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Brazilin attenuates methylglyoxal‑induced endothelial injury by enhancing eIF5A hypusination and inhibiting the AMPK/mTOR‑mediated autophagy/apoptosis axis in vitro and in vivo

  • Authors:
    • Yu Liang
    • Jinxiang Chen
    • Haiyang Li
    • Junjing Xiong
    • Jingcan You
    • Liqun Wang
    • Jianbo Wu
    • Youkun Zheng
    • Mao Luo
  • View Affiliations / Copyright

    Affiliations: Basic Medicine Research Innovation Center for Cardiometabolic Diseases, Ministry of Education, Southwest Medical University, Luzhou, Sichuan 646000, P.R. China
    Copyright: © Liang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 273
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    Published online on: July 30, 2026
       https://doi.org/10.3892/ijmm.2026.5944
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Abstract

Methylglyoxal (MGO), a glycolytic metabolic byproduct and major precursor of advanced glycation end products (AGEs), accumulates and is critically involved in diabetes, particularly in diabetic vascular complications. In endothelial cells (ECs), MGO has been implicated in oxidative stress and inflammatory responses, and it can further induce autophagy and apoptosis. Brazilin (BZ), a natural compound, confers cytoprotection by allosterically activating deoxyhypusine hydroxylase (DOHH), thereby enhancing eukaryotic initiation factor 5A (eIF5A) hypusination, a distinctive post‑translational modification. Although BZ can mitigate vascular inflammation and regulate autophagy and apoptosis, its effects on MGO‑induced autophagy and apoptosis and the underlying mechanism remain elusive. In the present study, it was demonstrated that BZ pretreatment conferred cytoprotection by targeting DOHH and the downstream eIF5A signaling cascade, thereby suppressing activation of the AMP‑activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signaling pathway. Moreover, 3‑methyladenine (3‑MA) and Compound C strengthened the inhibitory effect of BZ on MGO‑induced autophagy and apoptosis. By contrast, ciclopirox, a specific inhibitor of DOHH, abolished the BZ‑mediated suppression of MGO‑induced cellular responses. The in vivo experiments further confirmed that BZ suppresses MGO‑triggered autophagy and apoptosis. Using db/db mice to mimic the diabetic microenvironment, it was further found that DOHH inhibition reversed the BZ‑mediated suppression of autophagy and apoptosis. Collectively, the findings of the present study reveal a DOHH/eIF5A‑AMPK/mTOR axis through which BZ antagonizes MGO‑induced autophagy and apoptosis, shedding new light on the pharmacological mechanism of BZ and underscoring its therapeutic potential for diabetic vascular complications.
View Figures

Figure 1

Stereostructures of MGO and BZ and
the results of the CCK-8 method for screening the appropriate
concentrations of MGO and BZ. (A and B) MGO and BZ stereoscopic
molecular structures. (C and D) The effects of varying MGO and BZ
concentrations on cell viability were assessed by CCK-8 assay. (E)
Human umbilical vein endothelial cells were exposed to BZ at 1, 2,
and 5 μM for 2 h before treatment with 100 μM MGO for
24 h, and cell viability was subsequently assessed.
###P<0.001 vs. Ctrl; *P<0.05 and
***P<0.001 vs. MGO. MGO, methylglyoxal; BZ, brazilin;
CCK-8, Cell Counting Kit-8.

Figure 2

BZ attenuates MGO-induced autophagy
in endothelial cells. Human umbilical vein endothelial cells were
exposed to BZ at 1, 2, and 5 μM for 2 h before treatment
with 100 μM MGO for 24 h. (A) Changes in the level of
autophagy were detected by transmission electron microscopy. Scale
bar, 1 μm. (B) Changes in autophagy were detected by LC3
immunofluorescence, with red fluorescence representing LC3 and blue
fluorescence representing DAPI. (C and D) The LC3-II/LC3-I ratio
and the protein expression of Beclin1 and p62 were evaluated by
western blot analysis. (E) The mechanism of action of BZ on DOHH.
(F and G) Hypusinated eIF5A, eIF5A and DOHH protein expression was
measured by western blot analysis. #P<0.05 and
##P<0.01 vs. Ctrl; *P<0.05 and
**P<0.01 vs. MGO. MGO, methylglyoxal; BZ, brazilin;
DOHH, deoxyhypusine hydroxylase; eIF5A, eukaryotic translation
initiation factor 5A.

Figure 3

MGO-induced apoptosis is reversed by
BZ. (A and B) After trypsinization, cells were stained with Annexin
V-FITC and PI for 20 min, followed by flow cytometric
quantification of apoptosis. (C-F) Bax, cleaved
caspase-3/caspase-3, Bcl-2, p-mTOR, mTOR, p-AMPK and AMPK protein
expression was assessed by western blot analysis.
#P<0.05 ##P<0.01 and
###P<0.001 vs. Ctrl; *P<0.05
**P<0.01 and ***P<0.001 vs. MGO. MGO,
methylglyoxal; BZ, brazilin; p-, phosphorylated.

Figure 4

Inhibitory effect of BZ on
MGO-induced autophagy and apoptosis in HUVECs was enhanced by 3-MA.
HUVECs were preincubated with BZ (5 μM) in the presence or
absence of 3-MA (5 mM) for 2 h, followed by exposure to MGO (100
μM) for 24 h. (A) Changes in autophagy levels were detected
by transmission electron microscopy. Scale bar, 1 μm. (B)
Autophagy was detected by LC3 immunofluorescence. (C-F) Protein
expression of LC3-II/LC3-I, Beclin1, p62, Bcl-2, Bax and cleaved
caspase-3/caspase-3 was evaluated by western blotting.
#P<0.05 and ##P<0.01 vs. Ctrl;
*P<0.05 and **P<0.01 vs. MGO;
&P<0.05 and &&P<0.01 vs.
MGO + BZ. MGO, methylglyoxal; BZ, brazilin; HUVECs, human umbilical
vein endothelial cells.

Figure 5

Dynamic time-course changes in
autophagy and apoptosis markers in HUVECs. (A and B) Western blot
analysis of the LC3-II/LC3-I and cleaved caspase-3/caspase-3 ratio
in HUVECs treated with MGO for 0, 6, 12 and 24 h.
#P<0.05, ##P<0.01 and
###P<0.001 vs. Ctrl. HUVECs, human umbilical vein
endothelial cells; MGO, methylglyoxal; ns, not significant.

Figure 6

Effect of MGO on autophagic flux in
endothelial cells. (A and B) Western blot analysis of LC3-II
protein levels in human umbilical vein endothelial cells treated
with MGO (100 μM) and/or CQ (10 μM) for 24 h.
#P<0.05, ##P<0.01 and
###P<0.001 vs. Ctrl. MGO, methylglyoxal; CQ,
chloroquine.

Figure 7

Effect of Compound C on BZ-mediated
inhibition of MGO-induced autophagy and apoptosis in HUVECs. HUVECs
were preincubated with BZ (5 μM) in the presence or absence
of Compound C (50 μM) for 2 h, followed by exposure to MGO
(100 μM) for 24 h. (A) Changes in the level of autophagy
were detected by transmission electron microscopy. Scale bar, 1
μm. (B and C) Apoptotic changes were assessed by flow
cytometry. (D and E) Protein expression of p-AMPK, AMPK, p-mTOR and
mTOR was evaluated by western blotting. #P<0.05,
##P<0.01 and ###P<0.001 vs. Ctrl;
*P<0.05, **P<0.01 and
***P<0.001 vs. MGO; &P<0.05 and
&&P<0.01 vs. MGO + BZ. BZ, brazilin; MGO,
methylglyoxal; HUVECs, human umbilical vein endothelial cells; p-,
phosphorylated.

Figure 8

BZ-mediated inhibition of MGO-induced
autophagy and apoptosis in HUVECs is reversed by ciclopirox. HUVECs
were preincubated with BZ (5 μM) or BZ (5 μM) + the
DOHH signaling pathway inhibitor ciclopirox (20 μM) for 2 h
and then combined with 100 μM MGO for 24 h. (A) Changes in
the level of autophagy were detected by transmission electron
microscopy. Scale bar, 1 μm. (B) Autophagy was detected by
LC3 immunofluorescence. (C and D) Apoptotic changes were assessed
by flow cytometry. (E and F) Hypusinated eIF5A, eIF5A and DOHH
protein expression was evaluated by western blotting.
###P<0.001 vs. Ctrl; *P<0.05 and
***P<0.001 vs. MGO; &P<0.05,
&&P<0.01 and
&&&P<0.001 vs. MGO + BZ. BZ, brazilin;
MGO, methylglyoxal; HUVECs, human umbilical vein endothelial cells;
DOHH, deoxyhypusine hydroxylase; eIF5A, eukaryotic translation
initiation factor 5A.

Figure 9

DOHH knockdown by si-DOHH reduces
hypusinated eIF5A levels and activates AMPK signaling. (A and B)
Western blot analysis of DOHH, hypusinated eIF5A, eIF5A, p-AMPK and
AMPK protein levels in cells transfected with si-NC or si-DOHH.
#P<0.05 and ##P<0.01 vs. siNC. DOHH,
deoxyhypusine hydroxylase; si-, small interfering; eIF5A,
eukaryotic translation initiation factor 5A; p-, phosphorylated;
NC, negative control.

Figure 10

Changes in the physiological features
of mice and the inhibition of MGO-induced aortic vascular injury
and apoptosis by BZ. C57BL/6J mice were intraperitoneally injected
with normal saline or MGO solution (50, 60, or 75 mg/kg/d), and BZ
or normal saline was administered by gavage from the third week.
(A) Experimental design, sampling strategy and treatment schedule
in mice. (B) The weight changes of each mouse were measured before
and after the experiment and recorded statistically. (C) Changes in
food intake before and after the experiment were measured and
statistically recorded. (D) Changes in serum MGO concentration were
determined by extracting serum from mice euthanized after drug
administration. (E-G) Expression changes in
autophagy/apoptosis-related factors, including LC3, p62 and cleaved
caspase-3, were evaluated. (H and J) H&E staining was performed
to assess vascular endothelial morphology. (I and K) TUNEL staining
was performed to evaluate apoptosis in aortic vascular tissues. The
number of mice in each group was ≥6; scale bar, 50 μm.
*P<0.05, **P<0.01 and
***P<0.001. MGO, methylglyoxal; BZ, brazilin; NS, not
significant.

Figure 11

BZ decreases the MGO-induced
autophagy and apoptosis of blood vessels. (A-J) Immunohistochemical
staining was performed for LC3, p62, Beclin1, Bax and Bcl-2. Scale
bar, 50 μm. (K-M) Beclin1 and Bax protein expression was
measured by western blot analysis. *P<0.05,
**P<0.01 and ***P<0.001. BZ, brazilin;
MGO, methylglyoxal.

Figure 12

Effects of MGO and BZ on changes in
DOHH and hypusinated eIF5A in vivo. (A) Changes in DOHH and
hypusinated eIF5A in the vascular endothelial cells of mice were
detected by immunofluorescence staining for three markers. Scale
bar, 50 μm. (B-D) Hypusinated eIF5A, eIF5A and DOHH protein
expression was measured by western blot analysis.
***P<0.001. MGO, methylglyoxal; BZ, brazilin; DOHH,
deoxyhypusine hydroxylase; eIF5A, eukaryotic translation initiation
factor 5A; ns, not significant.

Figure 13

Effects of BZ and DOHH inhibition on
autophagy and apoptosis in db/db mice. (A-F) Hypusinated
eIF5A, eIF5A, Beclin1 and Bax protein expression was measured by
western blot analysis. *P<0.05,
**P<0.01 and ***P<0.001. BZ, brazilin;
DOHH, deoxyhypusine hydroxylase; eIF5A, eukaryotic translation
initiation factor 5A; wt, wild-type; ns, not significant.

Figure 14

Summary diagram. BZ confers
protection against MGO-triggered cellular autophagic and apoptotic
responses and vascular endothelial injury by enhancing eIF5A
hypusination, thereby regulating the downstream DOHH/eIF5A
signaling cascade and the AMPK/mTOR signaling pathway. BZ,
brazilin; MGO, methylglyoxal; DOHH, deoxyhypusine hydroxylase;
eIF5A, eukaryotic translation initiation factor 5A; HUVECs, human
umbilical vein endothelial cells.
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Copy and paste a formatted citation
Spandidos Publications style
Liang Y, Chen J, Li H, Xiong J, You J, Wang L, Wu J, Zheng Y and Luo M: Brazilin attenuates methylglyoxal‑induced endothelial injury by enhancing eIF5A hypusination and inhibiting the AMPK/mTOR‑mediated autophagy/apoptosis axis <em>in vitro</em> and <em>in vivo</em>. Int J Mol Med 58: 273, 2026.
APA
Liang, Y., Chen, J., Li, H., Xiong, J., You, J., Wang, L. ... Luo, M. (2026). Brazilin attenuates methylglyoxal‑induced endothelial injury by enhancing eIF5A hypusination and inhibiting the AMPK/mTOR‑mediated autophagy/apoptosis axis <em>in vitro</em> and <em>in vivo</em>. International Journal of Molecular Medicine, 58, 273. https://doi.org/10.3892/ijmm.2026.5944
MLA
Liang, Y., Chen, J., Li, H., Xiong, J., You, J., Wang, L., Wu, J., Zheng, Y., Luo, M."Brazilin attenuates methylglyoxal‑induced endothelial injury by enhancing eIF5A hypusination and inhibiting the AMPK/mTOR‑mediated autophagy/apoptosis axis <em>in vitro</em> and <em>in vivo</em>". International Journal of Molecular Medicine 58.4 (2026): 273.
Chicago
Liang, Y., Chen, J., Li, H., Xiong, J., You, J., Wang, L., Wu, J., Zheng, Y., Luo, M."Brazilin attenuates methylglyoxal‑induced endothelial injury by enhancing eIF5A hypusination and inhibiting the AMPK/mTOR‑mediated autophagy/apoptosis axis <em>in vitro</em> and <em>in vivo</em>". International Journal of Molecular Medicine 58, no. 4 (2026): 273. https://doi.org/10.3892/ijmm.2026.5944
Copy and paste a formatted citation
x
Spandidos Publications style
Liang Y, Chen J, Li H, Xiong J, You J, Wang L, Wu J, Zheng Y and Luo M: Brazilin attenuates methylglyoxal‑induced endothelial injury by enhancing eIF5A hypusination and inhibiting the AMPK/mTOR‑mediated autophagy/apoptosis axis <em>in vitro</em> and <em>in vivo</em>. Int J Mol Med 58: 273, 2026.
APA
Liang, Y., Chen, J., Li, H., Xiong, J., You, J., Wang, L. ... Luo, M. (2026). Brazilin attenuates methylglyoxal‑induced endothelial injury by enhancing eIF5A hypusination and inhibiting the AMPK/mTOR‑mediated autophagy/apoptosis axis <em>in vitro</em> and <em>in vivo</em>. International Journal of Molecular Medicine, 58, 273. https://doi.org/10.3892/ijmm.2026.5944
MLA
Liang, Y., Chen, J., Li, H., Xiong, J., You, J., Wang, L., Wu, J., Zheng, Y., Luo, M."Brazilin attenuates methylglyoxal‑induced endothelial injury by enhancing eIF5A hypusination and inhibiting the AMPK/mTOR‑mediated autophagy/apoptosis axis <em>in vitro</em> and <em>in vivo</em>". International Journal of Molecular Medicine 58.4 (2026): 273.
Chicago
Liang, Y., Chen, J., Li, H., Xiong, J., You, J., Wang, L., Wu, J., Zheng, Y., Luo, M."Brazilin attenuates methylglyoxal‑induced endothelial injury by enhancing eIF5A hypusination and inhibiting the AMPK/mTOR‑mediated autophagy/apoptosis axis <em>in vitro</em> and <em>in vivo</em>". International Journal of Molecular Medicine 58, no. 4 (2026): 273. https://doi.org/10.3892/ijmm.2026.5944
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