Alleviation of doxorubicin-induced nephrotoxicity in breast cancer mice by using combination of green tea and moringa: Focus on antioxidant, apoptosis, inflammation, and histopathological insights

Doxorubicin (DXR)-induced nephrotoxicity remains a major concern in cancer treatment and calls for potential prevention of kidney injury. This study aims to evaluating the nephroprotective potentials of green tea and moringa used as 1% and 2% water extracts in DXR‑induced kidney damage in female Balb/C mice with breast cancer. Thirty six female Balb/C mice were divided into six groups as follows: healthy control; 4T1 cells cancer-induced; healthy mice with DXR treatment; cancer-induced with DXR treatment; cancer-induced under DOX and treated with 1% green tea and moringa combination; cancer-induced under DOX and treated with 2% green tea and moringa combination. The variables of the experiment were body weight, tumor volume, antioxidant enzyme activities (CAT, GPx, SOD), oxidative stress markers (TOS, TAC, OSI), pro-inflammatory cytokines (IL‑1, TNF‑α), and apoptosis and inflammation-related genes (BAX, BCL2, NLRP3, NFKB). Histological analysis of the kidneys was also done to check for cellular changes. DXR treatment led to a decrease in the body weight and an increase in kidney enzymes, which is an indication of kidney damage. The levels of these enzymes were significantly lowered by the combination of herbal extracts, especially at 2%, indicating nephroprotective properties. The herbal extracts brought back the antioxidant enzyme activities to normal and reduced the oxidative stress markers in the kidney through raising the CAT, GPx, and SOD and decreasing the TOS and OSI levels. Furthermore, the herbal treatment also decreased the levels of pro-inflammatory cytokines and affected the apoptosis related gene expressions; the BAX was down-regulated and BCL2 was up-regulated, which helped in increasing the cell survival and decreasing inflammation. The extracts also reduced the NLRP3/NFKB in the kidneys of DXR‑treated mice in a dose dependent manner. Based on these results, 1% and 2% mixture of green tea and moringa leaf aqueous extracts (1:1 ratio) can be considered an appropriate combination to reduce DOX‑induced nephrotoxicity and kidney injury in cancer patients.

1. Islamuddin, M., Qin, X. (2024). Renal macrophages and NLRP3 inflammasomes in kidney diseases and therapeutics. Cell Death Discovery, 10(1), Article 229. https://doi.org/10.1038/s41420-024-01996-3

2. Kubat, G. B., Özler, M., Ulger, O., Ekinci, Ö., Atalay, Ö., Çelik, E. et al. (2020). The effects of mesenchymal stem cell mitochondrial transplantation on doxorubicin-mediated nephrotoxicity in rats. Journal of Biochemical and Molecular Toxicology, 35(1), Article e22612. https://doi.org/10.1002/jbt.22612

3. Charles, I. J., Okayo, O. D. (2021). Prevention of doxorubicin-induce renal function abnormalities by turmeric in Wistar rats. GSC Biological and Pharmaceutical Sciences, 14(3), 143–156. https://doi.org/10.30574/gscbps.2021.14.3.0070

4. Peter, S., Alven, S., Maseko, R. B., Aderibigbe, B. A. (2022). Doxorubicin-based hybrid compounds as potential anticancer agents: A review. Molecules, 27(14), Article 4478. https://doi.org/10.3390/molecules27144478

5. Angela, I. F. D., Dalimunthe, A., Harahap, U., Satria, D. (2023). Effect of andaliman (Zanthoxylum acanthopodium DC.) ethanol extract on doxorubicin-induced toxicity on hematology in male rats. Journal of Drug Delivery and Therapeutics, 13(3), 27–29. https://doi.org/10.22270/jddt.v13i3.5975

6. Amarasiri, S. S., Attanayake, A. P., Arawwawala, L. D. A. M., Jayatilaka, K. A. P. W., Mudduwa, L. K. B. (2021). Nephroprotective activity of Vetiveria zizanioides (L.) Nash supplement in doxorubicin-induced nephrotoxicity model of Wistar rats. Journal of Food Biochemistry, 45(9), Article e13901. https://doi.org/10.1111/jfbc.13901

7. Furcea, D. M., Agrigoroaie, L., Mihai, C.-T., Gardikiotis, I., Dodi, G., Stanciu, G. D. et al. (2022). 18F-FDG PET/MRI imaging in a preclinical rat model of cardiorenal syndrome — an exploratory study. International Journal of Molecular Sciences, 23(23), Article 15409. https://doi.org/10.3390/ijms232315409

8. Teibo, J., Bello, S., Olagunju, A., Olorunfemi, F., Ajao, O., Fabunmi, O. (2020). Functional foods and bioactive compounds: Roles in the prevention, treatment and management of neurodegenerative diseases. GSC Biological and Pharmaceutical Sciences, 11(2), 297–313. https://doi.org/10.30574/gscbps.2020.11.2.0143

9. Anyene, I. C., Ergas, I. J., Kwan, M. L., Roh, J. M., Ambrosone, C. B., Kushi, L. H. et al. (2021). Plant-based dietary patterns and breast cancer recurrence and survival in the pathways study. Nutrients, 13(10), Article 3374. https://doi.org/10.3390/nu13103374

10. Al-Temimi, W. K. A., Al- Garory, N. H. S., Khalaf, A. A. (2020). Diagnose the bioactive compounds in flaxseed extract and its oil and use their mixture as an antioxidant. Basrah Journal of Agricultural Sciences, 33(1), 172–188. https://doi.org/10.37077/25200860.2020.33.1.13

11. Hussain, M. A., Abogresha, N. M., Kader, G. A., Hassan, R., Abdelaziz, E. Z., Greish, S. M. (2021). Antioxidant and anti-inflammatory effects of crocin ameliorate doxorubicin-induced nephrotoxicity in rats. Oxidative Medicine and Cellular Longevity, 2021(1), Article 8841726. https://doi.org/10.1155/2021/8841726

12. Owumi, S. E., Lewu, D. O., Arunsi, U. O., Oyelere, A. K. (2021). Luteolin attenuates doxorubicin-induced derangements of liver and kidney by reducing oxidative and inflammatory stress to suppress apoptosis. Human and Experimental Toxicology, 40(10), 1656–1672. https://doi.org/10.1177/09603271211006171

13. Shi, H., Zhao, X., Peng, Q., Zhou, X., Liu, S., Sun, C. et al. (2023). Green tea polyphenols alleviate kidney injury induced by Di(2-ethylhexyl) phthalate in mice. American Journal of Nephrology, 55(1), 86–105. https://doi.org/10.1159/000534106

14. Arabzadeh, E., Norouzi Kamareh, M., Ramirez-Campillo, R., Mirnejad, R., Masti, Y., Shirvani, H. (2022). Twelve weeks of treadmill exercise training with green tea extract reduces myocardial oxidative stress and alleviates cardiomyocyte apoptosis in aging rat: The emerging role of bnip3 and HIF‑1α/IGFBP3 pathway. Journal of Food Biochemistry, 46(12), Article e14397. https://doi.org/10.1111/jfbc.14397

15. Nishat, R. J., Halim, M. R., Islam, M. M., Hamid, T., Ahmed, K. N., Hasan, R. et al. (2022). Effect of green tea on gentamicin induced nephrotoxicity in Long Evans male rats. Bangladesh Critical Care Journal, 10(2), 127–134. https://doi.org/10.3329/bccj.v10i2.62206

16. Adeoye, S. W. A., Adeshina, O. S., Yusuf, M. G., Omole, A. (2022). Hepatoprotective and renoprotective effect of Moringa oleifera seed oil on dichlorvos-induced toxicity in male Wistar rats. Nigerian Journal of Physiological Sciences, 37(1), 119–126. https://doi.org/10.54548/njps.v37i1.15

17. Putri, I. S., Siwi, G. N., Budiani, D. R., Rezkita, B. E. (2023). Protective effect of moringa seed extract on kidney damage in rats fed a high-fat and high-fructose diet. Journal of Taibah University Medical Sciences, 18(6), 1545–1552. https://doi.org/10.1016/j.jtumed.2023.07.001

18. Lukiswanto, B. S., Wijayanti, H., Fadhila, Y. N., Yuniarti, W. M., Arimbi, A., Suprihati, E. et al. (2022). Protective effect of Moringa oleifera leaves extract against gentamicin induced hepatic and nephrotoxicity in rats. Iraqi Journal of Veterinary Sciences, 37(1), 129–135. https://doi.org/10.33899/ijvs.2022.133276.2197

19. Elsayed, F. I., Elgendey, F., Waheed, R. M., El-Shemy, M. A. (2021). Protective effect of moringa oleifera seed extract on cisplatin induced nephrotoxicity in rats. International Journal of Pharmacy and Pharmaceutical Sciences, 13(5), 78–82. https://doi.org/10.22159/ijpps.2021v13i5.41125

20. Sebastian, D., Shankar, K. G., Ignacimuthu, S., Fleming, A. T., Sebastian, D. (2019). Detection of synergistic effect of three plant extracts against pathogenic bacteria. International Journal of Research and Analytical Reviews, 6(2), 438-449.

21. Wang, Y., Yang, H., Chen, L., Jafari, M., Tang, J. (2021). Network-based modeling of herb combinations in traditional Chinese medicine. Briefings in Bioinformatics, 22(5), Article bbab106. https://doi.org/10.1093/bib/bbab106

22. Sojoodi, M., Wei, L., Erstad, D. J., Yamada, S., Fujii, T., Hirschfield, H. et al. (2020). Epigallocatechin gallate induces hepatic stellate cell senescence and attenuates development of hepatocellular carcinoma. Cancer Prevention Research, 13(6), 497–508. https://doi.org/10.1158/1940-6207.capr‑19-0383

23. Wu, Z., Sun, L., Chen, R., Wen, S., Li, Q., Lai, X. et al. (2022). Chinese tea alleviates CCl4-induced liver injury through the nf-κbornrf2signaling pathway in C57BL‑6J mice. Nutrients, 14(5), Article 972. https://doi.org/10.3390/nu14050972

24. Shubhangini, C., Jaiganesh, R., Rajeshkumar, S. (2023). Green synthesis of zinc oxide nanoparticles using chamomile and green tea extracts and evaluation of their anti-inflammatory and antioxidant activity: An in vitro study. Cureus, 15(9), Article e46088. https://doi.org/10.7759/cureus.46088

25. Zhang, Y., Qu, X., Gao, H., Zhai, J., Tao, L., Sun, J. et al. (2020). Quercetin attenuates NLRP3 inflammasome activation and apoptosis to protect INH‑induced liver injury via regulating SIRT1 pathway. International Immunopharmacology, 85, Article 106634. https://doi.org/10.1016/j.intimp.2020.106634

26. Feng, T., Wan, Y., Dai, B., Liu, Y. (2023). Anticancer activity of bitter melon-derived vesicles extract against breast cancer. Cells, 12(6), Article 824. https://doi.org/10.3390/cells12060824

27. Shao, M., Kuang, Z., Wang, W., Li, S., Li, G., Song, Y. et al. (2022). Aucubin exerts anticancer activity in breast cancer and regulates intestinal microbiota. Evidence-Based Complementary and Alternative Medicine, 2022, Article 4534411. https://doi.org/10.1155/2022/4534411

28. Mradu, G., Saumyakanti, S., Sohini, M., Arup, M. (2012). HPLC profiles of standard phenolic compounds present in medicinal plants. International Journal of Pharmacognosy and Phytochemical Research, 4(3), 162–167.

29. Kumar, K. P., Reddy, V. R., Prakash, M. G., Kumar, K. P. (2018). In vitro estimation of total phenolics and DPPH radical scavenging activity of Withania somnifera extract. The Pharma Innovation Journal, 7(3), 588–590.

30. Pandey, B., Rajbhandari, M. (2014). Estimation of total phenolic and flavonoid contents in some medicinal plants and their antioxidant activities. Nepal Journal of Science and Technology, 15(1), 53–60. http://doi.org/10.3126/njst.v15i1.12010

31. Phuyal, N., Jha, P. K., Raturi, P. P., Rajbhandary, S. (2020). Total phenolic, flavonoid contents, and antioxidant activities of fruit, seed, and bark extracts of Zanthoxylum armatum DC. The Scientific World Journal, 2020(1), Article 8780704. https://doi.org/10.1155/2020/8780704

32. Védékoi, J., Selestin, S. D., Abdoulaye, H., Justin, K., Djamilah, Z., Pierre, K. (2019). Investigation of antioxidant activity of the ethanol extract of the resin exudates of trunk bark of Boswellia dalzielii Hutch (Burseraceae). Journal of Materials and Environmental Sciences, 10(12), 1413–1419.

33. Gulcin, İ., Alwasel, S. H. (2022). Metal ions, metal chelators and metal chelating assay as antioxidant method. Processes, 10(1), Article 132. https://doi.org/10.3390/pr10010132

34. Rajaratinam, H., Rasudin, N. S., Safuan, S., Abdullah, N. A., Mokhtar, N. F., Fuad, W. E. M. (2022). Passage number of 4T1 cells influences the development of tumour and the progression of metastasis in 4T1 orthotopic mice. The Malaysian Journal of Medical Sciences, 29(3), 30–42. https://doi.org/10.21315/mjms2022.29.3.4

35. Sauter, B. V., Martinet, O., Zhang, W. -J., Mandeli, J., Woo, S. L. С. (2000). Adenovirus-mediated gene transfer of endostatin in vivo results in high level of transgene expression and inhibition of tumor growth and metastases. Proceedings of the National Academy of Sciences, 97(9), 4802–4807. https://doi.org/10.1073/pnas.090065597

36. Zeiss, C. J., Gatti, D. M., Toro-Salazar, O., Davis, C., Lutz, C. M., Spinale, F. et al. (2019). Doxorubicin-induced cardiotoxicity in collaborative cross (СС) mice recapitulates individual cardiotoxicity in humans. G3 Genes/Genomes/Genetics, 9(8), 2637–2646. https://doi.org/10.1534/g3.119.400232

37. Amiri, R., Tabandeh, M. R., Hosseini, S. A. (2021). Novel cardioprotective effect of L‑carnitine on obese diabetic mice: Regulation of chemerin and CMKLRI expression in heart and adipose tissues. Arquivos Brasileiros de Cardiologia, 117(4), 715–725. https://doi.org/10.36660/abc.20200044

38. Erel, O. (2005). A new automated colorimetric method for measuring total oxidant status. Clinical Biochemistry, 38(12), 1103–1111. https://doi.org/10.1016/j.clinbiochem.2005.08.008

39. Benzie, I. F., Strain, J. J. (1999). Ferric reducing/antioxidant power assay: Direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Chapter in a book: Methods in Enzymology. Academic press, 1999. https://doi.org/10.1016/S0076-6879(99)99005-5

40. Tabandeh, M. R., Jozaie, S., Ghotbedin, Z., Gorani, S. (2022). Dimethyl itaconic acid improves viability and steroidogenesis and suppresses cytokine production in LPS‑treated bovine ovarian granulosa cells by regulating TLR4/nfkβ, NLRP3, JNK signaling pathways. Research in Veterinary Science, 152, 89–98. https://doi.org/10.1016/j.rvsc.2022.07.024

41. Namal Senanayake, S. P. J. (2013). Green tea extract: Chemistry, antioxidant properties and food applications — A review. Journal of Functional Foods, 5(4), 1529–1541. https://doi.org/10.1016/j.jff.2013.08.011

42. Musial, C., Kuban-Jankowska, A., Gorska-Ponikowska, M. (2020). Beneficial properties of green tea catechins. International Journal of Molecular Sciences, 21(5), Article 1744. https://doi.org/10.3390/ijms21051744

43. Pȩkal, A., Dróżdż, P., Biesaga, M., Pyrzynska, K. (2012). Screening of the antioxidant properties and polyphenol composition of aromatised green tea infusions. Journal of the Science of Food and Agriculture, 92(11), 2244–2249.

44. Lorenzo, J. M., Munekata, P. E. S. (2016). Phenolic compounds of green tea: Health benefits and technological application in food. Asian Pacific Journal of Tropical Biomedicine, 6(8), 709–719. https://doi.org/10.1016/j.apjtb.2016.06.010

45. Peñalver, R., Martínez-Zamora, L., Lorenzo, J. M., Ros, G., Nieto, G. (2022). Nutritional and antioxidant properties of Moringa oleifera leaves in functional foods. Foods, 11(8), Article 1107. https://doi.org/10.3390/foods11081107

46. Ntshambiwa, K. T., Seifu, E., Mokhawa, G. (2023). Nutritional composition, bioactive components and antioxidant activity of Moringa stenopetala and Moringa oleifera leaves grown in Gaborone, Botswana. Food Production, Processing and Nutrition, 5(1), Article 7. https://doi.org/10.1186/s43014-022-00124-x

47. Sreelatha, S., Padma, P. R. (2009). Antioxidant activity and total phenolic content of Moringa oleifera leaves in two stages of maturity. Plant Foods for Human Nutrition, 64(4), 303–311. https://doi.org/10.1007/s11130-009-0141-0

48. Na, H.-K., Surh, Y.-J. (2008). Modulation of Nrf2-mediated antioxidant and detoxifying enzyme induction by the green tea polyphenol EGCG. Food and Chemical Toxicology, 46(4), 1271–1278. https://doi.org/10.1016/j.fct.2007.10.006

49. Mostafa-Hedeab, G., Hassan, M. E., Halawa, T. F, Wani, F.A. (2022). Epigallocatechin gallate ameliorates tetrahydrochloride-induced liver toxicity in rats via inhibition of TGFβ/p-ERK/p-Smad1/2 signaling, antioxidant, anti-inflammatory activity. Saudi Pharmaceutical Journal, 30(9), 1293–1300. https://doi.org/10.1016/j.jsps.2022.06.021

50. Lang, Y., Gao, N., Zang, Z., Meng, X., Lin, Y., Yang, S. et al. (2024). Classification and antioxidant assays of polyphenols: A review. Journal of Future Foods, 4(3), 193–204. https://doi.org/10.1016/j.jfutfo.2023.07.002

51. Patintingan, C. G., Louisa, M., Juniantito, V., Arozal, W., Hanifah, S., Wanandi, S. I. et al. (2023). Moringa oleifera leaves extract ameliorates doxorubicininduced cardiotoxicity via its mitochondrial biogenesis modulatory activity in rats. Journal of Experimental Pharmacology, 15, 307–319. https://doi.org/10.2147/jep.s413256

52. Lee, E.-H., Park, H.-J., Kim, B.-O., Choi, H.-W., Park, K.-I., Kang, I.-K. et al. (2020). Anti-inflammatory effect of Malus domestica cv. green ball apple peel extract on Raw 264.7 macrophages. Journal of Applied Biological Chemistry, 63(2), 117–123. https://doi.org/10.3839/jabc.2020.016

53. Heo, Y. J., Lee, N., Choi, S.-E., Jeon, J. Y., Han, S. J., Kim, D. J. et al. (2023). Amphiregulin induces iNOS and COX‑2 expression through NF‑κB and MAPK signaling in hepatic inflammation. Mediators of Inflammation, 2023, 1–11. https://doi.org/10.1155/2023/2364121

54. Kastl, L., Sauer, S. W., Ruppert, T., Beissbarth, T., Becker, M. S., Süss, D. et al. (2014). TNF‑α mediates mitochondrial uncoupling and enhances ROS‑dependent cell migration via NF‑κB activation in liver cells. FEBS Letters, 588(1), 175–183. https://doi.org/10.1016/j.febslet.2013.11.033

55. Singh, A., Yau, Y. F., Leung, K. S., El-Nezami, H., Lee, J. C. -Y. (2020). Interaction of polyphenols as antioxidant and anti-inflammatory compounds in brain–liver–gut axis. Antioxidants, 9(8), Article 669. https://doi.org/10.3390/antiox9080669

56. Park, H. J., Lee, J.-Y., Chung, M.-Y., Park, Y.-K., Bower, A. M., Koo, S. I. et al. (2012). Green tea extract suppresses NFκB activation and inflammatory responses in diet-induced obese rats with nonalcoholic steatohepatitis3. The Journal of Nutrition, 142(1), 57–63. https://doi.org/10.3945/jn.111.148544

57. Wang, Z., Sun, W., Sun, X., Wang, Y., Zhou, M. (2020). Kaempferol ameliorates Cisplatin induced nephrotoxicity by modulating oxidative stress, inflammation and apoptosis via ERK and NF‑κB pathways. AMB Express, 10(1), Article 58. https://doi.org/10.1186/s13568-020-00993-w

58. Hamza, A. A. (2010). Ameliorative effects of Moringa oleifera Lam seed extract on liver fibrosis in rats. Food and Chemical Toxicology, 48(1), 345–355. https://doi.org/10.1016/j.fct.2009.10.022

59. Abd-Elnaby, Y. A., ElSayed, I. E., AbdEldaim, M. A., Badr, E. A., Abdelhafez, M. M., Elmadbouh, I. (2022). Anti-inflammatory and antioxidant effect of moringa oleifera against bisphenol-a-induced hepatotoxicity. Egyptian Liver Journal, 12(1), Article 57. https://doi.org/10.1186/s43066-022-00219-7

60. Wang, Y., Zhou, P., Li, P., Yang, F., Gao, X.-q (2020). Long non-coding RNA H19 regulates proliferation and doxorubicin resistance in MCF‑7 cells by targeting PARP1. Bioengineered, 11(1), 536–546. https://doi.org/10.1080/21655979.2020.1761512

61. Lukiswanto, B. S., Wijayanti, H., Fadhila, Y., Yuniarti, W. M., Arimbi, A., Kurnijasanti, R. (2022). Protective effect of Moringa oleifera leaves extract against gentamicin induced hepatic and nephrotoxicity in rats. Iraqi Journal of Veterinary Sciences, 37(1), 129–135. https://doi.org/10.33899/ijvs.2022.133276.2197

62. Mostafa-Hedeab, G., Hassan, M. E., Halawa, T. F., Ahmed Wani, f. (2022). Epigallocatechin gallate ameliorates tetrahydrochloride-induced liver toxicity in rats via inhibition of TGFβ/p-ERK/p-Smad1/2 signaling, antioxidant, anti-inflammatory activity. Saudi Pharmaceutical Journal, 30(9), 1293–1300. https://doi.org/10.1016/j.jsps.2022.06.021

63. Tak, E., Park, G.-C., Kim, S.-H., Jun, D. Y., Lee, J., Hwang, S. et al. (2016). Epigallocatechin‑3-gallate protects against hepatic ischaemia — reperfusion injury by reducing oxidative stress and apoptotic cell death. Journal of International Medical Research, 44(6), 1248–1262. https://doi.org/10.1177/0300060516662735

64. Abdel Fattah, M. E., Sobhy, H. M., Reda, A., Abdelrazek, H. M. (2020). Hepatoprotective effect of Moringa oleifera leaves aquatic extract against lead acetate–induced liver injury in male Wistar rats. Environmental Science and Pollution Research, 27(34), 43028–43043. https://doi.org/10.1007/s11356-020-10161-z

65. Zhang, Y., Qu, X., Gao, H., Zhai, J., Tao, L., Sun, J. et al. (2020). Quercetin attenuates NLRP3 inflammasome activation and apoptosis to protect INH‑induced liver injury via regulating SIRT1 pathway. International Immunopharmacology, 85, Article 106634. https://doi.org/10.1016/j.intimp.2020.106634