Tempol Protects Against Radiation Injury in the Submandibular Gland Through the β‐Catenin Signaling Pathway

ABSTRACT

Background

Radiation damage severely impacts salivary gland function and cell survival, especially in head and neck radiotherapy. Tempol (TPL), a free radical scavenger, has shown protective effects against radiation damage. This research aimed to investigate the protective effects of TPL on radiation‐induced damage in Hs917.T cells and the submandibular gland (SMG) of C57BL/6 mice, along with the mechanisms involved.

Methods

Human parotid fibroblasts (Hs917.T) were pre‐treated with TPL and exposed to ionizing radiation (IR). Protective effects were evaluated using MTT, clonogenic survival assays, flow cytometry, and intracellular reactive oxygen species levels. In vivo, C57BL/6 mice were pre‐treated with TPL (275 mg/kg) and exposed to 15 gray (Gy). Effects were assessed by survival rates, body weight changes, histological analysis, and TUNEL staining. Changes in apoptosis‐related markers and β‐catenin signaling pathway were analyzed, and the role of TPL was verified using the β‐catenin inhibitor XAV939.

Results

TPL pre‐treatment increased cell survival, reduced apoptosis, alleviated cell cycle arrest, and decreased intracellular superoxide and hydrogen peroxide levels in Hs917. T cells. In C57BL/6 mice, pre‐treatment with TPL improved survival, mitigated weight loss, reduced SMG damage, and decreased apoptosis. TPL inhibited IR‐induced apoptosis by increasing Bcl‐2 expression and decreasing Bax and caspase‐9 levels. TPL exerted anti‐apoptotic and protective effects by upregulating the expression of β‐catenin, promoting its nuclear translocation, and inhibiting its phosphorylation. These protective effects of TPL were reversed by XAV939.

Conclusions

TPL exerted protective effects against IR‐induced damage in Hs917.T cells and the SMG of C57BL/6 mice through activating the β‐catenin signaling pathway, inhibiting cell apoptosis, and alleviating oxidative stress.

Abbreviations

ANOVA

analysis of variance

AQP5

aquaporin‐5

DMEM

Dulbecco's Modified Eagle's Medium

FITC

fluorescein isothiocyanate

Gy

gray

HE

Hematoxylin–eosin

HRP

horseradish peroxidase

IF

Immunofluorescence

IHC

Immunohistochemistry

IP

intraperitoneally

IR

ionizing radiation

MTT

3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide

PBS

phosphate‐buffered saline

PI

propidium iodide

ROS

reactive oxygen species

SD

standard deviation

SMG

submandibular gland

TPL

Tempol

TUNEL

Terminal deoxynucleotidyl transferase dUTP Nick End Labeling

1. Introduction

Ionizing radiation (IR) plays a crucial role in the medical field, particularly in cancer treatment. However, the damage it causes to normal tissues has long been a focus and challenge in research [1]. Current studies have shown that IR induces a sharp increase in the levels of reactive oxygen species (ROS) within cells, triggering oxidative stress responses. This process leads to the disruption of biomolecular structures within cells and the activation of apoptotic signaling pathways, ultimately resulting in cell death and tissue functional impairment [2, 3]. In particular, during head and neck radiotherapy, the salivary glands (especially the submandibular gland, SMG) are highly susceptible to radiation damage. This damage causes atrophy of the acinar cells and dilation of the ducts, leading to reduced saliva secretion, xerostomia (dry mouth), and associated complications, which markedly affect the patient's quality of life [4, 5]. Despite ongoing efforts by researchers to explore protective strategies against radiation damage, there is still a lack of highly effective, safe, and widely applicable protective measures. Therefore, in‐depth investigation into the mechanisms of radiation damage and protective methods for the SMG holds important clinical significance.

Tempol (TPL) is a synthetic 4‐hydroxy‐2,2,6,6‐tetramethylpiperidin‐1‐oxyl free radical scavenger, which possesses excellent antioxidant properties [6]. TPL maintains cell membrane integrity, mitochondrial function, and DNA stability through its antioxidant effects, thereby reducing cell apoptosis or necrosis [7, 8]. In recent years, TPL has demonstrated significant protective effects in radiation damage models of multiple organs, including the heart, brain, and lungs [9]. Studies have found that TPL could modulate redox balance, inhibit inflammatory responses and apoptosis, and promote the repair of damaged tissues [10, 11]. However, studies exploring TPL's protective role against IR damage in the SMG are still nascent. The precise protective effects and molecular mechanisms of TPL in this area are not yet fully understood and warrant further research to enhance its potential applications in radiation protection.

The β‐catenin signaling pathway is one of the crucial intracellular signaling pathways, involved in regulating diverse biological processes including cell proliferation, differentiation, apoptosis, and migration [12]. Studies have shown that nuclear translocation of β‐catenin promoted the transcription of anti‐apoptotic genes (such as Bcl‐2) and inhibited oxidative stress [13]. Under normal physiological conditions, β‐catenin expression and activity are strictly regulated, and its abnormal activation is strongly associated with various diseases development [12, 14]. In recent years, research has indicated that the β‐catenin signaling pathway also played a significant role in radiation damage [15]. Radiation could lead to downregulation of β‐catenin expression or impairment of its function, thereby affecting the cell's anti‐apoptotic and repair capabilities [16]. However, the specific mechanisms of the β‐catenin signaling pathway in radiation‐induced SMG damage, as well as its association with the potential protective agent TPL, have not yet been fully elucidated.

Based on the above background, the present study aims to explore the protective effects of TPL on human parotid fibroblasts (Hs917.T) and radiation‐induced damage to the SMG in C57BL/6 mice. We will delve into the molecular mechanisms by which TPL regulates apoptosis and alleviates oxidative stress, and clarify the crucial role of the β‐catenin signaling pathway in TPL‐mediated protection against radiation damage. This research is intended to offer a theoretical basis for developing TPL‐based strategies to protect the SMG from radiation injury.

2. Materials and Methods

2.1. Cell Culture and Treatment

Human parotid fibroblasts (Hs917.T; GuangZhou Jennio Biotech Co. Ltd.) were routinely cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and maintained at 37°C in a humidified incubator with 5% CO₂. For TPL dose–response experiments, cells were treated with TPL (0, 0.05, 0.125, 0.25, 2, and 8 mM; T6699, TargetMol Chemicals Inc.) and then exposed to ionizing radiation (IR; 6 gray (Gy)). For radiation dose–response experiments, cells were incubated with 8 mM TPL or vehicle control for 16 h and subsequently irradiated with 0, 2, 4, 6, or 8 Gy. For mechanistic experiments, cells were pre‐incubated with vehicle or 8 mM TPL for 10 min prior to irradiation with 6 Gy (groups: Vehicle, TPL, IR + V, and IR + TPL). Where indicated, cells were treated with the β‐catenin pathway inhibitor XAV939 (100 μM; HY‐15147, MedChemExpress) for 8 h, with or without 8 mM TPL for 10 min, prior to irradiation with 6 Gy (groups: IR + V, IR + TPL, and IR + TPL + XAV939). Cell irradiation was performed using a clinical linear accelerator (6 MV X‐rays; source‐to‐skin distance [SSD] 100 cm; dose rate 400 cGy/min).

2.2. Animals

Male C57BL/6 mice (8 weeks old; initial body weight 24–26 g) were provided by the Animal Experimental Center of Nantong University (license no. SCXK (Su) 2008‐0010). Animals were maintained under specific pathogen‐free conditions with a controlled environment (20°C–26°C; 50%–70% relative humidity; 12‐h light/dark cycle) and had ad libitum access to standard chow and water. After acclimatization, mice were randomly assigned to experimental groups.

For survival and body weight experiments, mice were allocated to four groups (n = 9–10 per group): Vehicle, TPL, IR + V, and IR + TPL. Mice received an intraperitoneal (IP) injection of vehicle or TPL (275 mg/kg) once daily for 1 day as previously reported [17]. The IR + V and IR + TPL groups then underwent irradiation with a single 15 Gy dose delivered using a Varian clinical linear accelerator (6 MV X‐rays; SSD 100 cm; dose rate 400 cGy/min; field size 8 × 20 cm) [18]. Eight mice were irradiated per session using a custom plastic cylindrical immobilization device to ensure consistent positioning. Sham‐irradiated controls underwent identical handling without beam delivery. For SMG‐focused experiments (histology, TUNEL staining, immunoblotting, immunofluorescence (IF), and immunohistochemistry (IHC)), an independent cohort (n = 3 mice per group per timepoint) received a 15 Gy single‐fraction head‐and‐neck irradiation using the same beam parameters; the remainder of the body was shielded with 12‐mm lead to minimize off‐target exposure. For this cohort, SMGs were harvested at 24 h (for IHC of Bax and Bcl‐2) or 3 days (for HE, TUNEL, and β‐catenin analyses) post‐irradiation. All tissue collection was performed after euthanasia; mice were not followed after gland excision. Bilateral SMGs were dissected, with one portion fixed in 10% neutral‐buffered formalin and the remaining portion snap‐frozen in liquid nitrogen and stored at −80°C until analysis. During irradiation and terminal procedures, mice were anesthetized with inhaled isoflurane (3%–4% induction; 1.5%–2% maintenance in oxygen). Adequate depth of anesthesia was confirmed by loss of corneal and pedal withdrawal reflexes and stable respiration. For survival monitoring, animals were observed for 30 days after irradiation and body weight was recorded daily. Animals were checked at least once daily (twice daily when clinical signs were observed). Humane endpoints were predefined according to institutional animal care guidelines, including > 20% body weight loss from baseline, persistent anorexia/dehydration, inability to ambulate or access food/water, labored breathing, or other signs of severe distress. Animals reaching humane endpoint criteria were euthanized and recorded as deaths for Kaplan–Meier analysis. Euthanasia was performed by isoflurane overdose, followed by confirmation of death (cessation of respiration and heartbeat) and cervical dislocation as a secondary method.

All animal experimental procedures followed the ARRIVE guidelines and the IACUC Handbook (third edition). All animal protocols were approved by the committee of The First Affiliated Hospital of Gannan Medical University (approval number: ZLSL2023115).

2.3. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide (MTT)

4‐Cells were initially plated at 5000 cells per well in a 96‐well plate and grown until they reached the logarithmic phase. Following treatment, 100 μL of MTT solution (5 mg/mL) was introduced into each well, and cells were incubated for another 4 h. Supernatant was then discarded, and dimethyl sulfoxide was used to dissolve the formazan crystals. The absorbance of each well was subsequently measured at 490 nm using a microplate reader. Cell viability was evaluated by comparing the absorbance values of the treated groups to those of the control group.

2.4. Clonogenic Survival Assay

Cells were initially plated at a density of 400 cells per well in 6‐well plates or dishes. After treatment, cells were cultured for 7–14 days until visible colonies formed. Cells were then stained with crystal violet solution (0.5% crystal violet, 2% ethanol) for 15–30 min. Afterward, cells were rinsed with deionized water to remove unbound dye. Finally, colonies larger than 2 mm in diameter were counted under a microscope. The colony formation rate was determined by the formula: (number of colonies/number of seeded cells) × 100%.

2.5. Detection of Apoptosis and Cell Cycle

Hs917.T cells were collected and stained with Annexin V‐fluorescein isothiocyanate (FITC) and propidium iodide (PI) to assess apoptosis via flow cytometry. The procedure involved washing cells with phosphate‐buffered saline (PBS), resuspending them in binding buffer, and adding Annexin V‐FITC and PI. Cells were incubated in the dark at room temperature for 15 min before being analyzed by flow cytometry. Simultaneously, treated cells were harvested, fixed in 70% cold ethanol overnight, and stained with PI the following day for cell cycle analysis to quantify the proportion of cells in G2/M phase.

2.6. Detection of Intracellular Superoxide Levels and Hydrogen Peroxide Levels

Cells were seeded in culture dishes or 96‐well plates, followed by staining with DHE (10 μM, for detecting intracellular superoxide) and DCFH‐DA (10 μM, for detecting intracellular hydrogen peroxide, reflecting the level of ROS). For DHE staining, cells were kept in the dark at 37°C for 30 min; for DCFH‐DA staining, cells were maintained in the dark at 37°C for 20 min. Following staining, cells were rinsed with PBS to eliminate any unbound dye. Finally, intracellular fluorescence intensity was measured. For DHE staining, the excitation wavelength was 488 nm and the emission wavelength was 590 nm. In contrast, for DCF staining, the excitation wavelength was also 488 nm, but the emission wavelength was 525 nm.

2.7. Hematoxylin–Eosin (HE) Staining

The histological changes in the SMG were detected using an HE staining kit (G1100, Solarbio). Mouse SMG tissues were fixed in 4% paraformaldehyde and processed through dehydration, embedding, and sectioning to prepare tissue slices. The slices were dewaxed twice in xylene (XW‐RS‐008, Guangzhou Xiuwei Technology Co. Ltd.) for 5–10 min each time. After dewaxing through graded ethanol (75%–100%) to water, the slices were stained with hematoxylin. Slices were differentiated in differentiating solution for 10–60 s, followed by two drops or immersions in tap water. Slices were then stained in eosin solution, and the excess stain was removed before rapid dehydration. The slices were immersed in 75% ethanol, 85% ethanol, 95% ethanol, and 100% ethanol (I) for 2–3 s each. The slices were immersed in 100% ethanol (II) for 1 min, cleared twice in xylene for 1 min, and mounted with neutral gum (XW‐RS‐015, Guangzhou Xiuwei Technology Co. Ltd.) for observation under a microscope.

2.8. Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL)

Mouse SMG tissues were fixed in 10% neutral‐buffered formalin, dehydrated, paraffin‐embedded, and sectioned. After antigen retrieval, sections were incubated with 3% H₂O₂ for 10 min to quench endogenous peroxidase activity, followed by permeabilization with proteinase K. TUNEL staining was then performed according to the manufacturer's instructions using a TUNEL kit (G1502, Servicebio). Slides were counterstained with hematoxylin, and TUNEL‐positive cells were visualized and quantified.

2.9. Western Blot

Total protein was extracted using radio‐immunoprecipitation assay lysis and quantified using a bicinchoninic acid protein assay kit (BL521A, Biosharp). Equal amounts of protein were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (15% gels) and transferred to polyvinylidene fluoride membranes (ISEQ00010, Merck Millipore). Membranes were blocked with 5% non‐fat milk and incubated overnight at 4°C with primary antibodies against Bax (50599‐2‐Ig, Proteintech), Bcl‐2 (60178‐1‐Ig, Proteintech), β‐catenin (51067‐2‐AP, Proteintech), phospho‐β‐catenin (Ser33/37/Thr41; #9561, Cell Signaling Technology), and β‐actin (66009‐1‐Ig, Proteintech). Working dilutions for all antibodies are provided in Table 1. After washing, membranes were incubated with horseradish peroxidase (HRP)‐conjugated secondary antibodies (goat anti‐rabbit IgG‐HRP, BL003A, Biosharp; goat anti‐mouse IgG‐HRP, BL001A, Biosharp). Immunoreactive bands were visualized using an ultrasensitive ECL substrate (Advansta, K‐12045‐D50) and imaged using the Chemiscope 6100 system (CLINX).

TABLE 1.

Antibodies used in this study and working dilutions.

Target	Supplier	Catalog no.	Application	Working dilution
Bcl‐2	Proteintech	60178‐1‐Ig	WB; IHC	1:1000 (WB); 1:200 (IHC)
Bax	Proteintech	50599‐2‐Ig	WB; IHC	1:20000 (WB); 1:1000 (IHC)
AQP5	Proteintech	20334‐1‐AP	IHC	1:2500–1:10000 (IHC)
β‐catenin	Proteintech	51067‐2‐AP	WB; IF	1:5000 (WB); 1:500 (IF)
phospho‐β‐catenin (Ser33/37/Thr41)	Cell Signaling Technology	#9561	WB	1:2000
Caspase‐9	Proteintech	10380‐1‐AP	IF	1:50
β‐Actin	Proteintech	66009‐1‐Ig	WB	1:5000
Goat anti‐rabbit IgG‐HRP	Biosharp	BL003A	WB; IHC	1:5000
Goat anti‐mouse IgG‐HRP	Biosharp	BL001A	WB; IHC	1:5000

2.10. Immunofluorescence (IF)

Immunofluorescence (IF) was used to detect caspase‐9 expression and the nuclear/cytoplasmic localization of β‐catenin in Hs917.T cells, as well as β‐catenin localization in mouse SMG sections. Briefly, cells grown on coverslips (or tissue sections) were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X‐100, and blocked with 5% bovine serum albumin. Samples were incubated overnight at 4°C with primary antibodies against caspase‐9 (10380‐1‐AP, Proteintech) or β‐catenin (51067‐2‐AP, Proteintech) (see Table 1 for working dilutions), followed by fluorophore‐conjugated secondary antibodies for 1 h at 37°C in the dark. Nuclei were counterstained with 4′,6‐Diamidino‐2‐Phenylindole. Images were acquired using a fluorescence microscope at 400× magnification unless otherwise specified.

2.11. Immunohistochemistry (IHC)

Immunohistochemistry (IHC) was performed to assess Bax, Bcl‐2, and aquaporin‐5 (AQP5) expression in mouse SMG sections. After fixation and paraffin embedding, sections were deparaffinized, rehydrated, and subjected to antigen retrieval. Endogenous peroxidase activity was quenched with 3% H₂O₂ for 10 min. Sections were blocked with 5% bovine serum albumin and incubated overnight at 4°C with primary antibodies against Bax (50599‐2‐Ig, Proteintech) or Bcl‐2 (60178‐1‐Ig, Proteintech) (see Table 1 for working dilutions). After PBS washes, sections were incubated with HRP‐conjugated secondary antibodies for 30 min, developed with 3,3′‐diaminobenzidine, counterstained with hematoxylin, and imaged by light microscopy (200×; representative high‐power insets at 400×).

2.12. Image Acquisition and Quantitative Analysis

For HE, TUNEL, IF, and IHC analyses, images were acquired using standardized acquisition settings. HE‐ and TUNEL‐stained sections were imaged at 200× magnification, with representative regions additionally shown as 400× insets where indicated. IF images (cells and tissue sections) were acquired at 400× magnification. IHC images were acquired at 200×, with representative 400× insets. Where semi‐quantitative comparisons were performed, positive signal area, mean fluorescence intensity, or integrated optical density (IOD) was quantified using ImageJ (NIH) from at least 10 randomly selected fields per section, and the average value per animal was used for statistical analysis.

2.13. Antibodies and Working Dilutions

Primary antibodies, suppliers, catalog numbers, applications, and working dilutions are summarized in Table 1.

2.14. Statistical Analysis

All statistical analyses were performed using GraphPad Prism 8.0. Data distribution was assessed using the Shapiro–Wilk normality test prior to inferential testing. For comparisons between two groups, a two‐tailed Student's t‐test was used when data met assumptions of normality; otherwise, the Mann–Whitney U test was applied. For comparisons among multiple groups, one‐way analysis of variance followed by Tukey's post hoc test was used for normally distributed data; otherwise, the Kruskal–Wallis test followed by Dunn's multiple‐comparisons test was applied. Kaplan–Meier survival curves were compared using the log‐rank (Mantel–Cox) test. Quantitative results are presented as mean ± standard deviation (SD) unless otherwise stated, and exact p values are reported whenever possible. A two‐sided p < 0.05 was considered statistically significant. Complete numerical results underlying each figure panel (mean, SD, n, test statistic, exact p values, and 95% confidence intervals) are provided in Tables S1–S5.

3. Results

3.1. Protective Effects of TPL Pre‐Treatment on IR‐Induced Damage in Hs917.T Cells

To explore the protective effects of TPL on IR‐induced damage in Hs917.T cells, we treated these cells with different concentrations of TPL and different radiation doses. Figure 1A showed the molecular structure of TPL. Hs917.T cells were pre‐treated with varying concentrations of TPL prior to irradiation at 6 Gy. The results indicated that TPL treatment notably enhanced Hs917.T cell viability (Figure 1B). Additionally, Hs917.T cells treated with 8 mM TPL for 16 h were subjected to varying doses of radiation (0, 2, 4, 6, and 8 Gy). The surviving fraction was consistently higher in the TPL‐treated group than in the vehicle group, demonstrating that TPL treatment significantly improved the clonogenic survival ability of the cells (Figure 1C). Together, these results indicate that TPL improves short‐term viability and enhances clonogenic survival after irradiation. Subsequently, Hs917.T cells were treated with 8 mM TPL for 10 min before exposure to 6 Gy of IR. After 24 h, cells were stained with Annexin V‐FITC and PI, and flow cytometry analysis indicated that, in comparison with Vehicle group, apoptosis increased in IR + V group, while TPL treatment substantially reduced IR‐induced apoptosis (Figure 1D,E). Cell cycle analysis indicated that, in contrast to Vehicle group, the percentage of cells in G2/M phase substantially increased in IR + V group, whereas TPL treatment significantly decreased G2/M cell cycle arrest (Figure 1F,G). Finally, we assessed intracellular superoxide and hydrogen peroxide levels. In comparison with Vehicle group, these levels were substantially elevated in IR + V group, while TPL treatment notably reduced these levels (Figure 1H,I). These findings suggested that pre‐treatment with TPL markedly increased the survival rate of Hs917.T cells after IR exposure, reduced apoptosis, alleviated G2/M cell cycle arrest, and decreased intracellular ROS levels, thereby exerting a protective effect against radiation‐induced damage in Hs917.T cells.

FIGURE 1.

Protective effects of Tempol (TPL) pre‐treatment on IR‐induced damage in Hs917.T cells. (A) Molecular structure of TPL. (B) Cells were treated with the indicated concentrations of TPL and then irradiated with 6 Gy; cell viability was assessed by the MTT assay at 24 h post‐irradiation and expressed as a percentage of the vehicle control (0 Gy). Data are mean ± SD (n = 3 independent experiments); one‐way ANOVA with Tukey's post hoc test (see Table S2 for exact p values). (C) Clonogenic survival of Hs917.T cells treated with vehicle or 8 mM TPL for 16 h and irradiated with 0, 2, 4, 6, or 8 Gy. Data are mean ± SD (n = 3 independent experiments); two‐tailed unpaired t tests were performed at each dose (see Table S2). (D–G) Apoptosis (D and E) and cell‐cycle distribution (F and G) were assessed by Annexin V‐FITC/PI staining and flow cytometry 24 h after 6 Gy irradiation with or without 8 mM TPL pre‐treatment (10 min). Representative plots and quantification are shown (mean ± SD, n = 3 independent experiments). (H, I) Intracellular superoxide and hydrogen peroxide levels were measured by DHE and DCF assays, respectively, at 24 h post‐irradiation (mean ± SD, n = 3 independent experiments). Abbreviations: IR + V, irradiation + vehicle; IR + TPL, irradiation + TPL.

3.2. Protective Effects of TPL Pre‐Treatment on IR‐Induced Damage in C57BL/6 Mice

To explore the protective effects of TPL on IR‐induced damage in C57BL/6 mice, the mice were IP injected with TPL (275 mg/kg) or vehicle for 1 day, followed by 15 Gy total‐body irradiation. The findings revealed that the survival rate of irradiated mice in the IR + V group was lower than that of the Vehicle group. In contrast, TPL treatment markedly enhanced the survival rate of irradiated mice (Figure 2A). Additionally, while the body weight of mice in the IR + V group significantly dropped after 15 Gy of IR compared to the Vehicle group, TPL treatment led to a notable increase in body weight (Figure 2B). This indicated that TPL pre‐treatment could mitigate the impact of IR on mouse body weight and maintain their growth status. In a separate head‐and‐neck irradiation cohort (15 Gy with body shielding), SMGs were excised 3 days after irradiation for tissue analyses. HE staining revealed histological changes in the SMG, with TPL pre‐treatment significantly reducing IR‐induced acinar atrophy and ductal dilation (Figure 2C). Normal duct, acinus, destructed duct, and vacuolization are indicated by saffron, green, blue, and yellow arrows, respectively. Moreover, apoptosis cells in the SMG, indicated by red arrows, were examined using the TUNEL kit. Compared to the Vehicle group, an increase in apoptosis was observed in the SMG of the IR + V group. However, TPL treatment significantly reduced apoptosis in the SMG (Figure 2D). In addition, immunohistochemistry for AQP5, an acinar cell marker closely linked to salivary secretory function, showed that irradiation markedly reduced AQP5 staining in the SMG, whereas TPL pre‐treatment preserved AQP5 expression (Figure 2E). Together, these findings indicate that TPL pre‐treatment improves systemic outcomes (survival and body weight) after irradiation and, in the SMG‐focused cohort, mitigates histological injury, reduces apoptosis, and preserves AQP5 expression, supporting an SMG‐protective effect of TPL.

FIGURE 2.

Protective effects of TPL pre‐treatment on IR‐induced damage in C57BL/6 mice. For systemic outcomes, eight‐week‐old C57BL/6 mice received intraperitoneal injections of TPL (275 mg/kg) or vehicle for 1 day prior to a 15 Gy total‐body irradiation; mice were weighed daily and monitored for pre‐morbid signs (n = 9–10 per group). (A) Kaplan–Meier survival curves. (B) Average body weight changes after irradiation (mean ± SD). For SMG‐focused analyses, an independent cohort received a 15 Gy single‐fraction head‐and‐neck irradiation with lead shielding of the remainder of the body, and SMGs were harvested 3 days after irradiation (n = 3 mice per group). (C) Representative HE‐stained SMG sections (200×; representative 400× insets) showing histological alterations; saffron, green, blue, and yellow arrows indicate a normal duct, normal acinus, destructed duct, and vacuolization, respectively. (D) Representative TUNEL staining (200×; representative 400× insets) and quantification of TUNEL‐positive cells per SMG (mean ± SD, n = 3). (E) Representative AQP5 immunohistochemistry in SMG sections with semi‐quantitative staining analysis (mean ± SD, n = 3 mice per group; 200× with representative 400× insets where indicated). Abbreviations: IR + V, irradiation + vehicle; IR + TPL, irradiation + TPL. Exact p values are provided in Table S3.

3.3. The Apoptotic Regulatory Effects of TPL on IR‐Induced Damage in Hs917.T Cells and Mouse SMG

To explore the regulatory effects of TPL on apoptosis in Hs917.T cells and IR‐induced damage in the mouse SMG, we assessed apoptosis‐related markers levels. In Hs917.T cells, in contrast to Vehicle group, Bcl‐2 levels were decreased, while Bax and caspase‐9 levels were increased in IR + V group. This indicated that IR induced apoptosis by activating Bax and caspase family proteins. However, TPL treatment increased Bcl‐2 levels and decreased Bax and caspase‐9 levels, thereby inhibiting IR‐induced apoptosis (Figure 3A,B). Green arrow and saffron arrow point to the cytoplasmic caspase‐9 and nuclear caspase‐9, respectively. This suggested that TPL could protect cells by regulating the balance of Bcl‐2 and Bax expression and inhibiting activation of caspase‐9. The animal experiments further validated the results from the cell experiments. In the SMG of C57BL/6 mice, in comparison with Vehicle group, Bcl‐2 levels were decreased and Bax levels were increased in IR + V group. However, TPL pre‐treatment significantly increased Bcl‐2 levels and decreased Bax levels, thereby inhibiting IR‐induced apoptosis (Figure 3C). These results indicated that TPL inhibited the IR‐induced apoptosis process by increasing Bcl‐2 levels and decreasing Bax and caspase‐9 levels.

FIGURE 3.

TPL regulates apoptosis in IR‐treated Hs917.T cells and mouse SMG. (A) Hs917.T cells were treated with or without 8 mM TPL for 10 min prior to 6 Gy irradiation and harvested 6 h later; proteins were analyzed by Western blotting for Bcl‐2 and Bax. (B) Caspase‐9 expression in Hs917.T cells was evaluated by immunofluorescence at 24 h post‐irradiation; representative images are shown (400×) together with quantitative fluorescence analysis (mean ± SD, n = 3 independent experiments). (C) Bcl‐2 and Bax expression in mouse SMG was assessed by immunohistochemistry 24 h after 15 Gy head‐and‐neck irradiation with or without TPL pre‐treatment; representative images are shown (200×; representative 400× insets) together with quantitative integrated optical density (IOD) analysis (mean ± SD, n = 3 mice per group). Exact p values are provided in Table S4.

3.4. TPL Protected Hs917.T Cells and Mouse SMG From IR‐Induced Damage by Inhibiting Apoptosis Through β‐Catenin Signaling Pathway

To investigate whether TPL exerted its anti‐apoptotic and protective effects through the β‐catenin signaling pathway, we first assessed the variations in β‐catenin levels. In Hs917.T cells, in contrast to Vehicle group, β‐catenin levels were decreased in IR + V group, while TPL treatment substantially upregulated β‐catenin expression (Figure 4A). IF further revealed that TPL treatment markedly elevated nuclear expression of β‐catenin, suggesting that TPL may exert its protective effects by promoting nuclear translocation of β‐catenin (Figure 4B). Red arrow and saffron arrow point to the cytoplasmic β‐catenin and nuclear β‐catenin, respectively. The animal experiments further validated the results from the cell experiments. In the SMG of C57BL/6 mice, in comparison with Vehicle group, β‐catenin levels were decreased in IR + V group. However, TPL pre‐treatment notably upregulated β‐catenin expression in SMG (Figure 4C). IF further showed that TPL pre‐treatment markedly elevated nuclear expression of β‐catenin, suggesting that TPL may exert its protective effects by promoting nuclear translocation of β‐catenin (Figure 4D). Next, Hs917.T cells were treated with 8 mM TPL for 10 min and/or 100 μM β‐catenin signaling pathway inhibitor XAV939 for 8 h prior to irradiation with 6 Gy. Western blot revealed that, in comparison with IR + V group, β‐catenin levels were elevated and phospho‐β‐catenin levels were decreased in IR + TPL group. However, after treatment with XAV939, β‐catenin levels were decreased and phospho‐β‐catenin expression was increased, suggesting that TPL may stabilize β‐catenin expression by inhibiting its phosphorylation (Figure 4E). IF further revealed that, in contrast to IR + TPL group, nuclear β‐catenin levels were reduced in IR + TPL + XAV939 group, indicating that TPL may exert its protective effects by promoting nuclear translocation of β‐catenin (Figure 4F). Additionally, compared to the IR + TPL group, increased apoptosis was observed in the IR + TPL + XAV939 group, indicating that TPL exerted its anti‐apoptotic and protective effects through the β‐catenin signaling pathway (Figure 4G). These results suggested that TPL exerted its anti‐apoptotic and protective effects by upregulating β‐catenin expression, promoting its nuclear translocation, and inhibiting its phosphorylation. β‐catenin signaling pathway inhibitor XAV939 reversed the protective effects of TPL, further confirming that TPL exerted its protective effects through the β‐catenin signaling pathway.

FIGURE 4.

TPL protects Hs917.T cells and mouse SMG from IR‐induced injury by modulating β‐catenin signaling. (A) Representative Western blot analysis of total β‐catenin in Hs917.T cells treated with or without 8 mM TPL for 10 min prior to 6 Gy irradiation; cells were harvested 24 h post‐irradiation. (B) Nuclear/cytoplasmic localization of β‐catenin in Hs917.T cells assessed by immunofluorescence (400×) with quantitative analysis (mean ± SD, n = 3 independent experiments). (C) SMGs were harvested 3 days after irradiation for immunoblotting; representative blots are shown. (D) β‐catenin localization in irradiated SMG sections assessed by immunofluorescence (400×) with quantitative analysis (mean ± SD, n = 3 mice per group). Abbreviations: IR + V, irradiation + vehicle; IR + TPL, irradiation + TPL. Where indicated, Hs917.T cells were treated with 8 mM TPL (10 min) and/or 100 μM XAV939 (8 h) prior to 6 Gy irradiation. (E) Representative Western blots of β‐catenin and phospho‐β‐catenin. (F) β‐catenin localization assessed by immunofluorescence (400×) with quantitative analysis (mean ± SD, n = 3 independent experiments). (G) Apoptosis assessed by Annexin V‐FITC/PI staining and flow cytometry; representative plots and quantification are shown (mean ± SD, n = 3 independent experiments). Exact p values are provided in Table S5.

4. Discussion

This study investigated the radioprotective effects of TPL on ionizing radiation (IR)‐induced injury in the SMG and explored the underlying mechanism. Collectively, our in vitro and in vivo results indicate that TPL mitigates IR‐induced injury by activating the β‐catenin signaling pathway, thereby suppressing apoptosis and oxidative stress.

Radiation‐induced salivary gland damage is a common complication of head and neck radiotherapy and can result in reduced saliva secretion and xerostomia, substantially impairing patient quality of life [19]. In this study, we combined cellular assays with mouse irradiation models to evaluate both mechanistic and tissue‐level effects of TPL. At the cellular level, TPL pre‐treatment improved the survival of Hs917.T cells after IR, reduced apoptosis, alleviated G2/M cell‐cycle arrest, and decreased intracellular superoxide and hydrogen peroxide levels, supporting an antioxidant and anti‐apoptotic mode of action consistent with prior studies in other radiation‐injury models [20, 21, 22, 23, 24]. In vivo, two complementary irradiation paradigms were used to support interpretability. First, a 15 Gy total‐body irradiation model was used to assess systemic radioprotection, in which TPL pre‐treatment improved 30‐day survival and attenuated body weight loss. Second, a head‐and‐neck irradiation model with lead shielding of the remainder of the body was used to focus on SMG injury; in this setting, TPL preserved SMG histoarchitecture and reduced apoptosis in glandular tissue. These results support both an overall radioprotective effect and an SMG‐specific protective effect. We selected early post‐irradiation harvest timepoints (24 h and day 3) to capture acute molecular and histopathological responses, which are critical for mechanistic interrogation of apoptosis and β‐catenin signaling. We acknowledge, however, that clinically relevant xerostomia is typically a late effect developing over weeks. Therefore, salivary gland‐specific functional and phenotypic endpoints, such as pilocarpine‐stimulated salivary flow rate and the acinar marker aquaporin‐5 (AQP5), would further strengthen the translational relevance of these findings. Notably, prior work has demonstrated that Tempol can preserve salivary flow in mice after 15 Gy irradiation at both acute (day 3) and later (day 30) timepoints [25], supporting the plausibility that the structural and anti‐apoptotic protection observed here may translate into functional benefit. Future studies will incorporate longitudinal functional readouts and additional gland‐specific markers to further substantiate SMG protection.

IR‐induced apoptosis is one of the mechanisms of radiation‐induced cytotoxicity [26]. Further investigation into the protective mechanisms of TPL revealed that its regulation of apoptosis‐related proteins is a key aspect. IR induced upregulation of Bax and downregulation of Bcl‐2 within cells, activating caspase‐9 and thereby initiating the apoptotic program. TPL, however, upregulated Bcl‐2 and downregulated Bax and caspase‐9, disrupting the pro‐apoptotic balance induced by IR. This finding is consistent with other studies on antioxidants that inhibit apoptosis by modulating Bcl‐2 family proteins [27, 28]. Bcl‐2, an anti‐apoptotic protein, inhibited changes in mitochondrial membrane permeability and prevented cytochrome c release, thereby suppressing the caspase cascade. In contrast, Bax, a pro‐apoptotic protein, promoted increased mitochondrial membrane permeability, released cytochrome c, and activated the caspase cascade, thus promoting apoptosis [29]. By regulating these two proteins, TPL effectively inhibited IR‐induced apoptosis. This indicated that TPL could protect cells by regulating the expression balance of apoptosis‐related proteins and inhibiting the activation of caspase‐9.

The β‐catenin signaling pathway is pivotal in regulating cell proliferation, differentiation, apoptosis, and migration [30]. Under basal conditions, β‐catenin is predominantly localized in the cytoplasm and is targeted for phosphorylation‐dependent degradation. Upon pathway activation, β‐catenin becomes stabilized and translocates to the nucleus, where it interacts with transcription factors to regulate downstream gene expression [31]. In this study, we found that TPL increased β‐catenin protein levels, promoted its nuclear accumulation, and reduced β‐catenin phosphorylation, consistent with activation of β‐catenin signaling. Importantly, pharmacological inhibition of β‐catenin signaling using XAV939 reversed the anti‐apoptotic and protective effects of TPL, supporting the conclusion that β‐catenin activation is functionally required for TPL‐mediated protection. Following nuclear translocation, β‐catenin may regulate transcription of anti‐apoptotic genes (e.g., Bcl‐2) and genes involved in oxidative stress responses, thereby acting in concert with the free‐radical scavenging activity of TPL to mitigate IR‐induced injury.

Several limitations should be acknowledged. First, we used the Hs917.T cell line and a C57BL/6 mouse model; additional validation in primary SMG cells and other in vivo systems will be important. Second, although we demonstrate that β‐catenin signaling is required for the observed protection, the downstream transcriptional programs and potential upstream regulators of β‐catenin activation by TPL remain to be defined. In particular, high‐throughput omics approaches (e.g., transcriptomics, proteomics, or phosphoproteomics) could further validate whether β‐catenin signaling is central to the observed effects, identify upstream regulators, and reveal more specific molecular targets. Third, while we focused on acute injury timepoints to interrogate early molecular mechanisms, longer‐term functional endpoints (e.g., salivary flow rate) and gland‐specific phenotypic markers (e.g., AQP5) were not directly assessed in the present work and should be addressed in future studies to strengthen translational relevance.

This study provides a theoretical basis for the clinical application of TPL as a radioprotective agent. TPL significantly mitigated IR‐induced damage to the SMG through activating the β‐catenin signaling pathway, inhibiting apoptosis, and alleviating oxidative stress. Future research can further investigate the mechanisms of TPL in other radiation damage models and conduct preclinical trials to ascertain the safety and effectiveness of TPL for human use. Additionally, combining TPL with other antioxidants or anti‐apoptotic drugs may further enhance its protective effects, offering novel insights for developing more effective radiation protection strategies.

5. Conclusions

In summary, this study has confirmed that TPL exerted significant protective effects against IR‐induced damage in the SMG through activating the β‐catenin signaling pathway, inhibiting apoptosis, and alleviating oxidative stress. These findings offer novel insights and a potential therapeutic strategy for the protection of head and neck radiotherapy patients from radiation‐induced damage.

Author Contributions

Jinfeng Wang, Mingjun Xu, Jinghua Zhong, Kui Zhong, Zheng Chen, Chunyu Liu, Chao Liu: conceptualization, data curation, methodology, formal analysis, investigation, writing – original draft, and writing – review and editing.

Funding

This work was supported by Ganzhou Science and Technology Plan Project (Grant number GZ2023ZSF115).

Ethics Statement

All animal experimental procedures followed the ethical guidelines of ARRIVE guidelines and the IACUC Handbook (third edition). All animal protocols were approved by the committee of The First Affiliated Hospital of Gannan Medical University (approval number: ZLSL2023115).

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1: Normality assessment (Shapiro–Wilk test). All outcomes passed Shapiro–Wilk normality testing (alpha = 0.05). Where applicable, parametric tests were used. If normality assumptions were violated, the planned alternatives would be Mann–Whitney U (two groups) or Kruskal–Wallis with Dunn's multiple comparisons (three or more groups).

Table S2: Statistical reporting for Figure 1.

Table S3: Statistical reporting for Figure 2.

Table S4: Statistical reporting for Figure 3.

Table S5: Statistical reporting for Figure 4.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.