Oral delivery of nerolidol alleviates cyclophosphamide-induced renal inflammation, apoptosis, and fibrosis via modulation of NF-κB/cleaved caspase-3/TGF-β signaling molecules

Abstract

Cyclophosphamide (CP) is one of the most extensively used antineoplastic drug, but the nephrotoxicity caused by this drug is a major limiting factor for its use. Nerolidol (NERO) is a natural bioactive compound with diverse pharmacological actions. In Vitro and in vivo study was performed using HK-2 renal cells and Swiss Albino mice. Cell lines and animals were treated with NERO 25 and 50 µM + 30 µM CP (in vitro), 200 and 400 mg/kg, p.o. NERO from day 1 to day 15 + 200 mg/kg, i.p. CP on day 17 as single intraperitoneal injection (in vivo). The makers of oxidative stress, renal-specific injury markers, inflammation, apoptosis, fibrosis, and histopathological changes were studied. The study’s outcome showed a significant reduction in the level of malonaldehyde and interleukin-6 (p < 0.01), tumor necrosis factor-α, IL-1β (p < 0.001), and an increase in the superoxide dismutase, catalase, glutathione and interleukin-10 level (p < 0.01), in the in vivo study when treated with NERO 400 and compared with CP 200. In Vitro study showed reduced expression of nuclear factor kappa light chain enhancer of activated B cells, cleaved caspase-3, kidney injury molecule-1 and transforming growth factor-β-1 (p < 0.001), when treated with NERO 50 µM whereas NERO 25 µM only reduced the level of cleaved caspase-3 (p < 0.05) when compared with 30 µM. NERO 400 also reduced uric acid (p < 0.05), urea (p < 0.01), blood urea nitrogen, and serum creatinine levels (p < 0.001) and increased the level of blood-urea-nitrogen/creatinine ratio (p < 0.001). Additionally, the level of fibrosis-specific markers such as transforming growth factor-β1, hyaluronic acid (p < 0.01), 4-hydroxyproline, a collagen-rich area in Masson’s’ trichome stain, and Smad3 expression was also significantly reduced (p < 0.001). Furthermore, the outcome of multiple renal staining showed structural reversal aberrations, reduction of the thick basement membrane, and glycogen level toward normal when treated with NERO 400. Thus, the study showed a novel mechanistic modality of NERO against cyclophosphamide-induced renal toxicity. The outcome of this study can be considered a step closer to the development of an adjuvant to mitigate cyclophosphamide-induced renal toxicity among patients treated with cyclophosphamide.

Keywords:

• Nephrotoxicity
• oxidative stress
• renal fibrosis and inflammation

Acknowledgments

The authors are thankful to Jamia Hamdard for providing the infrastructure and necessary facilities to perform the experimental work.

Author contributions

For research articles with several authors, a short paragraph specifying their individual contributions must be provided. The following statements should be used Conceptualization, Syed Ehtaishamul Haque and Mansoor Ali Syed.; methodology, Ashif Iqubal.; software, Ashif Iqubal.; validation, Abul Kalam Najmi., Javed Ali. and Shadab Md.; formal analysis, Javed Ali.; Huda Mohammed Alkreathy.; investigation, Ashif Iqubal.; resources, Mansoor Ali Syed and Huda Mohammed Alkreathy.; data curation, Ashif Iqubal.; writing Ashif Iqubal; writing—review and editing, Syed Ehtaishamul Haque.; supervision, Syed Ehtaishamul Haque.; project administration, Syed Ehtaishamul Haque.; funding acquisition, Shadab Md. All authors have read and agreed to the published version of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Ethical approval statement

All institutional and ARRIVE guidelines for the care and use of laboratory animals were followed. Study was approved by the Institutional Review Board and Ethics Committee of Jamia Hamdard University, New Delhi. Experimental animals were used in the study since, the study was designed as preclinical in vivo model and Swiss Albino mice are suitable such studies. Experimental animals were provided by the central animal house facility of Jamia Hamdard after approval from the Committee for Control and Supervision of Experiments on Animals (CPCSEA).

The number or ID of the ethics approval: IAEC/JH/1484

Animals were maintained on a standard pellet diet and water ad libitum. The mice used in the present study was kept under room temperate of 20 ± 0.5 °C, humidity of 50 ± 10% and 12 h light/dark cycle. At the end of study, the animals were sacrificed following anesthesia via intraperitoneal injection of mixture of ketamine and xylazine (0.1 ml/20 gm).

Additional information

Funding

This research work was funded by Institutional Fund Projects under grant no. (IFPIP-768-166-1443). Therefore, the authors gratefully acknowledge technical and financial support from the Ministry of Education and King Abdulaziz University, DSR, Jeddah, Saudi Arabia.