MORPHOLOGICAL CHANGES IN LUNG TISSUE AFTER STANDARD CHEMOTHERAPY WITH ADDITIONAL CORRECTION USING POMEGRANATE SEED OIL

SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 4 ISSUE 11 NOVEMBER 2025 ISSN: 2181-3337 | SCIENTISTS.UZ 39 MORPHOLOGICAL CHANGES IN LUNG TISSUE AFTER STANDARD CHEMOTHERAPY WITH ADDITIONAL CORRECTION USING POMEGRANATE SEED OIL M.R. Shomurodova Bukhara State Medical Institute https://doi.org/10.5281/zenodo.17744517 Abstract. This study is devoted to investigating the morphological and morphometric changes in lung tissue when standard chemotherapy is supplemented with correction using pomegranate seed oil. In the experimental model on white outbred rats, toxic alterations were induced by intravenous administration of the chemotherapeutic agent cisplatin. After chemotherapy, the lung tissue showed thickening of the alveolar walls, interstitial inflammation, degenerative changes around blood vessels, and dystrophic processes in the alveolar epithelium. Correction with pomegranate seed oil led to a significant reduction of these pathological processes, restoration of the integrity of alveolar structures, and improvement of microcirculatory parameters. Morphometric results confirmed the normalization of alveolar space diameter, interalveolar septum thickness, and vascular density. The obtained data indicate that pomegranate seed oil possesses antioxidant, anti-inflammatory, and reparative properties, suggesting its potential as an effective phytocorrector for reducing chemotherapy-induced lung damage. Keywords: lungs, chemotherapy, cisplatin, pomegranate seed oil, morphology, morphometry, phytocorrection. Introduction In modern oncological practice, cytostatic drugs such as cisplatin are widely used and belong to the group of highly effective antineoplastic agents. However, along with its therapeutic efficacy, cisplatin exerts a pronounced toxic effect on body tissues, particularly on the lung parenchyma, which represents a serious clinical problem. Prolonged or high-dose administration of cisplatin leads to enhanced oxidative stress, accumulation of free radicals, dystrophic changes in epithelial and endothelial cells, as well as the activation of inflammatory and fibrotic processes in the lung tissue. These alterations result in respiratory dysfunction, impaired gas exchange, and instability of overall homeostasis. In this regard, the development and scientific justification of phytocorrection methods aimed at reducing the toxic effects of chemotherapy while preserving its therapeutic efficacy are of great importance today. Pomegranate seed oil has significant practical potential in this context, as it contains polyphenols, phytosterols, tocopherols, conjugated linoleic acids, and other antioxidant compounds that can reduce oxidative tissue damage, suppress inflammation, and stimulate regeneration processes. From this perspective, studying the morphological and morphometric changes in lung tissue following standard chemotherapy with cisplatin, combined with correction using pomegranate seed oil, is of both scientific and clinical significance. The findings of this research will provide valuable information for reducing cisplatin-induced pulmonary toxicity, enhancing tissue defense mechanisms, and scientifically substantiating the efficacy of biocorrection SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 4 ISSUE 11 NOVEMBER 2025 ISSN: 2181-3337 | SCIENTISTS.UZ 40 approaches. Thus, the relevance of this study is determined by the necessity to expand the use of phytocomponents in the complex treatment of oncological diseases, mitigate the damaging effects of cisplatin on the lungs, and strengthen the body’s antioxidant defense system. Purpose of the research: To investigate the morphological and morphometric changes in lung tissue that occur under the toxic effects of cisplatin, used as a standard chemotherapy drug, when phytocorrection with pomegranate seed oil is performed. Materials and Methods: Within the framework of the study, laboratory rats were divided into 4 groups (n = 40): 1. Control group (n = 10): Healthy white outbred rats were used for comparison with experimental groups. 2. Main experimental Group 1 (n = 10): Breast cancer was induced in rats using the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). 3. Main experimental Group 2 (n = 10): Rats with induced breast cancer received cisplatin intravenously at a dose of 0.4 mg/kg. 4. Main experimental Group 4 (n = 10): Rats with induced breast cancer received cisplatin intravenously at a dose of 0.4 mg/kg, along with pomegranate seed oil administered intragastrically via a gastric metal probe at a volume of 0.7 ml daily for 21 days. All groups were formed simultaneously for the experiment. The laboratory animals (white outbred rats) were uniform in age, sex, weight, and feeding conditions. Experiments with laboratory animals were conducted in accordance with the special approval letters from the Ethics Committee of the Ministry of Health of the Republic of Uzbekistan (No. 4/1439 dated September 21 and Protocol No. 4 dated August 26, 2020). At the appropriate time, the experimental animals were euthanized by decapitation. After opening the abdominal cavity, lung tissue was isolated, and the lung width was measured using a millimeter ruler. Lung tissue samples from the selected animals served as biomaterial for histological examination. The size of the tissue fragments did not exceed 5 × 3 × 3 mm. Immediately after sampling, the fragments were placed in a fresh fixative, with the volume of the fixative being 25 times greater than the volume of the tissue fragments. For staining the lung tissue micropreparations, the most commonly used method, hematoxylin and eosin (H&E), was applied. Hematoxylin served as the main stain, coloring the cell nuclei, while eosin, an acidic dye, stained the cytoplasm of cells and some acellular structures. For layer staining, hematoxylin prepared according to the Ehrlich method was used. Eosin was prepared by dissolving 0.1 g in 100 ml of 96° ethanol. After successful staining, the tissue fragments were dehydrated in alcohol and then mounted with Canada balsam. Results and Conclusions In rats aged 9 and 18 months that received cisplatin, pronounced disturbances in substance metabolism, inflammatory changes, and tissue damage were observed in the lung tissue. These alterations are associated with the activation of metabolic imbalance and inflammatory cascades in the tissues caused by the drug. The height of the mesothelium of the connective tissue surrounding the lung root was measured at 3.81 ± 0.16 nm in 9-month-old rats and 4.02 ± 0.16 nm in 18-month-old rats. Signs of vacuolization in mesothelial cells, intensely stained nuclei, and occasional destruction of epithelial cells were noted. SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 4 ISSUE 11 NOVEMBER 2025 ISSN: 2181-3337 | SCIENTISTS.UZ 41 Figure 1. Microscopic appearance of the lung tissue of a 9-month-old white outbred rat after experimental induction of breast cancer and subsequent standard chemotherapy (cisplatin administration). Stained with hematoxylin-eosin, objective 20×10. 1. Inflammation in arterial vessels (vasculitis) 2. Increased number of leukocytes around the arteries 3. Atelectasis due to alveolar collapse 4. Emphysematous alveoli 5. Thickening of alveolar septa Figure 1A. Microscopic appearance of the lung tissue of a 9-month-old white outbred rat after experimental induction of breast cancer and standard chemotherapy (cisplatin administration). Stained with hematoxylin-eosin, objective 20×40. 1. Inflammation in arterial vessels (vasculitis). Around the arterial vessels, light-colored (almost white) leukocytes are clearly visible, indicating pronounced perivascular infiltration. Collagen fibers are stained dark violet, with fibrosis occupying 67.9% of the tissue area. In a 497.89 × 412.74 px field, 216 leukocytes were counted. SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 4 ISSUE 11 NOVEMBER 2025 ISSN: 2181-3337 | SCIENTISTS.UZ 42 These changes are associated with impaired trophic function of the mesothelium in response to cisplatin’s toxic effects. The thickness of the serous layer was reduced to 17.81 ± 0.96 nm in 9-month-old rats and 19.25 ± 0.75 nm in 18-month-old rats, below normal values. This condition develops as a result of microcirculation disturbances and impaired fluid exchange in the tissues. Severe structural alterations were observed in the alveoli: their walls were partially or completely lost, the number of collapsed (airless) alveoli increased, and in the surrounding interstitial tissue, there was a rise in lymphocytes and an intensification of fibrosis. Figure 2. Microscopic appearance of the lung tissue of an 18-month-old white outbred rat after experimental induction of breast cancer and standard chemotherapy (cisplatin administration). Stained with hematoxylin-eosin, objective 20×10. 1. Inflammation and fullness in arterial vessels (vasculitis). 2. Increased number of leukocytes in the bronchial epithelium (bronchatrophy). 3. Atelectasis due to alveolar collapse. 4. Reduction in bronchial diameter. These changes are explained by the nonspecific toxic effects of cisplatin on lung tissue, its antiproliferative properties, and its effect in enhancing autoimmune responses. In 9-month-old rats, the thickness of the alveolar septa in peripheral regions was 14.31 ± 0.71 nm, and in the central regions 12.56 ± 0.54 nm; in 18-month-old rats, it increased to 15.88 ± 0.75 nm peripherally and 14.19 ± 0.71 nm centrally. Alveolar collapse and atelectasis in the surrounding tissue were clearly pronounced, which limits gas exchange and leads to hypoxia. Figure 2A. Microscopic appearance of the lung tissue of an 18-month-old white outbred rat after experimental induction of breast cancer and standard chemotherapy (cisplatin administration). Stained with hematoxylin-eosin, objective 20×100. SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 4 ISSUE 11 NOVEMBER 2025 ISSN: 2181-3337 | SCIENTISTS.UZ 43 1. Atypical cells 2. Diapedetic hemorrhage Figure 2B. Microscopic appearance of the lung tissue of an 18-month-old white outbred rat after experimental induction of breast cancer and standard chemotherapy (cisplatin administration). Stained with hematoxylin-eosin, objective 20×100. In a field of 435.61 × 410.46 px, 109 leukocytes were counted. In the intramural triangular region, fat tissue infiltration and lipid accumulation were markedly increased. This is associated with cisplatin’s effect on lipid metabolism and the suppression of antioxidant system activity. Although the number of LALCs (lipid-associated lymphoid cells) did not change significantly, their area increased sharply due to the rise in the number of lipid-containing cells. LALCs were observed in 29% of cases in 9-month-old rats and 34% in 18-month-old rats. Their diameters were 27.06 ± 2.88 nm and 38.62 ± 1.92 nm, and their areas were 642.88 nm² in 9-month-old rats and 964.62 nm² in 18-month-old rats. These changes are related to the increase in lipid-associated antigens due to cisplatin and the consequent enhancement of autoimmune reactions. Around the LALCs, there was extensive lymphogicytic infiltration, indicating an increase in lymphocyte numbers. Figure 3. Microscopic appearance of the lung tissue of a 9-month-old white outbred rat after experimental induction of breast cancer and standard chemotherapy (cisplatin administration). Stained with hematoxylin-eosin, objective 20×10. 1. Accumulation of leukocytes in the interstitium SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 4 ISSUE 11 NOVEMBER 2025 ISSN: 2181-3337 | SCIENTISTS.UZ 44 2. Filling of the lung tissue with fibrous tissue Fibrosis in the adventitia of the bronchial wall, inflammatory elements, and signs of bronchial wall inflammation were more pronounced. These changes are associated with the activation of cytokines and endothelial damage in the tissues. The bronchial diameter was 813.56 ± 38.96 nm in 9-month-old rats and 887.25 ± 23.83 nm in 18-month-old rats, while the muscle layer thickness was 63.38 ± 1.75 nm and 69.19 ± 1.79 nm, respectively. In the muscle tissue, disturbances in substance metabolism, destruction of myocytes, and an increase in scattered leukocytes were observed, which are associated with toxic effects directed at mitotically active cells. Figure 3A. Microscopic appearance of the lung tissue of a 9-month-old white outbred rat after experimental induction of breast cancer and standard chemotherapy (cisplatin administration). Stained with hematoxylin-eosin, objective 20×100. 1. Anucleated (necrotic) cells 2. Layering of vessel walls due to edema In the blood vessels, endothelial swelling, perivascular infiltration, intimal edema, and occasional diapedetic microhemorrhages were observed. These microangiopathic changes are associated with the direct toxic effect of cisplatin on endothelial cells. The diameter of the arteries, increased due to inflammation, was 22.88 ± 1.83 nm in 9-month-old rats and 27.75 ± 2.83 nm in 18-month-old rats. Figure 4. Microscopic appearance of the lung tissue of an 18-month-old white outbred rat after experimental induction of breast cancer and standard chemotherapy (cisplatin administration). Stained with hematoxylin-eosin, objective 20×40. SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 4 ISSUE 11 NOVEMBER 2025 ISSN: 2181-3337 | SCIENTISTS.UZ 45 1. Thickening of the bronchial muscle layer 2. Filling of the bronchi with serous fluid 3. Increase in the number and area of lipid-containing cells (LALCs) These changes are associated with vasculopathic reactions and ischemic-oxidative tissue damage. In the terminal bronchioles, trophic disturbances of the epithelium, dense staining of cell nuclei, and signs of granular cytoplasmic dystrophy were observed. These alterations develop due to the mechanism of action of the drug affecting cell division. The amount of serous material in the mucous membrane was significantly reduced, and in some areas, the basal membrane structure was disrupted, with increased infiltration. These findings indicate the negative impact of cisplatin on lung tissue. The use of pomegranate seed oil as corrective therapy effectively reduced the toxic effects of cisplatin on lung tissue. Morphological analysis of the lungs in 9and 18-month-old rats in the experimental group revealed several positive changes. The mesothelium height was 3.74 ± 0.08 nm in 9-month-old rats and 3.94 ± 0.08 nm in 18-month-old rats, close to control values. Vacuolization in mesothelial cells was minimal, dense nuclear staining was rare, and epithelial cell necrosis was almost absent, indicating the positive effect of pomegranate seed oil in maintaining trophic function. The thickness of the serous layer was 18.3 ± 0.27 nm in 9-month-old rats and 20.6 ± 0.30 nm in 18-month-old rats, reflecting the stabilization of microcirculation and fluid exchange. Signs of improvement were also observed in alveolar structure: the alveolar septa in 9-month-old rats measured 12.2 ± 0.18 nm peripherally and 10.6 ± 0.13 nm centrally, while in 18-month-old rats, they were 14.8 ± 0.22 nm peripherally and 12.4 ± 0.20 nm centrally, with partial preservation of normal architecture. Alveolar collapse and atelectasis were markedly reduced, indicating improved gas exchange and reduced risk of hypoxia. In the interstitial tissue, lymphocytic infiltration and fibrosis were significantly decreased. The tissue structures around blood vessels were more uniform, and the vascular walls maintained their shape. The arterial diameter was 23.0 ± 0.65 nm in 9-month-old rats and 32.8 ± 0.80 nm in 18-month-old rats, with minimal vasculopathic changes observed. Figure 5. Microscopic appearance of the lung tissue of a 9-month-old white outbred rat after standard chemotherapy with the addition of pomegranate seed oil. Stained with hematoxylineosin, objective 20×40. 1. Slight reduction in alveolar septa thickness SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 4 ISSUE 11 NOVEMBER 2025 ISSN: 2181-3337 | SCIENTISTS.UZ 46 2. Decrease in inflammation around alveoli and in the interstitial tissue In the intramural triangular region, fat tissue infiltration decreased, and the amount of lipid deposits remained low. In the area of LALCs, the number of lipid-containing cells in 9-month-old rats had an average diameter of 20.0 ± 0.44 nm and an area of 490.3 ± 12.3 nm², while in 18month-old rats, the diameter and area were 33.5 ± 0.40 nm and 610.5 ± 14.9 nm², respectively. The diameter of medium-sized bronchi was 709.5 ± 15.0 nm in 9-month-old rats and 827.0 ± 17.6 nm in 18-month-old rats, and the thickness of the bronchial muscle layer was 46.1 ± 0.9 nm and 56.8 ± 1.2 nm, respectively. The integrity and architecture of the bronchial walls were relatively preserved. Myocytes in the muscle tissue were maintained, and scattered leukocyte elements were minimal. In the blood vessels, the endothelial cell structure was intact, and there were no signs of significant perivascular infiltration. Microcirculation remained stable. In the terminal bronchioles, the trophic state of epithelial cells was well preserved, and the nuclear and cytoplasmic structures appeared normal. The amount of serous material in the mucous membrane was close to normal, and the basal membrane structure was intact. Figure 5. Microscopic appearance of the lungs of 9and 18-month-old white outbred rats after conventional (standard) chemotherapy with pomegranate seed oil treatment. Stained with hematoxylin-eosin, objective 20×10. 1. LALC (lipid-associated lymphoid clusters) 2. Lipids within LALC In 9-month-old rats, the bronchial mesothelium thickness was normally 4.65 nm. In the experimental group, it decreased to 3.62 m (–22.2%), and after cisplantin administration, it dropped further to 3.05 nm (–34.4%), indicating increased dystrophic changes in the tissue. With thymolin treatment, the thickness partially recovered to 3.56 nm (–23.4%), while pomegranate seed oil restored it to 3.74 nm (–19.6%), approaching normal values. In 18-month-old rats, a similar trend was observed: normal thickness 5.16 nm, experimental 3.83 nm (–25.8%), postcisplatin 3.25 nm (–37.0%), thymolin 3.76 nm (–27.1%), and pomegranate oil 3.94 nm (–23.6%), demonstrating the superior trophic support of pomegranate seed oil for the epithelium. Conclusion. Serous layer thickness showed a similar pattern. 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