Application of Zebrafish Model in the Suppression of Drug-Induced Cardiac Hypertrophy by Traditional Indian Medicine Yogendra Ras

Figures

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  Figure 1. Experimental design of D. rerio exposure to cardiac hypertrophy induction by erythromycin. Duration of the effective dose study was 7 days, and for therapeutic dose study it was 14 days.

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  Figure 2. Scanning electron microscope (SEM) and electron dispersive X-ray (EDX) analysis of Yogendra Ras (YDR). (A) SEM analysis of the YDR powder showed the presence of a heterogeneous mass of particles of varying shapes and sizes. Particles were present individually or as aggregates. (B) Electron dispersive X-ray analysis showed the presence of various elements on the surface of the YDR particles. (C) Quantitatively highest elemental concentrations were detected for mercury (Hg), tin (Sn), oxygen (O), calcium (Ca) and iron (Fe). Several other elements such as silicon (Si), magnesium (Mg), sodium (Na), aluminum (Al), and zinc (Zn) were also detected but at ≤ 1% w/w concentrations.

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  Figure 3. X-ray diffraction (XRD) and size distribution analysis of dry Yogendra Ras (YDR) sample. (A) XRD analysis showed the presence of cinnabar (HgS), metacinnabar (HgS), pure gold (Au), hematite (Fe2O3), arsenic trioxide (As4O6), and arsenic-iron alloy (AsFe). (B) Dynamic light scattering analysis of the YDR showed them to be present in a bimodal phase when suspended in pure MilliQ water. The size distribution showed the presence of particles with a hydrodynamic diameter of 1116 ± 102.7 d.nm along with a high polydispersity index (PdI) value representing an unstable colloidal suspension. When the YDR particles were suspended in cell culture media containing 2% FBS, the particle size distribution changed to a monomodal distribution showing the hydrodynamic diameter of 486 ± 46.95 d.nm and a lower PdI value representing a stable colloidal suspension.

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  Figure 4. Electrocardiogram (ECG) analysis and whole heart histological analysis of erythromycin (ERY)-stimulated cardiac hypertrophy. (A) Schematic diagram of Danio rerio heart: sinus venosus = SV, atrium = Atr, ventricle = Ven, bulbus arteriosus = BA, epicardium = Epc, myocardium = Myc, endocardium = End (graphics adapted from Yacoub et al. [32]). (B) In the effective dose screening analysis, ECG and heart anatomy of the D. rerio were studied for (i) normal control; (ii) disease control (25 µg/Lt erythromycin (ERY)); (iii) low dose (0.6 µg/kg of YDR); (iv) medium dose (4 µg/kg of YDR); and (v) high dose (18 µg/kg of YDR). The results indicated induction of CH by ERY. No changes were detected in the CH parameters following the 7 days of effective dose treatment with YDR at the low and medium doses. Mild changes were observed at the highest tested dose of YDR. (C) In the therapeutic dose treatment study, D. rerio were grouped as (i) normal control; (ii) disease control (25 µg/Lt ERY); (iii) standard drug (4 µg/kg of verapamil); (iv) therapeutic dose of YDR (18 µg/kg of YDR). The results indicated that, following 14 days of treatment, both the verapamil and YDR successfully ameliorated the…

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  Figure 5. Biomarker analysis for effective dose screening and therapeutic dose treatment of Yogendra Ras (YDR) in Danio rerio stimulated with erythromycin. (A) Effective dose screening of YDR in Danio rerio showed no change in the septum wall thickness of the ERY-stimulated D. rerio compared to the disease control. (B) ERY stimulation of the D. rerio significantly increased the systemic release of the inflammatory C-reactive protein. This was reduced in a dose-dependent manner through the treatment of the ERY-stimulated fish. (C) ERY stimulation of the D. rerio significantly decreased the platelet aggregation time. YDR treatment of the ERY-stimulated D. rerio at varying concentrations recovered the platelet aggregation time back to normal. (D) Therapeutic treatment of the ERY-stimulated D. rerio with YDR (18 µg/kg) and standard of care drug, verapamil (4 µg/kg) significantly reduced the CH-associated increase in septum wall thickness. (E) Therapeutic treatment of the ERY-stimulated D. rerio with YDR (18 µg/kg) significantly reduced the release of the inflammatory C-reactive protein. Verapamil did not induce any quantitative change in the release of the inflammatory protein. (F) Therapeutic…

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  Figure 6. Dose screening and oxidative stress analysis in H9C2 cells exposed to isoproterenol (ISP) and Yogendra Ras (YDR). (A) MTT-based dose screening in the H9C2 cells showed a dose-dependent loss of cell viability following exposure to the ISP (IC20 = 50 µM and IC50 = 122.4 µM) and YDR (IC20= 30 µg/mL and IC50 = 205 µg/mL). (B) Stimulation of the H9C2 cells with ISP (50 µM) induced up-regulation in the production of intracellular reactive oxygen species and its reduction following treatment with YDR. (C) Mitochondrial membrane potential (MMP) increment was detected in the ISP-stimulated H9C2 cells. It was reduced following co-treatment with YDR. (D) YDR also reduced the ISP-stimulated generation of superoxide ions (O−2) in H9C2 cardiomyocytes as indicated through the intracellular formation of formazan in the NBT assay. (E) Reduction in the generation of O−2 in the ISP-stimulated H9C2 cells following treatment with YDR was confirmed through the recovery in the intracellular levels of superoxide dismutase (SOD) enzyme. (F) Intracellular catalase enzyme levels were also recovered in the ISP-stimulated H9C2 cells following treatment with YDR. All the experiments were performed in…

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  Figure 7. Down-regulation of cardiac-hypertrophy-associated non-clinical and clinical molecular biomarkers by Yogendra Ras (YDR) in the isoproterenol (ISP)-stimulated H9C2 cells. H9C2 cells stimulated with ISP showed an increase in the mRNA expression levels of the pro-inflammatory mediator genes: (A) Cyclooxygenase-2 (COX-2), (B) NADPH oxidase-2 (NOX-2). and (C) NADPH oxidase-4 (NOX-4) using quantitative real-time PCR. Significant down-regulation of these pro-inflammatory genes was observed following a co-treatment of the ISP-stimulated H9C2 cells with YDR. Treatment of the H9C2 cells with ISP also induced an up-regulation of the expression of the fetal cardiac fetal gene (D) atrial natriuretic factor (ANF) and (E) cardiolipin (CRLS-1), (F) troponin I (TNN-I), and (G) troponin T (TNN-T). Expression of all the genes was normalized against the house-keeping gene β-actin. Co-treatment of the ISP-stimulated H9C2 cells with YDR significantly reduced the expression of all the up-regulated non-clinical and clinical genes. H) The heat map was generated to summarize the gene expression study. All the experiments were performed in biological and technical triplicates. Results represent mean ± SD.…