Water Extract of Capsella bursa-pastoris Mitigates Doxorubicin-Induced Cardiotoxicity by Upregulating Antioxidant Enzymes

Figures

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  Figure 1. Total ion chromatogram of hot-water extract of Capsella bursa-pastoris (CBW) obtained by ultra-performance liquid chromatography (UPLC) triple quadrupole mass spectrometry (UPLC-TQ-MS). Twenty micrograms of CBW were injected into the UPLC system followed by TQ-MS analysis, and a total ion chromatogram was acquired over a period of 21 min. The internal standard (IS), 13C3-catechin, was used with a retention time of 3.8 min.

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  Figure 2. CBW inhibits doxorubicin (DOX)-induced death of rat cardiomyocytes, H9c2, without affecting the death of MDA-MB-231 breast cancer cells. (A) CBW protects rat cardiomyocytes against DOX-induced cell death. H9c2 cells were pretreated with CBW for 24 h. Then, 2 μM DOX was added for another 48 h, and cells were observed using an optical microscope. (B) CBW blocked DOX-induced ATP loss. The values are presented as mean ± standard deviation (SD) of three independent experiments. #### p < 0.0001 (control vs. DOX) (Student’s t-test); **** p < 0.0001 (DOX vs. CBW) (one-way analysis of variance [ANOVA]). (C) CBW increased the viability of H9c2 cells with (left panel) or without (right panel) DOX treatment. The values are presented as mean ± SD of three independent experiments. #### p < 0.0001 (control vs. DOX) (Student’s t-test); **** p < 0.0001 (DOX vs. CBW) (one-way ANOVA) (left panel); *** p < 0.001 (control vs. CBW) (one-way ANOVA) (right panel). (D) Effect of CBW on DOX-induced death of MDA-MB-231 cells. MDA-MB-231 cells were pretreated with CBW for 24 h. Then, 2 μM DOX was added for another 48 h, and cell viability was measured. The values are presented as mean ± SD of three…

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  Figure 3. CBW reverses DOX-induced mitochondrial dysfunction. Mitochondrial reactive oxygen species (ROS) generation in H9c2 cells (200×). (A) Representative images demonstrating a dose-dependent decrease (left panel). Fluorescence intensity was quantified using ImageJ software (Ver. 1.53). The quantified intensities as the relative fold changes with respect to the control values are presented as mean ± SD of three independent experiments. #### p < 0.0001 (control vs. DOX) (Student’s t-test); *** p < 0.001 and **** p < 0.0001 (DOX vs. CBW) (one-way ANOVA). (B–E) CBW recovered DOX-induced decrease in mitochondrial respiration. Mitochondrial oxygen consumption rate (OCR) was analyzed in H9c2 cells. The arrow indicates the sequential addition of 1.5 μM oligo, 2 μM p-triflouromethoxyphenylhydrazone (FCCP), and 0.5 μM rotenone with 0.5 μM antimycin A (AA). OCR was expressed as pM O2/min/µg protein (B). Basal respiration was calculated as the difference between the pre-oligomycin and Rot + AA treatment values (C). Maximal respiration was converted with OCR between FCCP and Rot + AA treatment (D). ATP production was calculated by subtracting the pre-oligomycin treatment values from…

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  Figure 4. CBW prevents DOX-induced apoptotic cell death through Nrf2-mediated SOD activation in H9c2 cells. (A,B) CBW increased SOD1, SOD2, and HO-1 expression following DOX treatment in H9c2 cells. The results were demonstrated as the relative fold change with respect to the control values, and the values are presented as mean ± SD of three independent experiments. The means with different superscript letters are significantly different. #### p < 0.0001 (control vs. DOX) (Student’s t-test); * p < 0.05, *** p < 0.001, and **** p < 0.0001 (DOX vs. CBW) (one-way ANOVA) (A). Total protein was extracted from the cells and used for immunoblot assay (B). (C) CBW reversed DOX-induced Nrf2 reduction in H9c2 cells. Total protein was extracted from the cells and then used for immunoblot analysis. (D) CBW decreased the cleavage of caspase-3 and PARP in H9c2 cells. Total lysates were used for immunoblot analysis (left panel). Band intensities were quantified using Fusion analysis software (Ver. 16.07). The quantified expression was normalized with respect to that of the internal control, β-actin (right panel). The values are presented as mean ± SD of three independent experiments. The means with…

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  Figure 5. CBW protects cardiac function and diminishes cardiac injury in a DOX-induced cardiotoxicity in vivo model. (A) In vivo study scheme. (B) CBW ameliorated DOX-induced cardiotoxicity (DICT)-induced cardiac dysfunction. ECG was monitored without anesthesia using a non-invasive method. Heart rate, RR interval, QT prolongation, and ST prolongation were measured (n = 3/group). The values are presented as mean ± standard error (SE). # p < 0.05 and #### p < 0.0001 (control vs. DOX) (Student’s t-test); * p < 0.05 and ** p < 0.01 (DOX vs. CBW or Berberine [Ber]) (one-way ANOVA). (C) CBW improved DOX-induced toxicity indexes. The values are presented as mean ± SE. # p < 0.05, ## p < 0.01, and ### p < 0.001 (control vs. DOX) (Student’s t-test); ** p < 0.01 (DOX vs. CBW or Ber) (one-way ANOVA). (D,E) CBW administration inhibited DOX-induced cardiac injury. Cardiac fibrosis and DNA damage caused by DICT were determined using Masson’s Trichrome staining (D) and TUNEL staining (E), respectively. (magnification, 40×).

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  Figure 6. CBW reverses DOX-induced abnormal physiological changes in human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs). (A) Normal cord blood-cell-derived iPSCs were differentiated into cardiomyocytes. To validate hiPSC-CMs, cells were stained with the indicated antibodies, MLC2v or TNNT2, and analyzed using FACS. (B) Scheme of studies of hiPSC-CMs. (C) CBW improved the field potential in response to DOX. Field potential traces were recorded with the multielectrode array (MEA) exhibiting a mean spontaneous beating trace (right panel). Representative trace images were analyzed using field potential trace data (left panel). The values are presented as mean ± SD. ns, not significant (p < 0.05), * p < 0.05, *** p < 0.001, and **** p < 0.0001 (DOX vs. CBW) (one-way ANOVA). (D,E) CBW improved the decreased conduction velocity (D) and spike amplitude (E) by DOX treatment. Conduction velocity with percentage changes compared to that of the basal line was measured using MEA. The values are presented as mean ± SD. ns, not significant (p < 0.05), * p < 0.05, *** p < 0.001, and **** p < 0.0001 (DOX vs. CBW) (one-way ANOVA). (F) CBW recovered delayed beat period and irregular beat…

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  Figure 7. CBW ameliorates DOX-induced cardiotoxicity (DICT) by inhibiting the DOX-induced Nrf2 reduction which consequently increases the mRNA and protein expression of antioxidant enzymes, SOD1 and SOD2. This increased SOD activity suppresses ROS generation in mitochondria; hence, the DOX-induced disruption of mitochondrial membrane potential and respiration is subsequently restored. Furthermore, CBW administration inhibits collagen accumulation in cardiomyocytes and blocks apoptotic cell death, leading to protecting cardiac functions against DICT. Abbreviations: DOX, doxorubicin; CBW, water extract of Capsella bursa-pastoris; NRF2, nuclear factor erythroid-2-related factor 2; SOD, superoxide dismutase; ROS, reactive oxygen species; Casp-3, Caspase-3.