Esculetin Attenuates Doxorubicin-Induced Kidney Damage By Reducing Heat Shock Proteins Expression Levels

Öz

The effectiveness of Doxorobucin (DOX), a commonly used anti-cancer and immunosuppressive medication, is hindered by its potential for organ toxicity. Prolonged use of DOX is associated with severe hepatocellular toxicity. This study reveals fresh insights into the therapeutic impact of esculetin (E) on DOX-induced kidney cell damage. Esculetin demonstrates its remedial effects by modulating heat shock protein signaling pathways. In our research, we explored the impact of DOX and E on the expression of the 70 kDa HSP gene family, including Hspa1a, Hspa4, and Hspa5, which are small stress proteins in Rattus norvegicus. The study involved the assignment of five different groups (Control, DOX, E50 mg/kg, E100 mg/kg, DOX+ E50 mg/kg, and DOX+ E100 mg/kg). Subsequently, kidney tissues were collected from rats, and cDNA libraries were generated at the conclusion of the application process. The Real-Time PCR method was employed using these libraries to detect HSP70 genes. Analyses conducted on Hspa1a, Hspa4 and Hspa5 expression revealed a statistically significant increase in the DOX group compared to the control group. Additionally, the combination of DOX and esculetin demonstrates a reduction in the increase caused by DOX alone. The study suggests that esculetin could serve as a potential protective agent for shielding kidney tissue from oxidative damage and apoptosis.

Anahtar Kelimeler

• Nephrotoxicity
• Oxidative stress
• Doxorobucin
• Esculetin

Eskuletin, Isı Şoku Proteinlerinin İfade Düzeylerini Azaltarak Doksorubisinin Neden Olduğu Böbrek Hasarını Azaltıyor

Öz

Yaygın olarak kullanılan bir anti-kanser ve immünosupresif ilaç olan Doxorobucin'in (DOX) etkinliği, organ toksisitesi potansiyeli nedeniyle engellenmektedir. DOX'un uzun süreli kullanımı ciddi hepatoselüler toksisite ile ilişkilidir. Bu çalışma, eskuletin'in (E) DOX kaynaklı böbrek hücresi hasarı üzerindeki terapötik etkisine dair yeni bilgiler ortaya koymaktadır. Esculetin, ısı şoku protein sinyal yolaklarını modüle ederek iyileştirici etkilerini göstermektedir. Araştırmamızda, DOX ve E'nin Rattus norvegicus'ta küçük stres proteinleri olan Hspa1a, Hspa4 ve Hspa5 dahil olmak üzere 70 kDa HSP gen ailesinin ekspresyonu üzerindeki etkisini araştırdık. Çalışmada beş farklı grup (Kontrol, DOX, E50 mg/kg, E100 mg/kg, DOX+ E50 mg/kg ve DOX+ E100 mg/kg) oluşturulmuştur. Daha sonra sıçanlardan böbrek dokuları toplanmış ve uygulama sürecinin sonunda cDNA kütüphaneleri oluşturulmuştur. HSP70 genlerini tespit etmek için bu kütüphaneler kullanılarak Gerçek Zamanlı PCR yöntemi kullanılmıştır. Hspa1a, Hspa4 ve Hspa5 ekspresyonu üzerinde yapılan analizler, DOX grubunda kontrol grubuna kıyasla istatistiksel olarak anlamlı bir artış olduğunu ortaya koymuştur. Ek olarak, DOX ve eskuletin kombinasyonu, tek başına DOX'un neden olduğu artışta bir azalma olduğunu göstermektedir. Çalışma, eskuletin'in böbrek dokusunu oksidatif hasar ve apoptozdan korumak için potansiyel bir koruyucu ajan olarak hizmet edebileceğini göstermektedir.

Anahtar Kelimeler

• Doksorobusin
• Eskuletin
• Nefrotoksisite
• Oksidatif stres

Kaynakça

1. Albakova, Z., Siam, M. K. S., Sacitharan, P. K., Ziganshin, R. H., Ryazantsev, D. Y., & Sapozhnikov, A. M. (2021). Extracellular heat shock proteins and cancer: New perspectives. Translational Oncology, 14(2). https://doi.org/10.1016/j.tranon.2020.100995
2. Boopathy, L. R. A., Jacob-Tomas, S., Alecki, C., & Vera, M. (2022). Mechanisms tailoring the expression of heat shock proteins to proteostasis challenges. Journal of Biological Chemistry, 298(5). https://doi.org/10.1016/j.jbc.2022.101796.
3. Boussada, M., Hammami, I., Ben Ali, R., Ammar, A. B., Alves, M., Oliveira, P. F., Akacha, A. B., Abdelkarim, I. L., Zekri, S., & El May, M. V. (2022). In vivo exposure to a new 2-cyano-2-p-nitrophenyl-N-benzylthioamide decreases doxorubicin-triggered structural damages in the mature testis. Andrologia, 54(11). https://doi.org/10.1111/and.14634.
4. Ceylan, H., & Erdoğan, O. (2017). Cloning, expression, and characterization of human brain acetylcholinesterase in Escherichia coli using a SUMO fusion tag. Turkish Journal of Biology, 41(1), 77-87. https://doi.org/10.3906/biy-1602-83.
5. De Freitas, G. B., Penteado, L., Miranda, M. M., Filassi, J. R., Baracat, E. C., & Linhares, I. M. (2022). The circulating 70 kDa heat shock protein (HSPA1A) level is a potential biomarker for breast carcinoma and its progression. Scientific Reports, 12(1). https://doi.org/10.1038/s41598-022-17414-6
6. Desai, V. G., Herman, E. H., Moland, C. L., Branham, W. S., Lewis, S. M., Davis, K. J., George, N. I., Lee, T., Kerr, S., & Fuscoe, J. C. (2013). Development of doxorubicin-induced chronic cardiotoxicity in the B6C3F1 mouse model. Toxicology and Applied Pharmacology, 266(1), 109–121. https://doi.org/10.1016/j.taap.2012.10.025
7. Dos Santos, N. S., Gonçalves, D. R., Balbinot, B., & Visioli, F. (2023). Is GRP78 (Glucose-regulated protein 78) a prognostic biomarker in differents types of cancer? A systematic review and meta-analysis. Pathology-Research and Practice, 242. https://doi.org/10.1016/j.prp.2023.154301.
8. Dubrez, L., Causse, S., Borges Bonan, N., Dumétier, B., & Garrido, C. (2020). Heat-shock proteins: chaperoning DNA repair. Oncogene, 39(3), 516-529. https://doi.org/10.1038/s41388-019-1016-y.

9. Guo, H., Yi, J., Wang, F., Lei, T., & Du, H. (2023). Potential application of heat shock proteins as therapeutic targets in Parkinson's disease. Neurochemistry International, 162. https://doi.org/10.1016/j.neuint.2022.105453.
10. Hassanein, E. H., Sayed, A. M., Hussein, O. E., & Mahmoud, A. M. (2020). Coumarins as modulators of the Keap1/Nrf2/ARE signaling pathway. Oxidative Medicine and Cellular Longevity, 2020. https://doi.org/10.1155/2020/1675957
11. Hu, C., Yang, J., Qi, Z., Wu, H., Wang, B., Zou, F., Mei, H., Liu, J., Wang, W., & Liu, Q. (2022). Heat shock proteins: Biological functions, pathological roles, and therapeutic opportunities. MedComm, 3(3), e161. https://doi.org/10.1002/mco2.161
12. Julier, Z., Park, A. J., Briquez, P. S., & Martino, M. M. (2017). Promoting tissue regeneration by modulating the immune system. Acta Biomaterialia, 53, 13–28.
13. Kalmar, B., & Greensmith, L. (2009). Induction of heat shock proteins for protection against oxidative stress. Advanced Drug Delivery Reviews, 61(4), 310–318. https://doi.org/10.1016/j.actbio.2017.01.056.
14. Kirici, M., Kirici, M., Demir, Y., Beydemir, S., & Atamanalp, M. (2016). The effect of Al+3 and Hg+2 on glucose 6-phosphate dehydrogenase from capoeta umbla kidney. Applied Ecology and Environmental Research, 14(2), 253-264. http://dx.doi.org/10.15666/aeer/1402_253264.
15. Konstantinova, E. V., Chipigina, N. S., Shurdumova, M. H., Kovalenko, E. I., & Sapozhnikov, A. M. (2019). Heat Shock Protein 70 kDa as a Target for Diagnostics and Therapy of Cardiovascular and Cerebrovascular Diseases. Current Pharmaceutical Design, 25(6), 710–714. https://doi.org/10.2174/1381612825666190329123924.
16. Lan, Y., Wang, Y., Huang, K., & Zeng, Q. (2020). Heat Shock Protein 22 Attenuates Doxorubicin-Induced Cardiotoxicity via Regulating Inflammation and Apoptosis. Frontiers in Pharmacology, 11, 257. https://doi.org/10.3389/fphar.2020.00257.
17. Lang, B. J., Guerrero, M. E., Prince, T. L., Okusha, Y., Bonorino, C., & Calderwood, S. K. (2021). The functions and regulation of heat shock proteins; key orchestrators of proteostasis and the heat shock response. Archives of Toxicology, 95(6), 1943-1970. https://doi.org/10.1007/s00204-021-03070-8.
18. Lee, M. J., Chou, F. P., Tseng, T. H., Hsieh, M. H., Lin, M. C., & Wang, C. J. (2002). Hibiscus protocatechuic acid or esculetin can inhibit oxidative LDL induced by either copper ion or nitric oxide donor. Journal of Agricultural and Food Chemistry, 50(7), 2130–2136. https://doi.org/10.1021/jf011296a.
19. Liu, J. F., Chen, P. C., Ling, T. Y., & Hou, C. H. (2022). Hyperthermia increases HSP production in human PDMCs by stimulating ROS formation, p38 MAPK and Akt signaling, and increasing HSF1 activity. Stem Cell Research & Therapy, 13(1), 236. https://doi.org/10.1186/s13287-022-02885-1.
20. Liu, L., Zhang, X., Qian, B., Min, X., Gao, X., Li, C., Cheng, Y., & Huang, J. (2007). Over-expression of heat shock protein 27 attenuates doxorubicin-induced cardiac dysfunction in mice. European Journal of Heart Failure, 9(8),762–769. https://doi.org/10.1016/j.ejheart.2007.03.007.
21. Liu, P., Bao, H. Y., Jin, C. C., Zhou, J. C., Hua, F., Li, K., Lv, X. X., Cui, B., Hu, Z. W., & Zhang, X. W. (2019). Targeting Extracellular Heat Shock Protein 70 Ameliorates Doxorubicin-Induced Heart Failure Through Resolution of Toll-Like Receptor 2-Mediated Myocardial Inflammation. Journal of the American Heart Association, 8(20),e012338. https://doi.org/10.1161/JAHA.119.012338
22. Nguyen, H. C., Frisbee, J. C., & Singh, K. K. (2024). Different Mechanisms in Doxorubicin-Induced Cardiomyopathy: Impact of BRCA1 and BRCA2 Mutations. Hearts, 5(1), 54-74.
23. Oksala, N. K., Ekmekçi, F. G., Ozsoy, E., Kirankaya, S., Kokkola, T., Emecen, G., Lappalainen, J., Kaarniranta, K., & Atalay, M. (2014). Natural thermal adaptation increases heat shock protein levels and decreases oxidative stress. Redox Biology, 3, 25–28.
24. Omidi, A., Nazifi, S., Rasekh, M., & Zare, N. (2023). Heat-shock proteins, oxidative stress, and antioxidants in one-humped camels. Tropical Animal Health and Production, 56(1), 29.
25. Öztürk, N., Karaağaç, M. S., Yeşilkent, E. N., Isıyel, M., Tosun, H., Karataş, H., Ceylan, H., Demir, Y. (2024). Exploring Esculetin's Protective Role:Countering Doxorubicin-Induced Oxidative Stress in Rat Heart. Journal of Laboratory Animal Science and Practices, 4(1), 41-49.
26. Patel, U., Abernathy, J., Savani, B. N., Oluwole, O., Sengsayadeth, S., & Dholaria, B. (2022). CAR T cell therapy in solid tumors: A review of current clinical trials. EJHaem, 3, 24-31.
27. Rawat, P. S., Jaiswal, A., Khurana, A., Bhatti, J. S., & Navik, U. (2021). Doxorubicin-induced cardiotoxicity: An update on the molecular mechanism and novel therapeutic strategies for effective management. Biomedicine and Pharmacotherapy, 139, 111708.
28. Rehati, A., Abuduaini, B., Liang, Z., Chen, D., & He, F. (2023). Identification of heat shock protein family A member 5 (HSPA5) targets involved in nonalcoholic fatty liver disease. Genes and Immunity, 24(3), 124–129.
29. Shan, Q., Ma, F., Wei, J., Li, H., Ma, H., & Sun, P. (2020). Physiological functions of heat shock proteins. Current Protein and Peptide Science, 21(8), 751-760.
30. Shang, B. B., Chen, J., Wang, Z. G., & Liu, H. (2021). Significant correlation between HSPA4 and prognosis and immune regulation in hepatocellular carcinoma. PeerJ, 9, e12315.
31. Sojka, D. R., Hasterok, S., Vydra, N., Toma-Jonik, A., Wieczorek, A., Gogler-Pigłowska, A., & Scieglinska, D. (2021). Inhibition of the Heat Shock Protein A (HSPA) Family Potentiates the Anticancer Effects of Manumycin A. Cells, 10(6), 1418.
32. Szyller, J., & Bil-Lula, I. (2021). Heat Shock Proteins in Oxidative Stress and Ischemia/Reperfusion Injury and Benefits from Physical Exercises: A Review to the Current Knowledge. Oxidative Medicine and Cellular Longevity, 2021, 6678457.
33. Tian, Z., Yang, Y., Yang, Y., Zhang, F., Li, P., Wang, J., Yang, J., Zhang, P., Yao, W., & Wang, X. (2020). High cumulative doxorubicin dose for advanced soft tissue sarcoma. BMC cancer, 20(1), 1139.
34. Tien, Y. C., Liao, J. C., Chiu, C. S., Huang, T. H., Huang, C. Y., Chang, W. T., & Peng, W. H. (2011). Esculetin ameliorates carbon tetrachloride-mediated hepatic apoptosis in rats. International Journal of Molecular Sciences, 12(6), 4053–4067.
35. Toraman, A., Toraman, E., Özkaraca, M., & Budak, H. (2022). Increased nociceptive sensitivity is associated with periodontal inflammation and expression of chronic pain genes in gingival tissues of male rats. Chemico-Biological Interactions, 366, 110128.
36. Tukaj, S. (2020). Heat shock protein 70 as a double agent acting inside and outside the cell: insights into autoimmunity. International Journal of Molecular Sciences, 21(15), 5298.
37. Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B. C., Remm, M., & Rozen, S. G. (2012). Primer3—new capabilities and interfaces. Nucleic acids research, 40(15), e115-e115.
38. Van der Zanden, S. Y., Qiao, X., & Neefjes, J. (2021). New insights into the activities and toxicities of the old anticancer drug doxorubicin. The FEBS journal, 288(21), 6095-6111.
39. Vasques, M. T., Alves, M. A., Benetti, C., Aranha, A. C., Zezell, D. M., & Corrêa, L. (2013). Temperature measurement and Hsp47 immunoexpression in oral ulcers irradiated with defocused high-energy diode laser. Journal of Photochemistry and Photobiology. B, Biology, 118, 42–48.
40. Yesilkent, E. N., & Ceylan, H. (2022). Investigation of the multi-targeted protection potential of tannic acid against doxorubicin-induced kidney damage in rats. Chemico-Biological Interactions, 365, 110111.
41. Yin, B., Tang, S., Sun, J., Zhang, X., Xu, J., Di, L., Li, Z., Hu, Y., & Bao, E. (2018). Vitamin C and sodium bicarbonate enhance the antioxidant ability of H9C2 cells and induce HSPs to relieve heat stress. Cell Stress and Chaperones, 23(4), 735–748.