Research ArticleExperimental Studies
Open Access

Toll-like Receptor Agonist CBLB502 Protects Against Radiation-induced Intestinal Injury in Mice

QIONG WANG, JUNZHAO DUAN, JIAN HONG, KEXIN DING, FUMIN TAI, JIE ZHU, HANJIANG FU, XIAOFEI ZHENG and CHANGHUI GE
In Vivo July 2024, 38 (4) 1636-1648; DOI: https://doi.org/10.21873/invivo.13613

Abstract

Background/Aim: The small intestine is one of the organs most vulnerable to ionizing radiation (IR) damage. However, methods to protect against IR-induced intestinal injury are limited. CBLB502, a Toll-like receptor 5 (TLR5) agonist from Salmonella flagellin, exerts radioprotective effects on various tissues and organs. However, the molecular mechanisms by which CBLB502 protects against IR-induced intestinal injury remain unclear. Thus, this study aimed to elucidate the mechanisms underlying IR-induced intestinal injury and the protective effects of CBLB502 against this condition in mice. Materials and methods: Mice were administered 0.2 mg/kg CBLB502 before IR at different doses for different time points, and then the survival rate, body weight, hemogram, and histopathology of the mice were analyzed. Results: CBLB502 reduced IR-induced intestinal injury. RNA-seq analysis revealed that different doses and durations of IR induced different regulatory patterns. CBLB502 protected against intestinal injury mainly after IR by reversing the expression of IR-induced genes and regulating immune processes and metabolic pathways. Conclusion: This study preliminarily describes the regulatory mechanism of IR-induced intestinal injury and the potential molecular protective mechanism of CBLB502, providing a basis for identifying the functional genes and molecular mechanisms that mediate protection against IR-induced injury.

Key Words:

Ionizing radiation (IR) can cause DNA damage, lipid oxidation, and enzyme dysfunction, which can alter molecular structure and function, leading to functional impairments and metabolic disorders (1-3). Exposure of the small intestine to IR may cause acute radiation enteritis, which may result in abdominal pain, vomiting, bloody stools, and even death (4). IR-induced intestinal injury involves multiple complex pathophysiological processes, including intestinal necrosis, obstruction, and perforation (5). However, the underlying mechanisms remain unclear.

Toll-like receptor (TLR) agonist CBLB502, a truncated derivative of the Salmonella flagellin protein, exerts radioprotective effects and considerably reduces toxicity and immunogenicity by activating the nuclear factor-Embedded ImageB (NF-Embedded ImageB) pathway through the TLR5/MyD88 pathway (6). In the kidney, CBLB502 attenuates acute renal ischemic injury and failure by reducing the release of reactive oxygen species (7). In the liver, it increases hepatocyte resistance to acute liver injury via the TLR5-NF-Embedded ImageB and interleukin 22 (IL22)-signal transducer and activator of transcription 3 (STAT3) signaling pathways (8). In the lungs, it inhibits the apoptosis of pulmonary cells and ameliorates radiation pneumonitis and radiation pulmonary fibrosis via the TLR5/MyD88 pathway (9). In the testes, it alleviates IR-induced oxidative stress, inhibits lipid peroxidation, and decreases DNA strand break rates (10). In the head and neck area, it protects mice from dermatitis and oral mucositis caused by local IR (11). Evidence shows that CBLB502 also exerts protective effects on the intestine after IR (6, 12). In addition, CBLB502 plays a direct radioprotective effect in vitro via anti-apoptosis and promotes cell cycle recovery (13). However, the molecular mechanisms by which CBLB502 protects against IR-induced intestinal injury remain unclear.

Thus, this study aimed to investigate the mechanisms underlying IR-induced intestinal injury and the protective effects of CBLB502 against IR-induced intestinal injury in mice. RNA sequencing was performed to identify the differentially expressed genes (DEGs) mediated by CBLB502 and elucidate the potential molecular mechanisms underlying its radioprotective effects against IR-induced intestinal injury. In conclusion, this study provides novel insights into the functions and mechanisms of radiotherapy-related genes and may serve as a foundation for the further development of chemoradiotherapy.

Materials and Methods

Mice and radiation. C57BL/6 male mice (6-8 weeks old) were purchased from Vital River Experimental Animal Company (Beijing, PR China) and maintained in a specific pathogen-free room with a 12 h light/dark cycle. They were randomly divided into ten groups : irradiated at 6 Gy and sampled at 6 h (6 Gy6 h), irradiated at 6 Gy and sampled at 24 h (6 Gy24 h), irradiated at 6 Gy and sampled at 3 days (6 Gy3 d), irradiated at 15 Gy and sampled at 6 h (15 Gy6 h), irradiated at 15 Gy and sampled at 24 h (15 Gy24 h), irradiated at 15 Gy and sampled at 3 days (15 Gy3 d), pretreated with 0.2 mg/kg CBLB502 at 0.5 h before IR at 6/15 Gy (CBLB502+6/15 Gy), treated with 0.2 mg/kg CBLB502 (CBLB502) and phosphate-buffered saline (PBS) control (NC). The animals were irradiated with whole body 60Co γ-ray radiation at a dose rate of 72 R/min in the Beijing Institute of Radiation Medicine (Beijing, PR China). The use of animals and the study protocols were approved by the Institutional Animal Care and Use Committee of the Academy of Military Medical Sciences, PR China (permit number: IACUC-DWZX-2022-501).

Survival and body weight analysis. The CBLB502 polypeptide, derived from Salmonella flagellin, was prepared as described previously (10, 14), dissolved in PBS, and then injected intraperitoneally at a dose of 0.2 mg/kg bodyweight half an hour before IR. PBS was administered to control mice. The survival time of the irradiated mice was recorded daily for 30 days, and the body weights of the surviving mice were recorded on day 3.

Peripheral blood cell counts. Blood samples were collected from the tail veins of the mice on day 3 after IR or CBLB502 administration. The numbers of white blood cells (WBC), lymphocytes (LYM), and platelets (PLT) in the peripheral blood were counted using a hematology analyzer (DYMIND, DF52vet, Shenzhen, PR China).

Hematoxylin and eosin (H&E) staining. Small intestine samples of the mice were collected, fixed in 10% formalin, dehydrated, and then embedded in paraffin wax. The tissues were dissected, stained with H&E, and then mounted. The sections were analyzed under a light microscope, histological changes were recorded, and the area of the tissue lesion was indicated by arrows.

RNA extraction and sequencing. Total RNA was isolated from intestinal mucosal tissue at different time points (6 h, 24 h and 3 days) under different doses (6 and 15 Gy) using TRIzol (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The amount and purity of RNA were quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). Poly (A) RNA was purified from 1 μg of total RNA using Dynabeads Oligo (dT) 25-61005 (Thermo Fisher Scientific) with two rounds of purification. Then, poly (A) RNA was fragmented into small pieces using the Magnesium RNA Fragmentation Module [New England Biolabs (NEB), Ipswich, MA, USA]. Finally, RNA libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB) and performed on an Illumina Novaseq™ 6000 (LC-Bio Technology Co., Ltd., Hangzhou, PR China) following the manufacturer’s recommended protocol. RNA sequencing was uploaded to the GEO database with the GEO accession number GSE246176.

Bioinformatic analysis. DEGs were selected with |fold change (FC)|>2.0 (|FC|>1.5 was used in CBLB502+6/15 Gy vs. NC) and adjusted p-value<0.05 using the R package edgeR (15). The intersecting set of DEGs between groups was analyzed using Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/index.html). Gene Ontology (GO) pathway analyses of DEGs were conducted using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) online tool (http://david.ncifcrf.gov) (16). A protein-protein interaction (PPI) network for genes between groups was constructed using STRING (http://string-db.org) (17). A representative subnetwork containing nodes with high levels of interconnection was derived from the PPI network, with a defective confidence score of 0.400. Network graphs were generated using Cytoscape 3.7.1 (The Cytoscape Consortium, San Diego, CA, USA) (18).

Real-time polymerase chain reaction (RT-PCR). Total RNA was extracted from the intestine as described above. cDNA was generated using the HiScript® III All-in-one RT SuperMix Perfect (Vazyme Biotech Co., Ltd., PR China). RT-PCR was performed using the Stratagene Mx3000P (Agilent Technologies) and TOROGreen® HRM qPCR Master Mix (Torovid, PR China) in accordance with the manufacturer’s instructions. RT-PCR primer sequences were designed using NCBI Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The primer sequences used were listed in Table I. The threshold cycle (Ct) values of the target genes were normalized to those of the glyceraldehyde 3-phosphate dehydrogenase (Gapdh) control. Differential expression was calculated using the 2−ΔΔCT method (19).

View this table:
Table I.

Primers used for quantitative RT-PCR.

Statistical analysis. Data were presented as means±standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad, San Diego, CA, USA). Kaplan-Meier survival curves were compared using the log-rank test. The body weights and peripheral blood cell counts of the mice were compared using a two-tailed Student’s paired t-test. RT-PCR results were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test. Statistical significance was set at p<0.05.

Results

CBLB502 protects against IR-induced intestinal injury in mice. To investigate the effects of IR on intestine and protection of CBLB502 in intestine against IR, mice were irradiated with γ-rays at doses of 6 or 15 Gy, and were pre-treated with CBLB502 before IR. The results showed that mice irradiated at 15 Gy started to die on day 8 and all died by day 11, whereas those pre-treated with CBLB502 before IR at 15 Gy started to die on day 11 and all died by day 15 (p<0.01, Figure 1A). Therefore, the administration of CBLB502 before IR at 15 Gy prolonged the survival time of the mice. The body weights of the mice decreased after IR, especially at 15 Gy (p<0.0001), but this effect was reversed by pretreatment with CBLB502 (p<0.05, Figure 1B). Peripheral blood analysis showed that the WBC, LYM, and PLT counts significantly decreased in the IR group compared to the control group (p<0.0001), but they significantly recovered when CBLB502 was administered before IR (p<0.001, except for LYM: p<0.01; Figure 1C-E).

Figure 1.
Figure 1.

CBLB502 protects against ionizing radiation (IR)-induced intestinal injury in mice. (A) Kaplan-Meier survival curve of irradiated mice pretreated with 0.02 mg/kg CBLB502. (B) Body weights of surviving mice on day 3 (n=10). Peripheral blood counts of (C) white blood cells (WBC), (D) lymphocytes (LYM), and (E) platelets (PLT) on day 3 after IR (n=15-17). Data are presented as mean±standard error of the mean (SEM). The log-rank test or Student’s t-test was used to analyze the data. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. NC: Normal control; 502: CBLB502.

In addition, histopathological analysis showed abundant intestinal villi in the intestinal tissues, localized visible edema in the lamina propria, which was seen with more intestinal glands lost and replaced by proliferating connective tissue (indicated by yellow arrows in Figure 2) in the 6 Gy IR group compared to the control group, with no significant change over time. By contrast, multiple separations of intestinal epithelium from lamina propria in the mucosal layer were observed (indicated by black arrows in Figure 2) in the 15 Gy24 h treatment group, with localized epithelial cell shedding. Significant inflammation was observed in the 15 Gy3 d treatment group, with cells showing severe edema accompanied by punctate infiltration of lymphocytes (indicated by red arrows in Figure 2). Cytoplasmic basophilic enhancement was observed in a small number of epithelial cells (indicated by blue arrows in Figure 2). No apparent inflammation was observed in the CBLB502+15 Gy group (Figure 2). In summary, IR can damage mouse intestinal tissues in a dose-dependent manner. However, CBLB502 can protect against IR-induced intestinal injury.

Figure 2.
Figure 2.

Hematoxylin and eosin (H&E) staining of intestinal tissues treated with ionizing radiation (IR) and/or CBLB502. Mice were treated with CBLB502, IR, or CBLB502+IR, their intestinal tissues were harvested after 24 h or 3 days and stained with H&E. Representative images were shown. NC: Normal control; 502: CBLB502.

IR induces intestinal damage in mice in a dose-dependent manner. To further understand the potential regulatory mechanisms of IR-induced intestinal injury, RNA sequencing was performed under the doses of 6 and 15 Gy, which could lead to hematopoietic and intestinal acute radiation syndrome, respectively. Using RNA sequencing, we analyzed the effects of IR at 6 and 15 Gy on intestinal gene expression and generated a heatmap of DEGs (Figure 3A). A total of 1876 and 1490 DEGs were found in the intestinal tissue irradiated at 6 and 15 Gy, respectively, with 363 genes commonly regulated by both doses (Figure 3B). The biological process (BP) terms of DEGs in low-dose radiation were mainly involved in fatty acid metabolic process and transmembrane transport, whereas those in high-dose radiation were mainly involved in transcription regulation and DNA damage (Figure 3C and D). The BP terms of the common DEGs were mainly involved in response to bacterium, steroid metabolic process, and cellular iron ion homeostasis (Figure 3E), indicating that IR-induced general BPs were unrelated to the IR dose. The mRNA expression levels of acyl-CoA dehydrogenase long chain (Acadl), fatty acid amide hydrolase (Faah), major facilitator superfamily domain containing 4A (Mfsd4a), aquaporin 1 (Aqp1), cyclin dependent kinase inhibitor 1C (Cdkn1c), nipped-B-like (Nipbl), BCL2 associated X (Bax), and X-linked inhibitor of apoptosis (Xiap) in these BPs were validated by RT-PCR, after the RNA-seq results (Figure 4A-D). The expression levels of the genes related to DNA damage increased in a dose-dependent manner, but those of the genes related to the other BPs did not (Figure 4E). PPI network analysis revealed a significant subnetwork, indicating that most of the proteins involved in these BPs interacted with each other (Figure 4F-I). Taken together, these results indicate that the different IR doses induce different gene regulatory patterns in the mouse intestine, with an aggravating effect at a dose of 15 Gy.

Figure 3.
Figure 3.

Ionizing radiation (IR) induces intestinal damage in mice in a dose-dependent manner. (A) Hierarchical clustering of differentially expressed genes (DEGs) in the mouse intestine. Mice were treated with whole-body radiation at 6 or 15 Gy. (B) Venn diagram analysis showing the common DEGs in IR-treated intestine. The fold-change of DEGs was set to 2.0. (C-E) Biological process (BP) enrichment analyses of DEGs in the different treatment groups. The dotted line of the BP analysis indicates p=0.05. The length of the columns indicates the gene counts.

Figure 4.
Figure 4.

Identification of differentially expressed genes (DEGs) after ionizing radiation (IR) dependent on dose. (A-D) Validation of the expression of selected DEGs in the intestine. Real-time polymerase chain reaction (RT-PCR) of Acadl, Faah, Mfsd4a, Aqp1, Cdkn1c, Nipbl, Bax, and Xiap in the intestine. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used as the internal control. (E) Line graph shows the average FPKM expression levels of DEGs in each biological process (BP). (F-I) Protein-protein interaction network of DEGs from different BPs. The experiments were performed using three mice, and the data are presented as mean±standard error of the mean (SEM). DEGs from the RNA sequencing data were analyzed using the negative binomial distribution, with the DESeq2 R package. One-way ANOVA was used to analyze the RT-PCR data. **p<0.01, ***p<0.001 and ****p<0.0001. NC: Normal control.

IR induces intestinal damage in mice in a time-dependent manner. IR usually causes progressive tissue damage with increasing time. We then identified DEGs and analyzed the effect of IR duration on intestinal gene expression in enteral radiation sickness (Figure 5A). The numbers of DEGs at 6 h, 24 h, and 3 days after 15 Gy radiation were 1490, 727, and 2305, respectively, and 118 genes were commonly regulated (Figure 5B). The BP terms of the common DEGs were involved in response to bacterium, flagellated sperm motility, and steroid metabolic process, suggesting that these pathways were constantly regulated and played important roles during radiation (Figure 5C). The BP terms of the DEGs were mainly involved in transcription regulation at 6 h (Figure 3D), immune response at 24 h (Figure 5D), and cholesterol metabolic process at 3 days (Figure 5E), indicating stage-dependent regulation during radiation. The mRNA expression levels of important genes [lipin1 (Lpin1), enhancer of polycomb homolog 2 (Epc2), mannose binding lectin 2 (Mbl2), bone marrow stromal cell antigen 2 (Bst2), sterol O-acyltransferase 1 (Soat1), and ectonucleotide pyrophosphatase/phosphodiesterase 7 (Enpp7)] related to these BPs were verified by RT-PCR based on the RNA-seq analysis results (Figure 6A-C). PPI analysis revealed interactions between most of the proteins involved in these BPs (Figure 6D-E). Collectively, these results indicate that different IR durations induce different regulatory mechanisms in the mouse intestine.

Figure 5.
Figure 5.

Ionizing radiation (IR) induces intestinal damage in mice in a time-dependent manner. (A) Hierarchical clustering of differentially expressed genes (DEGs) in the mouse intestine. Mice were treated with whole-body radiation at different sampling time points (6 h, 24 h, and 3 days). (B) Venn diagram analysis indicated that the common DEGs were differentially altered in the IR-treated groups. The fold-change of DEGs was set to 2.0. (C-E) Biological process (BP) enrichment analyses of DEGs in different treatment groups in the intestines of IR-treated mice. The dotted line of the BP analysis indicates p=0.05. The length of the columns indicates the gene counts.

Figure 6.
Figure 6.

Identification of differentially expressed genes (DEGs) after ionizing radiation (IR) dependent on duration. (A-C) Validation of the expression of selected DEGs in the intestine. Real-time polymerase chain reaction (RT-PCR) of Lpin1, Epc2, Mbl2, Bst2, Soat1, and Enpp7 in the intestine. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used as the internal control. (D-E) Protein-protein interaction network of DEGs from different biological processes (BPs) is shown. The experiments were performed using three mice, and the data are presented as mean±standard error of the mean (SEM). DEGs from the RNA sequencing data were analyzed using the negative binomial distribution, with the DESeq2 R package. One-way ANOVA was used to analyze the RT-PCR data. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. NC: Normal control.

CBLB502 protects against intestinal damage mainly after radiation. The radioprotective drug CBLB502 has been reported to have protective effects on mice and primates after total body radiation (6). To further investigate the regulatory mechanisms involved in the protection of CBLB502 against IR-induced intestinal injury, we comparatively analyzed the DEGs between the IR (6 Gy, 15 Gy) and/or CBLB502 treatment groups. We then clarified the effects of CBLB502 on gene expression by comparing the DEGs between the CBLB502+IR group and the IR group i.e., CBLB502+IR/IR, while generally excluding IR-induced gene expression. Furthermore, we analyzed the direction of regulation (up-regulation/down-regulation) of DEGs in the CBLB502+IR/IR and CBLB502/NC groups relative to those in the IR-only group to uncover the effect of CBLB502 pretreatment on IR-induced DEGs in both unirradiated mice and mice that were irradiated but did not undergo IR-induced DEG changes. Our results showed that most DEGs in the CBLB502/NC group revealed the same regulatory direction (both up-regulated and down-regulated) as those in the IR-only (IR/NC) group, indicating that the DEGs induced by IR and CBLB502 were either up-regulated or down-regulated. Additionally, only a few DEGs demonstrated opposite regulatory directions, namely, up-regulation in the IR-only group and down-regulation in the CBLB502-only group, or vice versa (Figure 7A). The expression of these genes can be down-regulated by IR and up-regulated by pretreatment with CBLB502. By contrast, the majority of DEGs in the CBLB502+IR/IR group showed opposite regulatory directions to those in the IR/NC group, with most of them up-regulated in the 6 Gy group and down-regulated in the CBLB502+6 Gy group, while being down-regulated in the 15 Gy group and up-regulated in the CBLB502+15 Gy group (Figure 7B). Nine genes were common in the opposite DEGs between the CBLB502+6 Gy/6 Gy and CBLB502/NC groups compared with the 6 Gy/NC group, whereas six genes were common between the CBLB502+15 Gy/15 Gy and CBLB502/NC groups (Figure 7C and D). These results suggest that CBLB502 protects against IR-induced intestinal injury by reversing the expression of IR-induced DEGs during the IR protection process mainly after IR.

Figure 7.
Figure 7.

CBLB502 protects against intestinal damage mainly after radiation through varied pathway regulatory patterns. (A) Overlap of differential regulation between the CBLB502/NC and ionizing radiation (IR) (6 or 15 Gy)/NC groups in the intestine, revealing the effects of pretreatment with CBLB502-only on IR-induced differentially expressed genes (DEGs) in non-IR-treated mice. (B) Overlap of differential regulation between the IR+CBLB502/IR and IR/NC groups, revealing the effects of pretreatment with CBLB502 on IR-induced DEGs in mice after subtracting the effect of IR on DEGs. (C-D) Venn diagram analysis showing the number of opposite regulatory direction of DEGs in the intestine of the mice from the CBLB502 and IR+CBLB502 groups. (E-F) Biological process (BP) enrichment analyses of reserve DEGs in the intestine between the CBLB502+IR/IR and IR/NC groups. (G-H) Real-time polymerase chain reaction (RT-PCR) of Irgm1 and Bst2 in the intestine. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used as the internal control. The experiments were performed using three mice, and the data are presented as mean±standard error of the mean (SEM). DEGs from the RNA sequencing data were analyzed using the negative binomial distribution, with the DESeq2 R package. One-way ANOVA was used to analyze the RT-PCR data. *p<0.05, **p<0.01 and ****p<0.0001. NC: Normal control; 502: CBLB502.

CBLB502 protects the intestine from IR-induced injury through different pathway regulatory patterns. Although the total number of reverse DEGs involved in radiation protection was similar between the CBLB502+6 Gy/6 Gy and CBLB502+15 Gy/15 Gy groups (63 vs. 61), the regulatory pathways of their respective reverse DEGs were not consistent. IR-induced DEGs were regulated to protect against intestinal damage primarily through BPs, such as regulation of RNA splicing and lipid metabolic processes in the 6 Gy+CBLB502 group, and immune response and defense response to bacterium/virus in the 15 Gy+CBLB502 group (Figure 7E and F). Furthermore, the selection of DEGs was validated using RT-PCR analysis, which confirmed that their expression was consistent with the sequencing results (Figure 7G and H). Collectively, these results suggest that CBLB502 exhibits protective effects against different IR doses through varied mechanisms in the intestine.

Discussion

The present study showed that IR can cause intestinal injury by inducing different BPs in a dose- and time-dependent manner. However, IR can still induce some commonly regulated processes, including response to bacteria and the apoptotic signaling pathway, regardless of the dosage and duration of IR. Our study also revealed that CBLB502 played a protective role against IR-induced intestinal injury, mostly after but seldom before IR, by reversing IR-induced DEGs involved in immune response and metabolic processes.

IR can cause extensive tissue damage that worsens with increasing radiation dose (5). However, previous studies have shown that re-IR 12 weeks after the first IR produced similar biological effects in the normal intestine as observed after a single IR exposure (20). In the present study, although no obvious difference in hematology at the early IR stage was found between the groups irradiated with 6 and 15 Gy, the group irradiated with 15 Gy exhibited lower body weight and higher mortality at the early stage and more serious intestinal damage. Reportedly, when irradiated at doses ≤14 Gy, the mouse intestinal epithelia can regenerate, as indicated by animal survival following bone marrow transplant (21). By contrast, radiation at doses ≥15 Gy produces severe radiation syndrome, including intestinal damage and even mortality, which cannot be reversed by bone marrow transplant (22). These studies indicate that low-dose radiation causes much less damage to the intestine than high-dose radiation (5). The main BPs involved in intestinal injury under 6 Gy were lipid/fatty acid metabolism and transmembrane transport which were mostly related to cellular function and physiological stability (23-25), whereas those under 15 Gy were transcriptional regulation/DNA damage/apoptosis/cell cycle processes which were generally involved in cell death and intestine failure (26-29). Thus, these results strongly suggest that IR might induce preferential processes depending on the dosage. Interestingly, some processes, such as response to bacteria, were commonly regulated regardless of dosage, and bacteria could emerge as a critical driver of radiation-response (30), indicating that the intestinal basic function was easily affected upon radiation. Therefore, our data might preliminarily show a dose-dependent regulatory pattern in intestinal damage, which requires further investigation in the future.

Meanwhile, our results showed that IR might also regulate intestinal injury via time-dependent BPs. The main BPs involved in intestinal damage induced by radiation at 15 Gy were transcriptional regulation at 6 h, immune response at 24 h, and cholesterol metabolism at 3 days, which implied that these pathways were time-dependently involved in the intestinal injury. These pathways have been reported to be crucially being involved in IR-induced intestinal injury during the IR period (31-33). However, the temporal correlation and its evolutionary relationship on IR are still unclear, which also highlights the complexity of the duration effect on IR. Similar to dose, the response to bacteria was also regulated regardless of the stage, indicating that they were always present throughout IR duration. These results suggest a general regulatory mechanism of IR-induced injury in different radiation durations, which needs further exploration.

Interestingly, our results showed that both pathways in the 6 Gy6 h and 15 Gy3 d groups are involved in lipid metabolism. However, the intestinal injury in the 6 Gy6 h group was mainly associated with fatty acid metabolism, whereas in the 15 Gy3 d group, it primarily involved cholesterol metabolism. These results agree with previous reports stating that cutaneous fatty acid metabolism is altered in the early response to IR (23), while long-term radiation exposure could lead to cholesterol metabolism alterations (34). This also illustrates the complexity of IR-induced metabolic pathway alterations, where different lipid metabolism molecules may respond to different times or doses.

Flagellin has a hepatoprotective effect in the long term after IR (35). CBLB502 exerts radioprotective therapeutic effects on mice and rhesus monkeys exposed to lethal doses of IR; 87% of NIH Swiss mice survived after treatment with CBLB502 half an hour prior to 13 Gy radiation (36). In the present study, CBLB502 prolonged the survival time of irradiated mice, restored their body weight, and ameliorated IR-induced intestinal injury. Previous reports demonstrated that CBLB502 protected the bone marrow, lungs, liver, kidneys, and testes from damage by inhibiting normocellular apoptosis, reactive oxygen species generation, and inflammatory factor infiltration, and by increasing superoxide dismutase activity (7-10). Meanwhile, microarray and RNA sequencing revealed that CBLB502 exerted its protective effects in the liver, kidney, and bone morrow only after treatment with cisplatin or exposure to IR (37, 38). However, these results were not entirely consistent with our findings, and still a few DEGs were induced by CBLB502 alone and simply regulated against IR. Thus, CBLB502 possibly exerts complicated regulatory mechanisms to protect different tissues from IR-induced injury. Further studies are warranted to clarify this phenomenon.

CBLB502 exerts cellular protective effects within a few hours and responds rapidly to IR-induced stress (6) by interfering with genes involved in free radical clearance, cell cycle regulation, apoptosis, and inflammation via the NF-Embedded ImageB pathway (39, 40), thus achieving complex radiation-cell protection (10, 12, 41-43). The present findings indicate that CBLB502 induced diverse protective pathways against intestinal damage depending on the IR dose. Specifically, treatment with 6 Gy primarily induced intestinal injury through metabolism and other cellular pathways, whereas pretreatment with CBLB502 under 6 Gy IR exerted protective effects on the intestine through similar pathways. Meanwhile, treatment with 15 Gy induced intestinal injury mainly through immune response against external inflammatory viral infection, and pretreatment with CBLB502 under 15 Gy IR protected against this injury via the same pathway. Similarly, a previous study suggested that CBLB502 can indirectly eradicate colon cancer cells via immune response (44). In addition, it has been found that CBLB502 is involved in the regulation of kidney function through signaling pathways such as immunoglobulin production and immune response (45).

Several genes, including immunity-related GTPase family M protein 1 (Irgm1) and Bst2, regulate the protection against IR-induced intestinal injury. Existing reports show that Irgm1 can inhibit NOD-like receptor thermal protein domain associated protein 3 (NLRP3)-mediated inflammation in dextran sulfate sodium-induced colitis (46), and Irgm1 deficiency leads to inflammatory responses in the gut microbiota (47), intestinal inflammation, and impaired immune against pathogenic bacteria in mice (48). Furthermore, Bst2 has been found in macrophages, monocytes, plasmacytoid dendritic cells, and B and T-lymphocytes (49-51), and a high level of Bst2 has been significantly correlated with immune response (52). Our results preliminarily suggest that CBLB502 can up-regulate the expression of these genes, both of which are required for protecting the intestine. Therefore, further in vitro and in vivo studies are required to better understand the mechanism of regulation and the functions of these genes during radioprotection in intestinal injury by radioprotectants such as CBLB502.

Notably, we observed a significant difference in the protective mechanisms of CBLB502 against IR-induced intestinal and bone marrow damage. In our previous studies, the number of reverse DEGs for CBLB502 protection in the bone marrow was 367 (38), which was much higher than that in the intestine in this study. Additionally, CBLB502 exhibits various preferential regulatory patterns in protective effects between the liver and kidneys against cisplatin (37). This finding further illustrates the complexity of the regulatory mechanisms underlying the protective effects of CBLB502 against harmful external stimuli.

Conclusion

In summary, our data showed that IR at different doses and durations induces intestinal damage mostly through various regulatory mechanisms, whereas some important pathways are regulated regardless of the radiation dosage and duration. CBLB502 exhibits a protective effect on IR-induced intestinal injury mainly after exposure to IR by regulating immune processes and metabolic pathways. These findings provide novel insights into the functions and mechanisms of radiotherapy-related genes and lay a theoretical foundation for the further development of chemoradiotherapy.

Acknowledgements

We would like to thank Editage (www.editage.cn) for English language editing.

Footnotes

  • Authors’ Contributions

    CG and XZ conceived and designed the study; QW, JD, KD, FT, JZ, and HF performed the experiments; QW and JH analyzed the data; and QW wrote the manuscript with comments provided by CG. This study was approved by all the authors.

  • Supplementary Material

    The following supporting information can be downloaded at: https://doi.org/10.5281/zenodo.10817566

    Supplementary Material S1: The BP enrichment analyses of DEGs in the group of different doses. Supplementary Material S2: The BP enrichment analyses of DEGs in the group of different time points. Supplementary Material S3: The BP enrichment analyses of opposite regulated DEGs in the CBLB502+IR/IR group.

  • Conflicts of Interest

    The Authors declare no potential conflicts of interest with respect to the research, authorship, or publication of this article.

  • Received February 10, 2024.
  • Revision received March 15, 2024.
  • Accepted March 27, 2024.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Toll-like Receptor Agonist CBLB502 Protects Against Radiation-induced Intestinal Injury in Mice
QIONG WANG, JUNZHAO DUAN, JIAN HONG, KEXIN DING, FUMIN TAI, JIE ZHU, HANJIANG FU, XIAOFEI ZHENG, CHANGHUI GE
In Vivo Jul 2024, 38 (4) 1636-1648; DOI: 10.21873/invivo.13613
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