Introduction

Acute mesenteric ischemia is a life-threatening vascular emergency resulting from the interruption of intestinal blood flow, leading to rapid and potentially irreversible damage. Although its incidence is relatively low, the condition is associated with a high mortality rate, particularly in older adults and in cases with delayed diagnosis. The underlying etiologies include arterial embolism, thrombosis, and non-occlusive hypoperfusion [1]. Despite advances in surgical interventions, no pharmacological therapy has been established to limit cellular damage. There remains a critical need for adjunctive treatments that can preserve intestinal viability, prolong the therapeutic window, and improve surgical outcomes [2].

The pathogenesis involves a complex interplay between inflammation, oxidative stress, and apoptosis. Oxygen deprivation disrupts cellular metabolism, leading to ATP depletion, impaired ion transport, and early enterocyte damage. Reperfusion exacerbates injury by generating excessive reactive oxygen species (ROS) and releasing inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), which drive leukocyte infiltration and microvascular damage. These processes lead to lipid peroxidation, mitochondrial dysfunction, and activation of apoptotic pathways, ultimately undermining epithelial integrity and disrupting the intestinal barrier [3].

Phosphatidylserine is a naturally occurring phospholipid predominantly localized to the inner leaflet of the plasma membrane, where it plays an essential role in maintaining membrane integrity, signaling, and apoptotic regulation [4]. Endogenous phosphatidylserine contributes to the pathogenesis of mesenteric ischemia–reperfusion, with studies showing that disruption of its cellular trafficking exerts protective effects [5,6,7]. In contrast, exogenous phosphatidylserine has been reported to exert anti-inflammatory, antioxidant, and anti-apoptotic effects. It reduces pro-inflammatory cytokine release, neutrophil infiltration, and nuclear factor-kappa B (NF-κB) activation [8,9,10,11]. Phosphatidylserine also preserves mitochondrial membrane potential and mitigates lipid peroxidation by enhancing antioxidant defenses [12, 13]. Additionally, phosphatidylserine modulates intrinsic apoptotic pathways by regulating B-cell lymphoma 2 (Bcl-2) proteins and caspase activity [14, 15]. Emerging evidence also indicates that phosphatidylserine may act through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway [16,17,18], which regulates cell survival, metabolism, and inflammatory responses. These mechanisms highlight phosphatidylserine as a potential candidate for mitigating ischemia–reperfusion injury.

To explore the potential mechanisms, we employed a reverse docking approach to predict molecular targets of phosphatidylserine, revealing pathways relevant to ischemia–reperfusion injury. This study aimed to assess the effects of phosphatidylserine in a rat model of acute mesenteric ischemia–reperfusion, by evaluating inflammation, oxidative stress, and apoptosis, and exploring potential association with the Akt/mTOR pathway.

Results

Molecular docking

The list of 100 targets predicted using the SwissTargetPrediction tool, along with their binding affinities and enrichment analysis results, is available in the supplementary data. Based on enrichment analysis findings, ten key proteins, predominantly associated with the pathophysiology of ischemia–reperfusion injury as indicated by previous research, were identified for further investigation. These targets include:

  • Lysophosphatidic acid receptor Edg-7 (LPAR3)—UniProt ID Q9UBY5

  • Lysophosphatidic acid receptor 6 (LPAR6)—UniProt ID P43657

  • Lysophosphatidic acid receptor Edg-4 (LPAR2)—UniProt ID Q9HBW0

  • Lysophosphatidic acid receptor 5 (LPAR5)—UniProt ID Q9H1C0

  • Lysophosphatidic acid receptor 4 (LPAR4)—UniProt ID Q99677

  • PI3-kinase p110-alpha/p85-alpha (PIK3CA/PIK3R1)—UniProt IDs P42336, P27986

  • 3-phosphoinositide dependent protein kinase-1 (PDPK1)—UniProt ID O15530

  • Serine/threonine-protein kinase mTOR (MTOR)—UniProt ID P42345

  • Serine/threonine-protein kinase AKT1—UniProt ID P31749

The binding affinities of these targets ranged from -4.3 to -6.8 kcal/mol, suggesting possible interactions between phosphatidylserine and several of the identified proteins. Three proteins with the highest affinity interactions were examined further, as detailed in Table 1 and Fig. 1.

Table 1 Binding affinity and interaction between phosphatidylserine and the proteins active site.
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Fig. 1
figure 1

Phosphatidylserine -protein interactions. Right and left panels show 2D and 3D representations, respectively, of interactions of phosphatidylserine with (a–b) Lysophosphatidic acid receptor 6 (LPAR6), (c–d) 3-phosphoinositide dependent protein kinase-1, and (e–f) Lysophosphatidic acid receptor 4 (LPAR4).

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Binding affinity (kcal/mol) and amino acid residues of interaction between phosphatidylserine and the target proteins.

Histopathological evaluations

As shown in Fig. 2, mesenteric ischemia–reperfusion resulted in severe intestinal damage, corresponding to grade 3 injury (p < 0.001; Fig. 2a), characterized by marked villus blunting, epithelial vacuolization, and severe inflammation (Fig. 2e). Phosphatidylserine treatment did not alter the normal intestinal architecture in sham-operated rats; however, it attenuated ischemia–reperfusion-induced damage, with the 40 mg/kg dose reaching statistical significance (p < 0.05; Fig. 2a). This corresponded to grade 1 injury, characterized by sparse inflammation and mild vacuolization (Fig. 2e).

Fig. 2
figure 2

Histopathologic evaluations. (a) Histopathologic scores based on Macpherson and Pfeiffer scoring system for different groups (n = 6 rats in each group). Individual data points are presented, with the lines indicating median ± interquartile range (IQR). # indicates p < 0.05 compared to the Vehicle group. *, **, and *** denote p < 0.05, 0.01, 0.001, respectively, in comparison to Sham group. Statistical analysis was performed using the Kruskal–Wallis test followed by Dunn’s post-hoc test. Panels b–e show representative Hematoxylin and Eosin-stained ileal sections from groups treated with vehicle (b) and phosphatidylserine at 10 (c), 20 (d), and 40 (e) mg/kg under 1000 × magnification. Solid arrows show vacuolization and hollow arrows indicate infiltration of immune cells. PS: Phosphatidylserine.

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Oxidative and inflammatory biomarkers

Ischemia–reperfusion induced oxidative stress, as evidenced by elevated lipid peroxidation, with significantly increased malondialdehyde (MDA) levels (2.92 vs. 1.15 µmol/mg compared to Sham; p < 0.001; Fig. 3c), and by reduced intrinsic antioxidant defense, reflected in decreased superoxide dismutase (SOD) (310 vs. 861 U/mg; p < 0.0001; Fig. 3a) and glutathione peroxidase (GPx) activity (65 vs. 204 U/mg; p < 0.0001; Fig. 3b). Phosphatidylserine treatment had no significant effect on oxidative parameters under physiological conditions (p = 0.83, 0.97, and 0.74 for SOD, GPx and MDA, respectively). However, in ischemia–reperfusion injury, phosphatidylserine at 40 mg/kg significantly lowered MDA levels (1.38 µmol/mg; p < 0.05; Fig. 3c) and increased SOD (617 U/mg; p < 0.001; Fig. 3a) and GPx activity (125 U/mg; p < 0.01; Fig. 3b). Moreover, ischemia–reperfusion caused marked inflammation, as evidenced by increased IL-6 (55.1 vs. 21.1 pg/mg; p < 0.0001; Fig. 3d). Phosphatidylserine did not alter IL-6 levels under physiological conditions (p = 0.91); however, it significantly reduced IL-6 at both 20 mg/kg (40.4 pg/mg; p < 0.05) and 40 mg/kg (31.4 pg/mg; p < 0.001).

Fig. 3
figure 3

Oxidative and apoptotic biomarkers. (a) Superoxide dismutase (SOD) activity. (b) Glutathione peroxidase (GPx) activity. (c) Malondialdehyde (MDA). (d) Interleukin-6 (IL-6) levels. Rats were divided into six groups, undergoing either a sham operation or mesenteric ischemia–reperfusion, and received vehicle or Phosphatidylserine (PS) at doses of 10, 20, and 40 mg/kg. Individual data points are presented, with graphs indicating mean ± standard deviation (SD). n = 6 rats for each measurement in each group. *, **, ***, and **** indicates p < 0.05, 0.01, 0.001, and 0.0001 compared to the Sham group. #, ##, and ### denote p < 0.05, 0.01, 0.001, respectively, in comparison to the Vehicle group. Statistical analysis was performed using the ordinary (a, b, d) or welch (c) one-way ANOVA followed by Šidák (a, b, d) or Dunnett (c) multiple comparison post-hoc test.

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Inflammation, apoptosis and Akt/mTOR pathway

In line with IL-6 findings, ischemia–reperfusion elicited a marked inflammatory response, evidenced by a 3.54-fold increase in TNF-α levels compared to the sham group (p = 0.0019; Fig. 4a). This response was attenuated by phosphatidylserine treatment at 40 mg/kg, which produced a 49% reduction relative to the vehicle group (p = 0.026; Fig. 4a). Ischemia–reperfusion also triggered apoptosis, as indicated by a 3.82-fold increase in pro-apoptotic Bcl-2 Associated X-protein (BAX) expression (p < 0.0001; Fig. 4c) and a 61% reduction in anti-apoptotic Bcl-2 expression (p < 0.01; Fig. 4b). Phosphatidylserine at 40 mg/kg significantly mitigated apoptosis, lowering BAX expression by 55% (p < 0.001) and restoring Bcl-2 expression to near-sham levels (2.54-fold increase; p < 0.01). Furthermore, ischemia–reperfusion significantly suppressed Akt and mTOR phosphorylation (p = 0.0012 and p = 0.0014, respectively; Figs. 4d and e). Phosphatidylserine at both 20 and 40 mg/kg significantly restored Akt phosphorylation, whereas restoration of mTOR phosphorylation reached statistical significance only at 40 mg/kg (p = 0.13 for 20 mg/kg dose; Fig. 4e).

Fig. 4
figure 4

Inflammatory, apoptotic and Akt/mTOR pathway biomarkers. (a) tumor necrosis factor-alpha (TNF-α). (b) B-cell lymphoma 2 (Bcl-2). (c) Bcl-2 Associated X-protein (BAX) (d) protein kinase B (Akt). (e) mammalian target of rapamycin (mTOR). Rats were divided into six groups, undergoing either a sham operation or mesenteric ischemia–reperfusion, and received vehicle or Phosphatidylserine (PS) at doses of 10, 20, and 40 mg/kg. Individual data points are presented, with graphs indicating mean ± standard deviation (SD). n = 3 samples for each measurement in each group. *, **, and *** indicates p < 0.05, 0.01, and 0.001 compared to the Sham group. #, ##, and ### denote p < 0.05, 0.01, 0.001, respectively, in comparison to the Vehicle group. Statistical analysis was performed using the ordinary one-way ANOVA followed by Šidák multiple comparison post-hoc test. Original uncropped blots with markers are shown in Supplementary File 1.

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Discussion

This study is the first to demonstrate that phosphatidylserine protects against mesenteric ischemia–reperfusion injury by attenuating inflammation, apoptosis, and oxidative stress, while upregulating Akt/mTOR signaling. These findings are consistent with previous reports of the protective effects of phosphatidylserine in ischemia–reperfusion models. The observed activation of the Akt/mTOR pathway reinforces its role in ischemic injury and supports earlier evidence of the modulatory potential of phosphatidylserine [16,17,18]. Additionally, our reverse docking and molecular docking analyses further identified the PI3K/Akt/mTOR pathway as a potential target.

Reduced oxygen delivery during ischemia disrupts mitochondrial function and depletes cellular energy stores, leading to impaired membrane ion pump activity and electrolyte imbalance, particularly intracellular calcium overload. This cascade triggers oxidative stress, excessive ROS generation, and activation of intrinsic apoptotic pathways [19]. These events inactivate phosphatidylserine flippases, a group of enzymes that normally maintain phosphatidylserine on the inner leaflet, and activate scramblases that translocate it to the outer leaflet [20]. Surface-exposed phosphatidylserine acts as an apoptotic marker and immunologic signal, promoting phagocytosis by macrophages and T-cell activation [21]. Under oxidative conditions, oxidized phosphatidylserine further amplifies apoptotic and inflammatory signaling [5, 22]. Supporting this mechanism, previous studies have shown that limiting phosphatidylserine exposure on the cell surface, either through Annexin V binding or inhibition of phospholipid scramblase-1, attenuates ischemic injury, reduces inflammatory signaling, and improves endothelial barrier function in ischemia–reperfusion models [6, 7]. These findings highlight the pathological role of uncontrolled phosphatidylserine externalization during cellular stress.

Unlike endogenous phosphatidylserine, which promotes inflammatory and apoptotic signaling under stress conditions, the exogenous form may exert protective effects by modulating immune responses. Administration of phosphatidylserine liposomes has been reported to inhibit the immunogenic effects of endogenous phosphatidylserine, reducing lymph node mass, total leukocyte counts, and antigen-specific CD4 + T cell counts [23]. Moreover, activation of phosphatidylserine receptors on macrophages triggers a compensatory anti-inflammatory response, characterized by increased transforming growth factor-beta (TGF-β) release [23]. In microglial models, treatment with native phosphatidylserine liposomes or the phospho-L-serine head group suppressed lipopolysaccharide-induced production of pro-inflammatory cytokines, including TNF-α and interleukin-1 beta (IL-1β), while enhancing anti-inflammatory mediators such as TGF-β and prostaglandin E2 [24,25,26,27].

Beyond the protective effects of native phosphatidylserine, its oxidative modification may also contribute to mitigating tissue injury. The serine head group of phosphatidylserine serves as an efficient substrate for oxidative enzymes and ROS, leading to the formation of byproducts such as acetic acid, acetamide, phosphatidic acid, and hydroperoxylacetaldehyde. This oxidative process consumes ROS, thereby alleviating oxidative stress, an effect reflected in our findings by reduced MDA levels and increased SOD and GPx activity [28]. Notably, phosphatidylserine treatment in sham-operated rats did not affect physiological oxidative parameters, suggesting a selective action that preserves normal redox homeostasis. Oxidized phosphatidylserine also exhibits anti-inflammatory properties [22]: it reduces neutrophil oxidative bursts by inhibiting myeloperoxidase activity [29], counteracts lipopolysaccharide-induced responses through Toll-like receptor 4 signaling in immune cells [30], suppresses interleukin-1β and TNF-α production in monocytes [31], and promotes vascularization [32]. Additionally, oxidized phosphatidylserine inhibits nitric oxide production and downregulates inducible nitric oxide synthase and IL-1β gene expression by modulating c-Jun N-terminal kinase phosphorylation and nuclear translocation of NF-κB, effects not observed with native phosphatidylserine [9]. These findings suggest a potential dual mechanism in which phosphatidylserine oxidation initially attenuates oxidative stress and subsequently limits inflammation.

Phosphatidylserine has shown protective effects in multiple ischemia–reperfusion models [10, 33, 34]. In an acute myocardial infarction model, it reduced infarct size while enhancing cardiomyocyte survival and cardiac function through activation of the protein kinase C pathway. Notably, adding pretreatment before ischemia onset did not improve outcomes, indicating that phosphatidylserine retains therapeutic efficacy when given after ischemia, which is an essential consideration in unpredictable conditions such as mesenteric ischemia [33, 35]. In a retinal ischemia model, phosphatidylserine improved neuronal survival by downregulating pro-inflammatory cytokines [8]. Similarly, in a stroke model induced by unilateral common carotid artery occlusion, phosphatidylserine alleviated post-stroke depressive-like behaviors, enhanced hippocampal neuron survival, and reduced TNF-α and IL-10 levels [10]. These findings are consistent with our results, which demonstrated improved histopathological outcomes following phosphatidylserine treatment, together with reduced inflammation and apoptosis, as evidenced by decreased BAX and increased Bcl-2 expression.

Various pathways have been proposed as molecular targets of phosphatidylserine. Our findings demonstrated phosphatidylserine treatment was associated with upregulation of the Akt/mTOR pathway, a finding further supported by reverse and molecular docking analyses, suggesting this pathway as a potential site of interaction. The Akt/mTOR signaling axis plays a critical role in the pathophysiology of ischemia–reperfusion injury. In a model of transient coronary artery ligation, transgenic mice overexpressing mTOR exhibited reduced mortality, fibrosis, and necrosis, along with improved cardiac function and decreased pro-inflammatory cytokines expression [36]. Similarly, in a liver ischemia–reperfusion model, mTOR-deficient mice or those treated with rapamycin displayed greater histopathological damage, elevated alanine transaminase and lactate dehydrogenase levels, and increased caspase-3 activation and NF-κB translocation [37]. More specifically, in both an in vivo model of intestinal ischemia–reperfusion and an in vitro model of hypoxia/reoxygenation in intestinal epithelial cells, inhibition of the mTOR pathway by rapamycin exacerbated tissue injury, whereas activation of p70S6K, which is a major downstream effector of mTOR, conferred significant protection [38]. These findings further support the involvement of Akt/mTOR pathway in ischemia–reperfusion injury.

Mesenteric ischemia is an unpredictable event, and definitive treatment relies on surgery or endovascular intervention. Reported delays of 10–14 h between admission and surgery [39, 40], underscore the potential benefit of immediate pharmacological interventions to limit tissue injury, extend the window before irreversible damage, and improve the chances of successful surgery. A major component of ischemia–reperfusion injury is the oxidative burst following reoxygenation, suggesting that pharmacological agents may be more effective in attenuating reperfusion injury than in targeting the ischemic phase itself. Although no clinical studies have evaluated pharmacological agents in mesenteric ischemia to date, except for limited reports on vasodilators [41], the plausibility of therapeutic benefit remains. Nevertheless, given the unpredictable nature of mesenteric ischemia, the clinical applicability of phosphatidylserine is likely to be limited.

Endogenous phosphatidylserine exposure is a well-known marker of cell damage and an “eat me” signal, particularly studied in erythrocytes [42,43,44]. In contrast, exogenous phosphatidylserine has shown a favorable safety profile. Oral supplementation studies have reported no changes in standard biochemical or hematological parameters [4, 45], and the U.S. Food and Drug Administration has classified phosphatidylserine as generally recognized as safe. In a typical Western diet, daily intake is approximately 100–200 mg. No toxic effects have been reported in the literature, apart from minimal corticomedullary mineralization observed at extremely high doses (3.4 g/kg/day), far exceeding those used clinically or in our study [46]. Overall, current evidence suggests a favorable safety profile for phosphatidylserine, although concerns remain regarding its potential incorporation into plasma and organelle membranes and its impact on endogenous biosynthesis, which may have diverse physiological implications, particularly given structural differences between administered compounds and human-synthesized phosphatidylserine.

Despite the encouraging results, this study has several limitations that warrant further investigation. While our findings provide exploratory evidence for the protective effects of phosphatidylserine in mesenteric ischemia and suggest an association with the Akt/mTOR pathway, establishing a direct causal relationship will require future studies employing specific pathway agonists and antagonists. Moreover, mesenteric ischemia involves a complex interplay among epithelial, endothelial, neuronal, and immune cells. Dissecting the cell-specific effects of phosphatidylserine would provide a more comprehensive understanding of its mechanisms. Another limitation is the small sample size (n = 3) used for Western blot analyses, which was due to financial constraints. While useful for exploratory purposes, this limited sample size restricted our ability to firmly link Akt/mTOR and apoptotic pathways to the protective effects of phosphatidylserine. Nevertheless, the consistency between biochemical and histopathological findings and the Western blot results supports the validity of our conclusions. Another limitation is that the reverse docking and computational predictions were entirely in silico, without experimental validation. These results should therefore be interpreted as exploratory, and future in vitro and in vivo studies will be required to confirm the predicted binding interactions. Moreover, the proposed involvement of receptor-mediated signaling and phosphatidylserine oxidation should be considered as hypothesis-generating, since these mechanisms were not directly examined in the present study. Future targeted approaches comparing oxidized and native phosphatidylserine, together with pathway-specific inhibitors, will be necessary to confirm their involvement and to delineate their respective contributions. Lastly, although statistical power was sufficient to detect group differences, the modest number of animals per group may still limit the generalizability of our findings. Further research exploring phosphatidylserine’s interactions across different tissue compartments and its downstream signaling targets will be essential to fully elucidate its therapeutic potential in mesenteric ischemia.

Conclusion

This preclinical study demonstrates, for the first time, that phosphatidylserine may exert protective effects against mesenteric ischemia–reperfusion injury by attenuating oxidative stress, inflammation, and apoptosis, while upregulating the Akt/mTOR signaling pathway. These findings suggest its potential as a therapeutic candidate for intestinal ischemic injury. However, given the experimental nature of this study, further research is necessary to elucidate its cell-specific mechanisms and to evaluate its efficacy and safety before any clinical application can be considered.

Methods

Computational approach

Ligand preparation

Phosphatidylserine was selected as the ligand for this study. Its 3D structure was retrieved from the PubChem database (CID: 9547096) and converted to Mol2 format using OpenBabel [47, 48]. The Mol2 file was then energy-minimized using Chem3D to ensure the optimal conformation for docking [49]. The finalized Mol2 structure was then used in computational analyses.

Target protein prediction

To identify potential protein targets, the SwissTargetPrediction platform (https://www.swisstargetprediction.ch/) was employed. This tool applies ligand-based similarity and known bioactivity data to predict likely protein targets within the human proteome. Phosphatidylserine was submitted, and the 100 proteins with the highest predicted binding probabilities were retrieved. These proteins were subsequently used in docking simulations and protein–protein interaction network analysis.

Protein–protein interaction network

The STRING database (https://string-db.org/) was used to construct a protein–protein interaction network from the top 100 predicted targets. The resulting network was analyzed for pathway enrichment, molecular functions, biological processes, tissue expression profiles, and associated pathways (including KEGG, Reactome, and WikiPathways).

Molecular docking

ReverseDock (https://www.reversedock.org/) was utilized to perform molecular docking of phosphatidylserine against the 10 prioritized protein targets. Docking was carried out with AutoDock Vina [50] using default parameters. For each target, the 3D structure was retrieved from the AlphaFold Protein Structure Database using the corresponding UniProt IDs. Both 2D and 3D interaction diagrams were created using Discovery Studio Visualizer (Ver.17.2) [51] and PyMol version 1. Level [52] for structural interpretation and ligand-binding site analysis.

In vivo experiment

Animals

Thirty-six male Wistar albino rats (seven weeks old, 200–230 g) were obtained from the Department of Pharmacology, Tehran University of Medical Sciences. Rats were housed under standard conditions: a 12 h light/dark cycle, temperature of 21–23 °C, and ad libitum access to food and water. Animals were acclimated for one week before experimentation. To minimize cage-related variability, four rats were housed per cage with randomized group distribution. Each rat was considered an independent experimental unit. Animals with visible abdominal abnormalities or excessive intraoperative bleeding were excluded. All procedures were conducted in compliance with the ARRIVE guidelines and approved by the Institutional Animal Ethics Committee of Tehran University of Medical Sciences (IR.TUMS.AEC.1403.159), in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Phosphatidylserine suspension preparation

Liposomal phosphatidylserine was formulated using a modified injection method developed in our laboratory [27]. Phosphatidylserine (Pharmin LLC, USA) was prepared in quantities of 100 mg, 200 mg, and 400 mg, and each was dissolved in 2 mL of anhydrous ethanol. The ethanolic solution was then slowly introduced into 10 mL of pre-warmed normal saline under continuous stirring at 700 rpm. Stirring continued until complete evaporation of ethanol from the suspension. The resulting suspension was then sonicated at low frequency to ensure the formation of homogenous liposomal vesicles. Vesicle formation was confirmed through optical microscopy. The vehicle control was prepared using the same procedure without the addition of phosphatidylserine. All injections were administered intraperitoneally at a volume of 1 mL/kg.

Mesenteric ischemia–reperfusion induction

Rats were fasted overnight with unrestricted access to water prior to surgery. Anesthesia was induced by intraperitoneal injection of ketamine hydrochloride (85 mg/kg, 10% w/v solution, Alfasan Pharmaceuticals, The Netherlands) and xylazine hydrochloride (15 mg/kg, 2% w/v solution, Bremer Pharma, Germany). Animals were positioned supine on a thermostatically controlled heating pad to maintain normothermia. As previously described [53] and confirmed in our pilot experiments, a 2.5-cm midline laparotomy was performed to access the abdominal cavity. Ischemia was induced by occluding the superior mesenteric artery and its distal anastomosis with the inferior mesenteric artery near the cecum using a non-traumatic microvascular clamp. Occlusion was maintained for 60 min and confirmed by intestinal pallor or cyanosis. Reperfusion was initiated by clamp removal and verified by return of normal coloration. Additional ketamine (20 mg/kg) was administered as needed. After 60 min of reperfusion, animals were euthanized and ileal samples were collected for biochemical and histopathological analysis [53]. Consistent with previous studies supporting the 60/60 min ischemia/reperfusion protocol [54], we conducted a pilot study testing 30/60, 60/60, 30/120, and 60/120 min (n = 3 per group). The corresponding mean histopathological scores were 1.8, 2.7, 2.2, and 3.0, respectively. Among these, the 60/60 protocol produced the greatest reproducible injury without causing excessive damage, allowing reliable assessment of potential protective effects.

Experimental groups

As shown in Fig. 5, thirty-six rats were randomly assigned to six experimental groups (n = 6 per group) using computer-generated block randomization in blocks of 12 animals, with two rats assigned to each group per block. Randomization was performed by an author (M.M.) who was not involved in treatment administration, outcome assessment, or data analysis. The Sham group underwent anesthesia and laparotomy without vascular occlusion and received vehicle treatment, while the Sham + phosphatidylserine group followed the same protocol but received phosphatidylserine. The Vehicle group was subjected to mesenteric ischemia–reperfusion followed by vehicle administration. The remaining groups underwent mesenteric ischemia–reperfusion followed by phosphatidylserine at doses of 10, 20, or 40 mg/kg. All treatments and vehicle were administered intraperitoneally 15 min before reperfusion. Doses were selected by translating clinical doses of phosphatidylserine used as dietary supplements (100–400 mg/day) into rat equivalents using an established scaling factor [55, 56], corresponding to approximately 10–40 mg/kg in rats. This range is further supported by prior studies reporting protective effects of phosphatidylserine at 5–50 mg/kg in ischemia–reperfusion models [10, 35]. The primary outcome was histological evaluation of the ileum using the Macpherson and Pfeiffer scoring system [57], while secondary outcomes included biochemical markers of oxidative stress, inflammation, and protein expression of Akt and mTOR. Sample size was determined based on prior studies employing similar mesenteric ischemia–reperfusion models [53] and further supported by a post hoc power analysis of histopathological scores, which yielded an effect size of Cohen’s f = 5.43 (η2 = 0.967). With n = 6 rats per group, the achieved power exceeded 0.99 at α = 0.05, confirming adequacy of the chosen sample size. Drug solutions were prepared in coded vials by a blinded author (S.A.) based on the randomization key (M.M.), and surgeries with intraperitoneal injections were performed by another author (A.M.) using only coded vials.

Fig. 5
figure 5

Experimental design and timeline for ischemia–reperfusion induction, treatments, and sample collection. IRI: ischemia–reperfusion injury; PS: Phosphatidylserine.

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Biochemical measurements

At the end of the reperfusion period, animals were euthanized with a high-dose intraperitoneal injection of ketamine and xylazine (200 mg/kg and 20 mg/kg, respectively), and ileal tissues were harvested and stored at – 80 °C for biochemical analysis. MDA (Teb Pazhouhan Razi, Iran) was measured using the thiobarbituric acid reactive substances assay, while SOD (Nasdox, Iran) activity was determined with a pyrogallol-based spectrophotometric method. IL-6 levels and GPx activity were measured using commercial ELISA kits (Karmania Pars Gene, Iran). All measurements were carried out according to the manufacturers’ protocols by an investigator blinded to group allocation (F.N.).

Western blot

Western blotting was performed to evaluate Akt and mTOR expression in ileal tissues. Samples were lysed in radioimmunoprecipitation assay buffer and centrifuged at 14,000 rpm for 20 min at 4 °C. Protein concentrations were determined using the Bradford assay (DNAbioTech, Iran). Equal amounts of protein (20 µg) were mixed with 2 × Laemmli buffer, boiled for 5 min, separated by SDS-PAGE, and transferred to 0.2 µm polyvinylidene difluoride membranes (Bio-Rad, USA). Membranes were blocked in 5% bovine serum albumin with 0.1% Tween 20 for one hour and then incubated with primary antibodies against TNF-α (1:1500, KRC3011, Invitrogen, USA), Bcl-2 Associated X-protein (BAX) (1:1000, BS-28034R, Bioss, USA), Bcl-2 (1:1000, MAB8272, R&D systems, USA), p-S2448-mTOR (1:2000, ab109268, Abcam, USA), p-S473-Akt (1:2000, ab8805, Abcam, USA), and β-actin (1:2500, Abcam, USA). After washing, membranes were probed with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000, Abcam, USA). Bands were visualized using enhanced chemiluminescence and quantified with Gel Analyzer software (NIH, USA). Expression levels were normalized to β-actin. The entire procedure was performed by an investigator blinded to group allocation (F.N.).

Histopathological evaluations

Following the 60 min reperfusion period, animals were euthanized with a high-dose intraperitoneal injection of ketamine and xylazine (200 mg/kg and 20 mg/kg, respectively). Ileal tissues were excised, fixed in 4% neutral-buffered formalin, processed using standard protocols, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin for microscopic evaluation. Mucosal inflammation was assessed using a modified scoring system based on Macpherson and Pfeiffer [57]. Score 0 represented normal histology; Score 1 indicated villus blunting, crypt loss, sparse inflammation, and mild vacuolization and edema; Score 2 reflected villus blunting with epithelial vacuolization, crypt necrosis, and moderate to intense inflammation and edema; Score 3 denoted marked villus blunting, epithelial vacuolization, crypt necrosis, and severe inflammation and edema. All evaluations were conducted at 400 × magnification by a pathologist (M.T.) blinded to group allocation. For ambiguous cases, the mean of the two closest scores was recorded to ensure consistency.

Statistical analysis

Statistical analyses and data visualization were performed using GraphPad Prism version 10 (GraphPad Software, USA). Parametric data are presented as mean ± standard deviation, and non-parametric data as median with interquartile range. Data normality and variance homogeneity were assessed using the Shapiro–Wilk and Levene’s tests, respectively. Ordinary or Welch one-way analysis of variance, using Šidák or Dunnett multiple comparison post-hoc test, was used for parametric comparisons, while non-parametric data, including histological scores, were analyzed using the Kruskal–Wallis test with Dunn’s post hoc test. A p value < 0.05 was considered statistically significant. All statistical analyses were performed by a blinded investigator (M.G.).