The Radioprotective Effects of (−)-Epigallocatechin-3-gallate (EGCG): Restoring the Gut Microbiota-Immune Axis to Ameliorate Radiation-Induced Intestinal Injury

Abstract

Objectives

To investigate the protective effect and mechanism of (−)-epigallocatechin-3-gallate (EGCG) against radiation-induced intestinal injury (RIII).

Methods

C57BL/6J mice were pretreated with 10 mg/kg EGCG for 10 weeks or 3 days before 10 Gy total body irradiation (TBI) or whole abdominal irradiation (WAI), focusing on survival, body weight, intestinal structure, inflammation, and gut microbiota. For mechanism exploration, peritoneal and RAW264.7 macrophages were pretreated with 0-50 μM EGCG before irradiation, followed by assessments of cell viability, inflammatory proteins, and related signaling.

Results

EGCG protects mice from TBI-induced toxicity and weight loss, and alleviates WAI-induced intestinal injury, characterized by preserved villus architecture and reduced crypt cell apoptosis. Mechanistically, EGCG inhibits RIII by suppressing pro-inflammatory cytokine production (TNF-α, IL-6) and maintains the composition of gut microbiota disrupted by irradiation. In vitro studies confirm EGCG directly regulates the release of radiation-induced inflammatory factors in macrophages via inhibiting toll-like receptor (TLR) signaling, independent of microbial effects. The radioprotective effect of EGCG is attributed to its dual actions: preserving macrophage function to dampen inflammatory responses and maintaining gut microbiota homeostasis.

Conclusion

These findings highlight EGCG as a potential therapeutic agent for RIII, offering a novel strategy to improve outcomes in irradiated patients.

Introduction

Radiation-induced intestinal injury (RIII) is a severe complication in patients undergoing radiotherapy or accidental radiation exposure, stemming from the high radiosensitivity of intestinal tissues.^1,2 Clinically characterized by nausea, diarrhea, intestinal bleeding, and even perforation, RIII poses a critical threat to patient survival and quality of life. ³ As conventional therapies for RIII remain limited, natural extracts with minimal side effects, simple extraction processes, and significant efficacy rooted in traditional Chinese medicine (TCM) principles have emerged as promising research targets.

(−)-Epigallocatechin-3-gallate (EGCG), the major bioactive catechin in green tea celebrated in TCM for its “heat-clearing and detoxifying” properties, exhibits multi-faceted biological activities, including antibacterial, anti-inflammatory, and radioprotective effects.^4,5 Clinical trials have demonstrated its efficacy in mitigating radiation-induced esophagitis, ⁶ oral mucositis, ⁷ and acute skin damage, ⁸ highlighting its translational potential. Xie et al. ⁹ identified reactive oxygen species (ROS) and nuclear factor erythroid 2-related factor 2 (Nrf-2) as key mediators in EGCG’s protective effects on intestinal epithelial cells, while Emami et al. ¹⁰ reported symptom relief of diarrhea and vomiting in radiotherapy patients consuming green tea. Despite these insights, the precise mechanisms by which EGCG alleviates RIII—particularly its interactions with immune-mediated inflammation and gut microbiota—remain unclear.

Current radioprotective research has primarily focused on repairing intestinal epithelial cell damage, ¹¹ yet the role of immunological barriers in RIII pathogenesis remains understudied. Macrophages, pivotal components of intestinal immune surveillance, maintain tissue homeostasis by regulating inflammation, facilitating epithelial repair, and interacting with gut microbiota.^12,13 Their crosstalk with microbiota is bidirectional: macrophages shape microbial composition, ¹⁴ while microbial metabolites enhance macrophage antimicrobial activity. ¹⁵ However, whether EGCG modulates macrophage-mediated inflammatory pathways and restores microbiota homeostasis to alleviate RIII has not been explored.

In this study, using murine models of total body and whole abdominal irradiation, we investigate the protective effects of EGCG against radiation-induced intestinal injury and explore its mechanisms involving macrophage-mediated inflammation and gut microbiota regulation. Our study reveals a novel mechanism whereby EGCG coordinates immune-microbial interactions, providing a scientific basis for integrating TCM-derived compounds into RIII clinical management.

Materials and Methods

Drug and Reagents

EGCG was purchased from Sigma-Aldrich (St Louis, MO, USA). For the animal experiments, EGCG was dissolved in 0.9% normal saline with a stock concentration of 5 mg/mL. For the cell experiments, EGCG was dissolved in PBS with a stock concentration of 5 mg/mL. The doses and schedules for administration of EGCG and radiation are provided in the relevant figure legends. Primary antibodies used included: anti-high mobility group box 1 (HMGB1, #66525-1-Ig, Proteintech), anti-toll-like receptor 4 (TLR4, #19811-1-AP, Proteintech), anti-nuclear factor kappa B p65 (NF-κB p65, #PB0073, Boster Bio), and anti-β-actin (#BM3873, Boster Bio) as the loading control. HRP-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Boster Bio) were used for detection.

Animal Model

Six-to eight-week-old male C57BL/6J mice (Shanghai Laboratory Animal Center, Chinese Academy of Sciences, Shanghai, China) were housed in polycarbonate cages under specific pathogen-free conditions: controlled temperature (22 ± 2°C), relative humidity (60% ± 5%), and a 12-h light/dark cycle, with ad libitum access to standard rodent chow and water throughout the study. Male mice of pure C57BL/6J genetic background were randomly assigned to experimental groups. All procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Soochow University Animal Welfare Committee (SUDA201906A099).

Irradiation and EGCG Treatment

A precision X-RAD 320 Irradiator (Precision X-ray Inc., USA) delivering 2.0 Gy/min was used for all irradiation procedures. For survival studies, mice received daily intraperitoneal (i.p.) injections of vehicle (0.9% saline), 2.5 mg/kg, or 10 mg/kg EGCG for 10 consecutive weeks prior to total body irradiation (TBI). All mice were then exposed to a single 10 Gy TBI dose, with groups designated as: (1) vehicle + TBI; (2) 2.5 mg/kg EGCG + TBI; (3) 10 mg/kg EGCG + TBI. ¹⁶ The survival status was monitored for 14 days post-irradiation, with body weight changes recorded during this period.

For mechanistic experiments, mice were randomized into 3 groups: (1) sham-irradiated control (CON); (2) whole abdominal irradiation + vehicle (WAI); (3) 10 mg/kg EGCG + WAI. EGCG (10 mg/kg) or saline was administered i.p. daily for 3 consecutive days, with a final dose given 30 min before WAI. Mice were euthanized in batches at 3.5- and 7-days post-irradiation for the collection of blood and intestinal samples. Whole abdominal irradiation was performed using lead shielding to restrict radiation to the abdominal region, while sham controls underwent identical handling without radiation exposure.

Histopathological Studies

Small intestinal samples were collected and fixed in 10% buffered formaldehyde–saline solution. Subsequently, 4-μm paraffin-embedded sections were prepared and stained with hematoxylin and eosin (H&E, Sigma-Aldrich) and periodic acid-Schiff (PAS, Servicebio) according to established protocols. ¹⁷ Tissue sections were imaged under a DMi8 microscope (Leica, Germany), and morphological assessments were performed by a board-certified pathologist blinded to experimental groups, including semi-quantitative scoring of villus architecture and epithelial integrity.

Enzyme-linked Immunosorbent Assay (ELISA)

Jejunal samples were homogenized at 4°C for 10 min using a tissue homogenizer, followed by centrifugation at 3000 rpm for 10 min. Supernatants were collected and stored at −80°C until analysis. Levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) in the supernatants were quantified using quantitative sandwich enzyme-linked immunosorbent assay (ELISA) kits (JYMBio, China) according to the manufacturer’s protocols. Optical density (OD) values were measured at 450 nm using a multimode plate reader (Synergy NEO, BioTek, USA), and cytokine concentrations were calculated from standard curves.

Intestinal Microbiota Analysis

Genomic DNA was extracted from intestinal samples and subjected to 16S rDNA gene amplification following established protocols. ¹⁷ Bacterial community diversity was assessed by sequencing the V3-V4 hypervariable region of the 16S rRNA gene using the Illumina MiSeq platform (Lc-Bio Technologies Co., Ltd., China).

Cell Culture and Treatment

The RAW264.7 macrophage cell line were obtained from the Cell Resource Center, Shanghai Institute for Biological Sciences. RAW264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, HyClone, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA). All cell lines were incubated in a humidified atmosphere of 5% CO₂ at 37°C. For radiation exposure, confluent cells were irradiated with 6 Gy X-rays using a Rad Source Technologies X-ray tube (USA) at a fixed dose rate of 1.15 Gy/min.

Peritoneal Macrophage Isolation

Peritoneal macrophages were isolated from C57BL/6J mice with slight modifications to previously described methods. ¹⁸ Briefly, mice were intraperitoneally injected with 5 mL of DMEM containing 75% fetal bovine serum (FBS). After 30 min, peritoneal lavage was performed by injecting 5 mL of ice-cold DMEM into the peritoneal cavity, followed by gentle massaging to harvest cells. The lavage fluid was collected, centrifuged at 1000 rpm/min for 10 min at 4°C, and the cell pellet was resuspended in DMEM supplemented with 10% FBS. Cells were plated in petri dishes and incubated at 37°C with 5% CO₂ for 2 h to allow macrophage adhesion. Non-adherent cells were then discarded, and the plates were washed twice with warm PBS to remove residual non-adherent cells. The remaining adherent cells were confirmed as peritoneal macrophages and used for subsequent experiments.

Cell Viability Assay

Cell viability was assessed using the CCK-8 assay. Briefly, cells (5 × 10³ cells/well) were seeded into 96-well plates and cultured for 24 h. Cells were then incubated with 0, 2.5, 5, 10, 25, or 50 μM EGCG for 30 min prior to exposure to 0 or 6 Gy X-ray radiation. After 24 h of post-irradiation incubation, 10 μL of CCK-8 reagent (Beyotime, China) was added to each well and incubated for 1 h. Absorbance at 450 nm was measured using a Synergy NEO microplate reader (BioTek, USA). All experiments were independently repeated at least 3 times.

Live-Dead Cell Viability Assay

Additionally, cell viability was assessed using a Live-Dead assay (Invitrogen, USA). Cells were incubated with or without 10 μM EGCG for 1 h prior to exposure to 6 Gy X-ray radiation or sham treatment. Briefly, the Live-Dead staining solution was prepared by mixing 1 mL of PBS with 4 μL of 2 mM ethidium homodimer-1 and 2 μL of 50 μM calcein AM. Following irradiation, 200 μL of the staining solution was added to each well and incubated at 37°C for 15 min in the dark. The staining solution was then removed, and cells were imaged under a DMi8 fluorescence microscope (Leica, Germany) using excitation filters at 494 nm (green, calcein) and 528 nm (red, ethidium homodimer-1).

Microarray Analysis and Data Preprocessing

Cytokine expression profiles in RAW264.7 macrophages and peritoneal macrophages were analyzed using the Mouse Inflammation Array GS1 (GSM-INF-1-1, RayBiotech, USA), which contains 40 cytokine-specific antibodies printed in quadruplicate on glass slides. Cell lysates and culture supernatants were used to identify differentially expressed cytokines. For sample preparation, cells were lysed in ice-cold cell lysis buffer containing a protease inhibitor cocktail for 1 h, followed by centrifugation at 13 000 rpm for 20 min at 4°C. Supernatants and culture medium samples were diluted 1:2 and added to the wells pre-coated with capture antibodies for overnight incubation at 4°C. Subsequently, arrays were washed and incubated with a biotinylated antibody cocktail for 2 h at room temperature. Following additional washes, Cy3-conjugated streptavidin was applied, and fluorescent signals were visualized using an InnoScan 300 Microarray Scanner (Innopsys, France) at a Cy3 wavelength (550 nm excitation). Raw data were normalized using the RayBiotech Analysis Tool, an array-specific Excel-based program for sophisticated background correction and signal quantification. Detailed protocols are provided in the Supplementary Materials.

Western Blot Analysis

Protein expression levels were analyzed by Western blotting as previously described. Briefly, peritoneal macrophages were lysed in ice-cold RIPA lysis buffer (Beyotime, China) containing protease inhibitors for 30 min at 4°C. Cell lysates were centrifuged at 13 000 rpm for 15 min at 4°C to pellet debris, and the supernatants were collected as total protein extracts. Proteins were separated by SDS-PAGE and transferred to PVDF membranes, which were blocked with 5% non-fat dry milk in TBST. Membranes were incubated with primary antibodies overnight at 4°C, followed by secondary antibodies for 1 h at room temperature. Protein bands were visualized using Superstar ECL Chemiluminescent Substrate (Beyotime) and detected with a chemiluminescence imaging system. Band intensities were quantified using ImageJ software (version 1.8).

Statistical Analysis

All experiments were independently repeated at least 3 times, and data are presented as the mean ± standard error of the mean (S.E.M.). Survival rates were analyzed using Kaplan-Meier survival curves with log-rank testing. For two-group comparisons, statistical significance was evaluated by Student’s t-test. Multiple-group comparisons were performed using one-way analysis of variance (ANOVA), followed by Fisher’s least significant difference (LSD) post-hoc tests to identify intergroup differences. Statistical analyses were conducted using SPSS 24.0 (IBM, USA), and differences were considered significant at P < .05.

Results

EGCG Improves Survival and Attenuates Weight Loss in Mice Following TBI

To evaluate EGCG’s radioprotective efficacy, C57BL/6J mice were exposed to a lethal 10 Gy dose of total body irradiation (TBI) and monitored for 14 days (Figure 1A). Survival analysis revealed 100% mortality in the vehicle and 2.5 mg/kg EGCG groups by day 14, whereas 50% of mice in the 10 mg/kg EGCG group survived (Figure 1B). Mean survival times were 6.6 ± 1.6, 8.1 ± 1.8, and 11.2 ± 3.3 days for the vehicle, 2.5 mg/kg, and 10 mg/kg EGCG groups, respectively (P < 0.05 for 10 mg/kg vs vehicle; Figure 1C). Additionally, EGCG treatment at 10 mg/kg significantly mitigated radiation-induced weight loss, with treated mice maintained stable body weight (∼27-28 g),while the vehicle group exhibited precipitous weight loss, declining to <24 g by Day 8. (Figure 1D). These data demonstrate that EGCG confers dose-dependent protection against TBI-induced mortality and weight decline in mice.

Figure 1.

EGCG improves survival and mitigates weight loss in lethally irradiated mice. (A) Mice received daily intraperitoneal injections of vehicle (saline), 2.5 mg/kg, or 10 mg/kg EGCG for 10 consecutive weeks prior to TBI, followed by a single 10 Gy TBI dose. (B) Kaplan-Meier survival curves of mice after 10 Gy TBI. Log-rank test showed no significant difference between the 2.5 mg/kg EGCG and vehicle groups (P = 1.0000), while the 10 mg/kg EGCG group exhibited significantly improved survival compared to vehicle (P = .0092; n = 6 per group). (C) Mean survival times in the 3 groups (n = 6 per group, P < .05 vs vehicle). Data are presented as box plots, with the box representing the interquartile range (25th–75th percentiles), the central line indicating the median, and whiskers denoting the minimum and maximum values. (D) Body weight changes in each group over 14 days (n = 6 per group). Data are shown as mean ± SEM

EGCG Mitigates Small Intestinal Injury in Mice Following WAI

To investigate EGCG’s protective effects against RIII, we utilized a nonlethal whole abdominal irradiation (WAI) mouse model. ¹⁹ Gross morphological analysis revealed severe acute intestinal damage in the WAI group, characterized by reduced wall elasticity, thinning, and luminal dilation at 3.5 days post-irradiation, progressing to marked intestinal distension by day 7 (Figure 2B). In contrast, EGCG pretreatment (EGCG + WAI group) preserved intestinal architecture, with minimal luminal air accumulation and near-normal macroscopic appearance.

Figure 2.

EGCG preserves small intestinal architecture in nonlethally irradiated mice. (A) Mice were anesthetized with 5% chloral hydrate and received 10 Gy WAI. EGCG (10 mg/kg) or saline was administered intraperitoneally for 3 consecutive days, with a final dose given 30 min prior to WAI. (B) Representative images of intestinal morphology in each group. (C, D) H&E staining (for structural integrity) and PAS staining (for goblet cells) of jejunal sections at 3.5 days and 7 days post-WAI (Scale bar = 200 μm). (E, F) Quantification of villus height and goblet cell numbers. Data are mean ± SEM (n = 6 per group), *P < .05, **P < .01, and ***P < .001

Histological assessment via H&E and PAS staining (Figure 2C, D) confirmed significant reductions in villus height and goblet cell counts in WAI-exposed mice at both time points (Figure 2E, F). Conversely, EGCG-treated mice exhibited preserved crypt-villus structures, with villus heights and goblet cell numbers comparable to sham controls. These findings indicate that EGCG effectively attenuates radiation-induced intestinal epithelial damage and maintains mucosal integrity.

EGCG Inhibits Radiation-Induced Inflammation

We investigated the role of EGCG in modulating immune responses following radiation injury. Mice in the WAI group exhibited significant reductions in spleen and thymus indexes, whereas the EGCG + WAI group showed notable recovery, particularly at 3.5 days post-WAI (Figure 3A, B). Blood routine analysis revealed that WAI caused marked decreases in white blood cell and monocyte counts. EGCG pretreatment significantly restored white blood cell numbers by day 7 post-WAI (Figure 3C), while monocyte counts remained unchanged between the WAI and EGCG + WAI groups (Figure 3D).

Figure 3.

EGCG attenuates radiation-induced inflammatory response following WAI. (A, B) Spleen and thymus indexes at 3.5- and 7-days post-WAI in sham control, WAI, and EGCG + WAI groups. Data are mean ± SEM (n = 6 per group). (C, D) Quantification of white blood cell and monocyte counts. Data are mean ± SEM (n = 6 per group). (E–G) ELISA analysis of serum inflammatory cytokines IL-6, TNF-α, and IFN-γ at 3.5 days post-WAI. Data are mean ± SEM (n = 5 per group), *P < 0.05, **P < .01, ***P < .001

Moreover, EGCG treatment substantially reduced serum levels of pro-inflammatory cytokines, including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) (Figure 3E–G). Collectively, these data demonstrate that EGCG mitigates acute radiation-induced immune injury and suppresses systemic inflammation in WAI-exposed mice.

EGCG Modulates Gut Microbiota Composition in WAI Mice

Gut microbial dysbiosis is well-documented to influence host radiosensitivity. ²⁰ To investigate EGCG’s effects on the gut microbiome after WAI, we conducted 16S rRNA gene high-throughput sequencing. Alpha-diversity analyses (Chao 1 index, observed species, and Shannon index) revealed no significant differences among the 3 groups (Figure 4A), indicating that 10 Gy WAI did not alter gut microbiota species richness or evenness.

Figure 4.

EGCG restores gut microbiota composition following WAI. (A) Alpha-diversity analysis of gut microbiota, including Chao1 index, observed species, and Shannon index. Data are presented as box plots (n = 5 per group), with no significant differences among groups (P > .05 vs all comparisons). The box represents the interquartile range (25th–75th percentiles), the central line denotes the median, and whiskers indicate the minimum and maximum values. (B) Beta-diversity analysis via principal component analysis (PCA). PC1 and PC2 explain 47.67% and 21.85% of total variation, respectively. Each point represents an individual sample, color-coded by group (n = 5 per group). (C) Cladogram from LEfSe analysis identifying significantly enriched microbial clades. Red, blue, and green nodes indicate taxa enriched in CON, WAI, and EGCG + WAI groups, respectively; yellow nodes denote no significant difference. Taxonomic levels are shown from phylum (inner) to genus (outer). (D) LEfSe-derived effect size (LDA score >4.5) of differentially abundant taxa. Significance was defined as P < .05 (Kruskal-Wallis test). (E) Relative abundances of dominant gut microbiota at the genus level. (F) Quantification of significantly differentially abundant genera (n = 5 per group, *P < .05, **P < .01, ***P < .001)

Principal component analysis (PCA) of beta-diversity showed that WAI exposure distinctly altered gut microbiota community structure, whereas EGCG treatment partially restored microbial composition toward sham control levels (Figure 4B). Linear discriminant analysis (LDA) effect size (LEfSe) further identified differentially abundant taxa across groups (Figure 4C, D). At the genus level, the WAI group exhibited significantly higher relative abundances of Escherichia_Shigella and Bacteroides and lower abundances of Muribaculaceae and Duncaniella compared to the sham control group. Notably, EGCG pretreatment reversed these radiation-induced shifts in microbial abundance (Figure 4E, F).

Collectively, these data demonstrate that EGCG preserves gut microbiota alpha-diversity while restoring radiation-disrupted microbial community structure, suggesting a role in rebalancing dysbiotic gut ecosystems after abdominal irradiation.

EGCG Enhances Macrophage Viability Following Radiation Exposure

To assess EGCG’s protective effects against radiation-induced cellular damage, we utilized peritoneal macrophages and RAW264.7 macrophage cell lines as in vitro models. CCK-8 assays were first performed to determine optimal EGCG concentrations for cell viability. Treatment with EGCG at concentrations ≥50 μM significantly inhibited growth in both macrophage types (Figure 5A), prompting us to test lower doses (2.5, 5, 10, 25 μM) for radioprotective efficacy.

Figure 5.

EGCG enhances radiation-exposed macrophage viability. (A) Cytotoxicity assessment of EGCG in peritoneal and RAW264.7 macrophages. Cells were treated with indicated EGCG concentrations (0-50 μM) for 24 h, and viability was measured by CCK-8 assay. Data are mean ± SEM (n = 6 per group), *P < .05, **P < .01 vs 0 μM control. (B, C) Protective effects of EGCG on radiation-exposed macrophages. RAW264.7 (B) and peritoneal (C) macrophages were pretreated with EGCG (2.5-25 μM) for 30 min before 6 Gy irradiation, followed by 24-hour culture. Viability was assessed via CCK-8 assay. Data are mean ± SEM (n = 6 per group), *P < 0.05, **P < .01, ***P < .001. (D) Live/Dead staining of macrophages after EGCG pretreatment and 6 Gy irradiation. Cells were treated with 0 or 10 μM EGCG for 30 min prior to sham or 6 Gy irradiation, then stained with calcein AM (green, live cells) and ethidium homodimer-1 (red, dead cells) after 24 h. Representative images show viable cell enrichment in the EGCG + irradiation group. Scale bar = 200 μm

Pre-treatment with EGCG dose-dependently mitigated radiation-induced viability loss. Notably, a 10 μM EGCG concentration consistently protected against post-irradiation viability decline after 24 h of culture, outperforming lower doses (Figure 5B, C). Live/Dead staining corroborated these findings, showing reduced necrotic (red) and increased viable (green) cells in the EGCG-treated group compared to radiation-only controls (Figure 5D). Collectively, these data indicate that EGCG attenuates radiation-induced damage in both primary and immortalized macrophages, with 10 μM emerging as the optimal protective dose.

EGCG Attenuates Radiation-Induced Inflammation in Macrophages

To investigate the mechanism behind EGCG’s protective effects against radiation-induced macrophage dysfunction, we profiled 40 inflammatory factors in the culture medium of irradiated macrophages using a Mouse Inflammation Array (Figure 6A; full protein data in Supplemental Fig. S1). Volcano plot analysis revealed that compared to the EGCG+6 Gy group, the 6 Gy-only group exhibited significant upregulation of Eotaxin-2, IL-6, TNF-α, and BLC, and downregulation of Eotaxin, IL-7, IL-17, and FP4 (fold change >1.5 or < −1.5, P < 0.05; Figure 6B).

Figure 6.

EGCG Suppresses Radiation-Induced Inflammation in Peritoneal Macrophages. (A) Macrophages were pretreated with 0 or 10 μM EGCG for 30 min before sham or 6 Gy irradiation, followed by 24-hour culture. Culture supernatants were concentrated and analyzed using the Mouse Inflammation Array GS1 to profile 16 inflammatory proteins. Representative array results are shown. (B) Volcano plot of differentially expressed proteins between the 6 Gy and EGCG+6 Gy groups. The X-axis represents log₂(fold change), and the Y-axis represents -log₁₀(P value). Red spots indicate upregulated proteins (>1.5-fold, P < .05), blue spots indicate downregulated proteins (<-1.5-fold, P < .05), and gray spots denote no significant difference. (C) KEGG pathway enrichment analysis of differentially expressed proteins (P < .05). The top 20 enriched pathways are shown, ranked by significance. (D) Western blot analysis of HMGB1, NF-κB, and TLR4 protein levels in macrophages pretreated with EGCG (10 μM) before sham or 6 Gy irradiation. β-actin served as the loading control

KEGG pathway enrichment analysis of differentially expressed proteins in peritoneal macrophages identified 20 significantly enriched pathways (Figure 6C). Focusing on the toll-like receptor signaling pathway, Western blot validation showed that EGCG pretreatment significantly inhibited radiation-induced upregulation of HMGB1, NF-κB, and TLR4 protein expression (Figure 6D; Supplemental Fig. S2). Collectively, these results indicate that EGCG mitigates radiation-induced inflammation in macrophages by suppressing key pro-inflammatory cytokines and inhibiting the TLR4/NF-κB signaling axis.

Discussion

EGCG, a prominent member of green tea catechins, has been widely recognized for its multifaceted health benefits and favorable safety profile.^21,22 Preclinical investigations across in vitro systems and animal models have demonstrated its ability to inhibit diverse oncogenic processes, while in vivo studies further highlight its dual role as a radiosensitizer to enhance cancer therapy efficacy and a modulator to mitigate treatment-related adverse effects.^6,23,24 Notably, prior research has shown that EGCG protects rodents from intestinal injury following TBI at doses up to 9 Gy, ⁹ prompting us to hypothesize its potential as a radioprotectant for radiation-induced intestinal injury (RIII) and to explore its mechanisms through an immunological lens.

In this study, we confirmed that EGCG administration improved survival rates in mice subjected to 10 Gy TBI. The 10-week pretreatment was chosen to ensure sufficient EGCG accumulation and avoid transient effects of short-term exposure. For dosage, we used intraperitoneal injection of 2.5 mg/kg and 10 mg/kg EGCG (instead of oral administration) to address its low oral bioavailability,^25,26 ensuring effective target tissue exposure, with the dosage gradient balancing efficacy and safety. Building on these findings, we next evaluated its effects on RIII using a whole-abdominal irradiation (WAI) murine model. ¹⁹ Our results demonstrated that EGCG preserved the crypt-villus architecture of jejunal tissue and reduced the systemic release of proinflammatory cytokines after 10 Gy WAI. Although the pathogenesis of RIII remains incompletely understood, accumulating evidence implicates a complex interplay of epithelial damage, vascular dysfunction, and microbial-immune interactions in driving severe radiation enteropathy. ²⁷ The gut microbiota, critical for maintaining intestinal barrier integrity, immune homeostasis, and repair mechanisms,^28,29 has emerged as a key contributor to radiation enteropathy progression. ³⁰ For instance, Wang et al. ³¹ demonstrated that microbiota derived from radiation-injured intestines induced epithelial inflammation and barrier disruption via TNF-α and IL-1β upregulation in bacterial-epithelial co-culture systems. Consistent with these findings, our 16S rDNA sequencing revealed that WAI-induced gut dysbiosis characterized by increased abundances of Escherichia_Shigella and Bacteroides, and decreased Muribaculaceae and Duncaniella populations in mice, whereas EGCG treatment restored microbial balance by reducing harmful taxa such as Escherichia_Shigella.

Notably, recent studies have shown that Shigella employs multiple strategies to activate inflammasomes, ³² thereby triggering rapid macrophage cell death. As the intestinal tract harbors the body’s largest immune compartment, macrophages play a pivotal role in maintaining mucosal immunity, including bacterial clearance and cytokine-mediated regulation of local immune homeostasis. ³³ These cells include Ly6Chi monocyte-derived macrophages and long-lived resident macrophages. ³⁴ Following radiation exposure, macrophages sense damage-associated molecular patterns (e.g., HMGB1) via TLR4, initiating downstream signaling cascades that culminate in proinflammatory cytokine release and systemic inflammation. ³⁵ Our data show that EGCG protects peritoneal macrophages from radiation-induced cytotoxicity, regulates TLR signaling pathway activity, and attenuates the release of inflammatory mediators (e.g., Eotaxin-2, Eotaxin, IL-6, TNF-α). Collectively, these findings suggest that EGCG safeguards the intestinal system through dual mechanisms: a direct protective effect on intestinal stem cells and an indirect immunomodulatory effect via gut microbiota-driven macrophage regulation.

Several limitations warrant consideration in this study. First, although we established EGCG’s protective effects on macrophages and identified associated gut microbiota changes, the causal mechanisms underlying the “gut microbiota-macrophage” axis require further interrogation through targeted intervention approaches. Second, the relative contributions of EGCG’s direct actions on macrophages vs indirect effects mediated by the tissue microenvironment remain to be fully delineated. Third, our experimental design did not account for potential functional heterogeneity among macrophage subtypes, leaving open the question of whether specific subpopulations respond differentially to EGCG treatment following radiation exposure.

Conclusion

By demonstrating EGCG’s ability to protect the intestinal immune microenvironment and mitigate RIII in WAI mice, this study expands our understanding of its radioprotective mechanisms and highlights the gut microbiota-immune axis as a promising target for developing novel radiation protection strategies. The integration of natural compounds like EGCG with mechanistic insights into microbiota-immune interactions may offer new therapeutic avenues for radiation-related gastrointestinal injuries.

Supplemental Material

Supplemental Material - The Radioprotective Effects of (−)-Epigallocatechin-3-gallate (EGCG): Restoring the Gut Microbiota-Immune Axis to Ameliorate Radiation-Induced Intestinal Injury

Supplemental Material for The Radioprotective Effects of (−)-Epigallocatechin-3-gallate (EGCG): Restoring the Gut Microbiota-Immune Axis to Ameliorate Radiation-Induced Intestinal Injury by Jia Gu, Qing Guo, Qian Hua, Zhibing Tang, Ying Yang, Chen Ji, Jia Yu, Dongqian Zhu, Qiong Feng, Jiaying Xu, and Lin Zhao in Dose-Response