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Article Open Access

Ligustroflavone protects against acute kidney injury by inhibiting ferroptosis via acting on GSK3β/NRF2 signaling

  • Authors:
    • Jiayu Song
    • Long Wang
    • Yaru Wang
    • Jie Meng
    • Jingyi Sheng
    • Yongfeng Zhao
    • San Yan Xu
    • Juan Lei
    • Fangfang Cai
    • Yunwen Yang
  • View Affiliations / Copyright

    Affiliations: Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, Nanjing, Jiangsu 210008, P.R. China, Department of Cardiology, Translational Medicine Center, Westlake Laboratory of Life Sciences and Biomedicine, Affiliated Hangzhou First People's Hospital, Westlake University School of Medicine, Hangzhou, Zhejiang 310006, P.R. China, Department of Neurology, Jiangsu Key Laboratory of Brain Disease and Bioinformation, Changzhou Wujin People's Hospital, Wujin Clinical College of Xuzhou Medical University, Changzhou, Jiangsu 213017, P.R. China, Department of Pediatric Nephrology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210003, P.R. China, Department of Cell Biology, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, Jiangsu 211166, P.R. China
    Copyright: © Song et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 170
    |
    Published online on: April 30, 2026
       https://doi.org/10.3892/ijmm.2026.5841
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Abstract

Ferroptosis exerts a recognized role in the pathogenesis of acute kidney injury (AKI) and is considered a critical target for improving its prognosis. Emerging evidence indicates that ferroptosis serves a pivotal role in pathogenesis of AKI and targeting ferroptosis provides a promising therapeutic strategy in treatment of AKI. In the present study, ligustroflavone (LIG), which is a flavonoid with oral activity extracted from Ligustrum lucidum, was found to inhibit ferroptosis through activation of nuclear factor erythroid 2‑related factor 2 (NRF2) via inhibition of GSK3β in vivo and in vitro. In vivo, cisplatin (CDDP) and ischemia‑reperfusion injury (IRI)‑induced murine models of AKI were constructed to evaluate the possible effects of LIG. In vitro, the protective effects of LIG were assessed in cultured mouse renal proximal tubular epithelial cells (TKPTs). Immunostaining, reverse transcription‑quantitative PCR, western blot and lipid peroxidation assays were performed to detect renal tubular injury and ferroptosis. The results of the present study demonstrated that LIG administration significantly ameliorated CDDP or IRI induced renal damage in mice. Additionally, administration of LIG significantly ameliorated lipid peroxide accumulation and inhibited ferroptosis in the kidneys of AKI mice. In vitro, LIG treatment markedly ameliorated CDDP‑induced lipid peroxidation and ferroptosis in cultured TKPTs via GSK3β inhibition and NRF2 activation. Furthermore, knockout of GSK3β also protected against CDDP‑induced cell death and LIG exerted no additional protective effects in GSK3β‑knockout TKPTs. Together, the present findings offer a new potential strategy for AKI therapies by targeting ferroptosis.
View Figures

Figure 1

LIG mitigates acute kidney injury
induced by CDDP in a murine model. (A) SCr and BUN levels in mice.
(B) Calculation of tubular injury score based on PAS staining. (C)
PAS staining for renal histopathology (magnification, x200 and
x400). (D) Western blotting detection of the expression of tubular
injury molecules KIM-1 and NGAL and semi-quantitative analysis of
protein band intensities is presented on the right (n=6).
*P<0.05, **P<0.01 and
****P<0.0001, (one-way ANOVA or unpaired t-test).
LIG, ligustroflavone; CDDP, cisplatin; SCr, serum creatinine; BUN,
blood urea nitrogen; PAS, periodic acid-Schiff; KIM-1, kidney
injury molecule-1; NGAL, neutrophil gelatinase-associated
lipocalin; vehicle, control; ns, not significant.

Figure 2

LIG treatment reduces cell apoptosis
and inflammatory response in acute kidney injury. (A) TUNEL
staining and quantitative analysis were conducted, with
corresponding images generated. (B) The expression of cleaved
caspase-3 was assessed using western blotting. (C) The staining
distribution of F4/80 was evaluated using immunohistochemistry. (D)
The mRNA levels of inflammatory genes including TNF, IL1β, IL-6 and
MCP1 in kidneys of cisplatin-induced mice with or without LIG (30
mg/kg/day) treatment were analyzed using reverse
transcription-quantitative PCR. The results are expressed as the
mean ± SD of 6 mice in each group. *P<0.05,
**P<0.01 and ****P<0.0001 (one-way
ANOVA or unpaired t-test). LIG, ligustroflavone; CDDP, cisplatin;
P-, phosphorylated.

Figure 3

LIG activates NRF2 by inhibiting
GSK3β to improve acute kidney injury. (A) Molecular docking
analysis of the candidate targets with LIG. (B) A three-dimensional
molecular interaction model. (C) A cellular thermal shift assay was
performed to verify the direct binding of LIG to GSK3β. (D) WB of
GSK3β and its phosphorylation in TKPTs treatment with LIG (0, 5,
10, 15, 20, 30 μM) for 24 h, the semi-quantitative analysis
of protein band intensities is presented on Fig. S1B. (E) Subcellular localization
of NRF2 were analyzed by fraction western blot in TKPTs treated
with LIG (10, 20 μM) for 24 h, the semi-quantitative
analysis of protein band intensities is presented on Fig. S1C. (F) WB of GPX4 and HO-1 in
TKPTs treatment with LIG (10, 20 μM) for 24 h, the
semi-quantitative analysis of protein band intensities is presented
on Fig. S1D. (G) The protein
levels of NRF2 and p-GSK3β (Ser9) in mice kidneys daily gavage with
LIG (30 mg/kg/day) or vehicle for 72 h, the semi-quantitative
analysis of protein band intensities is presented on Fig. S1F. (H) Immunofluorescent
staining of NRF2, LTL and DAPI in mice kidneys daily gavage with
LIG (30 mg/kg/day) or vehicle for 72 h. LIG, ligustroflavone; NRF2,
nuclear factor erythroid 2-related factor 2; GSK3β, glycogen
synthase kinase 3β; TKPTs, mouse renal proximal tubular epithelial
cells; WB, western blotting; p-, phosphorylated; LTL, Lotus
tetragonolobus lectin.

Figure 4

LIG treats acute kidney injury by
inhibiting the ferroptosis pathway. (A) The concentration of
Fe2+ levels. (B) The levels of MDA. (C) The GSH/GSSG
ratio. (D) The expression of genes related to ferroptosis. (E) WB
analysis the protein levels of GPX4, MPO and p-GSK3β (Ser9) in mice
kidneys daily gavage with LIG (30 mg/kg/day) or vehicle then
treated with cisplatin (20 mg/kg) for 72 h. (F) WB analysis the
protein levels of ATPB and SOD2 in mice kidneys. (G)
Immunofluorescent staining for 4-HNE in mice kidneys. (H)
Transmission electron microscopy observation of mitochondrial
damage in mice kidneys, the ferroptosis-related damaged
mitochondria characterized by mitochondrial atrophy, reduced or
even disappearing cristae and increased membrane density is
indicated by red arrow. *P<0.05,
**P<0.01, ***P<0.001 and
****P<0.0001 (one-way ANOVA or two-way ANOVA). LIG,
ligustroflavone; MDA, malondialdehyde; GSH, reduced glutathione;
GSSG, glutathione disulfide; WB, western blotting; GPX4,
glutathione peroxidase 4; MPO, myeloperoxidase; p-, phosphorylated;
GSK3β, glycogen synthase kinase 3β; ATPB, ATP synthase subunit b;
SOD2, superoxide dismutase 2; 4-HNE, 4-hydroxynonenal; CDDP,
cisplatin.

Figure 5

LIG protects against CDDP-induced
damaged cells by regulating oxidative stress, lipid peroxidation
and the GSK3β pathway. (A) Cell viability performed by CCK-8 assay
in TKPTs treated with LIG (20 μM) and cisplatin (5
μg/ml) for 24 h. (B) LDH release was analyzed. (C) MDA
levels were detected. (D) The GSH/GSSG ratio of TKPTs was analyzed.
(E) Cisplatin induced lipid peroxidation in TKPTs were analyzed by
staining with a BODIPY 581/591 C11 probe (green, oxidized lipids;
blue, Hoechst; scale bar, 10 μm). (F) The successful
construction of GSK3β-knockout TKPTs was analyzed using western
blotting. (G) GSK3β-knockout and control TKPTs were treated with
cisplatin with or without LIG treatment, and the cell viability was
detected using CCK-8 assay. (H) TKPTs were treated with or without
cisplatin for 24 h with lipro-1 (1 μM), LIG (20 μM)
or LIG (20 μM) + lipro-1 (1 μM) treatment, and the
cell viability was detected using a CCK-8 assay. The data are
presented as the mean ± SD. **P<0.01,
***P<0.001 and ****P<0.0001 (one-way
ANOVA or two-way ANOVA). LIG, ligustroflavone; CDDP, cisplatin;
GSK3β, glycogen synthase kinase 3β; CCK-8, Cell Counting Kit-8;
TKPTs, mouse renal proximal tubular epithelial cells; LDH, lactate
dehydrogenase; MDA, malondialdehyde; GSH, reduced glutathione;
GSSG, glutathione disulfide; lipro-1, liproxstatin-1; ns, not
significant; NC, negative control.

Figure 6

LIG prevents IRI-induced AKI by
inhibiting ferroptosis. (A) BUN and SCr levels in IRI-induced AKI
mice. (B) MDA levels in kidneys of mice with IRI-induced mice. (C)
The concentration of Fe2+. (D) Periodic acid-Schiff
staining for renal histopathology. (E) Western blot analysis of
KIM-1, NGAL, NRF2 and GPX4 expression. (F) Transmission electron
microscopy observation of mitochondrial damage.
*P<0.05, **P<0.01,
***P<0.001 and ****P<0.0001 (one-way
ANOVA or two-way ANOVA). LIG, ligustroflavone; IRI,
ischemia/reperfusion injury; AKI, acute kidney injury; BUN, blood
urea nitrogen; SCr, serum creatinine; MDA, malondialdehyde; KIM-1,
kidney injury molecule-1; NGAL, neutrophil gelatinase-associated
lipocalin; NRF2, nuclear factor erythroid 2-related factor 2; GPX4,
glutathione peroxidase 4.

Figure 7

Schematic diagram of the present
study. Overview of the role and mechanism of ligustroflavone in
acute kidney injury. GSK3β, glycogen synthase kinase 3β; GPX4,
glutathione peroxidase 4; NRF2, nuclear factor erythroid 2-related
factor 2.
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Copy and paste a formatted citation
Spandidos Publications style
Song J, Wang L, Wang Y, Meng J, Sheng J, Zhao Y, Xu SY, Lei J, Cai F, Yang Y, Yang Y, et al: Ligustroflavone protects against acute kidney injury by inhibiting ferroptosis via acting on GSK3&beta;/NRF2 signaling. Int J Mol Med 58: 170, 2026.
APA
Song, J., Wang, L., Wang, Y., Meng, J., Sheng, J., Zhao, Y. ... Yang, Y. (2026). Ligustroflavone protects against acute kidney injury by inhibiting ferroptosis via acting on GSK3&beta;/NRF2 signaling. International Journal of Molecular Medicine, 58, 170. https://doi.org/10.3892/ijmm.2026.5841
MLA
Song, J., Wang, L., Wang, Y., Meng, J., Sheng, J., Zhao, Y., Xu, S. Y., Lei, J., Cai, F., Yang, Y."Ligustroflavone protects against acute kidney injury by inhibiting ferroptosis via acting on GSK3&beta;/NRF2 signaling". International Journal of Molecular Medicine 58.1 (2026): 170.
Chicago
Song, J., Wang, L., Wang, Y., Meng, J., Sheng, J., Zhao, Y., Xu, S. Y., Lei, J., Cai, F., Yang, Y."Ligustroflavone protects against acute kidney injury by inhibiting ferroptosis via acting on GSK3&beta;/NRF2 signaling". International Journal of Molecular Medicine 58, no. 1 (2026): 170. https://doi.org/10.3892/ijmm.2026.5841
Copy and paste a formatted citation
x
Spandidos Publications style
Song J, Wang L, Wang Y, Meng J, Sheng J, Zhao Y, Xu SY, Lei J, Cai F, Yang Y, Yang Y, et al: Ligustroflavone protects against acute kidney injury by inhibiting ferroptosis via acting on GSK3&beta;/NRF2 signaling. Int J Mol Med 58: 170, 2026.
APA
Song, J., Wang, L., Wang, Y., Meng, J., Sheng, J., Zhao, Y. ... Yang, Y. (2026). Ligustroflavone protects against acute kidney injury by inhibiting ferroptosis via acting on GSK3&beta;/NRF2 signaling. International Journal of Molecular Medicine, 58, 170. https://doi.org/10.3892/ijmm.2026.5841
MLA
Song, J., Wang, L., Wang, Y., Meng, J., Sheng, J., Zhao, Y., Xu, S. Y., Lei, J., Cai, F., Yang, Y."Ligustroflavone protects against acute kidney injury by inhibiting ferroptosis via acting on GSK3&beta;/NRF2 signaling". International Journal of Molecular Medicine 58.1 (2026): 170.
Chicago
Song, J., Wang, L., Wang, Y., Meng, J., Sheng, J., Zhao, Y., Xu, S. Y., Lei, J., Cai, F., Yang, Y."Ligustroflavone protects against acute kidney injury by inhibiting ferroptosis via acting on GSK3&beta;/NRF2 signaling". International Journal of Molecular Medicine 58, no. 1 (2026): 170. https://doi.org/10.3892/ijmm.2026.5841
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