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Calycosin attenuates mitochondrial damage and pyroptosis in heart failure via the Nrf2/ROS/TXNIP pathway

  • Authors:
    • Hua-Jing Yuan
    • Quan-Cheng Han
    • Yi-Ding Yu
    • Hui Yu
    • Xiu-Juan Liu
    • Yi-Tao Xue
    • Yan Li
  • View Affiliations / Copyright

    Affiliations: The First Clinical Medical College, Shandong University of Traditional Chinese Medicine, Jinan, Shandong 250014, P.R. China, Department of Cardiology, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, Shandong 250014, P.R. China
    Copyright: © Yuan et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 212
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    Published online on: September 30, 2025
       https://doi.org/10.3892/ijmm.2025.5653
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Abstract

Heart failure (HF) is a key public health concern worldwide due to its high morbidity and mortality rates. Calycosin (CA) is a flavonoid natural product that effectively treats HF with cardioprotective effects; however, its mechanism of action remains unclear. The present study aimed to investigate the therapeutic effect of CA on HF and its mechanism through in vivo and in vitro experiments, and to reveal the roles of pyroptosis and mitochondrial dysfunction in the pathophysiology of HF. The HF model was constructed 4 weeks after ligation of the left anterior descending artery in rats. Myocardial ischemia‑reperfusion injury was simulated using a hypoxia‑reoxygenation model and nuclear factor erythroid 2‑related factor (Nrf2) was silenced by transfection using small interfering RNA to further explore the therapeutic mechanism of CA. The results revealed that CA treatment improved cardiac function and myocardial injury, suppressed oxidative stress levels and improved mitochondrial ultrastructure in HF‑induced rats. CA downregulated the expression of relevant pyroptosis proteins via the Nrf2/reactive oxygen species (ROS)/thioredoxin‑interacting protein (TXNIP) pathway. In vitro experiments demonstrated consistent results confirming that CA ameliorated mitochondrial damage by reducing levels of ROS and inhibiting mitochondrial gasdermin D N‑terminal fragments activation. Silencing Nrf2 partially reversed the cardioprotective effects of CA, confirming the key therapeutic role of CA in Nrf2‑mediated anti‑pyroptosis. In conclusion, CA inhibits pyroptosis and improves mitochondrial damage in HF through the Nrf2/ROS/TXNIP pathway, which may disrupt the crosstalk between mitochondrial damage and pyroptosis, thereby exerting cardioprotective effects.
View Figures

Figure 1

CA effectively improves cardiac
function in HF. (A) Representative echocardiography, (B) H&E
staining and (C) Masson's trichrome staining images of each group.
In H&E staining, the yellow arrows indicate inflammatory
infiltration, the black arrows indicate loss of myocardial cells
and the blue arrows indicate interstitial fibrosis. (D) Statistical
analysis of LVEF and FS of rats in each group, as determined by
echocardiography (n=5). (E) Quantitative analysis of Masson's
trichrome staining (n=3). (F) Serum NT-proBNP levels in rats of
each group (n=5). Data are presented as the mean ± SD,
**P<0.01, ***P<0.001. CA, calycosin;
HF, heart failure; LVEF, left ventricular ejection fraction; FS,
fractional shortening; NT-proBNP, N-terminal pro B-type natriuretic
peptide; CA-L, calycosin low-dose; CA-H, calycosin high-dose; CAP,
captopril.

Figure 2

CA effectively improves mitochondrial
damage and cardiac oxidative stress in HF. (A) Representative
transmission electron microscopy images of rats in each group. (B)
Levels of oxidative stress-related indicators (n=5). Data are
presented as the mean ± SD, *P<0.05,
**P<0.01, ***P<0.001. CA, calycosin;
HF, heart failure; CA-L, calycosin low-dose; CA-H, calycosin
high-dose; CAP, captopril; LDH, lactate dehydrogenase; MDA,
malondialdehyde; SOD, superoxide dismutase; GSH, glutathione; GSSG,
GSH disulfide.

Figure 3

CA improves pyroptosis in HF through
the Nrf2/ROS/TXNIP pathway. (A) Representative Nrf2
immunohistochemistry images of each group and their quantitative
analysis (n=3). (B) Representative images of the expression levels
of Nrf2 and TXNIP in myocardial tissue of Wistar rats assessed by
western blotting. (C) Semi-quantitative analysis of Nrf2 and TXNIP
expressions (n=3). (D) Representative images of the expression
levels of NLRP3, ASC, pro- and cleaved-caspase-1, GSDMD, GSDMD-NT
in myocardial tissue of Wistar rats by western blotting. (E)
Semi-quantitative analysis of NLRP3, ASC, pro- and
cleaved-caspase-1, GSDMD, GSDMD-NT expressions (n=3). (F)
Representative images of the expression levels of IL-18 and IL-1β
in myocardial tissue of Wistar rats by western blotting. (G)
Semi-quantitative analysis of IL-18 and IL-1β expressions (n=3).
Data are presented as the mean ± SD, *P<0.05,
**P<0.01, ***P<0.001. CA, calycosin;
HF, heart failure; Nrf2, nuclear factor erythroid 2-related factor;
ROS, reactive oxygen species; TXNIP, thioredoxin-interacting
protein; CA-L, calycosin low-dose; CA-H, calycosin high-dose; ASC,
apoptosis-associated speck-like protein containing a CARD; GSDMD,
gasdermin D; GSDMD-NT, GSDMD N-terminal fragments.

Figure 4

CA protects cardiomyocytes and
inhibits pyroptosis. (A) Detection of CA cytotoxicity using a Cell
Counting Kit-8 assay (n=3). ###P<0.001, compared with
0, 5, 10, 20, 30 μM groups. (B) Cardiomyocyte protective
effects of CA (n=3). (C) Flow cytometric analysis of cell death
with Annexin V-FITC/PI staining. (D) Immunofluorescence staining of
cardiomyocytes for Nrf2. (E) Detection of intracellular ROS levels
in cardiomyocytes (n=3). Data are presented as the mean ± SD,
*P<0.05, ***P<0.001. CA, calycosin;
Nrf2, nuclear factor erythroid 2-related factor; ROS, reactive
oxygen species; H/R, hypoxia-reperfusion; LDH, lactate
dehydrogenase.

Figure 5

CA inhibits pyroptosis via the
Nrf2/reactive oxygen species/TXNIP pathway (n=3). (A)
Representative images of the expression levels and
semi-quantitative analysis of Nrf2 and TXNIP in cells by western
blotting (n=3). (B) and semi-quantitative analysis and
semi-quantitative analysis of IL-18 and IL-1β in cells by western
blotting and (n=3). (C) and semi-quantitative analysis and
semi-quantitative analysis of levels of NLRP3, ASC, pro- and
cleaved-caspase-1, GSDMD, GSDMD-NT in cells by western blotting
(n=3). Data are presented as the mean ± SD, *P<0.05,
**P<0.01, ***P<0.001. CA, calycosin;
Nrf2, nuclear factor erythroid 2-related factor; TXNIP,
thioredoxin-interacting protein; H/R, hypoxia-reperfusion, ASC,
apoptosis-associated speck-like protein containing a CARD; GSDMD,
gasdermin D; GSDMD-NT, GSDMD N-terminal fragments; NLRP3, NLR
family pyrin domain-containing protein 3.

Figure 6

CA improves mitochondrial damage in
cardiomyocytes. (A) Flow cytometric analysis of JC-1 staining was
used to detect changes in mitochondrial membrane potential (n=3).
(B) Representative intracellular ROS staining images of each group.
(C) Representative mitochondrial ROS staining images of each group.
(D) ECAR and OCR of cardiomyocytes were assessed (n=3). (E)
Expression levels of GSDMD, GSDMD-NT and cytochrome c
proteins were examined by western blotting (n=3). COX IV was used
as a loading control in mitochondria. Data are presented as the
mean ± SD, *P<0.05, **P<0.01,
***P<0.001. CA, calycosin; ROS, reactive oxygen
species; ECAR, extracellular acidification rate; OCR, oxygen
consumption rate; FCCP, Trifluoromethoxy carbonylcyanide
phenylhydrazone, Carbonyl cyanide
4-(trifluoromethoxy)phenylhydrazone; Rot/anti-A, rotenone/antimycin
A; GSDMD, gasdermin D; GSDMD-NT, GSDMD N-terminal fragments; H/R,
hypoxia-reperfusion.

Figure 7

Silencing Nrf2 inhibits the
therapeutic effects of CA. (A) Transfection efficiency of si-Nrf2.
(B) Detection of intracellular ROS levels in each group (n=3). (C)
Representative images and semi-quantitative analysis of Nrf2 and
TXNIP in cells by western blotting (n=3). (D) Representative images
and semi-quantitative analysis of NLRP3, ASC, pro- and
cleaved-caspase-1, GSDMD, GSDMD-NT in cells by western blotting
(n=3). (E) Representative images and semi-quantitative analysis
levels of IL-18 and IL-1β in cells by western blotting (n=3). Data
are presented as the mean ± SD, *P<0.05,
**P<0.01, ***P<0.001, ns, no
significance. si, small interfering; CA, calycosin; Nrf2, nuclear
factor erythroid 2-related factor; ROS, reactive oxygen species;
TXNIP, thioredoxin-interacting protein; ASC, apoptosis-associated
speck-like protein containing a CARD; NLRP3, NLR family pyrin
domain-containing protein 3; GSDMD, gasdermin D; GSDMD-NT, GSDMD
N-terminal fragments; H/R, hypoxia-reperfusion; NC, negative
control.

Figure 8

Molecular mechanisms of CA treatment
for HF. CA promotes Nrf2 expression and its nuclear translocation,
thereby alleviating mitochondrial damage and pyroptosis in HF via
the Nrf2/ROS/TXNIP pathway. This figure was generated by FigDraw.
CA, calycosin; HF, heart failure; LADL, left anterior descending
ligation; Nrf2, nuclear factor erythroid 2-related factor; ROS,
reactive oxygen species; TRX, thioredoxin; TXNIP,
thioredoxin-interacting protein; H/R, hypoxia-reoxygenation; NLRP3,
NLR family pyrin domain-containing protein 3; GSDMD, gasdermin D;
GSDMD-NT, GSDMD N-terminal fragments.
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Copy and paste a formatted citation
Spandidos Publications style
Yuan H, Han Q, Yu Y, Yu H, Liu X, Xue Y and Li Y: Calycosin attenuates mitochondrial damage and pyroptosis in heart failure via the Nrf2/ROS/TXNIP pathway. Int J Mol Med 56: 212, 2025.
APA
Yuan, H., Han, Q., Yu, Y., Yu, H., Liu, X., Xue, Y., & Li, Y. (2025). Calycosin attenuates mitochondrial damage and pyroptosis in heart failure via the Nrf2/ROS/TXNIP pathway. International Journal of Molecular Medicine, 56, 212. https://doi.org/10.3892/ijmm.2025.5653
MLA
Yuan, H., Han, Q., Yu, Y., Yu, H., Liu, X., Xue, Y., Li, Y."Calycosin attenuates mitochondrial damage and pyroptosis in heart failure via the Nrf2/ROS/TXNIP pathway". International Journal of Molecular Medicine 56.6 (2025): 212.
Chicago
Yuan, H., Han, Q., Yu, Y., Yu, H., Liu, X., Xue, Y., Li, Y."Calycosin attenuates mitochondrial damage and pyroptosis in heart failure via the Nrf2/ROS/TXNIP pathway". International Journal of Molecular Medicine 56, no. 6 (2025): 212. https://doi.org/10.3892/ijmm.2025.5653
Copy and paste a formatted citation
x
Spandidos Publications style
Yuan H, Han Q, Yu Y, Yu H, Liu X, Xue Y and Li Y: Calycosin attenuates mitochondrial damage and pyroptosis in heart failure via the Nrf2/ROS/TXNIP pathway. Int J Mol Med 56: 212, 2025.
APA
Yuan, H., Han, Q., Yu, Y., Yu, H., Liu, X., Xue, Y., & Li, Y. (2025). Calycosin attenuates mitochondrial damage and pyroptosis in heart failure via the Nrf2/ROS/TXNIP pathway. International Journal of Molecular Medicine, 56, 212. https://doi.org/10.3892/ijmm.2025.5653
MLA
Yuan, H., Han, Q., Yu, Y., Yu, H., Liu, X., Xue, Y., Li, Y."Calycosin attenuates mitochondrial damage and pyroptosis in heart failure via the Nrf2/ROS/TXNIP pathway". International Journal of Molecular Medicine 56.6 (2025): 212.
Chicago
Yuan, H., Han, Q., Yu, Y., Yu, H., Liu, X., Xue, Y., Li, Y."Calycosin attenuates mitochondrial damage and pyroptosis in heart failure via the Nrf2/ROS/TXNIP pathway". International Journal of Molecular Medicine 56, no. 6 (2025): 212. https://doi.org/10.3892/ijmm.2025.5653
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