Specifically, Mostafa et al.27 reported that PIRA at 300 mg/kg significantly attenuated CP-induced hepatotoxicity in rats using an identical CP dosing regimen (200 mg/kg, single i.p.
The remaining four rats per group provided additional serum and lung tissue, which were stored at − 80 °C for potential future analyses.
Lung tissue collectionThe superior lobe of the right lung from each rat was excised and preserved in 10% neutral-buffered formalin for histological investigation and immunohistochemistry examination.
Evaluation of biochemical parametersTotal protein concentration in BALFTotal protein concentration in BALF was determined using the BCA Protein Colorimetric Assay Kit (E-BC-K318-M, Elabscience, USA) following the manufacturer’s instructions.
This assay quantifies caspase-1 protein concentration via antibody-based recognition and does not assess its proteolytic (enzymatic) activity.
Medications and chemical agents
Cyclophosphamide (CP) was procured from Baxter Company, Germany, and was freshly dissolved in sterile normal saline (0.9% NaCl) immediately before each intraperitoneal injection, at a concentration adjusted to deliver 200 mg/kg in an injection volume not exceeding 1 mL/100 g body weight. Piracetam (PIRA) was acquired from Amoun Pharmaceutical Company, Egypt, and was freshly dissolved in distilled water immediately before oral gavage, at a concentration adjusted to deliver 300 mg/kg/day in a gavage volume not exceeding 1 mL/100 g body weight. The additional chemical reagents used in this study were all of the best grade that could be purchased for scientific research purposes.
Molecular docking
The crystallographic structures of five target proteins (NF-κB p65, NLRP3, caspase 1, caspase 3, and AMPK) were retrieved from the Protein Data Bank (PDB) repository. The three-dimensional molecular structure of piracetam was obtained from the PubChem database. Structural preparation of all protein targets was conducted through the removal of water molecules, cofactors, co-crystallized ligands, and other small organic molecules. Then, the protein structures and piracetam ligand were imported into the AutoDock. Structural preparation involved the addition of polar hydrogen atoms and assignment of Kollman atomic charges alongside atomic solvation parameters. Rotatable bonds within the piracetam structure were identified, and gasteiger energy was calculated. Both proteins and ligand were converted to the PDBQT format required for docking calculations. Grid box dimensions were optimized to encompass the entirety of the co-crystallized ligand binding site. The Lamarckian genetic algorithm was employed as the search methodology, with all computational parameters maintained at their default values. Following the docking process, the protein-ligand complex configuration demonstrating the most favorable binding affinity (lowest binding energy) was selected for subsequent analysis. These molecular interactions were visualized and analyzed using Discovery Studio Visualizer software to identify critical binding determinants.
Animals
A total of 40 male Wistar albino rats (7–9 weeks old and weighed 180–200 g) were used in this study, divided equally into 4 groups of 10 rats each; of these, 6 rats per group (n = 6/group; total n = 24) were included in the biochemical, functional, and histopathological analyses reported in this study. The remaining 4 rats per group were treated under the identical protocol but were not included in the present analyses; their serum and lung tissue were retained at − 80 °C for potential future separate investigations. We sourced our rats from Elnahda University. To help the rats adjust to their new environment, we provided them with free access to water and food pellets for one week prior to the experiment. In a controlled setting, the rats were kept under a 12-hour light/dark cycle, with a temperature of 25 ± 2 °C and a humidity level of 45 ± 5%34. Compliance with the ARRIVE standards was maintained during all animal experiments included in this study. Furthermore, the animal protocols in this work adhered to the NIH Guidelines for the use of animals. They were approved by the Institutional Ethical Committee Deraya Center for Scientific Research, Deraya University, Egypt (permission number: DCSR-04025-52).
Methodology of treatment allocation
Animals were randomly divided into four groups, with each group comprising ten rats at the time of treatment; of these, six animals per group constituted the effective sample size for the analyses reported in this study.
Control group:
Animals were administered normal saline for 15 days.
PIRA 300 group:
Animals received PIRA (300 mg/kg/day; p.o.) for 15 days.
CP group:
Animals were administered a single dosage of CP (200 mg/kg; i.p.) on the 12th day of the experiment.
CP+PIRA 300 group:
Animals were given PIRA (300 mg/kg/day; orally) for 15 days, concurrently receiving a single dosage of CP (200 mg/kg; intraperitoneally) on the 12th day of the experiment.
The dosage of PIRA (300 mg/kg/day; orally) for 15 days was selected based on previous studies demonstrating its pharmacological efficacy without observed toxicity in rat models. Specifically, Mostafa et al.27 reported that PIRA at 300 mg/kg significantly attenuated CP-induced hepatotoxicity in rats using an identical CP dosing regimen (200 mg/kg, single i.p. injection on day 15) to that adopted here, supporting both the efficacy and tolerability of this dose in the context of CP co-administration. Furthermore, El-Dessouki et al.30 demonstrated that PIRA (at comparable doses) exerted nephroprotective effects against cisplatin-induced toxicity through AMPK-related signaling modulation, mechanistically supporting its relevance to the AMPK pathway investigated in the current study. The administered CP dose (200 mg/kg, single i.p. injection) has been consistently shown to reliably induce pulmonary oxidative stress, inflammation, and histopathological injury in rats without excessive mortality, as validated by Saghir et al.20 and Şengül et al.5 in independent CP-induced lung toxicity models.
Sample allocation across outcome measures
All 40 rats (10 per group) survived to the end of the 15-day experimental period. Of these, 6 rats per group (n = 6/group) constituted the effective sample size for the biochemical, functional, and histopathological analyses reported in this study. Six rats per group were randomly designated for arterial blood gas analysis, BALF total protein quantification, lung oxidative stress markers (MDA, GSH, CAT), inflammatory cytokines (TNF-α, IL-18, IL-1β), caspase-1, Bax and Bcl-2 gene expression, and histopathological scoring (Table 2), since these assays required only limited aliquots of lung tissue or homogenate. Owing to the larger amount of high-quality protein lysate and tissue required for Western blotting and immunohistochemistry, a further random subset of three rats per group, drawn from the same six, was used for these two techniques. The remaining four rats per group provided additional serum and lung tissue, which were stored at − 80 °C for potential future analyses. This allocation is reflected in the sample size (n) reported in each figure legend and table.
Specimen collection
Arterial blood collection
At the end of the experiment, Rats were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) intraperitoneally, which were dissolved in distilled water35. Three hundred microliters of arterial blood were obtained from the carotid artery catheter port, which was secured with a heparinized dextrose solution, using a syringe in accordance with the vendor’s methodology. While still under the deep surgical plane of anesthesia induced by the ketamine/xylazine combination administered for blood collection, rats were then euthanized via decapitation, consistent with the institutionally approved animal care and use protocol (permission number: DCSR-04025-52). Blood gas analysis was promptly performed using an i-STAT VetScan Handheld Analyzer (Abaxis, Union City, California) after the collection of blood samples. The following parameters were assessed: The partial pressure of oxygen (PaO2) and the partial pressure of carbon dioxide (PaCO2) were measured to assess the state of acid-base balance36.
BALF collection
Bronchoalveolar lavage fluid (BALF) collection was performed via the left lung. Following a midline cervical incision, the trachea was exposed, and a sterile 18-gauge catheter was inserted and secured with a suture. BALF was obtained by instilling three sequential aliquots of 2 mL sterile, ice-cold phosphate-buffered saline (PBS, pH 7.4) into the lungs through the catheter. After each instillation, the fluid was gently aspirated back into sterile tubes. The recovered lavage fluids were pooled per animal, yielding approximately 5–6 mL of BALF, with a recovery rate exceeding 90%. Collected BALF samples were immediately placed on ice and centrifuged at 1500 × g for 10 min at 4 °C to pellet the cells. The supernatant was carefully collected for subsequent biochemical assays.
Lung tissue collection
The superior lobe of the right lung from each rat was excised and preserved in 10% neutral-buffered formalin for histological investigation and immunohistochemistry examination. The right lung’s lower lobe was excised and swiftly divided into two halves. One segment of the lower lobe was allocated for biochemical tests, while the remaining segment was designated for Western blot analysis.
Evaluation of lung wet-to-dry weight ratio
To ascertain the “wet” weight, the middle lobe of the right lung of rats was excised and weighed. A consistent “dry” weight was achieved by placing the lung tissues in an oven at 80 °C for 48 h. The pulmonary edema was assessed by calculating the lung wet-to-dry ratios.
Evaluation of biochemical parameters
Total protein concentration in BALF
Total protein concentration in BALF was determined using the BCA Protein Colorimetric Assay Kit (E-BC-K318-M, Elabscience, USA) following the manufacturer’s instructions. This assay is based on the bicinchoninic acid method, which measures protein-induced reduction of Cu²⁺ to Cu¹⁺, forming a purple complex quantifiable by absorbance at 562 nm.
Evaluation of lung content of MDA, GSH, and CAT
We followed the manufacturer’s protocols to determine the MDA (MD 25 29), GSH (GR 25 11), and CAT (CA 2517) in tissue homogenate using colorimetric kits (Bio Diagnostic, Egypt).
Evaluation of lung inflammatory mediators
Lung contents of TNF-α (E-EL-R2856), IL-18 (E-EL-R0567), and IL-1β (E-EL-R0012) were detected using ELISA kits (Elabscience, USA).
Evaluation of caspase-1
Lung caspase-1 level was quantified using a rat-specific sandwich ELISA kit (E-EL-R0371, Elabscience, USA) according to the manufacturer’s instructions. Briefly, samples and standards were added to pre-coated microplate wells and incubated with a biotinylated detection antibody specific for caspase-1, followed by an avidin-HRP conjugate; after substrate development, absorbance was read at 450 nm on a microplate reader, and caspase-1 concentration was calculated from a standard curve. This assay quantifies caspase-1 protein concentration via antibody-based recognition and does not assess its proteolytic (enzymatic) activity.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Lung tissue samples (100 mg) were homogenized in 1 mL of TRIzol reagent using a Branson Digital Sonifier ultrasonic cell homogenizer (SFX 550, USA) to extract total RNA. RNA yield was quantified, and sample purity was verified by the A260/A280 absorbance ratio; only RNA samples with a purity value exceeding 1.8 were carried forward for qRT-PCR analysis. Equal quantities of total RNA were reverse-transcribed into complementary DNA (cDNA) using the RevertAid First Strand cDNA Synthesis Kit (KI622, Thermo Fisher Scientific, USA). The resulting single-stranded cDNA was then amplified into double-stranded product by real-time PCR, using the same primer sets listed in Table 1. Amplification was performed with Thermo Scientific Maxima SYBR Green qPCR Master Mix (2X) (K0251, Thermo Fisher Scientific, USA) on a StepOne Real-Time PCR System (4369074, Applied Biosystems, Singapore)37. Primer sequences for the target genes are listed in Table 1. Using GAPDH as the internal reference gene, the 2-ΔΔ Ct technique was used to measure the levels of gene expression.
Table 1 The primer sequence. Full size table
Immunodetection by western blot
Lung tissues were homogenized in ice-cold SDS-PAGE sample buffer and denatured by boiling at 95 °C for 5 min. Protein concentration was determined using (Bradford assay kit). Equal amounts of total protein (40 µg/lane) were then separated on 12% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes via electrotransfer. Membranes were subsequently incubated with a blocking buffer with 5% non-fat dry milk in TBST for 1 h to prevent non-specific binding3. The following primary antibodies were used for detection: NLRP3 antibody (1:1000, ab263899, Abcam, UK); Rabbit Anti-P-AMPK alpha 1 (phospho T183) (1:1000, ab133448, Abcam, UK), Rabbit Anti-AMPK alpha 1 antibody (1:1000, ab32047, Abcam, UK). After primary antibody incubation, membranes were treated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (7074 S, Cell Signaling Technology, MA, USA). Immunoreactive bands were visualized and captured with the Chemiluminescence Azure 300 Bioanalytical Imaging System (Azure Biosystems, Dublin, CA, USA). Band densities were quantified using ImageJ software and normalized to β-actin as the loading control.
Immunohistochemical staining
Five-micrometer-thick sections were obtained from lung tissues of the various animal groups and subjected to immunohistochemical (IHC) analysis. Tissue sections were deparaffinized using xylene and rehydrated through a graded series of ethanol (99%) for 30 s at each concentration. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide (H₂O₂), followed by rinsing in phosphate-buffered saline (PBS). Then Sections were pre-treated in citrate buffer (pH 6.0) in a microwave for 20 min for antigen retrieval. Then, the slides were incubated in the humidity chamber at 4° C overnight with the caspase-3 (PA1-29157, Thermo Fisher Scientific Co., USA) and NF-κB p65 (bs-0465R, BiossAntibodies, China) primary antibodies. After that, the slides were incubated with biotinylated secondary antibody, followed by streptavidin-biotin for 30 min each. The slides were incubated with 3,3’-diaminobenzidine (DAB) for 4 min, then rinsed with distilled water before counterstaining with hematoxylin. Subsequently, the slides were dehydrated, mounted, and cover-slipped. Visualization was performed using a light microscope to evaluate the degree of positive immunoreactivity in lung tissues38.
Histological examination
Lung tissues were harvested from each animal and immediately fixed in 10% neutral-buffered formalin. The samples were then processed and stained using standard hematoxylin and eosin (H&E) techniques, followed by examination under a light microscope (Olympus, U.TV0.5XC-3)39. Lung injury scores were assessed in a blinded manner and determined based on four independent parameters: (i) Alveolar edema; (ii) Vascular congestion; (iii) infiltration of inflammatory cells; and (iv) thickened alveolar septum40.
Statistical analysis
All data were presented as mean ± standard deviation (SD). The Shapiro–Wilk test was used to assess data normality, a normal distribution exhibited p-value above 0.05. Given the normal distribution of the data, a one-way analysis of variance (ANOVA) was used to evaluate the findings, subsequently using the Tukey-Kramer test to assess the significant differences across groups. GraphPad Prism® was used to conduct statistical analysis. A p-value below 0.05 is considered suggestive of statistical significance. Moreover, for histopathological scoring data, which represents ordinal non-parametric data, the Kruskal-Wallis test was used to assess differences between groups, followed by Dunn’s multiple comparison test for pairwise comparisons.
