MGO was obtained from MilliporeSigma. BZ, Compound C and ciclopirox were supplied by MedChemExpress. 3-Methyladenine was purchased from APeXBIO. The apoptosis detection kits for Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) were supplied by BD Biosciences. MGO ELISA kits (cat. no. E03M0040) were obtained from Blue Gene Biotech. LC3 (cat. no. RX201696M), p62 (cat. no. RX202212M) and cleaved caspase-3 (cat. no. RX2D240566) ELISA kits were obtained from Ruixin Biotechnology Co., Ltd. A tricolor pre-dyed marker and Omni-ECL™ ultrasensitive chemiluminescence detection kits were purchased from Epizyme. Unless otherwise specified, the remaining chemicals and reagents were obtained from Beyotime Institute of Biotechnology. Information on the antibodies used in the present study is listed in Table I.

Primary HUVECs used in the present study were obtained from ATCC (cat. no. PCS-100-010) as passage 1 stock cells, and cells at passages 4-10 were used for all experiments. Mycoplasma contamination was assessed using a PCR-based detection kit (HUABIO, cat. no. K0103). The PCR products were separated by electrophoresis on a 1% agarose gel and visualized after staining with GelRed nucleic acid stain. No mycoplasma-specific amplification band was detected, confirming that the cells were mycoplasma-negative. HUVECs were cultured at 37°C under humidified conditions with 5% CO₂ in EC medium supplemented with 5% fetal bovine serum (FBS), 1% EC growth supplement, and 1% penicillin/streptomycin. All related reagents were purchased from ScienCell Research Laboratories: FBS (cat. no. 0025), EC growth supplement (cat. no. 1052) and penicillin/streptomycin (cat. no. 0503).

Changes in cell viability after treatment were detected in a Cell Counting Kit-8 (CCK-8) assay. A total of ~5×10³ cells were seeded into each well of 96-well plates and cultured for 24 h. The cells were then incubated with different concentrations of BZ (0, 1, 2, 5, 10 and 20 μM) for 2 h, followed by exposure to MGO (0, 10, 20, 50, 100 and 200 μM) for 24 h. Subsequently, 100 μl of diluted CCK-8 solution was added to each well, and the cells were incubated in the dark for 1 h. The optical density at 450 nm was measured using a microplate reader.

After treatment, the cells were collected and washed three times with 1X binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl₂). Each sample was then resuspended in 100 μl of 1X binding buffer, followed by staining with 5 μl Annexin V-FITC and 5 μl PI for 15-20 min at room temperature in the dark. The apoptotic rate was detected by flow cytometry, and data analysis was performed using Cell Quest software (version 5.2; BD Biosciences).

Following incubation, cells were fixed in 4% paraformaldehyde (PFA) for 30 min at room temperature and then permeabilized with 0.1% Triton X-100 for 10 min. After blocking with 5% BSA (cat. no. 36101ES; Shanghai Yeasen Biotechnology Co., Ltd.) for 1 h at room temperature, the cells were sequentially incubated with the primary antibody overnight at 4°C, followed by incubation with the appropriate fluorophore-conjugated secondary antibody for 1 h at room temperature in the dark. Nuclei were subsequently counterstained with DAPI (1 μg/ml) for 10 min at room temperature in the dark. After anti-fade mounting medium was added, fluorescence images were captured using an IX73 inverted fluorescence microscope (Olympus Corporation) (42).

Samples were initially fixed with 2.5% glutaraldehyde at 4°C overnight and subsequently post-fixed with 1% osmium tetroxide for 2 h at room temperature in the dark. After gradient ethanol dehydration, the samples were subjected to gradient infiltration and embedding using a dehydration agent and Epon-812 embedding agent. Ultrathin sections (60-90 nm thick) were prepared from the samples and mounted on copper grids. Following staining with uranyl acetate and lead citrate, the grids were dried overnight at room temperature, and images were acquired using a TEM (JEM-1400FLASH; JEOL, Ltd.) (43).

A BCA protein assay kit (Beyotime Institute of Biotechnology) was used to quantify protein concentration. Protein lysates were loaded at equal amounts of 30 μg per lane. SDS-PAGE was performed using 6%, 7.5%, 10%, 12.5%, or 15% separating gels selected based on target protein size. Proteins were subsequently transferred to PVDF membranes and blocked with 5% non-fat milk for 1 h at room temperature. The membranes were incubated with primary antibodies overnight at 4°C and then with secondary antibodies for 1 h at room temperature. Protein bands were visualized using ECL developer solution (Pierce; Thermo Fisher Scientific, Inc.). Densitometric analysis was conducted with ImageJ software (version 1.51j8; National Institutes of Health).

siRNA targeting DOHH (si-DOHH) and negative control siRNA (si-NC; cat. no. siN0000001-1) were synthesized by Guangzhou RiboBio Co., Ltd. The si-DOHH sequences were as follows: Sense, 5'-GCAGAGCUUUGGCGUCUAAtt-3'; antisense, 5'-UUAGACGCCAAAGCUCUGCgg-3'. HUVECs were seeded in 6-well plates and transfected with si-DOHH or si-NC at a final concentration of 50 nM using the riboFECT™ CP Transfection Kit (cat. no. C10511-05; Guangzhou RiboBio Co., Ltd.) according to the manufacturer's instructions when the cells reached ~60-70% confluence. At 48 h post-transfection, the cells were harvested for western blot analysis.

A total of 52 mice aged 6-8 weeks were used in the present study, including 24 C57BL/6 mice (18-25 g), 21 db/db mice (30-45 g), and 7 wild-type (WT) control mice (18-25 g). All mice were supplied by Huafukang Biological Technology Co., Ltd. and allowed to acclimate for 1 week before experimentation. Of these, data from 3 C57BL/6 mice were excluded from the final analysis due to incomplete data collection; therefore, data from 49 mice were included in the final analysis. The mice were maintained under standard laboratory conditions, including a 12/12-h light/dark cycle, 22°C temperature, and 60±10% humidity, with free access to food and water. Animals were randomly assigned to groups, and investigators were blinded to group allocation during outcome evaluation and data analysis. Sample sizes were determined based on previous studies to ensure adequate statistical power. Humane endpoints were predefined before the study. Mice were euthanized before the scheduled endpoint if they exhibited severe body weight loss exceeding 20% of baseline body weight, persistent anorexia or dehydration, severe lethargy, inability to access food or water, labored breathing, unrelieved pain or distress, or a moribund condition. No obvious signs of distress or morbidity were observed in any animal throughout the study. At the end of the 7-week experimental period, mice were anesthetized via intraperitoneal injection of 1% pentobarbital sodium at 50 mg/kg body weight, followed by blood collection via cardiac puncture, euthanasia by cervical dislocation, and harvesting of vascular tissues. The study was conducted in compliance with the ARRIVE guidelines (https://arriveguidelines.org). All animal procedures were approved by the Animal Ethics Committee of Southwest Medical University (approval no. 2020895; Luzhou, China).

Mice were intraperitoneally administered MGO 5 days per week for 7 consecutive weeks. The dose was set at 50 mg/kg body weight for the first 2 weeks, increased to 60 mg/kg for the next three weeks, and further raised to 75 mg/kg during the final 2 weeks (44). During the final five weeks, the mice received BZ by oral gavage at 30 mg/kg/day, with the dosage calculated from published effective regimens and IP/PO bioavailability conversion (45). DOHH inhibitor (25 mg/kg/day) were administered by oral gavage once daily, five times per week, for 5 consecutive weeks (46). Both the control and WT were treated with physiological saline.

MGO concentrations and serum levels of autophagy/apoptosis-related markers, including LC3, p62 and cleaved caspase-3, were quantified with commercial ELISA kits according to the prescribed protocol.

Tissue samples underwent fixation in 4% PFA, dehydration, embedding, processing and deparaffinization, followed by pretreatment, hematoxylin staining, bluing, and eosin staining. After dehydration through a graded ethanol series and clearing in xylene, the sections were mounted with neutral resin and scanned in bright-field mode using a whole-slide scanner (Pannoramic SCAN II; 3DHISTECH Ltd.).

Paraffin-embedded mouse artery tissue samples were washed twice with xylene, soaked once with gradient ethanol (95 and 75%), rinsed twice in PBS, and subsequently treated with proteinase K solution for 20 min at room temperature. After two PBS washes, TUNEL reaction solution was applied to the slides, which were then incubated for 1 h at 37°C. The slides were rinsed three times with PBS and incubated with Converter-POD for 30 min at 37°C. Following another three PBS washes, DAB substrate was added and allowed to react for 10 min at room temperature. After a final PBS wash series, the slides were counterstained with methyl green, cover-slipped with a mounting medium, and scanned in bright-field mode using a whole-slide scanner (Pannoramic SCAN II; 3DHISTECH Ltd.). A total of five randomly selected, non-overlapping fields of view encompassing the luminal endothelial layer were evaluated at ×400 magnification for the quantification of TUNEL-positive cells within the endothelial layer.

Aortic paraffin sections were deparaffinized using deparaffinization solution and absolute ethanol, and endogenous peroxidase activity was blocked with 3% hydrogen peroxide. After blocking with serum or BSA (cat. no. 36101ES; Shanghai Yeasen Biotechnology Co., Ltd.), the sections were sequentially incubated with primary and secondary antibodies, developed with DAB, counterstained with hematoxylin, and dehydrated through ethanol. The slides were finally sealed and scanned under bright-field mode using a whole-slide scanner (Pannoramic SCAN II; 3DHISTECH Ltd.).

Paraffin-embedded sections were dewaxed, rehydrated, and subjected to antigen retrieval. After the staining areas were outlined, endogenous peroxidase activity was inhibited with hydrogen peroxide, and serum blocking was performed to reduce nonspecific binding. The sections were incubated with primary and secondary antibodies, followed by tyramide signal amplification (TSA) fluorescence reactions using iF488-Tyramide (cat. no. G1231), iF555-Tyramide (cat. no. G1233) and iF647-Tyramide (cat. no. G1232), all purchased from Wuhan Servicebio Technology Co., Ltd. Between consecutive staining cycles, the sections underwent microwave treatment and another round of serum blocking before the subsequent staining cycle. Finally, nuclei were counterstained with DAPI (1 μg/ml), autofluorescence was quenched, and the sections were mounted for imaging.

Data analysis was performed using ImageJ and GraphPad Prism 9.0 software (Dotmatics). Results are expressed as the mean ± standard deviation (SD). Before statistical testing, normal distribution and homogeneity of variance were evaluated. Differences between two groups were analyzed using an unpaired two-tailed Student's t-test, whereas comparisons among multiple groups were conducted by one-way ANOVA followed by Tukey's HSD post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To establish a model of MGO-induced endothelial injury, the effects of MGO (Fig. 1A) and BZ (Fig. 1B) on HUVEC viability were first evaluated using a CCK-8 assay. Following 24 h of exposure to MGO at concentrations of 10, 20, 50, 100, or 200 μM, HUVEC viability decreased significantly, in agreement with previous findings (Fig. 1C) (19-21). Furthermore, after 2 h of treatment with BZ at concentrations of 1, 2, and 5 μM, no marked side effects were observed. However, when the concentration increased to 10 μM, cell viability began to decrease (Fig. 1D). Additionally, 2 h of pretreatment with BZ at 1, 2, or 5 μM dose-dependently restored HUVEC viability suppressed by MGO (Fig. 1E).

To determine whether BZ modulates MGO-induced autophagy, HUVECs were exposed to BZ at 1, 2, or 5 μM for 2 h before treatment with 100 μM MGO for 24 h. Changes in the level of intracellular autophagy were detected by TEM. Compared with controls, exposure to MGO markedly increased autophagic vesicle and autolysosome formation, whereas BZ supplementation attenuated these alterations in a dose-dependent manner. In particular, the number of autophagosomes/autophagolysosomes was minimized at a BZ concentration of 5 μM (Fig. 2A). Similarly, LC3 immunofluorescence experiments revealed that the fluorescence intensity was the strongest after the addition of MGO, and the autophagic flux reached a maximum. However, BZ progressively attenuated cellular autophagy, with the most pronounced reduction observed at 5 μM (Fig. 2B). Moreover, immunoblotting experiments were used to further verify the changes in autophagy-related protein levels. Relative to the control group, MGO significantly elevated the LC3-II/LC3-I ratio and Beclin1 levels while reducing p62 expression. By contrast, BZ treatment dose-dependently reversed these changes by lowering LC3-II/LC3-I and Beclin1 levels and restoring p62 expression (Fig. 2C and D). As illustrated in the schematic diagram in Fig. 2E, BZ has been reported to mediate its protective effects via DOHH, a target protein, and the downstream eIF5A signaling cascade (39).

The level of DOHH, a BZ target protein, was subsequently further examined by western blotting. Interestingly, treatment with either BZ or MGO did not significantly alter DOHH levels relative to the control group. Hypusinated eIF5A expression was assessed and the potential mechanism by which BZ exerts its effects through the DOHH/eIF5A axis was explored. Compared with the blank group, MGO stimulation did not markedly affect hypusinated eIF5A levels, whereas BZ pretreatment elevated hypusinated eIF5A, with a significant increase observed at 5 μM BZ (Fig. 2F and G). Therefore, these findings suggest that BZ may activate the DOHH/eIF5A signaling cascade to enhance eIF5A hypusination, thereby regulating MGO-induced cellular responses.

These findings confirmed that BZ attenuated MGO-induced autophagy in ECs. Therefore, it wss further examined whether BZ modulates MGO-triggered apoptosis. As aforementioned, HUVECs were preincubated with BZ at 1, 2, or 5 μM for 2 h and subsequently exposed to 100 μM MGO for 24 h. Annexin V-FITC/PI double staining was performed to quantify apoptosis by flow cytometry. MGO exposure significantly increased the apoptotic rate compared with controls, while BZ pretreatment progressively reduced apoptosis with increasing concentrations (Fig. 3A and B). Molecular analyses further corroborated these findings, revealing dose-dependent reductions in Bax and the cleaved caspase-3/caspase-3 ratio, alongside a concomitant increase in Bcl-2 expression (Fig. 3C and D). These findings collectively demonstrate that BZ not only mitigates MGO-induced autophagy but also effectively blocks the subsequent apoptotic cascade.

Combined flow cytometry and western blot data revealed that BZ was able to suppress autophagic and apoptotic responses elicited by MGO in HUVECs. As the AMPK/mTOR pathway is pivotal for autophagy regulation (47,48), it was hypothesized that BZ confers protection by modulating this pathway. Accordingly, it was first examined how MGO and BZ affected core pathway proteins at the phosphorylation level. Relative to the control group, MGO treatment elevated phosphorylated (p-)AMPK levels while reducing p-mTOR levels. Moreover, BZ pretreatment dose-dependently decreased p-AMPK levels while increasing p-mTOR levels (Fig. 3E and F). These findings suggest that MGO induces apoptosis and AMPK/mTOR signaling, which is modulated by BZ.

To further determine whether MGO-induced cellular autophagy and apoptosis are associated with the inhibitory effect of BZ on autophagy, HUVECs were pretreated with BZ (5 μM) + 3-MA (5 mM, an autophagy inhibitor) for 2 h and co-incubated them for 24 h with MGO (100 μM) (49). TEM was used to observe the changes in autophagosomes. BZ clearly reduced MGO-induced autophagy, while combined BZ and 3-MA pretreatment further suppressed this response, with reduced autophagosome and autolysosome formation (Fig. 4A). Further LC3 immunofluorescence analysis was conducted to determine whether 3-MA suppressed MGO-evoked autophagy in ECs and enhanced the protective effects of BZ. As observed in aforementioned results, the red fluorescence was strongest in the MGO-treated group. After the addition of BZ, autophagic flux was reduced. In addition, cotreatment with 3-MA and BZ further potentiated BZ-mediated suppression of MGO-induced autophagy in HUVECs (Fig. 4B).

Autophagy- and apoptosis-related protein expression was examined using western blot analysis. Relative to the control group, MGO significantly enhanced cellular autophagic and apoptotic responses. However, pretreatment with BZ reversed these effects. Combined treatment with BZ and 3-MA further enhanced this reversal, significantly downregulating the LC3-II/LC3-I ratio, the expression of Beclin1 and Bax, as well as the cleaved caspase-3/caspase-3 ratio, while upregulating the expression levels of Bcl-2 and p62 (Fig. 4C-F). In summary, BZ can decrease the levels of autophagy and apoptosis and further reduce MGO-induced autophagy and apoptosis when combined with the autophagy inhibitor 3-MA. These data suggest that 3-MA and BZ have synergistic effects on the protection of vascular ECs.

To further demonstrate that MGO-induced autophagy occurs before apoptosis, the time-course changes in autophagy and apoptosis markers in HUVECs were examined at 0, 6, 12 and 24 h after MGO treatment. As shown in Fig. 5A and B, the autophagy marker LC3-II was significantly upregulated as early as 6 h after MGO stimulation, whereas the cleaved caspase-3 began to increase significantly at 12 h and remained elevated at 24 h. In addition, a chloroquine (CQ) blockade assay was performed to evaluate autophagic flux. As revealed in Fig. 6A and B, treatment with either CQ or MGO alone significantly increased LC3-II levels compared with the control group. Notably, combined treatment with MGO and CQ further increased LC3-II levels compared with MGO treatment alone. The results suggest that MGO enhances autophagic flux, rather than merely causing passive accumulation of autophagosomes due to impaired lysosomal degradation.

Current data indicate that MGO stimulation significantly increases the levels of p-AMPK while decreasing the levels of mTOR and that BZ can reverse these effects. Therefore, it was hypothesized that BZ may regulate MGO-triggered autophagic and apoptotic responses through AMPK/mTOR signaling. Before exposure to 100 μM MGO for 24 h, HUVECs were preincubated for 2 h with BZ (5 μM) in the presence or absence of Compound C (50 μM, an AMPK inhibitor) (50). TEM revealed that BZ reduced MGO-induced autophagy. Furthermore, after cotreatment with BZ and Compound C, the numbers of autophagosomes and autolysosomes further decreased (Fig. 7A). Additionally, flow cytometric analysis was further performed to assess apoptosis following AMPK blockade. The data showed that BZ attenuated MGO-induced HUVEC apoptosis. Notably, cotreatment with BZ and Compound C also attenuated HUVEC apoptosis, which blocked AMPK signaling (Fig. 7B and C). Western blot analysis revealed that Compound C, an AMPK inhibitor, further enhanced the BZ-mediated reversal, as reflected by reduced p-AMPK levels and increased p-mTOR levels (Fig. 7D and E). Overall, the inhibitory effects of BZ on MGO-induced autophagy and apoptosis appear to be further enhanced by Compound C.

The aforementioned results reveal that BZ does not alter DOHH levels in MGO-treated HUVECs but significantly increases hypusinated eIF5A levels, suggesting that BZ may inhibit MGO-triggered autophagy and apoptosis by enhancing DOHH-mediated eIF5A hypusination. To verify the aforementioned hypothesis, HUVECs were pretreated for 2 h with BZ (5 μM) alone or with BZ (5 μM) combined with ciclopirox (20 μM), a DOHH signaling pathway inhibitor, and were subsequently stimulated with MGO (100 μM) (51,52). TEM revealed that BZ inhibited the MGO-induced increases in autophagic vesicles and autolysosomes, whereas combined treatment with BZ, ciclopirox and MGO markedly elevated autophagic activity (Fig. 8A). These results were further confirmed by LC3 immunofluorescence, which revealed that BZ reduced the extent of MGO-induced autophagy and decreased autophagic flux. However, cells receiving combined treatment with BZ, ciclopirox and MGO exhibited a marked increase in autophagic fluorescence intensity (Fig. 8B).

More importantly, as demonstrated in Fig. 8C and D, pretreatment with BZ reversed MGO-induced apoptosis, but the apoptotic rate increased after ciclopirox treatment. The aforementioned results suggest that the DOHH inhibitor ciclopirox could resist the protective effect of BZ and promote apoptosis. Subsequently, western blotting was performed to examine the BZ-associated downstream DOHH/eIF5A signaling cascade. DOHH levels were not significantly altered by BZ pretreatment or MGO exposure relative to the blank group; however, pretreatment with the DOHH inhibitor ciclopirox significantly reduced DOHH expression. Hypusinated eIF5A levels were not markedly altered by MGO treatment relative to the blank control group, whereas BZ pretreatment increased them; however, this response was significantly reversed by ciclopirox pretreatment (Fig. 8E and F). These findings indicate that ciclopirox promotes autophagic and apoptotic responses elicited by MGO in HUVECs through suppression of the DOHH/eIF5A signaling cascade and abolishment of the cytoprotective effects of BZ.

To exclude the potential off-target effects of ciclopirox, si-DOHH transfection was further performed in the present study to verify the specificity of DOHH inhibition and to investigate its relationship with AMPK activation. As revealed in Fig. 9A and B, si-DOHH transfection significantly reduced the expression levels of DOHH and hypusinated eIF5A, while significantly increasing p-AMPK levels. These results suggest that inhibition of the DOHH/eIF5A pathway may be closely associated with AMPK activation.

To assess whether BZ modulates MGO-evoked vascular autophagy and apoptosis in vivo, C57BL/6J mice were administered MGO and BZ. Body weight, food intake and serum MGO concentration were measured after intraperitoneal injection of MGO and intragastric administration of BZ. The experimental design, including the sampling strategy and treatment regimen for the mouse study, is presented in Fig. 10A. The results showed a modest increase in body weight across all mouse groups after treatment, with no statistically significant difference from pretreatment body weights, which may be attributable to normal physiological growth during the 7-week feeding period (Fig. 10B). Interestingly, the food intake of the mice did not significantly change after administration compared with that of the pre-administration mice (Fig. 10C). These data indicate that treatment with BZ and MGO does not significantly affect the physiological functions of mice. Next, serum MGO levels were quantified, revealing a significant elevation in the MGO group and a significant reduction following BZ treatment (Fig. 10D). Dynamic alterations in autophagy- and apoptosis-related factors, including LC3, p62 and cleaved caspase-3, were examined. As shown in Fig. 10E-G, MGO significantly elevated LC3 and cleaved caspase-3 levels while reducing p62 expression. However, these effects were significantly suppressed upon the addition of BZ.

Moreover, H&E and TUNEL staining were used to evaluate whether BZ affects MGO-induced vascular injury. H&E-stained aortic sections from the control group showed an intact, smooth, and regularly organized intima. Conversely, MGO treatment led to substantial thickening, irregularity, a darker color, and a more disordered shape, indicating pronounced vascular damage. Following BZ treatment, the dilatation and injury of the aortic intima were significantly ameliorated (Fig. 10H and J). Additionally, TUNEL staining was performed to further observe changes in EC apoptosis. MGO treatment significantly increased the apoptotic rate in mouse aortic vessels, but this effect was significantly reversed after BZ treatment (Fig. 10I and K).

Additionally, to determine whether BZ inhibits autophagy and apoptosis in vivo, immunohistochemical experiments were conducted. Relative to the control group, the MGO group showed elevated vascular autophagy levels, increased expression of LC3, Beclin1 and Bax, and reduced positivity rates for p62 and Bcl-2. However, these effects were reversed upon BZ treatment (Fig. 11A-J). Harvested mouse vascular tissue was subsequently analyzed by western blotting to further validate these findings. It was found that MGO elevated Beclin1 and Bax levels, whereas BZ counteracted these changes and attenuated autophagic and apoptotic responses (Fig. 11K-M). These data align with the cellular findings and indicate that BZ markedly suppresses vascular cell autophagic and apoptotic responses triggered by MGO, thereby supporting its vasoprotective effects.

The aforementioned in vitro findings revealed that BZ suppresses autophagic and apoptotic responses elicited by MGO in ECs through the DOHH/eIF5A pathway. Immunofluorescence localization was used to examine changes in DOHH and hypusinated eIF5A in vascular ECs. In the control group, ECs (red fluorescence), DOHH (green fluorescence) and hypusinated eIF5A (yellow fluorescence) were observed. Furthermore, the results from MGO treatment alone aligned with the in vitro findings, showing that MGO did not alter DOHH or hypusinated eIF5A positivity in ECs. Although DOHH positivity remained unchanged after MGO and BZ treatment, hypusinated eIF5A positivity was significantly increased (Fig. 12A). Consistently, western blot analysis of mouse vascular tissues showed that MGO did not affect DOHH or hypusinated eIF5A protein levels relative to the control group, whereas MGO and BZ cotreatment significantly elevated hypusinated eIF5A levels. These results further confirmed that BZ enhances the post-translational modification of the eIF5A protein by hypusine via allosteric activation of DOHH, thereby exerting its protective effects (Fig. 12B-D).

To more accurately mimic the pathophysiological characteristics of diabetes, db/db mice were used instead of MGO injection to investigate autophagy and apoptosis in vascular tissues as well as the involvement of the DOHH/eIF5A signaling pathway. Relative to the control group, DOHH and hypusinated eIF5A levels in vascular tissues from db/db mice showed no significant differences, whereas Beclin1 and Bax levels were elevated. This pattern was highly consistent with that observed in the MGO administration model, indicating that both diabetes and exogenous MGO stimulation increase the levels of autophagy and apoptosis in vascular tissues. After oral BZ administration to db/db mice, hypusinated eIF5A protein levels were significantly elevated, whereas DOHH expression remained unchanged, and autophagic and apoptotic responses were reduced. By contrast, DOHH inhibitor treatment significantly reduced DOHH and hypusinated eIF5A levels, elevated Beclin1 and Bax levels, and enhanced autophagic and apoptotic responses (Fig. 13A-F). These findings indicate that autophagic and apoptotic responses triggered by MGO are aligned with diabetes-related vascular pathological changes, while BZ may act as a promising therapeutic candidate for diabetic vascular injury.