Perillaldehyde mitigates ionizing radiation-induced intestinal injury by inhibiting ferroptosis via the Nrf2 signaling pathway

Abstract

Introduction

In recent years, there has been an increase in the application of ionizing radiation (IR), causing more radiation exposure risks. The gastrointestinal tract is highly sensitive to IR because it is a rapidly self-renewing system [1]. Abdominal or pelvic radiotherapy is one of the most common treatments for pelvic and abdominal tumors. However, the intestinal damage caused by radiation significantly limits the clinical application of radiotherapy [2]. Therefore, there is an urgent need for an effective drug to reduce radiation-induced gastrointestinal toxicity.

Volatile monoterpenoid perillaldehyde (PAH) is the major component and the most effective ingredient of the essential oil extracted from perilla plants. Perilla plants are used as leafy vegetables and medicament [3]. Perilla frutescens leaves are mostly eaten with pasta and sashimi in Japan and are currently consumed worldwide [4, 5]. Essential oil from the perilla plant has attracted significant attention recently because of its minimal side effects. PAH is thus certified as "generally recognized as safe" (GRAS) by the United States Food and Drug Administration and the Expert Panel of the U.S. Flavour and Extract Manufacturers Association [6]. In addition, PAH has antioxidant, anti-inflammatory, anti-microbial activity, antitumor and antidepressant effects [7–11]. Despite PAH’s multifunctionality, its role in preventing or alleviating IR-induced injuries remains unknown. Perilla frutescens leaf extract has been reported to protect the extracellular matrix of human skin fibroblasts and hairless mouse skin from damage by ultraviolet radiation [12]. PAH ameliorates intestinal inflammation through JNK-mediated cytokine regulation in a dextran sulfate sodium salt-induced intestinal inflammatory injury model [4]. Moreover, PAH alleviates doxorubicin-induced cardiotoxicity by suppressing NHE1 phosphorylation and stimulating PI3K/AKT phosphorylation [13].

Previous research postulates that IR can induce intestinal injury and various forms of cell death, including apoptosis, necroptosis, pyroptosis, autophagy-dependent cell death, and ferroptosis [14]. However, the main form of IR-induced cell death remains unclear. Ferroptosis is a newly identified iron-dependent form of cell death that involves destroying the intracellular antioxidative process and significantly accumulating intracellular reactive oxygen species (ROS) [15]. Ferroptosis plays an important role in radiation-induced intestinal injury [16, 17]. Inhibiting ferroptosis can increase radiation resistance and promote damage repair [18]. However, whether PAH has anti-ferroptosis effects against IR-induced intestinal injury remains unknown.

This study investigated whether PAH has protective effects against radiation-induced intestinal injury and its potential mechanisms. PAH treatment prolonged the survival time and attenuated radiation-induced intestinal injury in mice after total abdominal irradiation (TAI). PAH also had in vitro radioprotective effects in intestinal crypt organoids and human intestinal epithelial cells (HIEC). Moreover, PAH treatment activated the Nrf2 signaling pathway and protected against IR-induced ferroptosis.

Results

PAH promotes the proliferation and differentiation of crypt cells after TAI

Immunohistochemical (IHC) staining of intestinal sections was done to evaluate the radioprotective effect of PAH on intestinal crypt cells. An intestinal crypt is a tubular gland formed by sinking the intestinal epithelium to the lamina propria at the root of the villi opening between adjacent villi. Intestinal stem cells (ISCs) that maintain active and rapid circulation are located at the bottom of crypts. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) is an important marker of ISCs, which are indispensable for intestinal regeneration after radiation [19]. TAI significantly reduced the number of Lgr5⁺ ISCs per crypt compared to the controls. However, the number of Lgr5⁺ ISCs per crypt was partially recovered after treatment with PAH (Fig. 2A, B). Ki67 is a marker of cell proliferation and represents the regeneration of intestinal epithelial cells. The number of Ki67-positive cells per crypt sharply declined at day 3.5 post-irradiation. However, this phenomenon was reversed by PAH treatment (Fig. 2A, C). Furthermore, PAH increased the average number of Ki67-positive crypts per circumference, which decreased significantly after TAI (Fig. 2D). The number of periodic acid schiff (PAS)-positive goblet cells and lysozyme-positive paneth cells was further determined to evaluate the effect of PAH on the differentiation of crypt cells. Of note, the number of PAS-positive goblet cells and lysozyme-positive paneth cells after TAI was observably lower than those in the control group. However, PAH treatment attenuated these changes (Fig. 2E-G), suggesting that PAH benefited the differentiation of crypt cells. These results collectively indicated that PAH could promote the proliferation and differentiation of crypt cells after TAI in mice.

PAH attenuates radiation-induced apoptosis and DNA damage in intestinal crypts

The protective effect of PAH on radiation-induced cell death in intestinal crypts was investigated using the TUNEL assay at 6 h post-irradiation. The level of TUNEL-positive cells in the intestinal crypts of vehicle-treated mice was significantly increased compared to those of the control group (Fig. 3A). In contrast, PAH treatment reduced the upregulation of TUNEL-positive cells in irradiated mice (Fig. 3B). Immunofluorescence (IF) staining was subsequently performed to detect γ-H2AX foci, a biomarker of DNA double-strand breaks, to test whether PAH treatment preserved the genetic stability of intestinal crypt cells in irradiated mice. There were numerous γ-H2AX foci in the irradiated mice compared to the control group. However, PAH treatment observably decreased the γ-H2AX foci levels in the crypts (Fig. 3A, C). In parallel, 8-hydroxy-2′-deoxyguanosine (8-OHdG) IF staining was used to explore the role of PAH in radiation-induced DNA oxidative damage. Similarly, TAI resulted in an increased number of 8-OHdG-positive cells in intestinal crypts compared to the control group. However, PAH treatment reduced the number of 8-OHdG-positive cells in the crypts at 3.5 d post-irradiation (Fig. 3A, D). These data collectively suggested that PAH could attenuate radiation-induced apoptosis and DNA damage in the intestinal crypts of mice after TAI.

PAH protects against IR-induced intestinal barrier damage and intestinal permeability

TAI potentially causes damage to the intestinal barrier, leading to increased intestinal permeability [20]. Intestinal tight junction proteins, including Claudin-3, E-cadherin, and ZO-1, are key markers of epithelial integrity. The markers play an important role in maintaining the integrity of the intestinal barrier and preventing epithelial leakage [21]. IHC staining was thus used to analyze the tight junction proteins in the small intestinal tissues of mice at 3.5 days post-irradiation. Notably, the levels of tight junction proteins in the vehicle-treated mice were significantly reduced compared to the corresponding levels in the control group (Fig. 4A-C). Nonetheless, PAH treatment improved the expression of these tight junction proteins after TAI, indicating that PAH contributed to alleviating intestinal barrier damage after radiation. Mice were orally treated with fluorescein isothiocyanate (FITC)-dextran to measure intestinal permeability. FITC-dextran could not enter the blood through the intestinal tissues of normal mice because of the intestinal epithelial barrier. However, the function of the normal intestinal barrier was damaged after radiation, leading to increased intestinal permeability and the entry of FITC-dextran into the blood, which could be detected in vitro. PAH treatment significantly reduced the levels of FITC-dextran in the serum compared to the corresponding levels in the serum of vehicle-treated mice (Fig. 4D), indicating that PAH could ameliorate radiation-induced destruction of the intestinal structure and function. These data collectively demonstrated that PAH treatment enhanced the intestinal barrier of irradiated mice.

PAH protects intestinal crypt organoids against radiation-induced injury

An ex vivo intestinal crypt organoid culture system was developed to further analyze the influence of PAH on ISCs. X-ray irradiation at a dose of 6 Gy significantly induced damage to intestinal organoids, embodied by organoid expansion and even rupture, decreased budding, and restricted growth. Nevertheless, PAH treatment significantly improved the rate of budding organoids and the surface area of intestinal organoids after irradiation compared to the vehicle group (Fig. 5A-C), indicating that PAH had radioprotective effects on intestinal organoids and promoted the survival of ISCs. In addition, the vehicle-treated organoids had fewer Ki67-positive cells than the sham-irradiated organoids, but the number of Ki67-positive cells was significantly restored in the presence of PAH at 7 days post-irradiation (Fig. 5D, E). These results demonstrated that PAH treatment could mitigate radiation-induced injury by promoting the survival of ISCs and enhancing organoid growth.

PAH protects HIEC-6 against radiation-induced cell viability inhibition and death

HIEC-6 cells were used as an in vitro system to determine the potential mechanism of PAH in radiation-induced intestinal injury. CCK-8 assays showed that PAH treatment at concentrations lower than 100 µM had no adverse effects on the viability of HIEC-6 cells (Fig. 6A), with 100 µM PAH alleviating cell viability inhibition after exposure to 6 Gy X-ray irradiation (Fig. 6B). PAH at a concentration of 100 µM was thus used for the subsequent experiments. Colony formation assays revealed an increase in the survival and clonogenic potential of irradiated HIEC-6 cells upon PAH treatment, especially in the 4 Gy and 6 Gy dose groups, compared to the control group (Figs. 6C, D). These findings suggested that PAH had a radioprotective effect on HIEC-6 cells.

IF staining of HIEC-6 cells was subsequently done to explore the role of PAH on radiation-induced DNA damage by detecting the levels of γ-H2AX and 8-OHdG in HIEC-6 cells (Fig. 6E, G). Irradiation markedly increased the number of γ-H2AX foci and the fluorescence intensity of 8-OHdG in HIEC-6 cells. However, PAH treatment significantly reduced the number of γ-H2AX foci in HIEC-6 cells at several time points after irradiation (Fig. 6F). Similarly, PAH treatment markedly alleviated the fluorescence intensity of 8-OHdG in HIEC-6 cells at 2 h and 6 h after irradiation (Fig. 6H). PI staining revealed a significant increase in cell death in HIEC-6 cells after 6 Gy X-ray irradiation compared to the sham-irradiated HIEC-6 cells. In contrast, PAH treatment effectively reduced radiation-induced cell death in HIEC-6 cells (Fig. 6I). PAH treatment could alleviate radiation-induced DNA damage and cell death of HIEC-6 cells.

PAH mitigates radiation-induced ferroptosis by activating Nrf2 signaling pathways

IR induces excessive intracellular ROS and lipid peroxidation, leading to ferroptosis [22]. Ferroptosis plays an important role in radiation-induced intestinal injury [16, 17]. Indicators associated with ferroptosis, including intracellular ROS, lipid peroxidation, prostaglandin-endoperoxide synthase 2 (PTGS2), glutathione peroxidase 4 (GPX4), and glutathione (GSH), were thus examined. There was significant upregulation of the levels of intracellular ROS, lipid peroxidation, and the mRNA expression of ferroptosis marker PTGS2 in HEIC cells after 6 Gy X-ray irradiation compared to the sham-irradiated HIEC-6 cells (Fig. 7A-C). In contrast, there was a significant reduction in the mRNA levels of the antioxidant GPX4 and GSH, which could repress ferroptosis (Fig. 7D, E). Nonetheless, PAH treatment changed these trends significantly. The cells treated with PAH had a lower expression of intracellular ROS, lipid peroxidation, and mRNA level of PTGS2, and increased mRNA levels of GPX4 and GSH after irradiation, indicating that PAH mitigated radiation-induced ferroptosis in HIEC-6 cells. Oxidant and antioxidant indexes in the intestine of mice, including malonaldehyde (MDA) and superoxide dismutase (SOD), were also detected. MDA is one of the most important products of membrane lipid peroxidation [23]. Exposure to 13 Gy TAI induced a significant increase in the MDA content and a decrease in SOD activity compared to the control group (Fig. 7F, G). Notably, PAH treatment did not cause a significant change in SOD activity. However, it markedly decreased the MDA levels after IR, indicating the recovery of lipid peroxidation-induced damage. In summary, these results demonstrated that PAH could protect against IR-induced ferroptosis and damage in vitro and in vivo.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a critical regulator of intracellular oxidative homeostasis. Nrf2-related antioxidant stress is closely associated with ferroptosis suppression [24, 25]. Nrf2 silencing can thus significantly reduce the levels of antioxidant proteins, promote ferroptosis, and further aggravate cell damage [26]. An investigation of whether the Nrf2 signaling is involved in PAH-mediated radioprotective effects was thus done to explore the mechanisms of PAH against radiation-induced ferroptosis. Nrf2 and its downstream targets, including the antioxidative proteins solute carrier family 7 member 11 (Slc7A11), heme oxygenase 1 (HO-1), and GPX4, were detected using Western blot assays. Slc7A11 and GPX4 are key proteins in ferroptosis. Slc7A11 can promote the biosynthesis of GSH, which exhibits a strong antioxidant activity through GPX4 [27]. Herein, exposure to radiation increased the levels of Nrf2, Slc7A11, and HO-1 but decreased the levels of GPX4 (Fig. 8A). The expression of Nrf2, Slc7A11, HO-1 and GPX4 was further stimulated by PAH treatment post exposure, which suggested that PAH could enhance Nrf2-mediated antioxidant response. IHC staining further showed that PAH treatment promoted the upregulation of Nrf2 and GPX4 levels in the radiation-exposed small intestines of mice (Fig. 8B). These data indicated that PAH treatment possibly alleviated radiation-induced ferroptosis by activating the Nrf2 signaling pathway.

The Nrf2 inhibitor ML385 was used to suppress the expression of Nrf2 and its downstream targets to clarify the involvement of Nrf2 signaling in the radioprotective effects of PAH. The inhibitory effects of ML385 on Nrf2 activation were first verified in HIEC-6 cells using Western blot assays (Fig. 8C). The effect of ML385 on the survival of HIEC-6 cells was subsequently investigated through the colony formation assay and PI staining. ML385 partially blocked the radioprotective effects of PAH in HIEC-6 cells (Fig. 8D-F). The introduction of ML385 further reversed the PAH-mediated protective effects on radiation-induced ferroptosis, embodied by the detection of lipid peroxidation and GSH (Fig. 8G, H). These findings suggested that the radioprotective effects of PAH were Nrf2-dependent.

Discussion

IR is closely related to daily lives. The public is exposed to various sources of IR, such as natural radiation, medical procedures, and nuclear terrorist attack or accidents.

The intestine is a highly radiosensitive tissue, with the death rate of IR-induced gastrointestinal syndrome reaching almost 100%. Some candidate drugs, such as Amifostine (S-2-(3-aminopropyl)aminoethyl), with good radioprotective effects have been reported but are not widely used because of high toxicity [28] or are clinically insignificant because of limited radioprotection. It is thus urgent to find new radioprotective drugs with minimal toxicity and high efficacy. Natural botanical compounds have gained considerable attention in this field primarily because of having a wide range of effects, low toxicity, convenient administration, and affordable price, among other advantages [29]. PAH is one of the most important essential oils and metabolites extracted from Perilla frutescens. It has a long history of safe use as a food flavouring agent, raw material for perfume and spice production, and traditional Chinese medicine [5, 30, 31]. Previous studies postulate that PAH is generally safe with minimal adverse effects. Thus, using PAH at low and medium concentrations likely does not threaten people's health [5, 10, 32].

This study thoroughly evaluated the radioprotective effects of PAH on the intestinal tract and its mechanism. PAH treatment significantly prolonged the survival time and reversed radiation-induced intestinal injury in mice after TAI. The intestinal damage caused by IR originates from the complex effects of epithelial injury, including villi destruction, death of crypt epithelial cells, and the reduction of barrier integrity [33]. Notably, epithelial injury is the primary result of IR-induced elimination of ISCs residing at the bottom of the crypt [34]. High-proliferating and radiosensitive Lgr5⁺ ISCs can generate all types of mature intestinal epithelial cells in vitro and in vivo [35, 36] and are indispensable for radiation-induced intestinal regeneration [19, 37]. In this study, PAH-mediated radioprotection was closely associated with the number of Lgr5⁺ ISCs in the intestine after irradiation. PAH treatment prevented IR-induced loss of Lgr5⁺ ISCs and increased the number of surviving intestinal crypts. PAH also increased the Ki67⁺ descendant cells generated by Lgr5⁺ ISCs in mice and intestinal crypt organoids ex vivo, demonstrating that PAH benefited the regeneration of irradiated intestinal tissues.

The intestinal barrier is the first line of defense of the gastrointestinal tract. It prevents the body from being exposed to luminal pathogens, which are well-known causes of IR-induced intestinal toxicity [38]. The mucus secreted by goblet cells and tight junctions located on the cell membrane play vital roles in the intestinal barrier [39]. Intestinal tight junctions are highly sensitive to irradiation. Clinical studies postulate that receiving radiotherapy can lead to increased intestinal permeability and tight junction damage [40]. In this study, irradiation caused exhaustion of the goblet cells, disrupted tight junctions, and increased permeability. However, PAH treatment significantly mitigated these trends. These findings highlighted the radioprotective effect of PAH on the intestinal barrier.

Previous studies postulate that various forms of IR-induced cell death are the basis of intestinal injuries [14, 41]. While our results showed that PAH treatment alleviated radiation-induced DNA damage and cell death of HIEC-6 cells. Increasing evidence further postulates that ferroptosis is an important form of IR-induced cell death [42, 43]. Shen DY et al. [44] strongly suggested that ferroptosis plays a major role in IR-induced cell death. Mechanistically, IR induces the production of ROS and iron overload, resulting in increased lipid peroxidation and ferroptosis [22]. Ferroptosis depends on NCOA4-mediated ferritinophagy and is an important novel mechanism of IR-induced intestinal epithelial cytotoxicity [16]. The findings of this study were consistent with the results of recent studies, suggesting that IR can significantly increase the level of ferroptosis. In the same line, PAH treatment effectively relieved IR-induced ferroptosis by reversing the expression of ferroptosis markers in the irradiated HIEC-6 cells and mice.

Nrf2 is a key regulator of intracellular oxidative homeostasis. Many downstream target genes of Nrf2 can prevent or correct intracellular redox imbalance [45]. Irradiation increased the level of Nrf2 and its downstream proteins, Slc7A11 and HO-1, which possibly was the protective mechanism caused by the stress reaction. Recent studies postulate that Nrf2 is critical in alleviating lipid peroxidation and ferroptosis [24, 46]. Nrf2 inhibits ferroptosis by promoting Slc7A11-mediated cystine input, GSH biosynthesis, and GPX4-mediated lipid peroxide detoxification. Nrf2 is also involved in ferroptosis regulation in IR-induced intestinal injury [47]. PAH treatment increased the expression of Nrf2, Slc7A11, HO-1, and GPX4 after radiation. In addition, the Nrf2 inhibitor ML385 reverses the protective effects of PAH on ferroptosis, indicating that the radioprotective effects of PAH are Nrf2-dependent. In this study, the Nrf2 inhibitor ML385 partially reversed the protective effects of PAH, suggesting that the mechanism is potentially associated with other pathways. Chu L et al. [48] reported that orally administered PAH could effectively inhibit cGAS-STING signaling in mice. Attenuated cGAS-STING signaling protects mice from IR-induced intestinal damage and ferroptosis [49]. In the same line, Yin YL et al. [13] reported that orally administered PAH could stimulate PI3K/AKT phosphorylation in rats. The PI3K/AKT signaling pathway promotes cell survival, while PI3K/AKT-dependent pathways promote intestinal regeneration of mouse intestines after radiation [50]. These pathways may thus also contribute to the radioprotective effects of PAH, which is an important issue for future research.

Conclusions

In summary, this study suggested that PAH activates Nrf2 signaling to inhibit IR-induced ferroptosis and attenuate intestinal injury after irradiation. PAH is thus a promising radioprotective drug that can prevent intestinal side effects of radiotherapy and treat accidental radiation exposure.

Materials And Methods

Animal source and irradiation protocol

Male C57BL/6J mice (6–8 weeks old, 20–22 g) were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China) and housed under standard laboratory conditions in the Soochow University Animal Centre. All animal experiments were conducted following the protocols approved by the Animal Ethics Committee of Soochow University.

The mice were randomly divided into two groups (n = 10 / group): IR and IR + PAH, for survival experiments. PAH dissolved in normal saline was administered to mice in the IR + PAH group by oral gavage at a dose of 100 mg/kg daily for seven consecutive days before TAI (Fig. 1A) (4, 48). Mice in the IR group received an equal volume of normal saline by oral gavage and at the same frequency as the IR + PAH-treated mice. All mice were subjected to 13 Gy TAI using the X-RAD 320iX Biological Irradiator (Precision X-ray, North Branford, CT, USA) at a dose rate of 1.1 Gy / min. The mice were randomly divided into 3 groups (n = 6 / group): Control, IR, and IR + PAH, for the subsequent experiments. The processing procedures of mice in the IR and IR + PAH groups were similar to those described earlier. Mice in the control group were sham-irradiated and received an equal volume of normal saline by oral gavage and at the same frequency as the other groups.

Histological analysis, immunohistochemistry and immunofluorescence staining

The mice intestines were harvested and fixed in 4% paraformaldehyde at 6 hours or 3.5 days after TAI. The intestinal tissues were then paraffin-embedded and then cut into 4 µm sections for hematoxylin-eosin (H&E) staining and periodic acid-schiff (PAS) staining according to the manufacturer's instructions. H&E-stained sections were viewed under an optical microscope, followed by an analysis of the villus height using Image J software.

Immunohistochemistry (IHC) and immunofluorescence (IF) were performed as described by Li M et al.[51]. The primary antibodies used for IHC staining included anti-Lgr5 (1:200; 251487; Abbiotec, San Diego, CA, USA), anti-Ki67 (1:400; CST12202), anti-Nrf2 (1:50; CST12721), anti-E-Cadherin (1:200; CST14472) (Cell Signaling Technology, Beverly, MA, USA), anti-GPX4 (1:400; ab125066; Abcam, Cambridge, MA, USA), anti-ZO-1 (1:50; 61-7300), and anti-Claudin3 (1:100; 34-1700) (Invitrogen, Carlsbad, CA, USA) antibodies. The primary antibodies used for IF staining included anti-γ-H2AX (1:400; CST9718) (Cell Signaling Technology), anti-8-OHdG (1:200; NB600-1508), and anti-lysozyme (1:200; NBP2-6118) (Novus Biological, Littleton, CO, USA) antibodies.

Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay

The intestine sections at 6 h post-TAI were stained using an in situ Cell Death Detection Kit (Roche Diagnostic, Mannheim, Germany) according to the manufacturer's instructions.

Fluorescein isothiocyanate (FITC)-dextran permeability assay

Mice were randomly divided into 3 groups (n = 4 / group): Control, IR, and IR + PAH, and treated as described earlier. The mice were subjected to fasting for 8 hours at three days post-TAI and then administered with 0.2 ml of FITC-dextran (Sigma-Aldrich, Saint Louis, MO, USA) (50 mg/100 g body weight). Blood samples were collected 4 hours after FITC-dextran administration and centrifuged at 960 x g for 5 minutes to obtain the serum. The fluorescence level of FITC was measured using a microplate reader at a wavelength of 492 nm.

Crypt isolation and organoid culture

The small intestines of C57BL/6J mice were cut longitudinally and flushed with cold PBS. The tissues were cut into 2–3 mm pieces, washed 15–20 times with cold PBS, and incubated with Gentle Cell Dissociation Reagent (Stem Cell Technologies, Vancouver, BC, Canada) at room temperature on a rocking platform for 15 minutes. The supernatant was gently aspirated, and the pieces were subsequently resuspended in cold PBS supplemented with 0.1% BSA and passed through a 70 µm cell filter (BD Biosciences, San Diego, CA, USA) to remove the tissue fragments. Crypts for organoid culture were obtained by centrifuging the resuspended sections at 290 x g for 5 minutes at 4°C, followed by resuspending the sections in complete IntestiCult™ Organoid Growth Medium (Stem Cell Technologies) at a density of 500 crypts per 50 µl. The resuspended crypts were subsequently mixed with an equal volume of Matrigel® (BD Biosciences) and then seeded on a prewarmed 24-well plate at a density of 500 crypts per well. Complete IntestiCult™ Organoid Growth Medium (750 µl) was then added to each well. The resulting organoids were treated with complete medium with or without 100 µM PAH 4 hours pre-IR and then exposed to 6 Gy X-rays at a dose rate of 1.1 Gy / min or sham-irradiated. The organoids were finally viewed under an optical microscope, followed by an analysis of the organoids using Image J software. At least 50 organoids were counted.

Cell culture

The human intestinal epithelial cell line HIEC-6 was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in DMEM (HyClone, Hudson, NH, USA) supplemented with 10% FBS (Biological Industries, Cromwell, CT, USA) and 1% (v/v) penicillin-streptomycin (Beyotime, Shanghai, China) at 37°C in a humidified 5% CO₂ atmosphere. The cells were then treated with complete medium with or without 100 µM PAH 4 hours pre-IR.

Cell viability assay

The viability of the HIEC-6 cells was evaluated using the cell counting kit-8 (CCK-8; Beyotime) following the manufacturer's instructions. The HIEC-6 cells (2000 cells/well) were first seeded into 96-well plates and incubated for 24 hours. The cells were then subjected to varying treatments. CCK-8 solution (10 µL) was then added to the cells, followed by incubation for 2 hours at 37°C. The optical density (OD) of the cells was finally measured at 450 nm wavelength using a microplate reader.

Colony formation assay

HIEC-6 cells were seeded in 6-well plates in triplicate at densities of 200–2000 cells/well depending on the radiation dose. The cells were cultured overnight, treated with or without 100 µM PAH for 4 hours, and then subjected to 0, 2, 4, 6, and 8 Gy X-ray radiation. The medium containing drugs was then immediately replaced with fresh medium, and the cells were subsequently cultured at 37°C for 1–2 weeks to form colonies. The cell colonies were stained with crystal violet and viewed under a microscope. Viable colonies consisted of at least 50 cells were counted.

Cell death and lipid peroxidation assays

HIEC-6 cells were seeded in 6-well plates in triplicate. The cells were cultured overnight, treated with or without 100 µM PAH for 4 hours, and then subjected to 6 Gy X-ray radiation. The cells were then collected 48 h post-radiation and subjected to various assays. The cell death assay was done by staining the cells with PI (Beyotime) according to the manufacturer’s instructions. The lipid peroxidation assay was done by incubating the cells with 5 µM BODIPY™ 581/591 C11 fluorescence probe (D3861, Invitrogen, Carlsbad, CA, USA) for 30 minutes at 37°C. The level of cell death and lipid peroxidation was then analyzed by FACSVerse flow cytometry (BD Biosciences).

Intracellular ROS assay

HIEC-6 cells were seeded in 6-well plates in triplicate. The cells were cultured overnight, treated with or without 100 µM PAH for 4 hours, and then preincubated with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) (BD Biosciences, San Jose, CA, USA) for 30 min at 37°C according to the manufacturer’s instructions. The cells were then subjected to 6 Gy X-ray radiation and collected after 15 minutes, followed by an analysis of the intracellular ROS level using FACSVerse flow cytometry.

Oxidant and antioxidant index assay

Small intestine tissues of mice were harvested at 3.5 days post-radiation. The MDA content and SOD activity were then measured using commercially available kits (ZCIBIO Technology, Shanghai, China). The level of GSH in HIEC-6 cells was determined using a GSH and GSSG Assay kit (Beyotime) following the manufacturer’s instructions.

Western blot analysis

Protein levels of HIEC-6 cells were analyzed using the Western blot assay according to the published methods [52]. The primary antibodies used were anti-Nrf2 (1:1000; 16396-1-AP; Proteintech Group, Chicago, IL, USA), anti-Slc7A11 (1:1000; CST12691), anti-HO-1 (1:1000; CST43966) (Cell Signaling Technology), and anti-GPX4 (1:2000; ab125066; Abcam). The anti-tubulin antibody (1:1000; 66031-1-Ig; Proteintech Group) was used as the loading control.

Declarations

Acknowledgments

We thank the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and Jiangsu Provincial Key Laboratory of Radiation Medicine and Protection.

Author contributions statement

XM and ML conceived and designed the study. LFT and LWX coordinated and performed most experimental work. HZ performed statistical analyses. LFT wrote the manuscript, and ML provided critical review. All authors read and approved the final paper.

Funding

This work was supported by the National Natural Science Foundation of China (82192884) and Postgraduate Researh & Practice Innovation Program of Jiangsu Province (KYCX22_3221).

Conflict of interest

The authors declare no conflict of interest.

Declarations of interest

None.

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