Synergistic impacts of Montelukast and Klotho against doxorubicin-induced cardiac toxicity in Rats

Abstract

Doxorubicin (DOX) is a powerful antitumor agent with a well-known cardiaotoxic side effects. In the current study, the ameliorative combined impacts of montelukast (Mont) and Klotho against doxorubicin-induced cardiac toxicity were examined. Fifty-six adult male rats (2 months age and weighting 150-200 g) were grouped into 7 groups (8 rats per group). Animals received doxorubicin alone or in combination with either Mont or Klotho. After 2 weeks of treatments, serum samples were examined to assess the changes in cardiac activity biomarkers such as LDH, CK-MB, cardiac troponin-I (cTn-I), and heart fatty acid binding protein (H-FABP). Serum changes of IL-6, inducible nitric oxide synthase (iNOS), and caspase-3 levels were assayed. The oxidative stress biomarkers such as total antioxidant capacity (TAC) and inflammatory (rat IL-1β and rat TNF-α,) and anti-inflammatory (rat IL-10) cytokines were examined. Heart histology and transforming growth factor-β1 (TGF-β1) immunoreactivity were measured. DOX induced cardiomyopathy, which was reflected by the increases in all examined cardiac parameters. Real-time PCR confirmed that DOX upregulated the expression of TNF-α and IL-1β and decreased the expression of IL-10. Moreover, DOX showed marked elevation in the ST segment T wave complex, causing profound tachycardia. Heart histology assessments showed cardiac cell necrosis, inflammatory cell infiltration, interstitial congestion, and increased TGF-β1 immunoreactivity. Montelukast and Klotho administration ameliorated all the altered parameters when administered alone or in combination to DOX-intoxicated rats. Klotho was more effective compared with montelukast in terms of reductions in heart rate, ST segment T wave complex elevation, cardiac enzymes (lactate dehydrogenase; LDH, creatine kinase-MB; CK-MB, cardiac troponin I; cTn-I, heart fatty acid binding protein; H-FABP) cardiac histology, and caspase-3 levels and increases in TAC activity. Montelukast was more effective in reducing serum levels of IL6 and iNOS, expression of TNF-α and IL-1β, and the upregulation of IL-10 expression. The co-administration of both drugs led to significantly more synergistic results in terms of reducing cardiac toxicity. In conclusion, montelukast and Klotho either alone or in combination were confirmed to be effective in suppressing DOX-induced cardiac toxicity in rats.

Keywords: Klotho; cardiac biomarkers; cardiotoxicity; doxorubicin; gene expression; montelukast.

© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Figures

Fig. 1

Montelukast and Klotho ameliorate serum changes in A) H-FABP, B) LDH, C) CK-MB, and D) troponin-I in DOX-intoxicated cardiac toxicity. Data are expressed as means ± SD, (n = 6). ANOVA test was followed by post hoc multiple comparisons test. Values with different letters are significant at P < 0.05.

Fig. 2

Quantification of A) TNF-α, B) IL-1β, and C) IL-10 expression in rat cardiac tissue. Values expressed as means ± SEM (n = 6). ANOVA test was followed by post hoc multiple comparisons test. Values with different letters are significant at P < 0.05.

Fig. 3

Photomicrography of histopathological changes of rat cardiac tissues (H&E, ×400). Rats from the A) control, B) montelukast-treated group, and C) Klotho-treated group showed normal histology of cardiomyocyte cytoplasm (blue arrow) and centrally located oval nuclei (black arrow). D) Represents DOX-injected rats showing marked degeneration of cardiomyocytes (red arrow), interstitial inflammation (yellow arrow), and interstitial congestion (green arrow). E) Represents Mont + DOX-treated rats showing mild improvements in myocardium degeneration (red arrow) with moderate improvement in interstitial inflammation (yellow arrow) and interstitial congestion (green arrow). F) Represents Klotho + DOX-treated rats showing moderate improvements in myocardium degeneration (red arrow), interstitial inflammation (yellow arrow), and interstitial congestion (green arrow), although the letter 2 parameters were of a lesser degree than Mont + DOX-treated rats. G) Represents Mont + Klotho + DOX-treated rats showing marked improvements in myocardium degeneration (red arrow), interstitial inflammation (yellow arrow), and interstitial congestion (green arrow). In Fig. 3H, Values are mean ± SE for 6 slides/group. Values with different letters are statistically significant at P > 0.05.

Fig. 4

Immunohistochemical staining of transforming growth factor-β1 (TGF-β1) in the experimental rats’ cardiac cells (IHC, ×400). A–C) Represent control rats showing normal expression of TGF-β1 (brown color). D) Represents DOX-intoxicated rats showing marked increases in TGF-β1 expression in the form of multiple scattered foci of strong immune positivity. E) Represents Mont + DOX-treated rats and F) represents Klotho + DOX-treated rats showing moderate TGF-β1 expression. G) Represents Mont + Klotho + DOX-treated rats showing minimal TGF-β1 expression in the form of marked decreases in immune reactivity compared with e and f.

Fig. 5

Representative impacts of montelukast and Klotho against cardiac toxicity induced by doxorubicin.