Effects of Pomegranate Seed Oil on Lower Extremity Ischemia-Reperfusion Damage: Insights into Oxidative Stress, Inflammation, and Cell Death

Abstract

Aim: This study sought to clarify the therapeutic benefits and mechanisms of action of pomegranate seed oil (PSO) in instances of ischemia-reperfusion (IR) damage in the lower extremities. Materials and Methods: The sample size was determined, then 32 rats were randomly allocated to four groups: Control (C), ischemia-reperfusion (IR), low-dose PSO (IR + LD, 0.15 mL/kg), and high-dose PSO (IR + HD, 0.30 mL/kg). The ischemia model in the IR group was established by occluding the infrarenal aorta for 120 min. Prior to reperfusion, PSO was delivered to the IR + LD and IR + HD groups at doses of 0.15 mL/kg and 0.30 mL/kg, respectively, followed by a 120 min reperfusion period. Subsequently, blood and tissue specimens were obtained. Statistical investigation was executed utilizing Statistical Package for the Social Sciences version 20.0 (SPSS, IBM Corp., Armonk, NY, USA). Results: Biochemical tests revealed significant variations in total antioxidant level (TAS), total oxidant level (TOS), and the oxidative stress index (OSI) across the groups (p < 0.0001). The IR group had elevated TOS and OSI levels, whereas PSO therapy resulted in a reduction in these values (p < 0.05). As opposed to the IR group, TASs were higher in the PSO-treated groups. Histopathological analysis demonstrated muscle fiber degeneration, interstitial edema, and the infiltration of cells associated with inflammation in the IR group, with analogous results noted in the PSO treatment groups. Immunohistochemical analysis revealed that the expressions of Tumor Necrosis Factor-alpha (TNF-α), Nuclear Factor kappa B (NF-κB), cytochrome C (CYT C), and caspase 3 (CASP3) were elevated in the IR group, while PSO treatment diminished these markers and attenuated inflammation and apoptosis (p < 0.05). The findings demonstrate that PSO has a dose-dependent impact on IR injury. Discussion: This research indicates that PSO has significant protective benefits against IR injury in the lower extremities. PSO mitigated tissue damage and maintained mitochondrial integrity by addressing oxidative stress, inflammation, and apoptotic pathways. Particularly, high-dose PSO yielded more substantial enhancements in these processes and exhibited outcomes most comparable to the control group in biochemical, histological, and immunohistochemical investigations. These findings underscore the potential of PSO as an efficacious natural treatment agent for IR injury. Nevertheless, additional research is required to articulate this definitively.

Keywords: NF-κB; apoptosis; inflammation; ischemia–reperfusion; lower extremity; oxidative stress; pomegranate seed oil.

Figure 1

H&E-stained skeletal muscle sections were captured under a 200× in cross sectional plane and 400× magnifications in a longitudinal sectional plane. Black arrowheads: inflammatory cell infiltration; black arrows: muscle fibers exhibiting varying degrees of injury; asterisk: increased interstitial space between muscle fibers due to interstitial edema. IR: ischemia–reperfusion group; IR + LD: low-dose pomegranate seed oil-treated group; IR + HD: high-dose pomegranate seed oil-treated group; H&E: hematoxylin and eosin.

Figure 2

Micrographs representing the TNF-α and NF-κB immunostainings of the groups. Immunopositivity was revealed visually by DAB (in color brown) staining for both TNF-α and NF-κB. Since the nuclear translocation of NF-κB shows its activation state during inflammation, only nuclear positive staining (arrows) rather than cytoplasmic staining was considered for immunoreactivity. Unstained nuclei were indicated by arrowheads. IR: ischemia–reperfusion group; IR + LD: low-dose pomegranate seed oil-treated group; IR + HD: high-dose pomegranate seed oil-treated group. Magnification is 400× for all images.

Figure 3

Micrographs representing the CYT C and CASP3 immunostainings of the groups. Immunopositivity was revealed visually by DAB (brown) staining for both CYT C and CASP3. IR: ischemia–reperfusion group; IR + LD; low-dose pomegranate seed oil-treated group; IR + HD; high-dose pomegranate seed oil-treated group. Magnification is 400× for all images.