Introduction

Blunt chest trauma is a major cause of acute morbidity and mortality worldwide, frequently resulting in pulmonary contusion and acute lung injury (ALI)1. The pathophysiological cascade initiated by thoracic trauma extends far beyond the lung, as the damaged pulmonary parenchyma becomes a source of a systemic cytokine storm and oxidative stress mediators2. Circulating pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β), together with reactive oxygen species (ROS), propagate through the vascular system, leading to widespread endothelial injury, increased vascular permeability, and secondary tissue hypoxia3.

Although the lung represents the primary target organ, the systemic repercussions of trauma are increasingly recognized4. Among distal organs, female reproductive tissues are particularly susceptible due to their rich vascularization, endocrine regulation, and high dependency on oxygen delivery5. Endothelial dysfunction and hypoxia impair ovarian folliculogenesis, uterine receptivity, and tubal transport, processes tightly regulated by estrogen and oxytocin receptor signaling6,7,8,9. Disruption of these receptor pathways compromises reproductive tissue integrity and may have long-term implications for fertility8,9.

Cannabidiol (CBD), a non-psychotropic phytocannabinoid derived from Cannabis sativa, has attracted growing attention for its potent anti-inflammatory, antioxidant, and anti-apoptotic properties10,11. Previous studies have demonstrated its efficacy in reducing pulmonary inflammation and apoptosis in models of blunt chest trauma and sepsis, primarily via modulation of Bcl-2–Associated X protein (Bax)/ B-cell lymphoma 2 (Bcl-2)/Caspase (Cas) signaling12. However, whether CBD confers protection against secondary systemic organ injury, particularly in female reproductive tissues, remains unknown.

In this study, we hypothesized that blunt chest trauma induces secondary genital organ injury through cytokine storm–mediated endothelial dysfunction and hypoxia, leading to reduced estrogen receptor (ER) and dysregulated oxytocin receptor (OTR) expression. We further proposed that CBD administration mitigates these changes by suppressing systemic inflammation and restoring receptor homeostasis.

To illustrate the proposed mechanism, a graphical abstract (Fig. 1) has been prepared, summarizing the cascade from blunt chest trauma–induced pulmonary injury to systemic inflammatory and hypoxic responses, and subsequent alterations in genital organ tissues, including changes in ER, HIF-1α, and OTR expression. The figure also depicts the modulatory effects of CBD on these tissue changes.

Fig. 1
Fig. 1
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Proposed effects of cannabidiol (CBD) in blunt chest trauma. Schematic illustration of the proposed relationship between blunt chest trauma and systemic and genital organ alterations, and the potential effects of CBD. Blunt chest trauma induces lung injury accompanied by systemic inflammatory responses and hypoxic conditions. These systemic changes are associated with altered receptor expression and hypoxia-related signaling in genital organs, including reduced estrogen receptor (ER) expression and increased hypoxia-inducible factor-1α (HIF-1α) and oxytocin receptor (OTR) immunoreactivity. CBD administration is associated with attenuation of these alterations.

Methods

Ethical approval

All experimental procedures were conducted in accordance with the ARRIVE 2.0 guidelines. The study protocol was reviewed and approved by the Experimental Animal Production and Research Center of Burdur Mehmet Akif Ersoy University (MAKU-DEHUDAM, Burdur, Türkiye) and the Local Animal Research Ethics Committee (MAKU-HADYEK) under Approval No. 1580. All experiments were performed strictly in accordance with the approved protocols.

Blunt chest trauma model

A standardized blunt chest trauma model was employed to induce pulmonary contusion and systemic effects. Briefly, following anesthesia, each rat was placed in the supine position on a custom-designed trauma platform. Anesthesia was induced via intraperitoneal injection of ketamine (80 mg/kg; Ketalar, Pfizer, Türkiye) and xylazine (10 mg/kg; Xylazinbio 2%, Bioveta, Czech Republic) and maintained throughout trauma induction and terminal procedures.

Pulmonary contusion was generated by releasing a cylindrical weight (200 g) from a height of 1 m onto the right anterior thoracic wall, producing an impact energy of 1.96 J (E = mgh; m = 0.2 kg, g = 9.8 m/s2, h = 1 m). This thoracic trauma model was adapted from the method described by Raghavan et al. 13 and reliably produces reproducible pulmonary contusion characterized by alveolar rupture, intra-alveolar hemorrhage, interstitial edema, and inflammatory infiltration, closely resembling features of human blunt thoracic trauma. Trauma was applied once per animal, and all procedures were continuously monitored under anesthesia. Sham animals underwent identical anesthesia and handling without weight-drop application.

The cannabidiol (CBD) used in this study was provided by the Natural Products Application and Research Center (SUDUM) of Suleyman Demirel University. Its primary botanical source was Cannabis sativa L. (Cannabaceae). The extract contained a tetrahydrocannabidiol (THC) concentration of 0.001%, while CBD purity exceeded 99.9%. Independent analyses confirmed that residual solvents and heavy metal levels were within the limits specified by both the United States Pharmacopeia and the European Pharmacopeia14.

Animals and experimental groups

A total of 32 female Wistar Albino rats (12–14 weeks old, 300–350 g) were used in this study. The rats were randomly and equally assigned to four experimental groups (n = 8 per group). All animals were maintained under identical housing conditions and subjected to standardized fasting (8 h prior to procedures) and anesthesia protocols. The experimental groups and interventions were as follows:

1. Sham Group (Vehicle + Anesthesia only): Rats received an intraperitoneal injection of vehicle solution (0.1 mL; 0.9% NaCl + Tween-80). This group served as the negative control, representing baseline physiological conditions without trauma or CBD exposure.

2. TRA Group (Vehicle + Blunt Chest Trauma): Rats received intraperitoneal vehicle (0.1 mL). After 30 min, blunt chest trauma was induced using a standardized weight-drop model (200 g from 1 m height onto the right anterior thoracic wall, ~ 1.96 J). This group modeled the pathological consequences of thoracic trauma in the absence of therapeutic intervention13.

3. TRA + CBD Group (Cannabidiol Pre-treatment + Trauma): Rats received CBD (5 mg/kg, i.p., dissolved in 0.1 mL vehicle) 30 min prior to trauma induction14. Trauma was then applied using the same standardized blunt chest model. This group was designed to evaluate the potential protective effects of CBD against trauma-induced systemic and reproductive tissue injury.

4. CBD Group (Cannabidiol only, no Trauma): Rats received CBD (5 mg/kg, i.p., 0.1 mL vehicle) under anesthesia. No blunt chest trauma was applied. This group controlled for the direct effects of CBD on tissues in the absence of trauma.

At 48 h post-trauma, all animals were euthanized under deep anesthesia induced by intraperitoneal injection of ketamine (80 mg/kg; Ketalar, Pfizer, Türkiye) and xylazine (10 mg/kg; Xylazinbio 2%, Bioveta, Czech Republic). Genital tissues, including the ovaries, fallopian tubes, and uterus, were collected, and evaluated (Fig. 2)15.

Fig. 2
Fig. 2
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Experimental design of blunt chest trauma and cannabidiol (CBD) treatment in rats. Adult rats were randomly assigned to four groups: Sham, Trauma, Trauma + CBD, and CBD. Blunt chest trauma was induced under anesthesia by dropping a 200 g cylindrical weight from a height of 1 m onto the thoracic wall. CBD (5 mg/kg, i.p.) was administered 30 min prior to trauma in the Trauma + CBD group, and to non-traumatized rats in the CBD group. Forty-eight hours after trauma, animals were euthanized, and genital tissues (ovaries, uterus, fallopian tubes) were collected. Histopathological and immunohistochemical analyses were performed to evaluate estrogen receptor (ER), hypoxia-inducible factor-1α (HIF-1α), and oxytocin receptor (OTR) expression.

Histopathological examinations

Genital system tissues were harvested within 10 min following euthanasia and immediately fixed in 10% neutral buffered formalin for 48 h at room temperature. Small relieving incisions were made to facilitate fixation. After fixation, routine histological processing was performed using a fully automated tissue processor (Leica ASP300S; Leica Microsystems, Wetzlar, Germany). Tissues were then embedded in paraffin blocks. Paraffin-embedded tissues were sectioned at a thickness of 5 μm using a rotary microtome (Leica RM2155; Leica Microsystems, Wetzlar, Germany). For each organ and each animal, three nonconsecutive sections were obtained and mounted on glass slides. All slides were stained simultaneously in the same batch to minimize inter-batch variability. Hematoxylin–eosin (H&E) staining was applied to all sections. Histological evaluations were performed using a light microscope (Olympus BX51; Olympus Corporation, Tokyo, Japan) equipped with 4 × , 10 × , 20 × , and 40 × objective lenses. Observations were conducted under appropriate magnifications, and digital images were captured using an integrated Olympus DP72 camera system. All tissues were examined as whole-organ cross-sections taken from standardized anatomical regions of the ovaries, fallopian tubes, and uterus to ensure uniformity across samples. Histopathological alterations were evaluated by an experienced histopathologist who was blinded to the experimental groups. Lesions were graded using a semiquantitative scoring system based on hyperemia, edema, hemorrhage, inflammatory cell infiltration, degeneration, and epithelial loss. Each parameter was scored from 0 (no lesion) to 3 (severe lesion) according to the extent and severity of the histopathological changes15.

Immunohistochemical examinations

Sections obtained from genital tract tissues were mounted on poly-L-lysine–coated slides and subjected to immunohistochemical staining for ER-α, HIF-1α, and OTR. All primary antibodies used in this study, together with their host species, clonality, catalogue numbers, dilution ratios, documented reactivity for rat tissues, and the corresponding positive and negative control tissues, are summarized in Table 1. After routine deparaffinization and rehydration, sections were incubated with the respective primary antibodies for 60 min at room temperature. Subsequently, slides were treated with biotinylated secondary antibodies and a streptavidin–alkaline phosphatase conjugate. Immunoreactivity was visualized using the EXPOSE Mouse and Rabbit Specific HRP/DAB Detection IHC kit (ab80436, Abcam, UK), with diaminobenzidine (DAB) as the chromogen. Rat uterus tissue known to constitutively express ER-α and OTR, and rat kidney tissue for HIF-1α, were used as positive controls. For negative controls, primary antibodies were omitted and replaced with phosphate-buffered saline.

Table 1 Antibody sources and positive and negative controls used for immunohistochemical analysis.
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All evaluations were performed in a blinded manner. For each marker, the percentage of positively stained cells was determined by counting 20 cells in five randomly selected high-power fields (× 40 magnification), totaling 100 cells per sample15. Microphotographs were analyzed using ImageJ software (version 1.48, National Institutes of Health, Bethesda, MD, USA), and morphometric analyses were conducted with the CellSens Life Science Imaging Software (Olympus Corporation, Tokyo, Japan). The resulting data were subsequently subjected to statistical analysis.

Statistical analysis

All statistical analyses were performed using GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA, USA). The Shapiro–Wilk test was applied to assess the normality of data distribution. For data that met the assumption of normality (p > 0.05), intergroup comparisons were conducted using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test as the post hoc test. For data that did not conform to a normal distribution, the Kruskal–Wallis test was used, followed by Dunn’s multiple comparisons test for post hoc analysis. All results were expressed as mean ± standard deviation (SD), and a p < 0.05 was considered statistically significant.

Results

Histopathological findings

Histopathological evaluation demonstrated group-specific differences. In the Sham and CBD groups, ovarian follicles, fallopian tube ciliated epithelium, and uterine endometrial architecture were preserved, exhibiting normal morphology without detectable pathological alterations. In the Trauma (TRA) group, tissue changes were observed, including vascular congestion, interstitial edema, and inflammatory cell infiltration compared with Sham controls (p < 0.001). Structural alterations included disrupted ovarian follicular morphology, epithelial detachment and ciliary loss in fallopian tubes, and glandular degeneration with focal hemorrhage in the uterus (p < 0.001).

Co-administration of CBD (TRA + CBD group) mitigated these tissue alterations, with ovarian stroma, fallopian tube epithelium, and uterine endometrium appearing more similar to Sham controls (p < 0.001). Immunohistochemical analyses revealed parallel patterns: TRA animals exhibited increased expression of HIF-1α and OTR, along with reduced ER immunoreactivity, while CBD treatment in the TRA + CBD group attenuated HIF-1α and OTR expression and preserved ER levels. Semi-quantitative scoring confirmed these observations (Fig. 3, Table 2).

Fig. 3
Fig. 3
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Histopathological evaluation of genital tissues across experimental groups. Representative hematoxylin–eosin–stained sections of the ovaries (upper row), fallopian tubes (middle row), and uterus (lower row) are shown. (A) Sham group: preserved tissue architecture. (B) TRA group: vascular congestion, inflammatory cell infiltration, epithelial degeneration, ciliary loss in fallopian tubes, and endometrial alterations. (C) TRA + CBD group: attenuation of TRA-induced histopathological changes, with tissue morphology approaching that of Sham controls. (D) CBD group: tissue morphology comparable to Sham controls. Scale bar = 20 μm. Lower panels show semi-quantitative histopathological scores and immunohistochemical expression levels for ovaries, fallopian tubes, and uterus. Data are presented as mean ± standard deviation (SD; n = 8). Statistical significance: ***p < 0.001 versus Sham; ###p < 0.001 versus TRA.

Table 2 Statistical analysis results of histopathological scores of ovarian, fallopian and uterine tissues.
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Immunohistochemical findings in ovary

Immunohistochemical analyses revealed group-specific differences in ovarian expression of ER, HIF-1α, and OTR. In the Sham group, ovarian follicles and stromal cells exhibited moderate ER and OTR expression and low HIF-1α immunoreactivity, consistent with baseline physiological conditions. The CBD group showed similar immunoreactivity to Sham controls, indicating preserved receptor expression.

In contrast, the TRA group showed reduced ER expression, particularly in granulosa and stromal compartments, and increased OTR and HIF-1α immunopositivity (p < 0.001 versus Sham), reflecting trauma-associated hypoxic and inflammatory responses. These changes were accompanied by morphological alterations, including follicular disruption and stromal disorganization.

Co-administration of CBD in the TRA + CBD group restored ER immunoreactivity and significantly attenuated TRA-induced upregulation of HIF-1α and OTR (p < 0.001 versus TRA) (Fig. 4, Table 3). These results indicate that CBD treatment mitigated trauma-associated ovarian hypoxia and stress signaling and preserved estrogen receptor expression, reflecting maintenance of receptor-mediated tissue function in reproductive organs.

Fig. 4
Fig. 4
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Immunohistochemical evaluation of ovarian estrogen receptor, HIF-1α, and OTR expression across experimental groups. Representative ovarian sections show (A) Control group with normal immunostaining; (B) TRA group with decreased ER and intense HIF-1α/ OTR immunopositivity (arrows); (C) TRA + CBD group with restored ER and attenuated HIF-1α/ OTR expression; and (D) CBD group with staining patterns similar to Control. Scale bar = 20 μm. Lower panels: Quantitative immunoexpression analysis of ER, HIF-1α, and OTR. Data are expressed as mean ± SD (n = 8). ***p < 0.001, **p < 0.01.

Table 3 Results of statistical analysis of immunohistochemical scores for ER, HIF-1α, and OTR expression in ovarian tissue.
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Immunohistochemical findings in fallopian tubes

Immunohistochemical evaluation revealed distinct alterations in the expression of ER, HIF-1α, and OTR in the fallopian tubes across the groups. In the control group, epithelial and stromal layers showed strong ER and moderate OTR immunoreactivity with weak HIF-1α staining, reflecting normal physiological activity. The CBD group exhibited similar expression patterns, indicating no adverse impact of CBD alone. In contrast, the TRA group demonstrated significant pathological changes. ER immunoreactivity was markedly reduced in the tubal epithelium, while both HIF-1α and OTR expression levels were strongly elevated (p < 0.001 vs. Control). Prominent epithelial disruption and stromal disorganization were also evident (arrows). Notably, the TRA + CBD group displayed partial restoration of ER expression and a significant reduction in TRA-induced HIF-1α and oxytocin upregulation (p < 0.001 vs. TRA) (Fig. 5, Table 4).

Fig. 5
Fig. 5
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Immunohistochemical evaluation of ER, HIF-1α, and OTR expression in fallopian tubes. Representative images show: (A) Sham group: moderate ER and OTR staining, low HIF-1α expression. (B) TRA group: reduced ER expression and increased HIF-1α and OTR immunoreactivity (arrows). (C) TRA + CBD group: restored ER immunoreactivity and decreased HIF-1α and OTR expression. (D) CBD group: staining patterns similar to Sham controls. Scale bar = 20 μm. Lower panels show quantitative analysis of ER, HIF-1α, and OTR immunoexpression. Data are presented as mean ± SD (n = 8). Statistical significance: ***p < 0.001, **p < 0.01 versus Sham.

Table 4 Results of statistical analysis of immunohistochemical scores for ER, HIF-1α, and OTR expression in fallopian tissue.
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Immunohistochemical findings in uterus

Immunohistochemical analyses of the uterus revealed group-specific differences in ER, HIF-1α, and OTR expression. In the Sham group, endometrial glands and stromal cells exhibited moderate to strong ER staining, low HIF-1α, and moderate OTR immunoreactivity, consistent with baseline uterine physiology. The CBD group showed a similar staining pattern, indicating no detectable effect of CBD alone. In contrast, the TRA group showed reduced ER expression in both glandular and stromal compartments, accompanied by increased HIF-1α and OTR immunoreactivity (p < 0.001 versus Sham). Morphological changes included epithelial disorganization, glandular dilation, and stromal degeneration, with elevated HIF-1α. Co-administration of CBD (TRA + CBD group) partially restored ER expression and significantly attenuated TRA-induced upregulation of HIF-1α and OTR (p < 0.001 versus TRA) (Fig. 6, Table 5).

Fig. 6
Fig. 6
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Immunohistochemical evaluation of ER, HIF-1α, and OTR expression in the uterus. Representative photomicrographs show: (A) Sham group: moderate to strong ER and moderate OTR staining, low HIF-1α expression. (B) TRA group: reduced ER and increased HIF-1α and OTR immunoreactivity (arrows). (C) TRA + CBD group: partial restoration of ER and attenuation of HIF-1α and OTR expression. (D) CBD group: staining patterns comparable to Sham controls. Scale bar = 20 μm. Lower panels show quantitative analysis of ER, HIF-1α, and OTR immunoexpression in uterine tissues. Data are presented as mean ± SD (n = 8). Statistical significance: ***p < 0.001, **p < 0.01, *p < 0.05 versus Sham.

Table 5 Results of statistical analysis of immunohistochemical scores for ER, HIF-1α, and OTR expression in uterine tissue.
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These data demonstrate that CBD mitigates TRA-induced uterine hypoxia and inflammatory signaling and restores ER expression in uterine tissues.

Discussion

Trauma-induced lung injury is recognized as a systemic disorder rather than an isolated pulmonary event. Beyond its local manifestations, it can provoke a widespread inflammatory response with far-reaching physiological consequences16,17. Supporting this concept, Rowe et al. (2024) demonstrated that extracranial injury triggers broad cytokine-driven transcriptional changes in distant organs, including the brain, mediated through circulating inflammatory mediators18. These findings underscore that localized trauma can act as a systemic inflammatory stimulus capable of perturbing remote tissue homeostasis.

The excessive release of circulating pro-inflammatory cytokines and mitochondrial damage-associated molecular patterns (DAMPs) could plausibly propagate through the systemic circulation, contributing to endothelial dysfunction, vascular leakage, and secondary hypoxic stress in distal organs19,20. Given their dense vascularization and hormone receptor dependency, reproductive organs may be particularly susceptible to this inflammatory-hypoxic milieu, potentially predisposing them to receptor dysregulation and structural degeneration. This framework offers a biologically plausible explanation for the histopathological and immunohistochemical alterations observed in the trauma group.

HIF-1α is a pivotal transcription factor coordinating cellular responses to hypoxia and metabolic stress21. Increased HIF-1α expression under hypoxic conditions has been associated with enhanced inflammatory and apoptotic signaling in hypoxic tissues. Consistent with prior experimental observations, Kletkiewicz et al. (2024) reported that CBD suppresses HIF-1α accumulation in rodent models of global hypoxia, thereby attenuating downstream oxidative and inflammatory responses22. In our study, CBD administration was associated with reduced HIF-1α immunoreactivity in reproductive tissues. Although the underlying molecular mediators were not directly quantified, this observation supports the hypothesis that CBD may mitigate trauma-associated hypoxic stress and contribute to preservation of OTR expression and tissue integrity.

The potential involvement of mitochondrial and redox-sensitive pathways, including the SIRT-1/PGC-1α axis, therefore remains speculative in the present model. Nevertheless, based on existing literature23,24. CBD-induced modulation of these pathways could plausibly enhance mitochondrial resilience, limit reactive oxygen species generation, and indirectly support ER homeostasis, thereby contributing to cellular survival under hypoxic–inflammatory conditions.

Systemic cytokine release following trauma may further exacerbate tissue injury by disrupting vascular homeostasis. Li et al., (2024) comprehensively reviewed the role of pro-inflammatory cytokines in driving trauma-associated microcirculatory dysfunction and adverse outcomes25. In this context, the cytokine surge induced by blunt chest trauma may impair endothelial barrier function and tissue perfusion within the genital microvasculature, thereby promoting localized hypoxia and structural degeneration. The observed protective associations with CBD treatment may therefore reflect, at least in part, attenuation of systemic inflammatory burden and secondary vascular compromise.

Previous studies have proposed that CBD modulates trauma-associated inflammation through mitochondria-centered mechanisms involving the SIRT-1/PGC-1α axis NF-κB–dependent signaling pathways26, as well as through protective effects in cardiac and other systemic injury models27.While these pathways were not directly measured in the present study, they provide a biologically coherent mechanistic framework that supports the hypothesis that CBD may function as a systemic homeostatic modulator, potentially mitigating both primary pulmonary injury and secondary distal organ vulnerability following severe thoracic trauma.

Limitations

Despite the strengths of the experimental design and the consistency of the histopathological and immunohistochemical findings, several limitations of this study should be acknowledged. First, systemic inflammatory mediators such as circulating cytokines (e.g., TNF-α, IL-1β, IL-6) and endothelial dysfunction markers were not directly measured. As a result, the proposed link between pulmonary trauma–induced systemic inflammation and secondary reproductive tissue injury remains inferential rather than mechanistically confirmed. Second, the analysis was limited to a single post-trauma time point (48 h), which precludes evaluation of the temporal progression of injury, recovery dynamics, or potential delayed effects of CBD treatment. Third, only a single prophylactic dose and administration schedule of CBD was investigated. Therefore, conclusions regarding dose dependency, post-trauma therapeutic efficacy, and optimal treatment windows cannot be drawn.

Importantly, functional reproductive outcomes, such as fertility indices, estrous cyclicity, or hormonal serum levels, were not assessed. Consequently, the observed histological and receptor-level changes cannot be directly translated into functional reproductive implications. In addition, the estrous cycle stage of the female rats was not synchronized or recorded at the time of tissue collection. Although animals were randomly allocated and this variable was assumed to be evenly distributed across groups, hormonal fluctuations related to the estrous cycle may have influenced ER and OTR expression patterns. Finally, the study was conducted in a controlled experimental animal model of blunt thoracic trauma. While this model allows mechanistic insight, extrapolation of the findings to clinical trauma settings or human reproductive physiology should be made with caution. Future studies incorporating systemic inflammatory profiling, multiple time points, functional reproductive endpoints, and clinically relevant treatment paradigms are warranted.

Conclusions

In conclusion, the present demonstrates that blunt thoracic trauma is associated with histopathological alterations and changes in hypoxia- and hormone receptor–related immunoreactivity in female reproductive tissues. In this experimental model, cannabidiol administration was associated with attenuation of trauma-related tissue alterations, preservation of estrogen receptor expression, and reduced HIF-1α and oxytocin receptor immunoreactivity. These findings suggest that CBD may modulate secondary tissue responses following thoracic trauma, potentially through indirect effects on systemic inflammatory and hypoxic signaling pathways. However, the results do not establish a direct mechanistic link or therapeutic efficacy, and causal interpretations should be avoided. Overall, this study provides preliminary preclinical evidence supporting a potential modulatory role of CBD in trauma-associated secondary organ alterations. Further investigations incorporating molecular validation, functional reproductive outcomes, and clinically relevant dosing strategies are required before any translational or therapeutic implications can be considered.