Cell-Permeable Microprotein from Panax Ginseng Protects Against Doxorubicin-Induced Oxidative Stress and Cardiotoxicity

Figures

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  Figure 1. Ginsentide TP1 is a cystine-dense pseudocyclic peptide from ginseng. (A) Primary structure of ginsentide TP1 from Panax ginseng and P. notoginseng, showing disulphide linkages and its pseudocyclic structure with both of its terminals, C(I) and C(VIII), forming disulphides with internal cysteine. (B) The 3D structure of ginsentide TP1 (PDB: 2ML7) to support its compact and pseudocyclic structure. (C) Loops formed by four disulphides to highlight the pseudocyclic nature of TP1 with the N-terminal loop1 and the C-terminal loop6 in close proximity [see the top of TP1 3D structure in (B)]. Coloured circles indicate amino acid residues and connecting lines depicting disulphide bonds.

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  Figure 2. TP1 is nontoxic and prevents DOX-mediated cytotoxicity in H9c2 cardiomyocytes. (A) TP1 is nontoxic and nonmembranolytic—MTT-based cell viability analysis of H9c2 cells treated with 100μM of TP1 for 24 h. PBS was used as vehicle control, and 1% Triton X-100 (TX-100) was used as a positive control for cell death. n = 3; * p < 0.05 compared to PBS (left panel). LDH release-based cytotoxicity and membranolytic analysis of cells treated with 100 μM of TP1 for 24 h. n = 3; * p < 0.05 compared to 1% TX-100 (right panel). (B) TP1 prevents DOX-mediated cell death and increases cell viability. Cells were treated with PBS or 2 μM DOX with or without TP1 for 24 h, and viability was measured using an MTT assay. Data were presented as the percentage viability of the PBS control group. Data were presented as mean ± SEM (n = 4, analysis of variance (ANOVA) with Sidak’s multiple comparisons test). * p < 0.05 vs. only the DOX-treated group (left panel). TP1 mitigates DOX-induced cytotoxicity and membranolysis. A post-treated cell culture medium was assayed for LDH release. Data were represented as a percentage of LDH release in 1% Triton X 100-treated cells. Data were presented as mean ± SEM (n =…

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  Figure 3. Anti-apoptotic effect of TP1 in DOX-treated H9c2 cardiomyocytes. Cells were treated with DOX for 24 h with or without TP1. Apoptotic cells in the post-treated samples were examined using annexin V/PI staining-based flow cytometric analysis. (A) Respective images show only annexin V-positive early apoptotic cells (Q1), both annexin V and PI-positive late apoptotic cells (Q2), only PI-positive necrotic cells (Q3), and normal (Q4) cell population. (B) Apoptotic and necrotic cell populations in each group were expressed as a percentage of the total 20,000 cell population. Data presented as mean ± SEM (n = 3, analysis of variance (ANOVA) with Sidak’s multiple comparisons test). * p < 0.05 vs. PBS control group and # p < 0.05 vs. only DOX-treated group. (C) Microscopic images showing the nuclei morphology of H9c2 cells. Post-treated cells were stained with Hoechst 33342 and observed using a fluorescent microscope. DOX-treated cells displayed morphological changes such as fragmented nuclei (red arrow) and other distorted nuclei of varying nuclear shapes and sizes (yellow arrows) compared to the PBS/TP1-treated groups. Scale bars = 100 µm. (D) Representative Western blot images show that…

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  Figure 4. TP1 restores intracellular calcium (Ca2+) homeostasis and augments mitochondrial membrane potential (MMP) in DOX-treated H9c2 cells. (A) Representative microscopic images show that TP1 co-administration reduces DOX-mediated Ca2+ overload. Intracellular Ca2+ content was observed and calculated using a ratiometric Fluo-4-AM probe. Intracellular Ca2+ level was presented as the relative intensity of the Fluo-4 signal to PBS-treated control cells. Data were presented as representative microscopic images and mean values (±SEM) of about 100 cells in each group from experimental triplicates (analysis of variance (ANOVA) with Tukey’s multiple comparisons test). * p < 0.05 vs. PBS control group and # p < 0.05 vs. only DOX-treated group. Scale bars, 50 µm. (B) Representative microscopic images show that TP1 co-treatment restores MMP in DOX-treated cells. MMP of H9c2 cells was measured and quantified by Rhodamine 123 uptake. Scale bars = 200 µm. The relative MMP of respective experimental groups was calculated using ratiometric Rhodamine 123 uptake. Data in each group were normalised and expressed as a fold of the PBS-treated control group. Data were shown as mean values (± SEM) (n = 3,…

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  Figure 5. TP1 mitigates oxidative stress-linked inflammatory responses in DOX-treated H9c2 cells. (A) TP1 inhibits DOX-mediated ROS generation in H9c2 cells. Cells were treated with PBS or DOX with or without TP1 for 24 h, and ROS activity was observed using a DCFH-DA redox probe. Scale bar = 200 µm. Relative ROS levels were estimated through DCFH-DA-based fluorometric assay. Data were presented as mean ± SD (n = 3, analysis of variance (ANOVA) with Tukey’s multiple comparisons test). * p < 0.05 vs. PBS-treated control groups and # p < 0.05 vs. DOX-treated groups. (B) TP1 prevents DOX-induced inflammatory responses in H9c2 cells. Proinflammatory cytokines’ TNF-α and IL-6 expression and secretion were estimated with ELISA using respective cell lysates (CL) and condition media (CM). Data were presented as mean values (±SEM) (n = 3, ANOVA with Sidak’s multiple comparisons test). * p < 0.05 vs. PBS-treated control groups and # p < 0.05 vs. DOX-treated groups.

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  Figure 6. Ginsentide TP1 mitigates DOX-induced cardiotoxicity and restores cardiac function in zebrafish. In total, 24 hpf zebrafish embryos were used to visualise and evaluate the cardiac functions and 24 hpf embryos were treated with Sham control (Fish medium) or 50 µM of DOX with or without TP1 co-treatment. Six randomly selected larvae for each group from three cohorts were used for assessment. (A,B) Representative images of zebrafish embryos after 48 h and 72 h post-treatment. Red arrows indicate the cardiac sacs of zebrafish embryos. Enlarged cardiac sacs were detected in DOX-treated embryos, indicating the presence of pericardial oedema. Scale bar = 100 μm. (C) TP1 restores the heartbeat rates of DOX-treated zebrafish. The heartbeat of individual embryos was measured at 24 h, 48 h, and 72 h post-treatment. Data are presented as means ± SEM of 3 independent experiments (n = 45, analysis of variance (ANOVA) with Sidak’s multiple comparisons test). * p < 0.05 vs. Sham control group and # p < 0.05 vs. DOX-treated group.

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  Figure 7. Ginsentide TP1-cotreatment prevents DOX-induced ventricular dysfunction in zebrafish. Zebrafish transgenic line Tg (cmlc2: gCaMP) expressing a calcium-sensitive green fluorescent protein (GFP) in cardiomyocytes was used to visualise and evaluate the cardiac functions of zebrafish embryos. Twenty-four hpf embryos were treated with fish medium (Sham control) or 50 µM of DOX with or without TP1 co-treatment for 72 h. Data were collected from three cohorts, and six embryos were selected randomly from each group. (A) Representative images of hearts of pre-treated zebrafish embryos. Images show ventricular contractility of 72 h post-treated embryos. ‘V’ represents the ventricle and ‘A’ represents the atrium. Red dotted lines indicate the outlines of the ventricles. Scale bar = 50 μm (B) The ventricular ejection fraction (EF) was calculated from diastolic and systolic volume after 72 h post-treatment. Data were presented as means ± SEM (n = 18, analysis of variance (ANOVA) with Sidak’s multiple comparisons test). * p < 0.05 vs. Sham control group and # p < 0.05 vs. DOX-treated group.

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  Figure 8. Intraperitoneal administration of ginsentide TP1 mitigates DOX-induced acute myocardial injury in mice. Male ICR mice were prophylactically treated with resveratrol (RES) (50 mg/kg) and TP1 (20 mg/kg), one dose per day for 5 days. On experiment Day 2, 1 h after administration of RES or TP1, DOX was injected intraperitoneally to induce acute myocardial injury. On experiment Day 1, 3, and 5, the ECG of the animals were recorded, and serum was withdrawn accordingly. Eight mice were used for each group. Data represented as mean and standard error (±SE) and significance were calculated using analysis of variance (ANOVA) with Sidak’s multiple comparisons tests. (A) Ginsentide TP1 reduces serum levels of creatine kinase MB isoenzyme (CK-MB) and lactate dehydrogenase (LDH) in DOX-induced acute myocardial injury in mice (** p < 0.01 vs. control, # p < 0.05 and ## p < 0.01 vs. model). (B) Oral or intraperitoneal administration of RES and TP1, respectively, improved the heart rate of DOX-induced acute myocardial injury mice (** p < 0.01 vs. control, ## p < 0.01 vs. model). (C) Oral or intraperitoneal administration of RES and TP1, respectively, restored the QRS interval in DOX-treated mice…

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  Figure 9. Ginsentide TP1 is cardioprotective and mitigates DOX-induced cardiotoxicity.