Radiotherapy-Induced Digestive Injury: Diagnosis, Treatment and Mechanisms

Radiotherapy is one of the main therapeutic methods for treating cancer. The digestive system consists of the gastrointestinal tract and the accessory organs of digestion (the tongue, salivary glands, pancreas, liver and gallbladder). The digestive system is easily impaired during radiotherapy, especially in thoracic and abdominal radiotherapy. In this review, we introduce the physical classification, basic pathogenesis, clinical characteristics, predictive/diagnostic factors, and possible treatment targets of radiotherapy-induced digestive injury. Radiotherapy-induced digestive injury complies with the dose-volume effect and has a radiation-based organ correlation. Computed tomography (CT), MRI (magnetic resonance imaging), ultrasound (US) and endoscopy can help diagnose and evaluate the radiation-induced lesion level. The latest treatment approaches include improvement in radiotherapy (such as shielding, hydrogel spacers and dose distribution), stem cell transplantation and drug administration. Gut microbiota modulation may become a novel approach to relieving radiogenic gastrointestinal syndrome. Finally, we summarized the possible mechanisms involved in treatment, but they remain varied. Radionuclide-labeled targeting molecules (RLTMs) are promising for more precise radiotherapy. These advances contribute to our understanding of the assessment and treatment of radiation-induced digestive injury.


INTRODUCTION
Cancer is one of the greatest health problems in the 21st century. Approximately 29.8% of premature deaths (4.5 billion out of 15.2 billion) are attributed to cancer, ranking first or second in 134 of 183 countries (1). Radiotherapy, along with chemotherapy and surgery, is one of the three core methods of treating cancer. Nearly 50% of cancer patients receive radiotherapy (2). Compared with surgery, radiotherapy kills target tumor cells with less injury and is preferred when the target tumor tissue/organ cannot be removed. Compared with chemotherapy, radiotherapy limits the involved area and reduces lesions when the tumor is localized. However, radiotherapy is a doubleedged sword. That is, even though radiotherapy deals with tumor cells as planned, it may inevitably harm healthy cells.
The digestive system consists of the gastrointestinal tract and the accessory organs of digestion (the tongue, salivary glands, pancreas, liver and gallbladder). During eating, food is chewed by the oral cavity into small pieces and mixed with saliva, forming a bolus that passes through the esophagus into the stomach. Then, the stomach functions to store the food by receptive relaxation. In the stomach, gastric acid and pepsin are secreted, which, aided by the grinding of the stomach wall, turn food into chyme, helping in primary digestion until gastric emptying. Gastric emptying is regulated mainly by inhibitory feedback signals from the duodenum, including both enterogastric inhibitory nervous feedback reflexes and hormonal feedback by cholecystokinin, as well as partly by stomach factors (such as the degree of filling in the stomach and the excitatory effect of gastrin on stomach peristalsis). After the chyme passes into the small intestine, the pancreas secretes various digestive enzymes through the pancreatic bile tract, while the gallbladder releases bile secreted by the liver that breaks down nutrients into molecules to be absorbed in the small intestine. The length of the small intestine, as long as 10 to 16 feet, is helpful for fully absorbing carbohydrates, protein, fat and other nutrients. Then, indigestible food residue passes through the ileocecal valve into the large intestine and forms feces after dehydration. Defecation occurs as a result of reflex contraction of the rectum and relaxation of the anal sphincters (3).
Radiation-induced digestive injury is defined as acute or chronic lesions caused by ionizing radiation in the digestive organs, including the oral cavity, salivary glands, esophagus, stomach, intestines and anus. Radiotherapy, as one of the main methods of cancer treatment, accounts for almost all digestive injuries (4). The digestive system, as one of the most sensitive physiological organs to radiation therapy, usually suffers the most severe side effects from radiotherapy (4).

PHYSICAL CLASSIFICATION OF IONIZING RADIATION IN RADIOTHERAPY
Not all radiation can be applied to radiotherapy. Ionizing radiation refers to radiation carrying enough energy to ionize atoms and molecules and break chemical bonds. In a broad sense, ionizing radiation varies among different subjects. However, in biology, ionizing radiation is normally defined by the ionization energy of water, the main component of organisms. Nonionizing radiation refers to longer wavelength light including ultraviolet light, visible light, infrared light, microwaves and radiowaves, that cannot break bonds but can cause vibrations characterized as the heat effect. The specific numerical value of ionizing radiation's energy level is undefined but is usually approximately 12.4 eVs (corresponding wavelength of approximately 100 nm). Ionizing radiation can directly break bonds in DNA and protein. The shorter the wavelengths are, the higher the energy and corresponding radiation-induced damage. This is also true for energetic particles and magnetic waves (X-rays and g-rays). Energetic particles can be produced by unstable nuclei or by particle accelerators, usually including a-rays (helium), b-rays (electrons), proton rays, neuron rays and heavy ions ( Figure 1). These energetic particles have strong ionizing effects due to their relatively higher volume and/ or charge.

PATHOLOGICAL BASIS FOR RADIATION-INDUCED DIGESTIVE INJURY
DNA, proteins and lipids are the basis of cell survival. Their function relies on fine-tuned structure, meaning that there is a high risk of inactivation. Radiation may damage organisms as a result of direct effects, indirect effects and bystander effects. The direct effects refer to the collision of ionizing radiation causing destruction of DNA and/or protein structure, disturbing their functions (5). For indirect effects, both ionizing and nonionizing radiation produce free radicals and reactive oxygen species (ROS). However, compared with ionizing radiation, nonionizing radiation produces much less ROS via the heat effect. These highly active products subsequently react with DNA and proteins. The corresponding DNA damage includes single strand breaks, base damage, abasic sites, double strand breaks, non-double strand break clustered lesions, and complex double strand break, some of which are induced by DNA related protein (such as histone) damage (6). Radiation-induced RNA damage manifests as interference in transcription and accelerating in degradation. Both direct and indirect effects finally induce altered gene expression, protein modification, cell death/senescence, and genomic instability (7) (Figure 1). The bystander effect is defined from a different perspective. Regarding bystander effects, nonirradiated cells manifest biological changes resulting from transmitted signals from irradiated bystander cells, causing toxic radiation effects on adjacent nonirradiated tissues, usually genomic instability and chromosomal rearrangement (8). Originally, the effects of irradiated bystander cells are derived from direct effects and indirect effects. Both direct effects and indirect effects can function simultaneously, along with bystander effects, working together to induce radiation injury.
Radiotherapy utilizes various types of radiation rays. Each type of radiation ray has advantages and limitations. Compared with traditional photon radiotherapy, including X-rays and g-rays, protons and heavy ions have much longer wavelengths. As a result, the corresponding diffraction distances are on the same order of magnitude as the tissue size. Radiation diffraction converges on a peak named the Bragg peak (9). By refined calculation, the release of charged particle energy can be limited to the Bragg peak targeting tumor tissue, dramatically reducing the diffusion of radiation (10). Heavy ion therapy has an even narrower Bragg peak than proton therapy, making it more effective against cancer (11). Additionally, heavy ionradiated tissue manifests as clustered DNA double-strand breaks, enhancing therapeutic efficacy (12). However, protons and heavy ions have larger borders due to their longer wavelengths, making them difficult to locate (13). Comparatively, proton therapy and heavy ion therapy are superior to photon radiotherapy. Unfortunately, proton therapy and heavy ion therapy are severely limited due to their high cost (14). Future improvements in radiation methods for heavy ion therapy may further impel clinical application (15).

Clinical Features
The clinical characteristics of radiation-induced digestive injury are summarized in Figure 2. Salivary gland injury after radiation directly triggers hyposalivation. Subsequently, a lack of saliva induces xerostomia, mucositis, nutritional deficiencies, oral infections, and functional changes (such as difficulties with mastication, dysphagia and loss of taste) (16,17). In other digestive tract regions, including the esophagus, stomach, intestine and anus, radiation injury starts with mucous inflammation and is followed by diarrhea, constipation, and hemorrhage (4).

Assessment: Localized Radiation-Induced Digestive Injury a) Organ Correlation
Due to the need for precision medicine as well as reduced side effects, radiotherapy requires that the radiation be confined to the target area. Many studies have proven the efficacy of restricted radiation areas on reduced gastrointestinal side effects as well as enhanced dose tolerance in radiotherapy (18). Usually, radiation injury-related digestive system organs correlate with the surrounding radiotherapy. For example, anal radiotherapy and pancreas radiotherapy correlate with gastrointestinal side effects (19,20). Radiation of head and neck cancer induces dysphagia (21). Cervical cancer induces sigmoid stricture (22). Generally, periradiotherapy organs can help us locate the possible involved organs. Dose evaluation may help further reduce radiation-induced injury risks.

b) Dose-Volume Effect
Radiation-induced digestive injury manifests as a dosevolume effect, meaning that the extent of the lesion highly depends on the radiation dose and radiated volume (23). This theory has been verified in many studies in different organs, including the esophagus (21,24), stomach (25,26), small bowel (26,27), rectum (28)(29)(30)(31), and anus. On the basis of the dosevolume effect, radiotherapy-induced injury can be assessed by radiation dose and/or volume calculations. In this way, rectal toxicity (30,31), acute gastrointestinal toxicity (32,33), anal toxicity, and salivary gland injury were reported (34)(35)(36)(37)(38)(39)(40)(41)(42)(43) and precisely predicted (44). Conversely, Kim et al. found that a higher dose was not associated with cervical esophageal cancer radiotherapy-induced stenosis (45). This conclusion contradicts another study in nasopharyngeal carcinoma patients (46), probably because of the different tumor origins. Which symptoms correlate with dose and/or volume remains FIGURE 1 | Classification of radiation and mechanisms of radiation-induced injury. Radiation comprises of energetic particles and electromagnetic waves. Energetic particles and short wavelength electromagnetic waves (X rays and g rays) are classified as ionizing radiation. Longer wavelength electromagnetic waves (>100 nm) are categorized as nonionizing radiation. Ionizing radiation has enough energy to directly break DNA and protein. In addition, ionizing radiation can produce ROS (mainly by ionizing H 2 O), indirectly inducing DNA and protein damage. Nonionizing radiation may also produce little ROS. Impaired DNA and protein finally lead to cell mutation or death.
unknown. Clinical application lacks a detailed dose-volume standard assessing the radiation-induced risk of each complication. Systematic clinical evidence is necessary for evaluation guidance.

Computerized Tomography (CT)
CT provides a unique form of cross-sectional imaging. Threedimensional structures of "slices" of human tissue can be visualized, making CT an effective approach to predict radiation-induced injury. CT textural features could be used in combination with volume to characterize structural modifications of the parotid glands and to predict parotid shrinkage at the end of radiotherapy (47). By nonenhanced CT, a reduction in the volume of the parotid and submandibular glands and an increase in attenuation of the parotid gland can help grade radiation-induced salivary dysfunction (48). Parotid gland CT volume and density during head and neck cancer can also predict acute xerostomia (49). In summary, CT images of radiation-induced salivary injury are characterized by an increased mean gray value or density in the early stage, followed by shrinkage of the glands; texture analysis of CT is another indicator for assessing radiation-induced acute xerostomia (50) (Figure 3). Moreover, 18 F-FDG PET image biomarkers have considerably improved the prediction of late radiation-induced xerostomia (51), which is a promising method. Liver injury usually appears as CT imaging changes, and cases of CT assessing radiation-induced liver injury have been reported (52), suggesting that CT may help in the evaluation of radiation-induced liver injury. Although changes in CT images can be observed during radiotherapy, the variation in the liver is too small to diagnose, limiting CT to only prepared assessments that are started before radiation (53). Additional technologies may improve the CT diagnostic rate. For instance, single-photon emission CT imaging of mice precisely diagnosed radiation-induced liver disease (54). The diagnosis of other digestive organs by CT has rarely been reported.

Ultrasonic Histogram
Ultrasonic elastography, as a new ultrasound diagnostic technique, calculates the strain distribution by echo signals before and after compression and deformation of the tissue to obtain elastic (hardness) characteristic information for efficient clinical diagnosis. However, elastic noise usually interferes with imaging quality. Histogram matching algorithms can help suppress noise signals, accelerating the application of ultrasound histograms in many diseases. The efficacy of ultrasonic histogram analyses has been validated in salivary gland injury. Yang et al. used sonographic features as imaging signatures to assess radiation-induced parotid injury (55). They then summarized a family of sonographic features derived from echo histograms, including the peak intensity value, 23 dB intensity width, high intensity width and area of high intensity (56) (Figure 3). In addition, they further concluded that ultrasound histogram features (especially receiver operating characteristic curves) can be used to measure acute and late toxicity of the parotid glands after head and neck cancer radiotherapy, which may be developed into a low-cost imaging method for xerostomia monitoring and assessment (57). Salivary gland dysfunction, which relies on the blood supply, is easy to diagnose by ultrasound histogram. Other digestive organs, which have little external vascular variation compared with their surroundings, plus their deeper location, appear to have no distinguishable ultrasonic and CT distinctions.

Magnetic Resonance Imaging (MRI)
MRI, as a radiation-free medical imaging technique, is gradually replacing CT scans in clinical applications. MRI works by polarization of hydrogen atoms and has proven to be effective in diagnosing radiation-induced salivary gland injury, esophageal injury, liver injury, and rectal injury (58)(59)(60)(61). MRI images of radiation injury generally manifest as high signal intensity in T2, pathologically based on tissue edema. For acute radiation injury, an obvious shift in the T2 weighted imaging (T2WI) signal can be observed in the radiated area; for delayed radiation injury, the involved tissue may only exhibit a slight change on T2WI (62) (Figure 3).

Endoscopy
Early endoscopic findings deemed the Vienna rectoscopy score useful for predicting the possibility of late clinical radiation proctitis (63). Specific standards include congested mucosa, telangiectasia, ulceration, stricture, and necrosis ( Figure 3). Radiation-induced enteritidis can be diagnosed by wireless capsule endoscopy (64,65). Nevertheless, when it develops into obvious endoscopic manifestations, radiation injury is usually accompanied by other diagnostic clinical symptoms. Despite its low sensitivity in diagnosis, endoscopy may help in the prognosis as well as in essential treatment such as hemorrhage. The American Society for Gastrointestinal Endoscopy issued guidelines on the role of endoscopy for bleeding in chronic radiation proctopathy in 2019. These guidelines focused on currently available endoscopic therapies for managing patients with chronic radiation proctopathy, which include argon plasma coagulation, bipolar electrocoagulation, heater probe, radiofrequency ablation, and cryoablation (66). Further studies improving endoscopic standards to diagnose radiation proctopathy may lead to further refinement of these guidelines.

Gut Microbiota
The gut microbiota has become a new focus of various diseases, including chronic liver disease (67), type 2 diabetes mellitus (68), inflammatory bowel diseases (69), cardiovascular disease (70), sarcopenia (71) and cancer (72). Its correlation with radiation sensitivity has also been reported (73). A study in mice indicated that conventional intestinal microbiota composition may predict radiation injury (74). The control of bacterial translocation affects gastrointestinal acute radiation syndrome in mice (75). The prediction mechanism may involve pyrimidine and tryptophan pathways (76). Furthermore, a series of metabolic profile data of gut microbiota in cervical cancer patients summarized that radiation-induced acute intestinal symptoms are characterized by increased fecal concentrations of aketobutyrate, valine, uracil, tyrosine, trimethylamine N-oxide, phenylalanine, lysine, isoleucine, glutamine, creatinine, creatine, bile acids, aminohippurate, and alanine, accompanied by reduced concentrations of a-glucose, n-butyrate, methylamine, and ethanol (77). This study lays a solid foundation for the diagnosis and prediction of intestinal radioinjury. Analysis of the gut microbiota along with metabolic products is a promising method evaluating the severity of radiation-induced intestinal injury.

Other Predictive Factors
Moreover, some other factors should not be ignored. Substantial gland loss in the anterior rectal walls can predict radiationinduced late clinical proctitis (78). Single nucleotide polymorphisms and copy number variations were also reported to predict radiation rectal toxicity (79). Other metabolic-related nutrients, such as vitamin D (80) and citrulline (81), may serve as markers for radiation injuries. Besides, oral flora may also help diagnose radiation-induced injury, usually characterized by overgrowth of specific fungi such as Candida albicans (82,83).

Gland Transfer
Salivary glands have relatively separate structures and can be isolated for transplantation to avoid radiation injury. This theory has been proven by various studies, especially for head and neck cancer radiotherapy and nasopharyngeal carcinoma-induced xerostomia (84,85). Moreover, although fails to relieve dysphagia (86), gland transfer does not affect long-term treatment efficacy (85). A phase II study found that the technique of submandibular salivary gland transfer is reproducible in a multicenter setting (87). Further phase III randomized studies proved that submandibular salivary gland transfer is effective in curing radiation-induced xerostomia (88). Similar conclusions were reproduced in a meta-analysis (89). More phase III clinical studies may be required to evaluate the efficacy of gland transfer to promote the clinical application of gland transfer in radiation-induced salivary lesions.

Improvement in Radiotherapy a) Shielding
Shielding of the sensitive part of the target area is a traditional way to avoid radiation-induced injury. For example, partial shielding of the oral cavity in rhesus macaques may prevent oral mucositis (90). However, it is difficult to shield the visceral organs. Hydrogels precisely solve this problem. Hydrogels are three-dimensional cross-linked polymer networks that can absorb and retain large amounts of water, meaning that they are not poisonous to humans. This feature allows hydrogels to easily absorb radiation, similar to normal tissue. Implantation of hydrogel between the target tissue area and radiosensitive normal structure can effectively reduce the radiation volume of the normal structure. As proof, a simulation in cadaveric models of oropharynx cancer treated with intensity-modulated radiation therapy (IMRT) found that the hydrogel reduces the salivary gland radiation dose (91). Reductions in the radiated dose were verified in patients (92). In the clinic, rectum spacer hydrogel implantation prevents rectal injury in prostate cancer radiotherapy (93). Hydrogel spacers decreased duodenum radiation in pancreatic cancer radiotherapy (94). In addition, improvement in gastrointestinal syndrome was reported after prostate radiotherapy (95,96). Hydrogels have been widely used in clinical practice. Traditional hydrogels are preshaped and are usually implanted via operation. Compared with traditional hydrogels, injectable hydrogels have the advantages of eliminating operation limitations and drug administration but have accompanying high risks of inflammation and dislocation (97). Improvement in hydrogels, such as adding antiinflammatory drug components or using other inflammationfree hydrogels, may avoid inflammation. For instance, in situ photo-cross-linking hydrogels can restore the hypoxia-inducible factor 1-alpha pathway (98). Pectin/polyacrylamide hydrogels successfully deliver budesonide to the colon (99). Tannic acid acts as a cross linker and additionally enhances the antiinflammatory properties of hydrogels (100). Topical hydrogels containing Achyrocline satureioides oily extract can reduce inflammation (101). Dexamethasone-loaded thermosensitive hydrogels suppress inflammation in rheumatoid arthritis (102). All of these findings suggest promising application of improved injectable hydrogels in radiotherapy.

b) Dose Distribution
The dose distribution of radiotherapy influences radiationinduced injuries. High-dose-rate monotherapy can relieve radiation toxicity compared with low-dose-rate multitherapy (103). High-dose-rate boost treatment is associated with fewer side effects (104). Traditional radiotherapy is limited by dose administration to avoid radiotoxicity to normal tissues. Fractioned radiotherapy increases total dose tolerance and reduces the number of visits and the total cost of treatment without increasing radiotoxicity (105). In contrast, hypotreated prostate cancer patients suffered from significantly increased late genitourinary toxicity (106). In contrast, in the latest studies comparing hyperfractionated radiotherapy, conventionally fractionated radiotherapy, and hypofractionated radiotherapy, although relatively lower-fractionated radiotherapy may increase acute toxicity, there appears to be no significant difference in the long-term effects or late toxicity (105,(107)(108)(109)(110)(111)(112). More systematic studies are required to determine whether fractionated radiotherapy is superior to conventional radiotherapy.

Mesenchymal Stem Cells (MSCs)
MSCs are widely defined as a plastic-adherent cell population that can be directed to differentiate in vitro into osteogenic, chondrogenic, adipogenic, myogenic, and other lineages. MSC differentiation potential is widely used in tissue repair. MSCs have been proven to be able to restore radiation-induced injury (113,114). For example, adipose-derived stromal cells have the potential to restore salivary gland function after irradiation, as evidenced by the restoration of blood flow within submandibular gland tissue (115). Furthermore, human adipose tissue-derived stem cells alleviate radiation-induced xerostomia (116). Salivary gland stem cells can also ameliorate radiation-induced hyposalivation (117). Stem cell transplantation not only rescues hyposalivation but also restores tissue homeostasis in the irradiated gland, which is necessary for long-term maintenance of adult tissue (118). Administration of adiposederived stem cells immediately after radiation at a dose of 18 Gy can protect both the morphology and function of the salivary glands eight weeks after radiation in mice (119). In summary, MSCs can ameliorate radiation-induced salivary injury, including xerostomia (120,121).
Compared with radiation-induced salivary injury, the efficacy of MSCs in other digestive organs remains variable. Related research is summarized as follows: in a rat model of radiationinduced esophageal injury, dental pulp stem cell transplantation exhibited a therapeutic effect (122). For the colorectum, one study showed that MSCs may reverse radiation injury (123). Autologous bone marrow-derived mesenchymal stem cells may improve radiation-induced proctitis (124). Adipose-derived stem cells may facilitate the repair of defects in maxillofacial soft tissue (125). These cases alone hardly prove the viewpoint. Nonetheless, these results suggest that MSCs may have therapeutic potential for radiotherapy-induced tissue damage (126). Unfortunately, the specific mechanisms of MSC-based treatment have rarely been investigated among the studies, except that platelet-rich plasma improves the therapeutic efficacy of MSCs (127).
Chang et al. investigated the therapeutic mechanisms of MSCs and found that human adipose-derived mesenchymal stem cells (hAd-MSCs) had postradiation healing effects, including anti-inflammation, neovascularization and maintenance of epithelium homeostasis, as indicated by the elevated serum IL-10, upregulation of vascular endothelial growth factor, basic fibroblast growth factor and epidermal growth factor in irradiated intestine, mobilization of CD31positive hematopoietic stem cells or hematopoietic progenitor cells, and the prolonged presence of Bmi1-positive cells within crypts. The authors found that irradiated rats survived longer than nontreated animals (128). More related research is warranted in further studies.

Bone Marrow Transplantation
Bone marrow, similar to digestive system organs, is often involved in radiation-induced injury. Transplantation of bone marrow is a traditional way to cure bone marrow lesions. Improvement in bone marrow transplantation not only restores hematopoietic function but also alleviates other digestive symptoms (129,130). Bone marrow-derived cells can also reduce radiogenic oral mucositis (131). To further determine how bone marrow restores digestive symptoms, Tran et al. injected bone marrow soluble extract ("soup") into mice and found that bone marrow soup restored salivary flow rates to normal levels; protected salivary acinar, ductal, myoepithelial, and progenitor cells; increased cell proliferation and blood vessels; and upregulated the expression of tissue remodeling/repair/ regenerative genes. Bone marrow soup can be advantageously used to repair irradiation-damaged salivary glands rather than transplanting whole live bone marrow cells which carry the risk of differentiating into unwanted/tumorigenic cell types in the salivary glands (132). Further study suggests that bone marrow transplantation recruits host myelomonocytic cells and enhances intestinal stroma proliferation after radiation by secreting cytokines that enhance angiogenesis and chemotaxis (133). Bone marrow transplantation may share common mechanisms with MSCs in radiation-induced injury restoration. Controlled studies of MSCs and bone marrow transplantation may reveal interesting mechanisms.

Gut Microbiota
Since the gut microbiota can predict radiation injury, it is quite likely that modulation of the gut microbiota could minimize radiation injury. The gut microbiota plays a major role in the pathogenesis of radioinjury through the modification of intestinal barrier function, innate immunity and intestinal repair mechanisms (134). We determined the correlation between gut microbiota, metabolites, and radiation injury in Table 1 (135)(136)(137)(138)(139).
Characteristic changes in the structure of the gut microbiota after radiation (such as Bacteroides) can serve to predict radiation injury (140). Meanwhile, interference of gut microbiota may lessen radiation toxicity (141). Measures regulating gut microbiota include probiotics (142), a methionine diet (143), hydrogen-water oral gavage (144), and omega-3 polyunsaturated fatty acids (w-3 PUFAs) (145). Cui et al. reported the sex related effects for gut microbiota in relieving radiation injury (146). Notably, a large proportion of therapeutic drugs for radiation induced injury have effects on estrogen receptors and downstream effectors. This finding highlighted the importance of sex related receptors in treating radiation-induced injury. Nonetheless, these are all animal model studies with low reliability. Recently, Guo et al. transferred human and mouse radiation survivors' gut microbiota by fecal engraftment and dirty cage sharing and found improved radiation-induced injury related to Lachnospiraceae and Enterococcaceae. Two tryptophan pathway metabolites of these two bacteria, namely, 1H-indole-3-carboxaldehyde and kynurenic acid, provided long-term radioprotection. This is the first study proving the efficacy of gut microbiota modulation in humans, laying a foundation for clinical intervention of the human gut microbiota against radiation injury. All these cases prove that the gut microbiota presents opportunities to predict, prevent, and treat radiation lesions (147). Future targeting of patient-tailored restoration of optimal microbial composition could lead to a new era of radioprotection (148).

Related Therapeutic Drugs and Possible Mechanisms
The reported radioprotective agents are divided into several categories: free radical scavengers [such as thiols and amines (esp. aminothiols and phosphorothioates)], redox stabilizers (such as superoxide dismutase), antioxidant nutrients (vitamin A, B, C, E, and their related metabolites or analogues (such as bcarotene and folic acids), selenium derivatives, and phytochemicals (149). The overall effects of these drugs have been verified. With the development of modern biotechnology, many new drugs have proved their effectiveness in radiationinduced injury. We summarize representative mechanisms as well as updated drugs below.

a) Cell Death in Radiation-Induced Digestive Injury
Radiation-induced digestive injury induces cellular responses. These responses have mutual effects, and it is difficult to determine the dominant pathways. Cell autophagy, cell cycle arrest and even cell death have been reported in response to radiation . Here, we focused on cell death related pathways, especially apoptosis and ferroptosis in radiationinduced digestive injury.
Since p53 plays various roles in radiation-induced injury, it is unclear which effect is dominant. Coincidently, the summarized related drugs that attenuate radiation injury present clustering of p53, bcl-2/bcl-x, bax and caspase 3. Among antiapoptotic changes, including decreasing p53, decreasing caspase 3 and increasing bcl-2/bcl-x or bax, most drugs induce more than one effect ( Figure 4). This discovery strongly supports the p53/bcl-2/ bax pathway as dominant in radiation-induced digestive injury (174, 176-179, 181-186, 188) (Figure 4). Other p53-related drugs that have curative effects in radiation-induced digestive injury, such as Ex-RAD ( ® ) (173), may share the same pathway. Nevertheless, knockout of p53 or p21 paradoxically accelerates gastrointestinal damage and death, indicating that p53 may have a bidirectional effect in radiation-induced injury (198).

c) Ferroptosis in Radiation-Induced Digestive Injury
Ferroptosis is an iron-dependent type of programmed cell death initiated by lipid peroxide accumulation and depletion of plasma membrane polyunsaturated fatty acids (199). Traditionally, ferroptosis is regulated by amino acid and glutathione metabolism, lipid metabolism, and iron metabolism (200). Radiotherapy may also induce ferroptosis (201,202). Specific mechanisms involve promotion of lipid

d) Inflammation in Radiation-Induced Digestive Injury
Inflammation-related cytokines are another high-frequency group of anti-radiation drugs for digestive injury. IL-6-related anti-radiation drugs include ciprofloxacin (249), C-GSF (250), 18-b-glycyrrhetinic acid (251), CD28 mimetic peptide p2TA (252), delta-tocotrienol (253), and palmitoylethanolamide (254), suggesting that inflammatory inhibitors may also contribute to radiation injury ( Figure 4). Even so, the American Society for Gastrointestinal Endoscopy (ASGE) guidelines on the role of endoscopy for bleeding from chronic radiation proctopathy recommended not using antiinflammatory drugs because they lacked clinical evidence (66). The efficacy and safety of anti-inflammatory drugs and countermeasures warrant further investigation.

e) Natural Herbs (or Extractions) Against Radiation-Induced
Digestive Injuries In addition to modern synthetic drugs, traditional herbs play an indispensable role in curing radiation-induced digestive injury. Most of these effective herbs have been reported to regulate cell death (mainly apoptosis), including tea polyphenols (255), genistein (161), pachymic acid (172), sesamol (175), baicalein (180), acidic polysaccharide of Panax ginseng (154), explosively puffed ginseng (187), and resveratrol (256). Some herbs are involved in inflammation pathways, such as Vernonia cinerea L (257), fractions of diosmin + hesperidin (258), podophyllotoxin + rutin (259), Zhuye Shigao decoction (260), and rheinic acid (261) (Figure 4). Apocynin protects against radiation-induced injury by reducing apoptosis and oxidative stress-derived inflammation (165). Similarly, chamomile extract and walnut oligopeptides are also involved FIGURE 4 | Cell death-and inflammation-related drugs and countermeasures in radiation-induced digestive injury. Cell death-related drugs and countermeasures are divided into apoptosis and ferroptosis. In the left part of the table labeled "Apoptosis", all listed drugs prove to be effective in radiation-induced digestive injury. Grey part represents apoptosis inhibitors and inducers with no specific targets. The red part represents drugs that decrease p53, and the green part means drugs that decrease caspase3. Blue is for drugs that increase bcl-2/bcl-x or bax. Overlapping parts for two of the above three kinds of drugs are painted magenta, yellow and cyan respectively, meaning that drugs regulate both two factors. White stands for drugs with all of three functions. Clustering of drugs regulating p53, bcl-2/bcl-x, bax and caspase 3 implies that the p53 pathway is activated. The middle square painted red lists inducers and inhibitors of ferroptosis. The right square in blue lists inhibitors of inflammation that alleviate radiation-induced digestive injury. Natural herbs are selected with red boxes.
in both apoptotic and inflammatory pathways (170,262). Quercetin increases aquaporin 5 expression and calcium uptake, thus suppressing radiation-induced oxidative stress and inflammatory responses (263). Glycyrrhizin protects g-irradiated mice from gut bacteria-associated infectious complications by improving miR-222-associated Gas5 RNA reduction in macrophages at the bacterial translocation site (264). There are several curative herbs without corresponding mechanisms, with only morphological improvement, including Lagenaria siceraria extract (265), triphala (266), and resveratrol (267). Natural herbs are a great source of active compounds for reducing radiationinduced digestive injury. More research investigating the underlying mechanisms may reveal new therapeutic targets.

FUTURE PERSPECTIVES
Radiation-induced digestive injury remains a dominant problem since the application of radiotherapy. The current means of diagnosis and treatment are still far from satisfactory. Specific clinical guidelines supported by valid data are urgently needed. In diagnosis, artificial intelligence and deep learning can integrate comprehensive information including clinical features, imaging manifestations, and other predictive factors. Based on the antigen-antibody reaction and affinity interaction, specific biomarkers can be labeled by radionuclides and specifically targeted in diagnosis and treatment (268). For example, 89 Zrlabeled anti-gH2AX has successfully shown a radiobiological response in PET-CT (269). It is expected that radionuclidelabeled targeting molecules (RLTMs) may be used to precisely diagnose and evaluate radiation damage. Moreover, according to the biological effect of targeted biomarkers, aided by tissuespecific binding sites, RLTMs may act as radiotherapy sensitizers and radio-protectors. The combined application of RLTMs can provide an all-around assessment and strategies for multifunctional treatment. In precaution, novel regenerative peptide may prevent radiation-induced injury (270). In treatment, stem cell regeneration as well as gut metabolites application has shown their promise ameliorating radiotherapyinduced injury. However, there is still a long way from lab bench to bedside.

CONCLUSIONS
In general, radiation-induced digestive injuries during radiotherapy can be divided into two categories: salivary gland injury and digestive tract injury. For salivary gland injury, radiation damage derives from hyposalivation, followed by xerostomia, mucositis, nutritional deficiencies, oral infections, and functional changes. The unique anatomical structure of the salivary gland makes it easier to diagnose injury in these glands by CT, US, and MRI. Gland transfer is a promising method for preventing radiation damage. For digestive tract injury, the involved organ correlates with the radiated area, and the initial symptom is mucous inflammation, followed by diarrhea, constipation, and hemorrhage. Microbiota modulation may become an effective way of reducing radiation-induced gastrointestinal syndrome. Both salivary gland injury and digestive tract injury can be relieved by shielding, dose redistribution, mesenchymal stem cell transplantation and bone marrow transplantation. Inhibitors of cell death and inflammation may be an effective approach for reducing radiation-induced digestive injury. Natural herbs leave plenty of therapeutic potential to be discovered. We concluded that RLTMs are a promising technique in radiotherapy.

AUTHOR CONTRIBUTIONS
GC: Topic Presentation and Review Structure Control. SZ: Review Modification and Improvement. YH: Review Writing. HZ: Illustration design and Review Typeset. WT: Illustration design. All authors contributed to the article and approved the submitted version.