α-Bisabolol Attenuates Doxorubicin Induced Renal Toxicity by Modulating NF-κB/MAPK Signaling and Caspase-Dependent Apoptosis in Rats

Abstract

Doxorubicin (DOX) is a well-known and effective antineoplastic agent of the anthracycline family. But, multiple organ toxicities compromise its invaluable therapeutic usage. Among many toxicity types, nephrotoxicity is one of the major concerns. In recent years many approaches, including bioactive agents of natural origin, have been explored to provide protective effects against chemotherapy-related complications. α-Bisabolol is a naturally occurring monocyclic sesquiterpene alcohol identified in the essential oils of various aromatic plants and possesses a wide range of pharmacological properties such as antioxidant, anti-inflammatory, analgesic, cardioprotective, antibiotic, anti-irritant, and anticancer activities. The present study aimed to evaluate the effects of α-Bisabolol on DOX-induced nephrotoxicity in Wistar male albino rats. Nephrotoxicity was induced in rats by injecting a single dose of DOX (12.5 mg/kg, i.p.), and the test compound, α-Bisabolol (25 mg/kg) was administered intraperitoneally along with DOX as a co-treatment daily for 5 days. DOX-injected rats showed reduction in body weight along with a concomitant fall in antioxidants and increased lipid peroxidation in the kidney. DOX-injection also increased levels/expressions of proinflammatory cytokines namely tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) and inflammatory mediators like inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and activated nuclear factor kappa-B (NF-κB)/mitogen-activated protein kinases (MAPK) signaling in the kidney tissues. DOX also triggered apoptotic cell death, evidenced by the increased expression of pro-apoptotic markers like BCL2-Associated X Protein (Bax), cleaved caspase-3, caspase- 9, and cytochrome-C) and a decrease in the expressions of anti-apoptotic markers namely B-cell lymphoma 2 (Bcl2) and B-cell lymphoma-extra large (Bcl-xL) in the kidney. These biochemical alterations were additionally supported by light microscopic findings, which revealed structural alterations in the kidney. However, treatment with α-Bisabolol prevented body weight loss, restored antioxidants, mitigated lipid peroxidation, and inhibited the rise in proinflammatory cytokines, as well as favorably modulated the expressions of NF-κB/MAPK signaling and apoptosis markers in DOX-induced nephrotoxicity. Based on the results observed, it can be concluded that α-Bisabolol has potential to attenuate DOX-induced nephrotoxicity by inhibiting oxidative stress and inflammation mediated activation of NF-κB/MAPK signaling alongwith intrinsic pathway of apoptosis in rats. The study findings are suggestive of protective potential of α-Bisabolol in DOX associated nephrotoxicity and this could be potentially useful in minimizing the adverse effects of DOX and may be a potential agent or adjuvant for renal protection.

Keywords: NF-κB/MAPK signaling; apoptosis; doxorubicin; nephrotoxicity; phytochemicals; renal toxicity; α-Bisabolol.

Figure 1

Effect of α-Bisabolol on body weight, renal injury, and oxidative stress markers in normal and DOX-injected rats. (A,B). Effect of α-Bisabolol on body weight and serum creatinine levels. DOX-injected rats showed a significant (p < 0.05) decrease in body weight along with a significant (p < 0.05) increase in the levels of creatinine in the serum compared to normal control rats, whereas α-Bisabolol treatment in DOX-treated rats significantly (p < 0.05) decreased the body weight loss and serum creatinine levels compared to DOX-alone treated rats (C–F). Effect of α-Bisabolol on the levels/activities/concentrations of MDA, SOD, catalase, and GSH in the kidney of normal and DOX-injected rats. DOX-injected rats showed a considerable (p < 0.05) decrease in the activities/levels of enzymatic and non-enzymatic antioxidants in the kidney compared to normal control rats, while α-Bisabolol treatment showed a significant (p < 0.05) decrease in the activities/levels of enzymatic and non-enzymatic antioxidants in the kidney compared to DOX-alone treated rats. Each column is mean ± SEM for eight rats in each group; columns not sharing a common symbol (*, **) differ significantly with each other (* p < 0.05 vs. normal control, ** p < 0.05 vs. DOX control).

Figure 2

(A–C) Effect of α-Bisabolol on the levels of proinflammatory cytokines (TNF-α, IL-6, and IL-1β) in the kidney of normal and DOX-injected rats. DOX-injected rats showed a considerable (p < 0.05) increase in the levels of proinflammatory cytokines in the kidney compared to normal control rats, while α-Bisabolol treatment showed a significant (p < 0.05) decrease in the levels of proinflammatory cytokines in the kidney compared to DOX-alone treated rats. Each column is mean ± SEM for eight rats in each group; columns not sharing a common symbol (*, **) differ significantly with each other (* p < 0.05 vs. normal control, ** p < 0.05 vs. DOX control).

Figure 3

Effect of α-Bisabolol on the expressions of proinflammatory cytokines (TNF-α, IL-6, and IL-1β) in the kidney of normal and DOX-injected rats. (A). Representative images of Western immunoblot analysis for TNF-α, IL-6, and IL-1β. (B) Densitometric analysis of renal protein expressions of TNF-α, IL-6, and IL-1β assessed by Western blot analysis revealed that DOX-injected rats showed a considerable (p < 0.05) increase in the renal protein expressions of proinflammatory cytokines compared to normal control rats while α-Bisabolol treatment showed significant (p < 0.05) decrease in the expressions of cytokines in the kidney compared to DOX-alone treated rats. Immunoblotting analysis was done in duplicates; columns not sharing a common symbol (*, **) differ significantly with each other (* p < 0.05 vs. normal control, ** p < 0.05 vs. DOX control).

Figure 4

Histopathology of the kidney. Normal control and α-Bisabolol alone treated rat’s kidney showed no pathological changes. DOX-alone treated rats showed thinning and loss of apical cytoplasm, necrosis of individual tubular epithelial cells, loss of nuclei, adhesion, and denudation of tubular basement membrane, whereas α-Bisabolol treatment reinstates the near normal renal architecture in DOX-injected rats (Magnification; 20×).

Figure 5

Effect of α-Bisabolol on NF-κB/MAPK signaling in the kidney of normal and DOX-injected rats. (A). Representative images of Western immunoblot analysis for iNOS, COX-2, p-NF-κB-P65, IκBα, p-IκBα, P38, and p-P38. (B) Densitometric analysis of renal protein expressions of iNOS, COX-2, p-NF-κB-P65, IκBα, p-IκBα, P38, and p-P38 assessed by Western blot analysis. The expressions of renal proinflammatory mediators (iNOS and COX-2) and NF-κB/MAPK signaling proteins in DOX-alone treated rats considerably (p < 0.05) increased compared to normal rats while α-Bisabolol treatment considerably (p < 0.05) reduced the altered renal expressions of inflammatory mediators (iNOS and COX-2) and NF-κB/MAPK signaling proteins compared to DOX-alone treated rats. Immunoblotting analysis was done in duplicates. Columns not sharing a common symbol (*, **) differ significantly with each other (* p < 0.05 vs. normal control, ** p < 0.05 vs. DOX control).

Figure 6

Effect of α-Bisabolol on the intrinsic apoptosis pathway in the kidney of normal and DOX-injected rats. (A). Representative images of Western immunoblot analysis for Bax, Bcl2, Bcl-xL, procaspase-3, active caspase-3, procaspase-9, active caspase-9, cytochrome-C. Immunoblotting analysis was done in duplicates. (B). Densitometric analysis of renal protein expressions of Bax, Bcl2, Bcl-xL, active caspase-3, active caspase-9, cytochrome-C. DOX-injected rats showed significant (p < 0.05) increase in the renal protein expressions of pro-apoptotic proteins (Bax, cleaved caspase-3, cleaved caspase-9 and cytochrome-C) with considerable (p < 0.05) decrease in the expressions of anti-apoptotic proteins (Bcl2 and Bcl-xL) compared to normal control rats. Meanwhile, α-Bisabolol treatment to DOX-injected rats significantly (p < 0.05) downregulates the renal protein expressions of Bax, cleaved caspase-3, cleaved caspase-9, and cytochrome-C with significant (p < 0.05) increase in the expression of renal Bcl2 and Bcl-xL compared to DOX-alone injected rats. Columns not sharing a common symbol (*, **) differ significantly with each other (* p < 0.05 vs. normal control, ** p < 0.05 vs. DOX control).