Herbo-Mineral Medicine, Cardiogrit Gold, Exhibits Protective Effects in Caenorhabditis elegans Model of Doxorubicin-Induced Cardiotoxicity

Abstract

Doxorubicin, an effective antineoplastic agent, is often prescribed for the treatment of various carcinomas. However, the use of doxorubicin becomes limited due to its adverse effects like cardiotoxicity, dysmenorrhea, and leucopenia. Cardiogrit Gold (CG) is a herbo-mineral Ayurvedic medicine prescribed for the treatment of various cardiovascular ailments. The current study aimed to investigate the therapeutic potential of CG in imparting protection against doxorubicin-induced cardiotoxicity. Wild-type (N2) and genetically modified Caenorhabditis elegans(SJ4005 and DA597) were used as model organisms to assess the bioactivity of CG against doxorubicin-induced cardiotoxicity. Chemical characterization of CG was performed by HPLC-based analysis. Calcium, a key mineral component of CG, was measured in CG-treated C. elegans using inductively coupled plasma mass spectrometry (ICP-MS) analysis, as the marker of CG internalization in C. elegans. Toxicity induced by doxorubicin and its recovery upon CG treatment was determined by various toxicologically important endpoints. CG treatment rescued N2 C. elegans from doxorubicin-induced reduction in their growth, reproduction, locomotory behavior, pharyngeal pumping, feeding ability, and increased ROS generation. CG treatment modulated the expression of hsp-4 in SJ4005 C. elegans suggestive of decreased ER stress and normalized the pharyngeal grinder damage in DA597 C. elegans, indicating a robust induction of cardio-normalcy. Novel analytical methods were developed to detect and quantify doxorubicin in C. elegans on HPLC and UPLC/QToF-MS platforms. Interestingly, CG treatment decreased bioaccumulation of doxorubicin in C. elegans, robustly correlating with the observed cardioprotective effects. Taken together, CG has a strong cardioprotective profile against doxorubicin-induced damages and could be taken for further preclinical and clinical assessments.

Keywords: Caenorhabditis elegans; Cardiogrit Gold; cardiotoxicity; doxorubicin; pharyngeal pumping.

Figure 1

HPLC-based identification and quantification of phytometabolites present in Cardiogrit Gold (CG). Overlayed HPLC chromatograms of standard mix (blue color) and CG (pink color). Ellagic acid was quantified at 365 nm wavelength, and arjungenin and arjunic acid were quantified at 210 nm wavelength. Quantitative analysis of CG is shown in Table 2.

Figure 2

Effect of CG and doxorubicin on survival of N2 worms. (a) CG (1–300 μg/mL), (b) doxorubicin (0.1–10 μM), (c, d) effect of CG or doxorubicin on reproduction. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001 in comparison with control.

Figure 3

Accumulation of doxorubicin in N2 worms. (a) Overlayed HPLC chromatogram of pure doxorubicin standard, control untreated worms, and varying concentrations of doxorubicin (0.01–10 μM)-treated worms, structure of doxorubicin was sourced from ChemSpider (ChemSpider ID: 29400). (b) Bar diagram of doxorubicin accumulation. ^∗∗∗p < 0.001 in comparison with control.

Figure 4

CG decreased the uptake and accumulation of doxorubicin in N2 worms. (a) Brightfield images of C. elegans pharynx (magnification = 200×) (I) untreated control, (II) doxorubicin (10 μM), (III–VI) worms co-treated with doxorubicin (10 μM) and varying concentrations of CG (1–30 μg/mL). (b) Doxorubicin red intensity was quantified by ImageJ software. (c) Overlayed UPLC/QToF-MS chromatogram of the reference standard (200 ppb), control worms, 10 μM doxorubicin alone, and co-treatment with CG (1–30 μg/mL). (d) CG (1–30 μg/mL) co-treatment reduced the accumulation of doxorubicin in C. elegans as detected by UPLC/QToF-MS. (e) CG (30 μg/mL) increased calcium accumulation in N2 worms as detected by ICP-MS. ^∗p < 0.05 and ^∗∗∗p < 0.001 in comparison with doxorubicin; and ^###p < 0.001 in comparison with control.

Figure 5

CG enhanced the growth of N2 worms. (a) Brightfield images of C. elegans (magnification = 100×) (I) control, (II) doxorubicin, (III–VI) worms co-treated with doxorubicin (10 μM) and varying concentrations of CG (1–30 μg/mL). (b) CG (1–30 μg/mL) increased the length of N2 worms. (c) CG recovered the reproductive ability of the worms. ^∗p < 0.05; ^∗∗p < 0.01; and ^∗∗∗p < 0.001 in comparison with doxorubicin and ^###p < 0.001 in comparison with control.

Figure 6

Effect of CG on the behavior of N2 worms. CG (1–30 μg/mL) recovers (a) locomotory behavior, (b) pharyngeal pumping behavior, and (c) feeding ability in N2 worms. ^∗p < 0.05; ^∗∗p < 0.01; and ^∗∗∗p < 0.001 in comparison with doxorubicin and ^###p < 0.001 in comparison with control.

Figure 7

CG recovers GFP intensity in SJ4005 worms. (a) CG (1–30 μg/mL) reduced hsp-4::GFP expression localized in the pharynx of SJ4005 worms (magnification = 200×), (I) control, (II) doxorubicin, (III–VI) worms co-treated with doxorubicin (10 μM) and varying concentrations of CG (1–30 μg/mL). (b) Bar diagram of mean fluorescence intensity of hsp-4::GFP as quantified by ImageJ software. (c) CG treatment decreased the ROS generation (fold change) in doxorubicin-exposed worms. (d) CG (1 and 30 μg/mL) recovers pharyngeal grinder damage in DA579 worms (magnification = 200×). ^∗∗p < 0.01 and ^∗∗∗p < 0.001 in comparison with doxorubicin and ^###p < 0.001 in comparison with control.

Figure 8

Summary of the protective effect of CG against doxorubicin-induced cardiotoxicity in C. elegans.