Combination of Spirulina, Ganoderma Lucidum and Moringa Oleifera improves cardiac functions and reduces pro-inflammatory biomarkers in preclinical models of short-term doxorubicin-mediated cardiotoxicity

Introduction: Anthracyclines are essential adjuvant therapies for a variety of cancers, particularly breast, and gastric and esophageal cancers. Whilst prolonging cancer-related survival, these agents can induce drug-related cardiotoxicity. This causes an increased risk of heart failure. Cardioprotective agents used to mitigate cardiotoxicity, such as angiotensin antagonists, angiotensin receptor blockers and beta‐blockers, are often poorly tolerated in these patients due to intravascular volume fluctuations, which are further escalated by the hemodynamic side effects of these agents. Spirulina, Reishi (Ganoderma Lucidum) and Moringa are three nutaceuticals with anti-inflammatory effects that are currently used in cancer patients as Complementary and Alternative Medicines to improve quality of life and fatigue.Purpose:  We hypothesize that the nutraceutical combination of Spirulina, Reishi and Moringa (Singo) could reduce inflammation and cardiotoxicity induced by anthracyclines. Methods: Female C57Bl/6 mice were untreated (Sham, n=6) or treated for 10 days with short-term doxorubicin i.p at 2.17 mg/kg (DOXO, n=6), Singo at 12 mg/kg (Singo, n=6) or doxorubicin combined to Singo (DOXO-Singo, n=6). Ejection fraction, radial and longitudinal strain were analyzed through transthoracic echocardiography (Vevo 2100, Fujifilm). Myocardial expression of NLRP3, DAMPs (galectine 3 and calgranulinS100) and 13 cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL17-α, IL-18, IFN-γ, TNF-α, G-CSF, and GM-CSF) were quantified through selective mouse ELISA methods. Myocadial fibrosis, necrosis and hypertrophy were analyzed through Immunohistochemistry (IHC). Human cardiomyocytes (HFC cell line) were exposed to subclinical concentration of doxorubicin (200 nM) alone or in combination to Singo ( at 10, 25 and 50 µg/ml)  for 24 and 48h. Cell viability studies were performed through MTS assay. Quantification of malondialdehyde and 4-hydroxynonenal were performed through spectrophotometric methods. Anti-inflammatory studies (expression of NLRP3 and p65/NF-kB) were made through selective ELISA methods. Intracellular concentration of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL17-α, IL-18, IFN-γ, TNF-α, G-CSF, and GM-CSF were also performed. Results: In preclinical models of short-term DOXO cardiotoxicity, Singo improved significantly EF and FS and prevented the reduction of radial and longitudinal strain after 10 days of treatment with DOXO. A reduced expression of myocardial NLRP3 and NF-kB levels in cardiac tissues were seen in DOXO-Singo group compared to DOXO mice (p<0.05). Myocardial expression of pro-inflammatory cytokines were significantly reduced after treatment with Singo indicating anti-inflammatory properties. Myocardial levels of Calgranulin S100 and galectine-3 were strongly enhanced in DOXO group; on the other hand their expression were reduced in Singo-DOXO group vs DOXO (p<0.05). Immunohistochemistry analysis indicates that Singo reduces fibrosis and hypertrophy in myocardial tissue of mice during exposure to DOXO. In human cardiomyocytes exposed to DOXO, IL1α-β, IL-6, IL-17α, IL-18 and TNF-α levels were strongly enhanced compared to untreated cells. When co-incubated with Singo, cytokine levels were significantly reduced in cells exposed to Singo compared to only DOXO-treated cells. An opposite behavior was seen for IL-10 intracellular levels in cells co-incubated with Singo and DOXO, compared to DOXO. Conclusion: In preclinical models of DOXO-induced cardiotoxicity, Singo is able to improve cardiac function and reduce biomarkers involved in heart failure and fibrosis. Cardioprotective properties of Singo are mediated by the reduction of lipid peroxidation products and expression of NLRP3-NF-kB –cytokine pathways. The overall picture of this study indicates that Singo could be a potential complementary and alternative medicine for primary prevention of cardiomyopathies induced by anthracyclines in cancer patients.