HUVECs were obtained from Procell (Wuhan, China). The cell line was authenticated by short tandem repeat (STR) profiling and tested negative for mycoplasma. The cells were seeded in culture dishes containing complete DMEM medium supplemented with 10% fetal bovine serum (FBS; Aoqing, Beijing, China). After thorough mixing, the dishes were placed in a cell culture incubator (Thermo, Waltham, MA, USA) maintained at 37 °C with 5% CO₂. Upon reaching 80% confluence, the supernatant was discarded, and the cells were rinsed with PBS (NCM, Suzhou, China). Subsequently, 1.5 mL of trypsin (Gibco, Grand Island, NY, USA) was added to initiate digestion, which was terminated after 3 minutes. The cells were then resuspended and subcultured at a 1:4 split ratio.

HUVECs were irradiated using an X-ray irradiator (KUBTEC, Stratford, CT, USA) at doses of 0, 3, 6, 9, and 12 Gy, with a dose rate of 1 Gy/min. After irradiation, the HUVECs were cultured in an incubator for subsequent experiments.

Tec was dissolved in DMSO to prepare a stock solution at a concentration of 2 mg/mL (6.66 mM) and subsequently diluted to the desired working concentrations using basal DMEM medium. HUVECs were pretreated with Tec working solution for 24 h prior to irradiation exposure, and assays were performed 72 h post-irradiation.

MTK458 (MCE, South Brunswick, NJ, USA) and U0126 (MCE, USA) were dissolved in DMSO to prepare stock solutions and subsequently diluted with basal DMEM medium to working concentrations of 100 µM. HUVECs were treated with the working solutions for 48 h to induce PINK1 overexpression or inhibit mitophagy, respectively. Similarly, HUVECs were transfected with PINK1 siRNA (GenePharma, Suzhou, China) for 48 h prior to subsequent experimental procedures.

Cell viability was assessed using the CCK-8 assay (Beyotime, Shanghai, China). Briefly, HUVECs were seeded into 96-well plates (Corning, New York, NY, USA) at a density of 5 $\times$ 10³ cells per well. After cell attachment, the culture medium was aspirated and replaced with CCK-8 working solution. Following incubation for 2 h at 37 °C, absorbance was measured at 450 nm using a microplate reader (Thermo, USA).

HUVECs were harvested and centrifuged at 1500 rpm for 3 min using a high-speed centrifuge (Sigma, Osterode, Lower Saxony, Germany). The supernatant was carefully aspirated, and the cell pellet was resuspended in 4% paraformaldehyde solution. Cells were fixed at room temperature in the dark for 2 h, followed by storage at 4 °C. Subsequently, the fixed cells were post-fixed with 1% osmium tetroxide at 4 °C for 2 h. Dehydration was performed using a graded ethanol series (50%, 70%, 80%, 90%, and 100%). After dehydration, samples were embedded in resin and polymerized at 60 °C for 48 h. Ultrathin sections (80 nm) were prepared, mounted on copper grids, and stained. Cellular ultrastructure was observed under a transmission electron microscope, with particular emphasis on analyzing mitochondrial morphological changes and autophagosome formation. For quantitative analysis, the independent experiments were examined. Mitochondria were classified as damaged if they exhibited swelling, cristae disruption, cristae loss, or vacuolization.

HUVECs were seeded into 6-well plates (Corning, USA) at a density of 1 $\times$ 10⁵ cells per well, with 3 replicate wells per experimental condition. Cells were collected and incubated with JC-1 staining working solution in the dark for 20 min, followed by 2 washes to remove unbound probes. Fluorescence was then analyzed using a flow cytometer (Beckman Coulter, Brea, CA, USA).

HUVECs were seeded into confocal dishes (Corning, USA) at a density of 1 $\times$ 10⁵ cells per dish. Upon reaching 80% confluence, cells were washed with PBS and incubated with JC-1 working solution in the dark for 20 min. After incubation, unbound probes were removed by washing, and the samples were mounted using Antifade Mounting Medium with DAPI (Beyotime, China). Fluorescent signals were visualized under a confocal laser scanning microscope (Leica, Wetzlar, Hessen, Germany).

HUVECs were seeded into 6-well plates (Corning, USA) at a density of 1 $\times$ 10⁵ cells per well, with 3 replicate wells per experimental condition. MitoSOX (MCE, USA) was prepared as a 5 µM working solution. Cells were harvested and incubated with the working solution in the dark for 30 min, followed by 2 washes with PBS to remove unbound probes. The samples were then resuspended in PBS and kept on ice until analysis. Fluorescence intensity was measured using a flow cytometer.

HUVECs were seeded into confocal culture dishes at a density of 1 $\times$ 10⁵ cells per dish. When cell confluence exceeded 80%, the supernatant was discarded, followed by washing the cells with PBS. HUVECs were stained with MitoSOX Red (5 µM) and MitoTracker Green (1 µM) in buffer for 30 minutes at room temperature in the dark. After washing away unbound probes, the cells were mounted using Antifade Mounting Medium with DAPI. Finally, red fluorescence was observed using a confocal laser scanning microscope.

HUVECs were lysed in RIPA lysis buffer (Beyotime, China) supplemented with 1% protease inhibitor (NCM, China). Following 30 min of lysis on ice, the lysates were centrifuged at 14,000 rpm for 15 min at 4 °C using a high-speed centrifuge. The supernatant was collected, and total protein concentration was determined using the BCA method. Protein samples were mixed with loading buffer (Servicebio, Wuhan, China) and heated at 95 °C for 10 min. Proteins were separated by SDS-PAGE (Bio-Rad, Hercules, CA, USA) and transferred onto PVDF membranes (Bio-Rad, USA). After blocking with 5% non-fat milk for 1 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies against PINK1 (Cat No.23274-1-AP, Proteintech, Wuhan, China; 1:1000), Parkin (ab324566, Abcam, Cambridge, UK; 1:1000), and $\beta$-actin (Ab8227, Abcam, UK; 1:1000). Following washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized and analyzed using a gel imaging system (Bio-Rad, USA). Densitometric analysis was performed using ImageJ software (Java 1.8.0, NIH, Bethesda, MD, USA). The gray value of each target protein band was normalized to that of $\beta$-actin from the same sample, and the relative expression levels were expressed as fold change compared to the control group.

HUVECs were seeded into confocal dishes at a density of 1 $\times$ 10⁵ cells per dish. Upon reaching 80% confluence, cells were washed with PBS after aspiration of the culture medium. HUVECs were then fixed with 4% paraformaldehyde at room temperature for 20 min. Subsequently, the cells were permeabilized with 0.3% Triton X-100 (Beyotime, China) at room temperature, followed by blocking with 5% goat serum for 1 h. The samples were incubated overnight at 4 °C with primary antibodies against LC3B (Abcam, USA) and TOM20 (CST, USA). After washing, the samples were incubated with Alexa Fluor 594-conjugated goat anti-mouse IgG (H+L) secondary antibody (Proteintech, China) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) secondary antibody in the dark at room temperature for 1 h. Finally, the slides were mounted using Antifade Mounting Medium with DAPI (Beyotime, China), and fluorescence was visualized under a confocal laser scanning microscope.

HUVECs were seeded into 6-well plates at a density of 1 $\times$ 10⁵ cells per well, with 3 replicate wells per experimental group. Cells were harvested using EDTA-free trypsin (Gibco, USA), washed with PBS, and resuspended in 100 µL of Annexin V binding buffer. Subsequently, 2.5 µL of Annexin V-FITC and 2.5 µL of propidium iodide (PI) staining solution were added, and the cells were incubated in the dark at room temperature for 15 min. After incubation, 400 µL of Annexin V binding buffer was added to each sample, and fluorescence was immediately measured using a flow cytometer. Early apoptotic cells were defined as Annexin V-positive/PI-negative, while late apoptotic cells were defined as Annexin V-positive/PI-positive. The total apoptotic rate was calculated as the sum of early and late apoptotic percentages.

HUVECs were seeded into 6-well plates at a density of 1 $\times$ 10⁵ cells per well, with three replicate wells per experimental group. DHE was prepared as a 5 µM working solution in serum-free medium. Cells were harvested, incubated with the DHE working solution in the dark for 30 min at 37 °C, and then washed twice with PBS to remove unbound probes. The samples were resuspended in PBS and kept on ice until analysis. Fluorescence intensity was measured using a flow cytometer.

HUVECs were seeded into confocal dishes and cultured until reaching approximately 80% confluence. The culture medium was then aspirated, and cells were incubated with DHE working solution (5 µM prepared in serum-free medium) in the dark for 30 min at 37 °C. After incubation, unbound probes were removed by washing with PBS. Samples were then mounted using Antifade Mounting Medium with DAPI (Beyotime, China). Fluorescence was visualized under a fluorescence microscope (Leica, Germany).

Mitophagic activity was evaluated using a Mitophagy Detection Kit (DOJINDO, Kumamoto Prefecture, Japan). HUVECs were seeded into confocal dishes at a density of 1 $\times$ 10⁵ cells per dish. Following the designated treatments, the culture medium was aspirated, and the samples were gently rinsed 3 times with PBS. The samples were then incubated in the dark for 30 min with a staining solution containing 100 nM Mitophagy Dye and 1 µM LysoTracker. Subsequently, the staining solution was removed, and the samples were washed 3 times with PBS. Finally, the samples were mounted using Antifade Mounting Medium with DAPI and visualized under a laser scanning confocal microscope.

HUVECs were seeded into 6-well plates at a density of 1 $\times$ 10⁵ cells per well, with 3 replicate wells per experimental group. Following treatment, the cells were harvested and collected into 15 mL centrifuge tubes, then centrifuged at 1500 rpm for 5 minutes. The supernatant was carefully aspirated, and the samples were resuspended in a freshly prepared staining solution containing 100 nM Mitophagy Dye and 1 µM LysoTracker. Samples were incubated in the dark for 30 minutes. After incubation, the samples were washed 3 times with PBS to remove unbound probes. Finally, the samples were resuspended in PBS and immediately analyzed by flow cytometry.

HUVECs were seeded into 6-well plates at a density of 1 $\times$ 10⁵ cells per well, with 3 replicate wells per experimental group. After the designated treatments, the culture medium was aspirated. Subsequently, 200 µL of lysis buffer was added to each well. Following lysis, the lysates were collected and centrifuged at 12,000 rpm for 5 minutes at 4 °C. Aliquots of ATP standards and samples were transferred to a 96-well white opaque plate, and the luminescence was measured using a luminometer. ATP concentrations in the samples were calculated based on a standard curve generated from known ATP standards.

All statistical analyses were conducted using GraphPad Prism 9.5 (GraphPad Software, La Jolla, CA, USA). Data are presented as mean $\pm$ standard deviation (SD). Differences among multiple groups were assessed using one-way analysis of variance (ANOVA), with pairwise comparisons performed by Tukey test. Statistical significance was set at a p-value 0.05.

To establish an optimal irradiation dose for subsequent experiments, HUVECs were exposed to increasing doses of irradiation (3, 6, 9, and 12 Gy), and cellular injury was assessed. As shown in Fig. 1, irradiation induced dose-dependent increases in apoptotic rate, reactive oxygen species (ROS) production, and reductions in cell viability. Annexin V/PI double staining revealed that the apoptotic rate was significantly increased in the 3 Gy group compared to the control (Con) group, with further progressive increases observed at 6 Gy and 9/12 Gy (Fig. 1A,B). CCK-8 assays demonstrated that cell viability decreased progressively with increasing irradiation doses relative to the Con group (Fig. 1C). Flow cytometric analysis of DHE fluorescence intensity, an indicator of superoxide levels, showed a significant increase in the 3 Gy group compared to the Con group, with significantly higher levels in the 6 Gy group than in the 3 Gy group, and further elevation in the 9 Gy and 12 Gy groups compared to the 6 Gy group (Fig. 1D,E). DHE fluorescence staining further confirmed enhanced red fluorescence signals in all irradiated groups relative to the Con group (Fig. 1F). Western blot analysis revealed that protein expression levels of PINK1 and Parkin increased progressively with escalating irradiation doses (Fig. 1G–I). Notably, 9 Gy irradiation induced the most pronounced cellular injury, while increasing the dose to 12 Gy did not result in statistically significant exacerbation of damage compared to the 9 Gy group. Therefore, a radiation dose of 9 Gy was selected for all subsequent experiments.

Fig. 1.

Irradiation-induced injury in HUVECs. (A) Representative flow cytometry plots of Annexin V/PI staining for apoptosis detection. (B) Quantitative analysis of apoptotic rates (n = 8). (C) Cell viability assessed by CCK-8 assay (n = 8). (D) Representative flow cytometry histograms of DHE fluorescence intensity. (E) Quantitative analysis of DHE fluorescence intensity (n = 8). (F) Representative images of DHE fluorescence staining (red). Scale bar = 20 µm. (G) Western blot analysis of PINK1 and Parkin protein expression. (H) Quantitative analysis of PINK1 expression levels normalized to $\beta$-actin (n = 3). (I) Quantitative analysis of Parkin expression levels normalized to $\beta$-actin (n = 3). Data are presented as mean $\pm$ SD. Statistical analysis was performed using one-way ANOVA followed by Tukey test. *: p 0.05; NS: p $>$ 0.05. HUVECs, human umbilical vein endothelial cells; PI, propidium iodide; CCK-8, Cell Counting Kit-8; DHE, Dihydroethidium; PINK1, PTEN induced kinase 1; SD, standard deviation; ANOVA, analysis of variance.

To further investigate the effects of 9 Gy irradiation on mitochondrial function, both mitochondrial function and morphology were assessed. Flow cytometry showed that irradiated cells exhibited a decrease in JC-1 aggregates and an increase in JC-1 monomers, indicating mitochondrial membrane potential ($\mathrm{\Delta }$$\mathrm{\Psi }$m) depolarization (Fig. 2A–C). This loss of $\mathrm{\Delta }$$\mathrm{\Psi }$m was corroborated by confocal microscopy, which demonstrated a marked attenuation of red fluorescence (JC-1 aggregates) and an intensification of green fluorescence (JC-1 monomers) following irradiation (Fig. 2D). Flow cytometry showed a significant increase in MitoSOX fluorescence intensity in irradiated cells (Fig. 2E,F). Consistent with this, confocal microscopy confirmed enhanced red fluorescence (MitoSOX) that co-localized with MitoTracker Green stained mitochondria, confirming the mitochondrial specific oxidative stress (Fig. 2G). Ultrastructural analysis by TEM further uncovered severe irradiation-induced morphological injury Mitochondria in Con group cells exhibited a regular morphology with clearly defined and orderly cristae (green arrows). In contrast, mitochondria from irradiated cells displayed pronounced swelling and vacuolization (purple arrows), with a substantial proportion exhibiting cristae that were disorganized, fragmented, or entirely absent (red arrows) (Fig. 2H). Finally, immunostaining revealed a significant increase in red fluorescence (LC3B) in irradiated cells, which robustly co-localized with TOM20, suggesting an augmentation of mitophagic activity in response to irradiation-induced injury (Fig. 2I).

Fig. 2.

9 Gy irradiation induces mitochondrial injury at 72 h post-exposure. (A) Flow cytometry detection of mitochondrial membrane potential. (B) Quantitative analysis of the JC-1 aggregates proportion (n = 9). (C) Quantitative analysis of the JC-1 monomers proportion (n = 9). (D) JC-1 fluorescence staining. Scale bar = 20 µm. (E) Flow cytometry detection of MitoSOX fluorescence intensity. (F) Quantitative analysis of MitoSOX fluorescence intensity (n = 9). (G) MitoSOX and MitoTracker co-localization fluorescence staining. Scale bar = 20 µm. (H) Observation of cellular ultrastructure under TEM, Green arrows: Normal mitochondria; Red arrows: Mitochondria with disorganized, fragmented, or absent cristae; Purple arrows: Mitochondria with swelling and vacuolization. Scale bar = 1 µm or 500 nm. (I) LC3B and TOM20 co-localization fluorescence imaging. Scale bar = 20 µm. Data are represented as mean $\pm$ SD. Statistical analysis between two groups was performed using Tukey test. *: p 0.05. TEM, Transmission Electron Microscopy.

To evaluate the protective effects of Tec against 9 Gy irradiation-induced injury, HUVECs were divided into 5 experimental groups: the Con group, the Ir group, and Ir groups treated with Tec at concentrations of 0.5, 1.0, and 2.0 µg/mL (1.67, 3.33, and 6.66 µM). As shown in Fig. 3, Annexin V/PI double staining revealed that the apoptotic rate was significantly increased in the Ir group compared to the Con group (p 0.05), while treatment with Tec at 1.67, 3.33, and 6.66 µM significantly reduced irradiation-induced apoptosis relative to the Ir group (p 0.05) (Fig. 3A,B). CCK-8 assays demonstrated that cell viability was significantly suppressed in the Ir group compared to the Con group (p 0.05), an effect markedly reversed by Tec treatment at all three concentrations (p 0.05 vs. Ir group) (Fig. 3C). Flow cytometric analysis of DHE fluorescence intensity showed a significant elevation in the Ir group relative to the Con group (p 0.05), which was significantly attenuated by Tec treatment at 1.67, 3.33, and 6.66 µM (p 0.05 vs. Ir group) (Fig. 3D,E), and DHE fluorescence staining further confirmed visibly reduced red fluorescence in all Tec-treated groups compared to the Ir group (Fig. 3F). Collectively, these results demonstrate that Tec protects HUVECs from irradiation-induced injury by enhancing cell viability and reducing apoptosis and oxidative stress, with the most pronounced protective effect observed at 3.33 µM. As increasing the concentration to 6.66 µM did not confer statistically significant additional benefit compared to 3.33 µM, this concentration was selected for all subsequent experiments.

Fig. 3.

Tec attenuated 9 Gy irradiation-induced injury in HUVECs at 72 h following irradiation exposure. (A) Cell apoptosis detected by Annexin V/PI double staining. (B) Quantitative analysis of apoptotic rates (n = 9). (C) Cell viability under treatment with different concentrations of Tec measured by the CCK-8 assay (n = 9). (D) DHE fluorescence intensity analyzed by flow cytometry. (E) Quantitative analysis of DHE fluorescence intensity (n = 9). (F) DHE fluorescence staining. Scale bar = 20 µm. Data are represented as mean $\pm$ SD. Statistical analysis was performed using one-way ANOVA followed by Tukey test. *: p 0.05; NS: p $>$ 0.05.

To further investigate the effect of Tec on mitochondrial function following 9 Gy irradiation, both mitochondrial function and morphology were assessed. Flow cytometric analysis of mitochondrial oxidative stress revealed that MitoSOX fluorescence intensity was significantly increased in the Ir group compared to the Con group, while Tec treatment markedly attenuated this elevation (Fig. 4A,B). Colocalization analysis of MitoSOX and MitoTracker further confirmed that red fluorescence (MitoSOX) was significantly enhanced in the Ir group relative to the Con group, accompanied by colocalization with green fluorescence (MitoTracker). In contrast, the Ir+Tec group exhibited markedly diminished red fluorescence and decreased colocalization with green fluorescence compared to the Ir group (Fig. 4C). Flow cytometric assessment of mitochondrial membrane potential demonstrated that irradiation induced depolarization, as evidenced by a significant reduction in the proportion of JC-1 aggregates in the Ir group relative to the Con group. Conversely, Tec treatment significantly increased the proportion of JC-1 aggregates compared to the Ir group (Fig. 4D,E). JC-1 staining further confirmed that red fluorescence (JC-1 aggregates) was markedly diminished in the Ir group compared to the Con group. Conversely, the Ir+Tec group exhibited significantly enhanced red fluorescence relative to the Ir group, while green fluorescence (JC-1 monomers) displayed an opposite trend (Fig. 4F). TEM revealed distinct ultrastructural alterations in mitochondria across experimental groups (Fig. 4G). In the Con group, mitochondria exhibited intact morphology with well-defined cristae structures (green arrows). In contrast, the Ir group displayed marked pathological changes, characterized by disorganized, fragmented, or absent cristae (red arrows). Notably, mitophagic vacuoles were observed in the Ir group (purple arrows), indicating irradiation-induced activation of mitophagy. Importantly, Tec treatment ameliorated these irradiation-induced mitochondrial injuries, partially restoring cristae integrity and promoting a more regular mitochondrial morphology (blue arrows) (Fig. 4G). Colocalization analysis of LC3B and TOM20 revealed that mitophagic activity was significantly enhanced in the Ir group compared to the Con group, as evidenced by markedly increased red fluorescence (LC3B) and its colocalization with green fluorescence (TOM20). In contrast, Tec treatment significantly attenuated red fluorescence relative to the Ir group (Fig. 4H). Dual staining of Mitophagy and Lysosomes was performed to assess mitophagic activity. Fluorescence staining revealed that red fluorescence (Mitophagy) was markedly increased in the Ir group compared to the Con group, accompanied by green fluorescence (Lysosomes), indicating enhanced mitophagic activity. In contrast, red fluorescence was significantly attenuated in the Ir+Tec group relative to the Ir group (Fig. 4I). Flow cytometric analysis of the mitophagy rate revealed that the proportion of mitophagy-positive cells was significantly increased in the Ir group compared to the Con group, while Tec treatment markedly attenuated this elevation (Fig. 4J,K). Western blot analysis revealed that protein expression levels of PINK1 and Parkin were significantly increased in the Ir group compared to the Con group, while Tec treatment attenuated this elevation (Fig. 4L–N).

Fig. 4.

Tec protects mitochondria from injury induced by 9 Gy irradiation at 72 h post-exposure. (A) Representative flow cytometry histograms of MitoSOX fluorescence intensity for mitochondrial oxidative stress assessment. (B) Quantitative analysis of MitoSOX fluorescence intensity (n = 8). (C) Representative confocal images of MitoSOX (red) and MitoTracker (green) colocalization. Scale bar = 20 µm. (D) Representative flow cytometry dot plots of JC-1 staining for mitochondrial membrane potential. (E) Quantitative analysis of the JC-1 aggregate/monomer ratio (n = 8). (F) Representative images of JC-1 fluorescence staining (red: aggregates, green: monomers). Scale bar = 20 µm. (G) Representative TEM images of mitochondrial ultrastructure. Scale bar = 2 µm or 1 µm. Green arrows, normal mitochondria; red arrows, pathologically altered mitochondria; purple arrows, mitophagic vacuoles; blue arrows, mitochondria with improved pathological changes. (H) Representative immunofluorescence images of LC3B (red) and TOM20 (green) colocalization. Scale bar = 20 µm. (I) Representative confocal images of dual staining for Mitophagy (red) and Lysosomes (green). Scale bar = 20 µm. (J) Representative flow cytometry histograms of mitophagy rate. (K) Quantitative analysis of mitophagy rate (n = 6). (L) Western blot analysis of PINK1 and Parkin protein expression. (M) Quantitative analysis of PINK1 expression levels normalized to $\beta$-actin (n = 3). (N) Quantitative analysis of Parkin expression levels normalized to $\beta$-actin (n = 3). Data are presented as mean $\pm$ SD. Statistical analysis was performed using one-way ANOVA followed by Tukey test. *: p 0.05.

To investigate whether the radioprotective effect of Tec against 9 Gy irradiation is mediated through suppression of mitophagy activation. HUVECs were treated with the mitophagy inhibitor U0126 following irradiation exposure, and the results are shown in Fig. 5. Flow cytometric analysis revealed that irradiation significantly increased DHE fluorescence intensity, which was markedly attenuated by both Tec and U0126 treatment, with the combination of Tec and U0126 (Ir+Tec+U0126) exhibiting the lowest DHE fluorescence among irradiated groups (Fig. 5A,B). DHE fluorescence staining further confirmed that red fluorescence (DHE) was significantly attenuated by both Tec and U0126 treatment compared to the Ir group, with the Ir+Tec+U0126 group displaying the faintest red fluorescence among all irradiated groups (Fig. 5C). CCK-8 assays demonstrated that both Tec and U0126 significantly restored irradiation-induced reduction in cell viability, with the Ir+Tec+U0126 group exhibiting the highest cell viability among all irradiated groups (Fig. 5D). Flow cytometric analysis revealed that both Tec and U0126 treatment suppressed the irradiation-induced increase in apoptotic rate, with the Ir+Tec+U0126 group exhibiting the lowest apoptosis levels among all irradiated groups (Fig. 5E,F). Colocalization analysis of mitophagy (red) and lysosomes (green) revealed that the red fluorescence signal was significantly enhanced following irradiation, while both Tec and U0126 treatment attenuated this enhancement, with the most pronounced reduction observed in the Ir+Tec+U0126 group (Fig. 5G). Flow cytometric analysis further confirmed that both Tec and U0126 significantly reduced the proportion of mitophagy-positive cells compared to the Ir group, with the Ir+Tec+U0126 group exhibiting the lowest percentage among all irradiated groups (Fig. 5H,I). ATP assays showed that irradiation-induced ATP depletion was reversed by both Tec and U0126, with the combination treatment maintaining the highest ATP levels among all irradiated groups (Fig. 5J). MitoSOX fluorescence analysis revealed that irradiation significantly increased MitoSOX fluorescence intensity compared to the Con group, while both Tec and U0126 treatment attenuated this effect, with the Ir+Tec+U0126 group exhibiting the lowest levels among all irradiated groups (Fig. 5K,L). MitoSOX and MitoTracker colocalization further confirmed that both Tec and U0126 significantly reduced red fluorescence (MitoSOX) intensity compared to the Ir group, with the Ir+Tec+U0126 group exhibiting the faintest red fluorescence among all irradiated groups (Fig. 5M). JC-1 staining revealed that irradiation-induced loss of mitochondrial membrane potential, characterized by decreased red fluorescence (JC-1 aggregates) and increased green fluorescence (JC-1 monomers), was reversed by treatment with both Tec and U0126, with the combination group exhibiting the highest red fluorescence (Fig. 5N). Flow cytometric quantification confirmed that U0126, similar to Tec, significantly increased the proportion of JC-1 aggregates following irradiation, with the Ir+Tec+U0126 group exhibiting the highest JC-1 aggregate proportion among all irradiated groups (Fig. 5O–Q).

Fig. 5.

Tec protects HUVECs from 9 Gy irradiation-induced injury at 72 h post-exposure by inhibiting mitophagy. (A) Representative flow cytometry histograms of DHE fluorescence intensity. (B) Quantitative analysis of DHE fluorescence intensity (n = 6). (C) Representative images of DHE fluorescence staining (red). Scale bar = 20 µm. (D) Cell viability assessed by CCK-8 assay. (E) Representative flow cytometry plots of Annexin V/PI staining for apoptosis detection. (F) Quantitative analysis of apoptotic rates (n = 6). (G) Representative confocal images of dual staining for Mitophagy (red) and Lysosomes (green). Scale bar = 20 µm. (H) Flow cytometric analysis of mitophagy rate. (I) Quantitative analysis of mitophagy rate (n = 6). (J) ATP levels measured by ATP assay. (K) Representative flow cytometry histograms of MitoSOX fluorescence intensity. (L) Quantitative analysis of MitoSOX fluorescence intensity (n = 6). (M) Representative confocal images of MitoSOX (red) and MitoTracker (green) colocalization. Scale bar = 20 µm. (N) Representative images of JC-1 fluorescence staining (red: aggregates, green: monomers). Scale bar = 20 µm. (O) Representative flow cytometry dot plots of JC-1 staining for mitochondrial membrane potential. (P) Quantitative analysis of JC-1 aggregates proportion (n = 6). (Q) Quantitative analysis of JC-1 monomers proportion (n = 6). Data are presented as mean $\pm$ SD. Statistical analysis was performed using one-way ANOVA followed by Tukey test. *: p 0.05; NS: p $>$ 0.05. ATP, Adenosine triphosphate.

To further explore whether Tec protects HUVECs against 9 Gy irradiation-induced injury by regulating mitophagy, we examined protein expression levels by Western blotting, along with apoptosis and oxidative stress levels. Western blot analysis confirmed that PINK1 expression was suppressed by PINK1 siRNA (Fig. 6A–E), while MTK458 significantly upregulated PINK1 expression (Fig. 6F–H). Flow cytometric analysis of apoptosis revealed that the apoptotic rate was significantly increased in the Ir group compared to the Con group, whereas Tec treatment markedly reduced this elevation. Notably, inhibition of PINK1 expression (Ir+Tec+siRNA) further decreased the apoptotic rate relative to the Ir+Tec group, while overexpression of PINK1 (Ir+Tec+MTK) completely abolished the protective effect of Tec, resulting in a significantly increased apoptotic rate (Fig. 6I,J). CCK-8 assays demonstrated that Tec significantly enhanced cell viability following irradiation-induced injury, an effect further potentiated by PINK1 inhibition. Conversely, PINK1 overexpression abrogated the protective effect of Tec, as evidenced by a marked reduction in cell viability (Fig. 6K). Flow cytometric analysis of oxidative stress revealed that DHE fluorescence intensity was significantly increased in the Ir group compared to the Con group, while Tec treatment attenuated this increase. Relative to the Ir+Tec group, inhibition of PINK1 expression further reduced DHE fluorescence intensity, whereas PINK1 overexpression significantly exacerbated oxidative stress levels, as evidenced by enhanced DHE fluorescence (Fig. 6L,M). DHE fluorescence staining further confirmed that red fluorescence (DHE) was markedly diminished in the Ir+Tec group compared to the Ir group. Relative to the Ir+Tec group, inhibition of PINK1 expression further attenuated the red fluorescence signal, while PINK1 overexpression completely abolished the protective effect of Tec, resulting in enhanced red fluorescence (Fig. 6N). Collectively, these findings demonstrate that PINK1 plays a critical role in mediating the radioprotective effects of Tec in HUVECs.

Fig. 6.

Tec protects HUVECs from 9 Gy irradiation-induced injury at 72 h post-exposure by inhibiting PINK1. (A) Western blot analysis of PINK1 expression following siRNA-mediated knockdown. (B) Quantitative analysis of PINK1 and Parkin expression levels normalized to $\beta$-actin (n = 3). (C) Western blot analysis of PINK1 and Parkin protein expression. (D) Quantitative analysis of PINK1 expression levels normalized to $\beta$-actin (n = 3). (E) Quantitative analysis of Parkin expression levels normalized to $\beta$-actin (n = 3). (F) Western blot analysis of PINK1 overexpression following MTK458 treatment. (G) Quantitative analysis of PINK1 expression levels (n = 3). (H) Quantitative analysis of Parkin expression levels (n = 3). (I) Representative flow cytometry plots of Annexin V/PI staining for apoptosis detection. (J) Quantitative analysis of apoptotic rates (n = 6). (K) Cell viability assessed by CCK-8 assay. (L) Representative flow cytometry histograms of DHE fluorescence intensity. (M) Quantitative analysis of DHE fluorescence intensity (n = 6). (N) Representative images of DHE fluorescence staining (red). Scale bar = 20 µm. Data are presented as mean $\pm$ SD. Statistical analysis was performed using one-way ANOVA followed by Tukey test. *: p 0.05; NS: p $>$ 0.05.

To further explore whether Tec regulates PINK1 to influence mitochondrial outcomes following 9 Gy irradiation-induced injury, mitochondrial function was assessed. To assess mitophagic activity, dual staining of mitophagy and lysosomes was performed. Confocal microscopy revealed that red fluorescence (mitophagy) was significantly enhanced in the Ir group compared to the Con group, accompanied by green fluorescence (lysosomes), while Tec treatment markedly attenuated this enhancement. Relative to the Ir+Tec group, inhibition of PINK1 expression further diminished the red fluorescence signal, whereas overexpression of PINK1 led to a pronounced increase in red fluorescence (Fig. 7A). Flow cytometric quantification of the mitophagy-positive cells further confirmed these findings. The proportion of mitophagy-positive cells was significantly increased in the Ir group compared to the Con group, while Tec treatment markedly reduced this proportion relative to the Ir group. Furthermore, inhibition of PINK1 expression in combination with Tec treatment further decreased the ratio of mitophagy-positive cells compared to the Ir+Tec group, whereas PINK1 overexpression significantly increased this proportion (Fig. 7B,C). ATP levels were significantly decreased in the Ir group compared to the control (Con) group. Conversely, Tec treatment significantly restored ATP levels relative to the Ir group. Relative to the Ir+Tec group, inhibition of PINK1 expression further elevated ATP levels, whereas overexpression of PINK1 abolished the protective effect of Tec, resulting in reduced ATP production (Fig. 7D). Flow cytometric analysis of mitochondrial membrane potential revealed that the proportion of JC-1 aggregates was significantly decreased in the Ir group compared to the Con group. Conversely, Tec treatment significantly increased the proportion of JC-1 aggregates relative to the Ir group. Relative to the Ir+Tec group, inhibition of PINK1 expression restored mitochondrial membrane potential, as evidenced by an increased proportion of JC-1 aggregates. In contrast, overexpression of PINK1 reversed the protective effect of Tec, resulting in a significant reduction in JC-1 aggregates. The proportion of JC-1 monomers exhibited an opposite trend (Fig. 7E–G). JC-1 staining further confirmed that Tec treatment significantly enhanced red fluorescence (JC-1 aggregates) compared to the Ir group. Furthermore, inhibition of PINK1 expression in combination with Tec treatment further restored mitochondrial membrane potential, as evidenced by a marked increase in red fluorescence. In contrast, overexpression of PINK1 reversed the protective effect of Tec, resulting in significantly diminished red fluorescence, indicating a loss of mitochondrial membrane potential (Fig. 7H). Flow cytometric analysis of mitochondrial oxidative stress levels revealed that MitoSOX fluorescence intensity was significantly increased in the Ir group compared to the Con group, while Tec treatment markedly attenuated this increase. Relative to the Ir+Tec group, inhibition of PINK1 expression further reduced mitochondrial oxidative stress, as evidenced by decreased MitoSOX fluorescence. In contrast, overexpression of PINK1 reversed the protective effect of Tec, resulting in significantly enhanced MitoSOX fluorescence (Fig. 7I,J). Colocalization analysis of MitoSOX and MitoTracker further confirmed that red fluorescence (MitoSOX) was markedly diminished in the Ir+Tec group compared to the Ir group, accompanied by decreased colocalization with green fluorescence (MitoTracker). Relative to the Ir+Tec group, inhibition of PINK1 expression further attenuated red fluorescence, indicating reduced mitochondrial oxidative stress levels. In contrast, overexpression of PINK1 reversed the protective effect of Tec, as evidenced by significantly enhanced red fluorescence and its colocalization with green fluorescence (Fig. 7K).

Fig. 7.

Tec preserves mitochondrial function against 9 Gy irradiation-induced injury at 72 h post-exposure via PINK1 inhibition. (A) Representative confocal images of dual staining for Mitophagy (red) and Lysosomes (green). Scale bar = 20 µm. (B) Flow cytometric analysis of mitophagy rate. (C) Quantitative analysis of mitophagy rate (n = 6). (D) ATP levels measured by ATP assay (n = 3). (E) Representative flow cytometry dot plots of JC-1 staining for mitochondrial membrane potential. (F) Quantitative analysis of JC-1 monomer proportion (n = 6). (G) Quantitative analysis of JC-1 aggregate proportion (n = 6). (H) Representative images of JC-1 fluorescence staining (red: aggregates, green: monomers). Scale bar = 20 µm. (I) Representative flow cytometry histograms of MitoSOX fluorescence intensity. (J) Quantitative analysis of MitoSOX fluorescence intensity (n = 6). (K) Representative confocal images of MitoSOX (red) and MitoTracker (green) colocalization. Scale bar = 20 µm. Data are presented as mean $\pm$ SD. Statistical analysis was performed using one-way ANOVA followed by Tukey test. *: p 0.05; NS: p $>$ 0.05.