Protective Potential of Morin in Ifosfamide-Induced Lung Toxicity: Modulation of Oxidative Stress, Inflammation and Apoptosis Parameters

Öz

In this study, the protective effect of morin against lung toxicity induced by ifosfamide (IFO), a widely used drug in cancer treatment, was investigated. Thirty-five male Sprague Dawley rats were divided into five groups: Control, Morin (200 mg/kg), IFO and two different Morin doses (IFO + Morin 100 mg/kg and IFO + Morin 200 mg/kg). Rats were given morin 100 mg/kg or 200 mg/kg for 2 days and on the second day, IFO 500 mg/kg was administered as a single dose. Biochemical and molecular methods analyzed oxidative stress, inflammation, autophagy and apoptosis markers. According to the data obtained, IFO increased malondialdehyde (MDA) levels in lung tissue, while decreasing superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) activities and glutathione (GSH) levels. However, it was observed that morin administration decreased MDA levels and increased GSH levels and GPx, SOD, CAT activities. IFO administration inhibited the expression of B-cell lymphoma-2 (Bcl-2) and the nuclear factor erythroid 2-related factor 2 (Nrf-2) /heme oxygenase-1 (HO-1) pathway, while increasing levels of nuclear factor-kappa B (NF-κB), inducible nitric oxide synthase (iNOS), Bcl-2-associated x protein (Bax), and cysteine aspartate specific protease-3 (Caspase-3). However, Morin decreased NF-κB and iNOS levels and inhibited inflammation by activating the Nrf-2/HO-1 pathway and prevented apoptosis by decreasing Bax and Caspase-3 and increasing Bcl-2. It also caused a decrease in Beclin-1 levels. According to the findings, IFO by affecting various signaling pathways in lung tissue, causing cellular damage, while morin demonstrated protective qualities against this damage.

Anahtar Kelimeler

• Apoptosis
• Ifosfamide
• Inflammation
• Lung toxicity
• Morin
• Oxidative stress.

İfosfamid ile İndüklenen Akciğer Toksisitesinde Morin’in Koruyucu Potansiyeli: Oksidatif Stres, İnflamasyon ve Apoptoz Parametrelerinin Modülasyonu

Öz

Bu çalışmada, kanser tedavisinde yaygın olarak kullanılan bir ilaç olan İfosfamid (IFO)'in neden olduğu akciğer toksisitesine karşı morin’in koruyucu etkisi araştırıldı. Otuz beş erkek Sprague Dawley sıçanı beş gruba ayrıldı: Kontrol, Morin (200 mg/kg), IFO ve iki farklı Morin dozu (IFO + Morin 100 mg/kg ve IFO + Morin 200 mg/kg) uygulandı. Sıçanlara 2 gün boyunca Morin 100 mg/kg veya 200 mg/kg verildi ve ikinci gün IFO 500 mg/kg tek doz olarak uygulandı. Oksidatif stres, inflamasyon, otofaji ve apoptoz belirteçleri biyokimyasal ve moleküler yöntemlerle analiz edildi. Elde edilen verilere göre, IFO akciğer dokusunda malondialdehit (MDA) seviyelerini artırırken, süperoksit dismutaz (SOD), katalaz (KAT), glutatyon peroksidaz (GPx) aktiviteleri ve glutatyon (GSH) seviyelerini azalttı. Ancak morin uygulamasının MDA düzeyini azalttığı, GSH seviyeleri ve GPx, SOD, KAT aktivitelerini arttırdığı gözlemlendi. IFO uygulaması, B-hücreli lenfoma-2 (Bcl-2) ve nükleer faktör eritroid 2 ile ilişkili faktör 2 (Nrf-2) /hem oksijenaz-1 (HO-1) yolunun ekspresyonunu inhibe ederken, nükleer faktör-kappa B (NF-κB), indüklenebilir nitrik oksit sentaz (iNOS), Bcl-2 ile ilişkili x proteini (Bax) ve sistein aspartat spesifik proteaz-3 (Kaspaz-3) seviyelerini artırdı. Ancak morin NF-κB ve iNOS seviyelerini azalttı ve Nrf-2/HO-1 yolağını aktive ederek inflamasyonu inhibe etti ve Bax ve Kaspaz-3’ü azaltıp Bcl-2’yi artırarak apoptozu engelledi. Ayrıca Beklin-1 düzeylerinde azalmaya neden oldu. Bulgulara göre, IFO akciğer dokusunda çeşitli sinyal yollarını etkileyerek hücresel hasara neden olurken, morin bu hasara karşı koruyucu özellik gösterdi.

Anahtar Kelimeler

• Akciğer toksisitesi
• Apoptoz
• İfosfamid
• İnflamasyon
• Morin
• Oksidatif stres.

Kaynakça

1. 1.Özdemir S, Kucukler S, Çomaklı S, et al. The protective effect of Morin against ifosfamide-induced acute liver injury in rats associated with the inhibition of DNA damage and apoptosis. Drug Chem Toxicol. 2022;45(3):1308-1317.
2. 2.Kuzu M, Yıldırım S, Kandemir FM, et al. Protective effect of morin on doxorubicin-induced hepatorenal toxicity in rats. Chem Biol Interact. 2019;308:89-100.
3. 3.Beyoğlu D, Hamberg P, IJzerman NS, et al. New metabolic insights into the mechanism of ifosfamide encephalopathy. Biomedicine & Pharmacotherapy. 2025;182:117773.
4. 4.Idle JR, Beyoğlu D. Ifosfamide - History, efficacy, toxicity and encephalopathy. Pharmacol Ther. 2023;243:108366.
5. 5.Çelik H, Kucukler S, Çomaklı S, et al. Morin attenuates ifosfamide-induced neurotoxicity in rats via suppression of oxidative stress, neuroinflammation and neuronal apoptosis. Neurotoxicology. 2020;76:126-137.
6. 6.Patel JM. Metabolism and pulmonary toxicity of cyclophosphamide. Pharmacol Ther. 1990;47(1):137-146.
7. 7.Balaha MF, Alamer AA, Aldossari RM, et al. Amentoflavone Mitigates Cyclophosphamide-Induced Pulmonary Toxicity: Involvement of -SIRT-1/Nrf2/Keap1 Axis, JAK-2/STAT-3 Signaling, and Apoptosis. Medicina (B Aires). 2023;59(12):2119.
8. 8.Rahimi-Madiseh M, Lorigoini Z, Zamani-Gharaghoshi H, et al. Berberis vulgaris: Specifications and traditional uses. Iran J Basic Med Sci. 2017;20(5):569-587.

9. 9.Varışlı B, Caglayan C, Kandemir FM, et al. The impact of Nrf2/HO-1, caspase-3/Bax/Bcl2 and ATF6/IRE1/PERK/GRP78 signaling pathways in the ameliorative effects of morin against methotrexate-induced testicular toxicity in rats. Mol Biol Rep. 2022;49(10):9641-9649.
10. 10. Zan G, He H, Wang X, et al. Morin reactivates Nrf2 by targeting inhibition of keap1 to alleviate deoxynivalenol-induced intestinal oxidative damage. Int J Mol Sci. 2025;26(3):1086.
11. 11. Althagafy HS, Hassanein EHM. Morin mitigates 5-fluorouracil-induced nephrotoxicity by activating Nrf2/HO-1 and FXR, and suppressing ERK/VCAM-1 and NF-κB pathways. Int Immunopharmacol. 2025;148:114092.
12. 12. Ginis Z, Ozturk G, Albayrak A, et al. Protective effects of caffeic acid phenethyl ester on ifosfamide-induced central neurotoxicity in rats. Toxicol Ind Health. 2016;32(2):337-343.
13. 13. Yıldız MO, Çelik H, Caglayan C, et al. Neuromodulatory effects of hesperidin against sodium fluoride-induced neurotoxicity in rats: Involvement of neuroinflammation, endoplasmic reticulum stress, apoptosis and autophagy. Neurotoxicology. 2022;90:197-204.
14. 14. Sedlak J, Lindsay RH. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with ellman’s reagent. Anal Biochem. 1968;25:192-205.
15. 15. Aebi H. Catalase in vitro. In Methods in enzymology. 1984;105:121-126.
16. 16. Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem. 1988;34(3):497-500.
17. 17. Lawrence RA, Burk RF. Glutathione peroxidase activity in selenium-deficient rat liver. Biochem Biophys Res Commun. 1976;71(4):952-958.
18. 18. Placer ZA, Cushman LL, Johnson BC. Estimation of product of lipid peroxidation (malonyl dialdehyde) in biochemical systems. Anal Biochem. 1966;16(2):359-364.
19. 19. Lowry Oh, Rosebrough Nj, Farr Al, et al. Proteın measurement with the folin phenol reagent. JBC. 1951;193(1):265-275.
20. 20. Lee MY, Yoon SY, Kim KH, et al. Pulmonary toxicities of molecular targeted antineoplastic agents: a single-center 10-year experience. Korean J Intern Med. 2021;36(3):689-698.
21. 21. Dimopoulou I, Bamias A, Lyberopoulos P, et al. Pulmonary toxicity from novel antineoplastic agents. Annals Oncol. 2006;17(3):372-379.
22. 22. Sun JD, Liu Q, Ahluwalia D, et al. Comparison of hypoxia-activated prodrug evofosfamide (TH-302) and ifosfamide in preclinical non-small cell lung cancer models. Cancer Biol Ther. 2016;17(4):371-380.
23. 23. El-Gendy HF, Tahoun EA, Elfert AY, et al. Trial for decreasing ifosfamide-induced hematological toxicity, oxidative stress, inflammation, and hepatotoxicity by beetroot extract in male albino rats. Comp Clin Path. 2022;31(4):699-712.
24. 24. Cakmak F, Kucukler S, Gur C, et al. Morin provides therapeutic effect by attenuating oxidative stress, inflammation, endoplasmic reticulum stress, autophagy, apoptosis, and oxidative DNA damage in testicular toxicity caused by ifosfamide in rats. Iran J Basic Med Sci. 2023;26:1227-1236.
25. 25. Semis HS, Gur C, Ileriturk M, et al. Investigation of the anti-inflammatory effects of caffeic acid phenethyl ester in a model of λ-Carrageenan–induced paw edema in rats. Hum Exp Toxicol. 2021;40(12):721-738.
26. 26. Aksu EH, Kandemir FM, Küçükler S, et al. Improvement in colistin‐induced reproductive damage, apoptosis, and autophagy in testes via reducing oxidative stress by chrysin. J Biochem Mol Toxicol. 2018;32(11):e22201.
27. 27. Aksu EH, Kandemir FM, Yıldırım S, et al. Palliative effect of curcumin on doxorubicin‐induced testicular damage in male rats. J Biochem Mol Toxicol. 2019;33(10):e22384.
28. 28. Al-Khawalde AA marddyah A, Abukhalil MH, Althunibat OY, et al. Taxifolin mitigates cisplatin-induced testicular damage by reducing inflammation, oxidative stress, and apoptosis in mice. Tissue Cell. 2025;93:102767.
29. 29. Ozturk G, Ginis Z, Kurt SN, et al. Effect of alpha lipoic acid on ifosfamide-induced central neurotoxicity in rats. Int J Neurosci. 2014;124(2):110-116.
30. 30. Lowenberg D, Thorn CF, Desta Z, et al. PharmGKB summary. Pharmacogenet Genomics. 2014;24(2):133-138.
31. 31. Chen N, Aleksa K, Woodland C, et al. N ‐Acetylcysteine prevents ifosfamide‐induced nephrotoxicity in rats. Br J Pharmacol. 2008;153(7):1364-1372.
32. 32. Prahalathan P, Kumar S, Raja B. Morin attenuates blood pressure and oxidative stress in deoxycorticosterone acetate-salt hypertensive rats: A biochemical and histopathological evaluation. Metabolism. 2012;61(8):1087-1099.
33. 33. Han HY, Choi MS, Yoon S, et al. Investigation of ifosfamide toxicity induces common upstream regulator in liver and kidney. Int J Mol Sci. 2021;22(22):12201.
34. 34. Negm WA, El-Kadem AH, Hussein IA, et al. The mechanistic perspective of bilobetin protective effects against cisplatin-induced testicular toxicity: role of Nrf-2/Keap-1 signaling, inflammation, and apoptosis. Biomedicines. 2022;10(5):1134.
35. 35. Amanat S, Shal B, Kyoung Seo E, et al. Icariin attenuates cyclophosphamide-induced cystitis via down-regulation of NF-кB and up-regulation of Nrf-2/HO-1 signaling pathways in mice model. Int Immunopharmacol. 2022;106:108604.
36. 36. Kannan G, Paul BM, Thangaraj P. Stimulation, regulation, and inflammaging interventions of natural compounds on nuclear factor kappa B (NF-kB) pathway: a comprehensive review. Inflammopharmacology. 2025;33(1):145-162.
37. 37. Liu T, Zhang L, Joo D, et al. NF-κB signaling in inflammation. Signal Transduct Target Ther. 2017;2(1):17023.
38. 38. Şimşek H, Akaras N, Gür C, et al. Beneficial effects of Chrysin on Cadmium‐induced nephrotoxicity in rats: Modulating the levels of Nrf2/HO‐1, RAGE/NLRP3, and Caspase-3/Bax/Bcl-2 signaling pathways. Gene. 2023;875:147502.
39. 39. de Fatima Pinheiro Rangel G, Cajado AG, Falcao Pereira A, et al. Uroprotective effect of a protein isolated from seed of Morinda citrifolia (McLTP 1) on hemorrhagic cystitis induced by ifosfamide in mice. BioRxiv. 2023;2.
40. 40. Cai Z, Gao L, Hu K, et al. Parthenolide enhances the metronomic chemotherapy effect of cyclophosphamide in lung cancer by inhibiting the NF-kB signaling pathway. World J Clin Oncol. 2024;15(7):895-907.
41. 41. Bachewal P, Gundu C, Yerra VG, et al. Morin exerts neuroprotection via attenuation of ROS induced oxidative damage and neuroinflammation in experimental diabetic neuropathy. BioFactors. 2018;44(2):109-122.
42. 42. Han C, Zhu Y, Yang Z, et al. Protective effect of Polygonatum sibiricum against cadmium-induced testicular injury in mice through inhibiting oxidative stress and mitochondria-mediated apoptosis. J Ethnopharmacol. 2020;261:113060.
43. 43. Yang B, Johnson TS, Thomas GL, et al. A shift in the Bax/Bcl-2 balance may activate caspase-3 and modulate apoptosis in experimental glomerulonephritis. Kidney Int. 2002;62(4):1301-1313.
44. 44. Kızıl HE, Caglayan C, Darendelioğlu E, et al. Morin ameliorates methotrexate-induced hepatotoxicity via targeting Nrf2/HO-1 and Bax/Bcl2/Caspase-3 signaling pathways. Mol Biol Rep. 2023;50(4):3479-3488.
45. 45. Asadi M, Taghizadeh S, Kaviani E, et al. Caspase‐3: Structure, function, and biotechnological aspects. Biotechnol Appl Biochem. 2022;69(4):1633-1645.
46. 46. Öztürk AB, Şimşek H, Akaras N, et al. Effects of morin on the wnt, notch1/hes1, ki-67/3-nitrotyrosine and damage signaling pathways in rats subjected to experimental testicular ischemia/reperfusion. Bratisl Med J. 2025;1-20.
47. 47. Gencer S, Gür C, İleritürk M, et al. The ameliorative effect of carvacrol on sodium arsenite‐induced hepatotoxicity in rats: Possible role of Nrf2/HO‐1, RAGE/NLRP3, Bax/Bcl‐2/Caspase‐3, and Beclin‐1 pathways. J Biochem Mol Toxicol. 2024;38(10):e23863.
48. 48. Ciechomska IA, Goemans GC, Skepper JN, et al. Bcl-2 complexed with Beclin-1 maintains full anti-apoptotic function. Oncogene. 2009;28(21):2128-2141.
49. 49. Liu Z, Zeng Y, Li R, et al. Treatment of chronic obstructive pulmonary disease by traditional Chinese medicine Morin monomer regulated by autophagy. J Thorac Dis. 2024;16(9):6052-6063.