we used small-capacity osmotic pumps to provide continuous subcutaneous treprostinil delivery. The animals were briefly subjected to anesthesia and analgesia with ketamine hydrochloride, dexmedetomidine and tramadol, and osmotic minipumps filled with treprostinil were aseptically implanted on the dorsum. Published data have demonstrated that osmotic minipumps in rats constitute a viable alternative for delivering drugs at stable doses over a long period and that the surgical stress associated with implantation does not affect recovery or development[61].

Recent studies have reported encouraging results regarding the experimental and clinical use of treprostinil in HIRI and liver transplantation. Specifically, hepatic damage during renal ischemia-reperfusion injury occurs as a result of the depletion of hepatic antioxidants and the upregulation of toll-like receptor-9, which recognizes pathogen-associated molecular patterns and damage-associated molecular patterns and triggers inflammatory reactions. Continuous subcutaneous administration of treprostinil at a dose of 100 ng/kg/minute via osmotic minipumps to rats for 18-24 hours before renal ischemia-reperfusion improved hepatic function, alleviated oxidative stress and lipid peroxidation, reduced hepatic toll-like receptor-9, inhibited the inflammatory response and restored mitochondrial function in hepatocytes[62]. In another experimental study, rats that underwent orthotopic liver transplantation were previously treated with continuous subcutaneous treprostinil at a dose of 100 ng/kg/minute using osmotic minipumps for 24 hours before surgery. Animals treated with treprostinil presented improved liver function, increased postreperfusion liver graft blood flow, preserved microcirculation, reduced neutrophil accumulation in liver grafts and minimal structural changes in liver sinusoidal endothelial cells[52]. A year later, Ghonem et al[63] reported similar results, supporting the previous findings. In accordance with the experimental protocol, rat donors and recipients received subcutaneous treprostinil (100 ng/kg/minute) for 24 hours before and up to 48 hours after transplantation. Treprostinil significantly reduced serum transaminase levels after liver transplantation and downregulated TNF-α and interferon-γ mRNA expression in liver grafts. Furthermore, treprostinil significantly improved cytochrome P450 activity, which is an important indicator of liver graft function[63]. In a recent study, Almazroo et al[33] used an isolated perfused rat liver model to investigate the effect of treprostinil supplementation on ischemia-reperfusion injury. Rat livers were harvested and preserved in cold organ preservation solution supplemented with treprostinil (20 ng/mL) for 24 hours and then reperfused with or without the abovementioned drug. The presence of treprostinil during liver preservation and reperfusion was positive, as the mRNA expression levels of certain efflux drug transporters (Abcb11 and Abcg2) increased, whereas the differential mRNA expression of major hepatic uptake drug transporters (Slc22a1m) remained similar to that in the control group. The regulation of important drug transporter gene transcription improved ischemia-reperfusion-mediated dysfunction of liver grafts[33]. This preclinical study offers initial evidence that treprostinil reduces HIRI and helps maintain hepatic drug transporter expression, warranting further investigation in large experimental and clinical models. To fully establish its therapeutic potential, future research should include protein-level validation, functional analyses of transporter activity, cytokine profiling, and evaluation of long-term graft performance.

A prospective, pilot, single-center, open-label, nonrandomized, dose-escalation study conducted by Almazroo et al[30] investigated the safety and efficacy of continuous intravenous treprostinil administration in adult liver transplant patients. Deceased donor liver transplant recipients received intravenous treprostinil at various dosages (from 2.5-7.5 ng/kg/minute for approximately 5 days) during the perioperative period. The maximum tolerable dose associated with better outcomes was 5 ng/kg/minute administered for 120 hours. A group of liver transplant patients who received treprostinil showed a rapid reduction in serum transaminase levels, improved excretory function and a lower probability of acute kidney failure. Moreover, no episodes of primary graft nonfunction were observed in the treprostinil group, and all patients demonstrated a reduced need for ventilation support and hospitalization time in general, with 100% graft and patient 6-month survival. Based on previous preclinical studies, the present study used continuous 100 ng/kg/minute treprostinil[52,63]. In future studies, a dose-response analysis, evaluation of different administration timings and assessment of dynamic functional parameters will provide valuable information regarding the optimal therapeutic window and maximize clinical relevance. However, although continuous intravenous treprostinil administration is considered safe and well tolerated, the small sample size and absence of underlying mechanistic analysis limit the interpretation of the proposed protective role of this drug; thus, phase III trials are needed to confirm the use of treprostinil in liver transplantation.

With respect to the effects of treprostinil on HIRI and myocardial injury in our study, treprostinil treatment reduced hepatocellular and myocardial damage compared with HIRI, as revealed by the significantly lower serum concentrations of SGOT (P < 0.001), SGPT (P = 0.001), LDH (P < 0.001) and troponin I (P < 0.001). Similarly, the treprostinil group presented decreased CPK serum levels, but the difference was not statistically significant (P = 0.07). Analysis of systemic inflammatory response markers revealed a significant decrease in cytokine expression in the treprostinil group compared with the HIRI group. Treprostinil treatment reduced serum TNF-α (P < 0.001), serum ET-1 (P = 0.001), serum and tissue IL-1β (P < 0.001), and tissue protein carbonyl (P < 0.001) concentrations. However, treprostinil did not affect protein carbonyl concentration in the serum compared with HIRI (P = 0.95). Serum protein carbonyls are commonly employed as surrogate markers of systemic oxidative stress and are relevant for clinical biomonitoring. Despite a marked reduction in hepatic tissue levels, the absence of a significant systemic response suggests that treprostinil may primarily exert localized hepatic antioxidant effects with initially limited systemic impact. Moreover, protein carbonyls often exhibit a delayed systemic response following HIRI; therefore, the brief 120-minute observation window may not have been sufficient to detect downstream systemic alterations. This observation highlights the need for further investigations to clarify the translational importance of serum biomarkers in evaluating hepatoprotective interventions. Histopathological examination of the liver demonstrated significant reductions in hepatocellular necrosis (P < 0.001) and inflammation (P < 0.001) in treprostinil-treated animals compared with HIRI. Mallory-like bodies and hemorrhagic lesions were also decreased in the treprostinil group, although the difference was not statistically significant (P = 0.10 and P = 0.83, respectively). Histopathological evaluation of the heart did not identify any significant differences in cardiomyocyte necrosis, edema or wavy fibers (P = 0.86, P = 0.52 and P = 0.96, respectively) compared with the HIRI group. The incidence rates of loss of striation and inflammatory cell aggregates were lower in the treprostinil group than in the HIRI group, although the differences were not statistically significant (P = 0.08 and P = 0.26, respectively).

Our findings are consistent with previously reported observations, as treprostinil administration reduced hepatocellular damage, as demonstrated by decreased serum transaminase and LDH levels and hepatocellular necrosis and inflammatory cell infiltration of the liver. In addition, treprostinil significantly reduced serum and tissue cytokine levels and attenuated the inflammatory reaction. Myocardial damage was assessed using serum troponin 1 and CPK levels and histopathological examination of heart tissue samples. Compared with the HIRI group, the treprostinil group presented reduced myocardial damage, as evidenced by lower troponin 1 and CPK levels. Although treprostinil treatment tended to reduce myocardial damage according to the histopathological examination of heart tissue samples, there were no significant differences in the scores between the treprostinil and untreated HIRI groups.

We did not observe any complications or adverse events during this experimental procedure, as the average duration of the operation was approximately 3 hours. Treprostinil is approved for the treatment of patients with pulmonary arterial hypertension and has a known side effect profile consisting of infusion site pain, diarrhea, nausea, vomiting, headache, flushing, jaw pain, dizziness and extremity pain, which may be challenging to manage in a surgical setting[24,64,65]. Adverse events may vary depending upon the patient, dose and therapy duration, although the dosage scheme applied in liver surgery and transplantation is short-term. In our experimental model, there was no indication of harmful effects on any of the parameters studied.

Hepatic IP is an intraoperative technique that consists of brief repetitive periods of hepatic inflow occlusion followed by reperfusion, which increases the resistance of the hepatic parenchyma to subsequent prolonged ischemia[66]. There is no ideal preconditioning protocol; therefore, the duration of the procedure may vary depending on the surgical context, presence of preexisting liver conditions and whether total or partial ischemia is applied[67,68]. The goal of IP in liver surgery is to protect the liver parenchyma from HIRI, and it is often used before major hepatectomy and transplantation to reduce postoperative liver damage. Notably, IP has a biphasic protective effect, an early phase that occurs within minutes and a delayed phase that emerges hours later, both of which are mediated by distinct molecular pathways, including kinase activation and stress protein expression. Sikalias et al[69] used five experimental groups of rats with steatotic livers subjected to different periods of ischemia-reperfusion. Intermittent IP, consisting of three ischemic occlusions of 10 minutes followed by 20 minutes of reperfusion each prior to HIRI and extensive hepatectomy, improved postoperative liver function and increased 30-day survival. In an experimental study conducted by Kim et al[70], IP was applied as 10 minutes of hepatic occlusion, followed by 15 minutes of reperfusion before prolonged ischemia. The IP group presented significantly lower serum transaminase levels, decreased expression of the proinflammatory cytokine IL-6 and minimal histological grades of hepatic injury. Tian et al[34] applied an IP experimental protocol consisting of 10 minutes of hepatic ischemia followed by 10 minutes of reperfusion while investigating HIRI at the molecular level. Serum SGOT and SGPT levels were significantly decreased, and TNF-α and IL-6 expression levels were downregulated in the IP group, whereas the analysis of circular RNAs, microRNAs and mRNAs provided novel suggestions for understanding the underlying mechanisms of IP protection. Boyko et al[71] reported improved histopathological structures and reduced hepatic functional disorders when an intermittent IP protocol consisting of five cycles (5 minutes of ischemia, 5 minutes of reperfusion, 10 minutes of ischemia, 5 minutes of reperfusion and 15 minutes of ischemia) was applied. Although further clinical studies are needed to standardize an optimal IP protocol, several reports have described the effectiveness of hepatic IP and the underlying mechanisms involved. The main protective mechanisms include a reduction in oxidative stress; activation of endothelial NO synthase and increased NO production; inhibition of cytokine production and attenuation of the inflammatory response; maintenance of ATP production; modulation of mitochondrial function; activation of cell survival pathways (PI3K/Akt, extracellular signal-regulated kinase 1/2); activation of autophagic mechanisms to remove damaged cell organelles; inhibition of apoptosis; and induction of heat shock protein expression (HSP70, HSP27)[72-76]. In particular, HSP70 and HSP27 appear to play protective roles in the delayed phase of IP by stabilizing mitochondrial function and reducing cellular injury. Although their expression levels increase after IP, the exact mechanisms by which HSPs contribute to ischemic tolerance are still under investigation. Autophagy also contributes to the late phase of IP, supporting cytoprotection, metabolic stabilization and the modulation of the inflammatory response via the activation of AMP-activated protein kinase/mammalian target of rapamycin, Beclin-1, light chain 3, mitogen-activated protein kinase, PTEN-induced putative kinase 1/Parkin proteins, and NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome-mediated signaling pathways[16,37].

In our study, IP pretreatment significantly reduced hepatocellular damage in the HIRI group compared with the sham group, as evidenced by markedly lower concentrations of serum SGOT (P < 0.001), SGPT (P = 0.003), and LDH (P < 0.001). Histopathological analysis of liver tissue samples revealed a significant decrease in portal inflammatory cell infiltration (P = 0.011), but we did not detect a significant difference in the extent of hepatocyte necrosis (P = 0.11) or the presence of liver hemorrhage (P = 0.60), although the damage scores tended toward reduced injury. However, the presence of Mallory-like bodies was greater in the IP group than in the HIRI group (P = 0.32), which, in this context, may be associated with more prolonged exposure of the hepatic parenchyma to ischemia and reperfusion in the IP group. According to our results, animals pretreated with IP presented greater myocardial damage after HIRI than after sham surgery, as evidenced by lower serum concentrations of troponin I (P = 0.32) and CPK (P = 0.10), but the difference did not reach statistical significance. Histopathological assessment of heart tissue samples revealed downward trends in histological injury markers, such as edema (P = 0.38), inflammatory cell infiltration (P = 1.00), wavy fibers (P = 0.84), and loss of striation (P = 0.13), although the differences did not reach statistical significance. The extent of cardiomyocyte necrosis was higher in the IP group than in the HIRI group (P = 0.20). This may reflect the systemic stress induced by repeated hepatic ischemia-reperfusion cycles in the IP group, potentially triggering increased myocardial damage. The evaluation of inflammatory biomarkers revealed attenuation of acute inflammation and oxidative stress in the IP group compared with the HIRI group, as evidenced by reduced serum TNF-α (P < 0.001), ET-1 (P < 0.001), IL-1β (P < 0.001) and tissue IL-1β (P < 0.001) concentrations. Protein carbonyl concentrations were also lower in the IP group than in the HIRI group, but a statistically significant difference was only detected in liver tissue levels (serum, P = 0.14; tissue, P < 0.001).

The mechanisms of hepatic IP can be classified into two phases of protection against HIRI, early and late, depending on different time frames and key molecular events[77]. The early phase immediately follows IP and lasts for 2-3 hours, where the reaction is mediated by existing proteins and signaling pathways and offers immediate cytoprotection. The late phase begins 12-24 hours after IP and lasts for approximately 48-72 hours. Late preconditioning mechanisms are primarily based on gene transcription and new protein synthesis[78]. The biphasic protection pattern of IP may explain the delayed cardioprotective effect observed, as neither the serum biomarkers nor the histopathological findings showed definitive significance, despite a positive trend.

The experimental design of the present study included a sham group (surgical control), an untreated HIRI group (negative control), and an IP group (positive control). By incorporating both negative and positive control groups, we could assess the effectiveness of treprostinil compared to baseline injury and a recognized protective technique. A key limitation of this experimental design is the short reperfusion duration, which may not adequately reflect the full protective potential of IP. The 120-minute reperfusion period primarily captures the early, transient effects of the IP while overlooking its delayed protective phase. Owing to the short duration of our experimental model, it is possible that late-phase responses and delayed protective effects of IP were not properly evaluated. This limitation may lead to an underestimation of the full therapeutic potential of IP. Future studies should incorporate extended observation periods and multiple time-point assessments to evaluate both the early and late phases of hepatic IP, more comprehensively capturing its local and remote protective effects.

In this study, both treprostinil and IP were evaluated for their potential protective effects against liver and myocardial damage in the context of HIRI. The comparison between the two protective methods revealed no statistically significant differences in the concentrations of serum biomarkers indicative of hepatocellular function, such as SGOT (P = 0.53), SGPT (P = 1.00), and LDH (P = 0.19). These findings are consistent with the results from the assessment of inflammatory response markers, such as TNF-α (P = 0.09), ET-1 (P = 0.35), serum IL-1β (P = 0.42), tissue IL-1β (P = 0.26), and protein carbonyl concentrations (serum, P = 0.60; tissue, P = 1.00). However, as evident from the histopathological evaluation, treprostinil appeared to exert a stronger hepatoprotective effect than IP, as reflected by significantly lower scores for inflammatory cell infiltration (P = 0.012) and Mallory-like body formation (P = 0.027). Although both treatments decreased hepatocyte necrosis and hemorrhage scores, the differences were not statistically significant (necrosis, P = 0.05; liver hemorrhage, P = 0.62). With respect to myocardial tissue damage, treprostinil exerted a stronger cardioprotective effect than IP, as evidenced by the significant reduction in troponin I (P = 0.006), whereas IP failed to achieve a marked decrease. Although treprostinil and IP decreased the CPK concentration compared to HIRI, the differences did not reach statistical significance (P = 1.00). Histologically, the treprostinil-treated group decreased the presence of wavy fibers (P = 0.80), inflammatory cell aggregation (P = 0.75), and loss of striation (P = 0.82), although these changes did not reach statistical significance. These findings suggest that the short-term experimental model may not be sufficient to capture the full extent of the delayed protective effects associated with either treatment.

Several key mechanisms have been identified to explain the cytoprotective actions of treprostinil and IP. As a PGI₂ analog, treprostinil has been shown to reduce oxidative stress and the expression of proinflammatory cytokine genes (TNF-α and IL-6), leading to decreased neutrophil accumulation in liver tissue[50,62]. IP has also been reported to attenuate ROS generation and decrease proinflammatory cytokine production. Furthermore, IP can modulate the programmed cell death rate by increasing antiapoptotic protein expression, suppressing caspase-3 activation and activating cell survival pathways (PI3K/Akt and extracellular signal-regulated kinase 1/2)[37-39]. There is also emerging evidence that IP activates autophagy-related pathways, such as Beclin-1 and light chain 3-II, contributing to mitochondrial protection and increased cell survival[37]. While the current study focused on functional and biochemical endpoints to evaluate the efficacy of treprostinil and IP against HIRI, future studies exploring the underlying molecular mechanisms are warranted to fully elucidate these pathways.

There are several limitations regarding treprostinil administration compared with IP in the context of HIRI management. IP is widely used in liver surgery, but clinical evidence regarding treprostinil treatment is still limited. Most studies assessing the effectiveness of treprostinil in liver surgery are experimental. Considerable cost differences exist between treprostinil therapy and IP, as the former involves high pharmaceutical expenses, including the cost of the drug, monitoring and extended intensive care unit support, whereas IP, performed via standard surgical clamping techniques, is already integrated into routine operative protocols without incurring additional costs. Treprostinil administration during liver surgery presents several logistical challenges that may hinder its broader clinical application. Subcutaneous or intravenous treprostinil administration requires a specialized hospital infrastructure, central line access and/or infusion pumps, trained personnel for dosage control and, in some cases, monitoring in intensive care units. Hospitals must be equipped with protocols for drug administration and adverse event management; thus, treprostinil use is not always feasible in resource-limited surgical centers. In contrast, IP is widely applied in clinical settings, with no need for specialized equipment or pharmacological agents, and adverse events are extremely limited when the method is properly timed. Nevertheless, although the high cost of treprostinil necessitates careful consideration and individualized use in chronic conditions, its administration in the acute setting of HIRI constitutes a short-term intervention rather than a prolonged treatment. In addition, some patients with advanced liver disease develop portopulmonary hypertension, a subtype of pulmonary arterial hypertension that occurs secondary to portal hypertension. Patients with portopulmonary hypertension are often managed with PGI₂ analogs such as treprostinil to optimize pulmonary hemodynamics, and their peri- and postoperative continuation might positively affect graft outcomes[79,80].

While the present study was conducted using rats, species-specific differences in hepatic drug metabolism and HIRI responses may affect the translation of these findings to clinical settings. However, the current clinical use of treprostinil in humans for pulmonary arterial hypertension provides a foundational pharmacological safety profile, supporting further investigations in the hepatic surgery setting. This experimental model is considered well established, providing safe and well-documented results. Additional clinical studies are needed to confirm these findings, support drug application in clinical practice and secure the necessary approval from the FDA. Nonetheless, our experimental findings were positive, as adequate anesthesia and analgesia were successfully achieved, and significant therapeutic effects of treprostinil and IP were observed in the study groups compared with the control group.

CONCLUSION

The findings of our study support the conclusion that HIRI induces oxidative stress and promotes cytokine overexpression, leading to impaired liver function and exacerbated myocardial injury. To our knowledge, this is the first study to evaluate the hepatoprotective effects of treprostinil and hepatic IP on myocardial damage in the context of HIRI. The protective effects of treprostinil may be attributed to its vasodilatory and anti-inflammatory mechanisms. Accordingly, the attenuation of oxidative stress and reduction in the inflammatory response constitute possible hepatoprotective pathways of IP. These results suggest that treprostinil and IP may exert beneficial effects in protecting the liver and heart against HIRI-induced damage. Notably, compared with IP, treprostinil appeared to offer slightly superior cardioprotection. Further large-scale experimental and clinical studies are essential to validate the safety and therapeutic efficacy of treprostinil administration in liver transplantation and liver surgery before its application in clinical practice.

ACKNOWLEDGEMENTS

The authors extend their appreciation to Taitzoglou I, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki for his assistance and guidance.