Fecal metabonomics-based radiation enteritis differential metabolite marker as well as screening method and application thereof

Abstract

The invention discloses a radiation enteritis differential metabolite marker based on faeces metabonomics and a screening method and application of the radiation enteritis differential metabolite marker. The radiation proctitis differential metabolite marker is prepared from (R)-lipoic acid salt, (S)-malic acid, 3-mercaptolactic acid, 4-acetaminobutyric acid, 4-guanidinobutyric acid, 5-hydroxyindoleacetic acid, 5-methoxy indoleacetic acid, isocitrate, L-homocysteine and L-proline. The excrement differential metabolite is used as the radiation proctitis biomarker, has important significance on clinical early diagnosis of radiation proctitis, and can be used for preparing a diagnosis tool for radiation proctitis, predicting the individual treatment effect of radiation proctitis patients and preparing a medicine for treating radiation proctitis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to differential metabolite markers of radiation enteritis based on fecal metabolomics, and screening methods and applications thereof. Background Art

[0002] There are more than 4 million new cases of pelvic malignant tumors worldwide, and more than 700,000 new cases of pelvic malignant tumors in my country. 20%-60% of patients with pelvic malignant tumors need to receive pelvic radiotherapy. However, radiotherapy can cause physical damage to normal tissues, leading to damage to pelvic and abdominal organs and tissue metabolic disorders, among which radiation proctopathy (RP) is the most common. The progressive development of RP can lead to chronic and refractory lesions. Chronic radiation proctitis is protracted, with stubborn symptoms and progressive aggravation. The course of damage is difficult to reverse, and finally leads to various late serious complications (such as intestinal tissue fibrosis, massive bleeding, perforation, obstruction, intestinal fistula, etc.). Clinical diagnosis and treatment are difficult, and the quality of life of patients is seriously affected. Therefore, there is an urgent need to explore metabolite markers for the detection of radiation proctitis.

[0003] Metabolites participate in the basic functions of the host, including energy metabolism, signal transduction and apoptosis. In addition to being directly produced by the host itself, metabolites can also come from diet and microorganisms. Non-targeted metabolomics is a cutting-edge research technology that uses modern analytical techniques to analyze the endogenous metabolic changes of the host under different pathophysiological states. It reveals the types and levels of metabolites and finds the relative relationship between metabolites and physiological and pathological changes through comprehensive and systematic qualitative and quantitative analysis of metabolite spectra. It has been a hot topic in recent years. Among them, fecal metabolites are the products of co-metabolism between intestinal microorganisms and hosts. Detecting metabolites in feces can provide rich metabolomics information and reflect the status of the host body and intestinal microorganisms. Through in-depth analysis of the metabolome data of host feces, it is possible to discover new molecules and pathways that change the disease state, providing an experimental basis for studying the prevention and treatment of radiation proctitis. At present, there are relatively few studies on the metabolite markers of radiation proctitis using fecal non-targeted metabolomics technology. Summary of the invention

[0004] In view of the lack of metabolite markers suitable for early screening and diagnosis of radiation proctitis in the prior art, the present invention provides a differential metabolite marker of radiation proctitis based on fecal metabolomics and a screening method and application thereof, and constructs a research paradigm for the metabolic mechanism of radiation proctitis based on the long-clawed gerbil radiation proctitis animal model, which is beneficial to the research on the pathological mechanism of radiation proctitis and drug development.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] The present invention first provides a method for screening differential metabolite markers of radiation proctitis based on fecal metabolomics, comprising the following steps:

[0007] (1) Long-clawed gerbils were selected and randomly divided into a normal control group and a radiation group. The radiation group was used to establish an animal model of radiation proctitis, and feces of the two groups of animals were collected;

[0008] (2) Fecal samples were analyzed by ultra-high performance liquid chromatography-mass spectrometry, and MS / MS data were extracted after filtering isotope peaks and compared with the metabolite database to obtain non-targeted metabolomics data;

[0009] (3) Using multivariate statistical analysis methods, we screened and identified endogenous metabolites with significant changes from multiple variables as potential differential metabolites associated with radiation proctitis;

[0010] (4) Metabolic pathway enrichment analysis was performed on the above non-targeted metabolomics data to obtain metabolic pathways significantly associated with radiation proctitis and differential metabolites in the metabolic pathways. The differential metabolites were screened by ROC curve analysis to obtain metabolite markers.

[0011] Preferably, in step (1), the construction of the radiation proctitis animal model is performed by using a single radiation dose of 30 Gy to model the long-clawed gerbil.

[0012] Preferably, in step (2), the chromatographic conditions of the ultra-high performance liquid chromatography-mass spectrometry analysis are:

[0013] The chromatographic column was BEH C18 2.1mm×10cm, 1.7μm; the mobile phase A was water containing 0.1% formic acid by volume, and the mobile phase B was acetonitrile containing 30% isopropanol by volume, and the following gradient was used for elution: 0-1min, 5% B liquid; 1-5min, 5%-30% B liquid; 5-9min, 30%-50% B liquid; 9-11min, 50%-78% B liquid, 11-13.5min, 78%-95% B liquid, 13.5-14min, 95%-100% B liquid, the above flow rate was 0.4mL / min; 14-16min, 100% B liquid, the flow rate was 0.6mL / min; 16-18min, 5% B liquid, the flow rate was 0.4mL / min; the column temperature was 40°C;

[0014] The mass spectrometry conditions were:

[0015] The ion source parameters were set as follows: ion source temperature: 400°C; ion spray voltage: 4500 V positive mode and -4500 V negative mode; ion source gas I, gas II and curtain gas were set to 60, 60 and 35 psi, respectively.

[0016] Preferably, in step (3), the orthogonal partial least squares discriminant method is used to analyze the non-targeted metabolomics data, and the following conditions are set to screen for differential metabolites: ① VIP of the OPLS-DA model ≥ 1, ② |Fold change| ≥ 1.2 or ≤ 0.83, ③ p-value < 0.05.

[0017] Preferably, in step (4), the metabolic pathways significantly associated with radiation proctitis include: tryptophan metabolism, glyoxylate and dicarboxylate metabolism, citric acid cycle, pyruvate metabolism, lipoic acid metabolism, arginine and proline metabolism, β-alanine metabolism, histidine metabolism, cysteine ​​and methionine metabolism, inositol phosphate metabolism and galactose metabolism.

[0018] Secondly, the present invention also provides a differential metabolite marker of radioactive proctitis obtained by the above screening method, wherein the differential metabolite marker of radioactive proctitis includes (R)-lipoic acid, (S)-malic acid, 3-mercaptolactic acid, 4-acetylaminobutyric acid, 4-guanidinobutyric acid, 5-hydroxyindoleacetic acid, 5-methoxyindoleacetic acid, isocitrate, L-homocysteine ​​and L-proline.

[0019] Furthermore, the present invention also provides the use of a reagent for detecting the above-mentioned differential metabolite markers of radiation proctitis in the preparation of a product for early screening and diagnosis of radiation proctitis.

[0020] Preferably, the product includes a reagent, a test paper, a test kit or an instrument.

[0021] Furthermore, the present invention also provides a product for early screening and diagnosis of radiation proctitis, which comprises a reagent for detecting the above-mentioned differential metabolite markers of radiation proctitis.

[0022] The present invention has the following beneficial effects:

[0023] (1) Based on non-targeted metabolomics data, the present invention screened 87 fecal differential metabolites related to the occurrence and development of radiation proctitis; further analyzed the pathway enrichment analysis of the differential metabolites, screened out 11 metabolic pathways related to radiation proctitis, and screened out 10 metabolite markers from the screening of metabolite markers; and laid a theoretical foundation and research platform for clarifying the pathogenesis and comprehensive prevention and treatment of subclinical radiation proctitis.

[0024] (2) The method of the present invention is simple and practical, and the 10 pure markers obtained are easy to detect and can be easily promoted and applied in the diagnosis and prevention of radiation proctitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1The results of the establishment and verification of the radiation proctitis long-clawed gerbil animal model in the embodiment are shown in Figure 1. A is a flow chart of the model establishment; B is an observation diagram of the appearance of the gerbil, colonoscopy, etc. at the end point of the model; C is a representative HE & Masson diagram of intestinal tissue pathology after radiation; D is the protein expression value of fibronectin in the control group and the radiation group gerbil; E is a representative IHC diagram of fibronectin (FN1), type I collagen, type II collagen, and type III collagen in the rectum of the gerbil in the control group and the radiation group.

[0026] Figure 2 It is the result analysis diagram of non-targeted metabolomics of the control group and the radiation group in the embodiment. A is the result diagram of orthogonal partial least squares discriminant analysis (OPLS-DA) of non-targeted metabolomics of the control group and the radiation group; B is the permutation test diagram of non-targeted metabolomics of the control group and the radiation group; C is the volcano diagram of 87 differential metabolites obtained by screening the control group and the radiation group by VIP value, P value, and FC multiple; D is the heat map of 87 differential metabolites obtained by screening the control group and the radiation group by VIP value, P value, and FC multiple; E is the pathway diagram obtained by metabolite pathway enrichment analysis of differential metabolites of the control group and the radiation group.

[0027] Figure 3 ROC curves of metabolite markers (R)-lipoate, (S)-malic acid, 3-mercaptolactate, 4-acetylaminobutyric acid, 4-guanidinobutyric acid, 5-hydroxyindoleacetic acid, 5-methoxyindoleacetic acid, isocitrate, L-homocysteine, and L-proline for predicting radiation proctitis. DETAILED DESCRIPTION

[0028] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0029] Unless otherwise specified, the reagents and consumables used in the following experimental examples can be obtained through commercial channels.

[0030] The gerbils used in the experiment were raised in a breeding facility provided by the Experimental Animal Center of the Sixth Affiliated Hospital of Sun Yat-sen University.

[0031] Example 1

[0032] 1. Construction of the Radiation Proctitis Model in Gerbils

[0033] (1) Gerbils were randomly divided into a control group and a radiation group, with 9 animals in each group. The radiation dose of the control group was 0 Gy, and the dose of the radiation group was 30 Gy.

[0034] (2) Use a lead plate mold to shield the area outside the abdomen and expose the lower pelvic area (1.5 cm 2), and then placed in the RS2000 small animal irradiator, with a total dose of 0 Gy of sham radiation or 30 Gy of single radiation.

[0035] (3) Figure 1 As shown, the model construction was evaluated, and eight weeks after radiation was the model endpoint ( Figure 1 A), observing the appearance of the skin at the anus of the gerbil and representative images of rectal tissue obtained by the ENDOQ colonoscopy system ( Figure 1 B), it can be seen that the radiation group gerbils have obvious hair loss and skin hardening around the anus, and the endoscopic image shows obvious fibrosis, blurred blood vessels and inflammation in the intestine after radiation; then, the intestinal pathological tissue was used as a pathological section for HE staining and Masson staining ( Figure 1 C), observing the degree of tissue damage and fibrosis, we can see that the intestinal epithelium is obviously damaged after radiation, inflammatory cells infiltrate, and fibrosis and thickening occur in the submucosal layer; in addition, Western blot and immunohistochemical staining detected that the expression of fibrosis markers such as Fibronectin, CollagenⅠ, CollagenⅡ and CollagenⅢ in the rectal tissue of irradiated gerbils was significantly increased, indicating that the intestinal tissue undergoes significant fibrosis after radiation ( Figure 1 D and E in ).

[0036] 2. Stool sample collection

[0037] At the end point of the model, the animals were placed in a feces collection box to quickly collect feces samples. Nine biological samples were stored in 2 mL cryovials in each group and stored in a -20°C freezer after a short storage period.

[0038] 3. Metabolite extraction

[0039] (1) Take out 80 mg of frozen stool sample and put it into a 1.5 mL EP tube, add 20 μL of internal standard (L-2-chlorophenylalanine, 0.3 mg / mL, prepared in methanol), and add 600 μL of methanol-water (V:V = 4:1);

[0040] (2) Then add two small steel balls, place in a -20°C refrigerator for 5 min, and grind in a grinder (60 Hz, 2 min);

[0041] (3) Ultrasonic extraction in an ice-water bath for 10 min, and then standing at -20°C for 30 min;

[0042] (4) Centrifuge for 10 min (13000 rpm, 4°C), take 200 μL of the supernatant and put it into an LC-MS injection vial to evaporate to dryness;

[0043] (5) Redissolve with 300 μL methanol-water (V:V = 1:4), add 10 μL of internal standard (Lyso PC17:0, 0.1 mg / mL, prepared in methanol), vortex for 30 s, and sonicate for 3 min;

[0044] (6) Stand at -20°C for 2 hours;

[0045] (7) Centrifuge for 10 min (13,000 rpm, 4°C), and take 150 μL of the supernatant and transfer it to an LC-MS injection vial with a foot-lined tube for subsequent analysis.

[0046] 4. Metabolite UPLC-MS Analysis

[0047] In this experiment, Waters ACQUITY UPLC I-Class Plus (Waters, USA) was used in tandem with QTRAP6500Plus high-sensitivity mass spectrometer (SCIEX, USA) for separation and quantitative detection of metabolites.

[0048] (1) Chromatographic conditions:

[0049] The chromatographic column used was BEH C18 (2.1 mm×10 cm, 1.7 μm, waters).

[0050] The mobile phases were an aqueous solution containing 0.1% formic acid by volume (liquid A) and an acetonitrile solution containing 30% isopropanol by volume (liquid B), and the following gradient was used for elution: 0-1 min, 5% liquid B; 1-5 min, 5%-30% liquid B; 5-9 min, 30%-50% liquid B; 9-11 min, 50%-78% liquid B, 11-13.5 min, 78%-95% liquid B, 13.5-14 min, 95%-100% liquid B, with a flow rate of 0.4 mL / min; 14-16 min, 100% liquid B, with a flow rate of 0.6 mL / min; 16-18 min, 5% liquid B, with a flow rate of 0.4 mL / min; the column temperature was 40°C.

[0051] (2) Mass spectrometry conditions:

[0052] For the QTRAP 6500Plus equipped with ESI Turbo ion spray interface, the ion source parameters were set as follows: ion source temperature: 400 °C; ion spray voltage (IS): 4500 V (positive mode) and -4500 V (negative mode); ion source gas I (GS1), gas II (GS2) and curtain gas (CUR) were set to 60, 60 and 35 psi, respectively. The MRM method was set in MRM mode, which contained the MRM parent and daughter ion pair information, collision energy (CE) and declustering potential (DP) and retention time of the target metabolites.

[0053] 5. Data preprocessing

[0054] Metabolite peak extraction and identification were performed on the original mass spectrometry data to obtain information such as metabolite peak area and identification results. rmetaX was used to further preprocess the software export results to obtain compounds and quantitative values ​​that can be used for formal analysis. The identification results of metabolites were annotated using the KEGG and HMDB databases, including KEGG ID, HMDBID, category, and KEGG metabolic pathway involved.

[0055] 6. Multivariate statistical analysis and identification of differential metabolites

[0056] A total of 481 fecal metabolites were detected based on non-targeted metabolomics analysis. In order to screen out the differential metabolites with significant changes in the radiation proctitis animal model, the pre-processed data were imported into metaX software. First, multivariate statistical analysis was performed on the non-targeted metabolomics data, such as Figure 2 As shown in Figure A, OPLS-DA was used to identify the differences in metabolites between the two groups. The metabolic clustering within each group was good, and the separation between the groups was significant. The fecal metabolic profiles of the control group and the radiation group animal model group showed a clear trend of separation, indicating that the fecal metabolites of radiation proctitis were different from those of the control group. Figure 2 As shown in B, the permutation test can verify the stability of the model, indicating that the model has good predictive and explanatory capabilities.

[0057] Then, the larger the VIP value of the metabolite in the OPLS-DA analysis, the greater the contribution of the substance to distinguishing the two groups; and the univariate analysis calculated the fold change of each metabolite in each comparison group and used Student's t-test to perform a significance test on the expression of each metabolite in each comparison group to obtain the p value, and the pvalue was used to evaluate the significance level of the difference between the two groups of samples. Differential metabolites were screened under the following conditions: 1) VIP ≥ 1 of the OPLS-DA model, 2) Fold Change ≥ 1.2 or ≤ 0.83, 3) p-value < 0.05, and finally 87 differential metabolites were screened. The changes in these metabolites were related to the occurrence and development of radiation proctitis. The results are as follows Figure 2 As shown in C and D.

[0058] The 87 differential metabolites included 2,4-dihydroxypyrimidine-5-carboxylic acid, γ-muricholic acid, 3-acetic acid ethyl ester, lithocholic acid methyl ester, 12-dehydrocholic acid methyl ester, homogentisic acid, malic acid, L-tyrosine, L-proline, L-aspartic acid, isocitrate, anserine, indoleacetic acid, inositol, orotic acid, uric acid, urocanic acid, 3-(3-hydroxyphenyl)propionic acid, vanillic acid, 4-hydroxybenzoic acid, 3-methylglutaric acid, 4,5-dihydroorotic acid, cinnamic acid, and cinnamyl alcohol. Acid, L-glutamine, glucaric acid, homoarginine, isovalerylglycine, caproylglycine, mandelic acid, hippuric acid, levulinic acid, indoleacrylic acid, homocysteine, 3-hydroxyisovaleric acid, lithocholic acid, 5-hydroxyindoleacetic acid, mannitol, p-hydroxymandelic acid, salicylic acid, pyrimethicone, monomethylglutaric acid, trigonelline, trimethylamine oxide, traumatic acid, ferulic acid, L-sorbose, L-norleucine, benzoic acid, 4-hydroxycinnamic acid, 3-mercaptolactic acid, 2,4-diaminobutyric acid, 5-hydroxymethyl-2-furancarboxylic acid, naringenin, shikimic acid, indole-3-carboxylic acid, N2-acetyloxamic acid, 4-guanidinobutyric acid, taurine amine, 4-acetylaminobutyric acid, 5-methoxyindole acetate, pyrrole-2-carboxylic acid, 4-hydroxy-3-methylbenzoic acid, N-acetylasparagine, N-acetylgluconate, 3-hydroxyhippuric acid, muconic acid, phenylacetylglutamine, p-methylhippuric acid, 2,6-dihydroxybenzoic acid, 4 -Hydroxyhippuric acid, lipoic acid, alanine glutamine, propylphenylalanine, glycyl valine, leucyl glycine, 2,4'-dihydroxyacetophenone, trihexanoin, 2-ethylhexanoic acid, 2-methylheptanoic acid, 2-acetylpyrazine, sinapinic acid, dimethyl succinate, 3,4-dihydro-2H-1-benzopyran-2-one, N6-cis-p-coumaryl serotonin, 3-indolyloxyacid, 10-hydroxydecanoic acid, 1-(10H-phenothiazin-2-yl)ethanone.

[0059] 7. Differential metabolite pathway enrichment analysis and screening of metabolite markers

[0060] MetaboAnalyst 6.0 (https: / / www.metaboanalyst.ca / ) analysis tool was used to perform functional pathway enrichment and topological analysis of the screened differential metabolites using the hypergeometric test algorithm, and the enriched significant pathways were visualized. The analysis process was as follows: upload data, data standardization, pathway analysis, select the Global Ancova enrichment method, and follow the defaults for the rest. The final significant metabolic enrichment pathways mainly include: tryptophan metabolism, glyoxylate and dicarboxylate metabolism, citric acid cycle (TCA cycle), pyruvate metabolism, lipoic acid metabolism, arginine and proline metabolism, β-alanine metabolism, histidine metabolism, cysteine ​​and methionine metabolism, inositol phosphate metabolism, galactose metabolism, and other 11 metabolic pathways ( Figure 2E); the differential metabolites in the significant pathways were (R)-lipoate, (S)-malic acid, 3-mercaptolactic acid, 4-acetylaminobutyric acid, 4-guanidinobutyric acid, 5-hydroxyindoleacetic acid, 5-methoxyindoleacetic acid, L-angesine, indole-3-acetic acid, isocitrate, L-aspartic acid, L-glutamine, L-homocysteine, L-proline, myo-inositol, uric acid and other 16 metabolites.

[0061] Further ROC analysis was performed on all differential metabolites obtained by the non-targeted metabolomics to verify the specificity and sensitivity of the above 16 molecular markers in predicting radiation proctitis. Figure 3 As shown in the data, a total of 10 metabolites were found with an area under the curve (AUC) greater than 0.75, namely: (R)-lipoic acid, (S)-malic acid, 3-mercaptolactic acid, 4-acetylaminobutyric acid, 4-guanidinobutyric acid, 5-hydroxyindoleacetic acid, 5-methoxyindoleacetic acid, isocitrate, L-homocysteine, and L-proline, indicating that the above 10 metabolites can be used as molecular markers of fecal metabolites of radiation proctitis damage.

[0062] The present invention links radiation proctitis with differential metabolites in feces and constructs a relationship diagram between the two, thereby screening out differential metabolites that contribute the most to the prevention and treatment of radiation proctitis, laying a theoretical foundation and research platform for clarifying the pathogenesis and comprehensive prevention and treatment of subclinical radiation proctitis in the future.

[0063] The above description is only a preferred embodiment of the present invention and is used to limit the present invention. Any modification, equivalent substitution, improvement, etc. made within the principle and content of the present invention should be included in the protection scope of the present invention.

Claims

1. A differential metabolite marker for radiation proctitis, characterized in that: The differential metabolite markers of radiation proctitis include (R)-lipoic acid, (S)-malic acid, 3-mercaptolactic acid, 4-acetylaminobutyric acid, 4-guanidinobutyric acid, 5-hydroxyindoleacetic acid, 5-methoxyindoleacetic acid, isocitrate, L-homocysteine ​​and L-proline.

2. Use of a reagent for detecting the differential metabolite marker of radiation proctitis according to claim 1 in the preparation of products for early screening and diagnosis of radiation proctitis.

3. The use according to claim 2, characterized in that The products include reagents, test strips, test kits or instruments.

4. A product for early screening and diagnosis of radiation proctitis, characterized in that: Contains a reagent for detecting the differential metabolite marker of radiation proctitis according to claim 1.

5. A method for screening differential metabolite markers of radiation proctitis as claimed in claim 1, characterized in that: The following steps are involved: (1) Long-clawed gerbils were selected and randomly divided into a normal control group and a radiation group. The radiation group was used to establish an animal model of radiation proctitis, and feces of the two groups of animals were collected; (2) Fecal samples were analyzed by ultra-high performance liquid chromatography-mass spectrometry, and MS / MS data were extracted after filtering isotope peaks and compared with the metabolite database to obtain non-targeted metabolomics data; (3) Using multivariate statistical analysis methods, we screened and identified endogenous metabolites with significant changes from multiple variables as potential differential metabolites associated with radiation proctitis; (4) Metabolic pathway enrichment analysis was performed on the above non-targeted metabolomics data to obtain metabolic pathways significantly associated with radiation proctitis and differential metabolites in the metabolic pathways. The differential metabolites were screened by ROC curve analysis to obtain metabolite markers.

6. The screening method according to claim 5, characterized in that In step (1), the construction of the radiation proctitis animal model is to use a single radiation dose of 30 Gy to model the long-clawed gerbil.

7. The screening method according to claim 5, characterized in that In step (2), the chromatographic conditions of the ultra-high performance liquid chromatography-mass spectrometry analysis are: The chromatographic column was BEH C18 2.1mm×10cm, 1.7μm; the mobile phase A was water containing 0.1% formic acid by volume, and the mobile phase B was acetonitrile containing 30% isopropanol by volume, and the following gradient was used for elution: 0-1min, 5% B liquid; 1-5min, 5%-30% B liquid; 5-9min, 30%-50% B liquid; 9-11min, 50%-78% B liquid, 11-13.5min, 78%-95% B liquid, 13.5-14min, 95%-100% B liquid, the above flow rate was 0.4mL / min; 14-16min, 100% B liquid, the flow rate was 0.6mL / min; 16-18min, 5% B liquid, the flow rate was 0.4mL / min; the column temperature was 40°C; The mass spectrometry conditions were: The ion source parameters were set as follows: ion source temperature: 400°C; ion spray voltage: 4500 V positive mode and -4500 V negative mode; ion source gas I, gas II and curtain gas were set to 60, 60 and 35 psi, respectively.

8. The screening method according to claim 5, characterized in that In step (3), the orthogonal partial least squares discriminant method is used to analyze the non-targeted metabolomics data, and the following conditions are set to screen the differential metabolites: ① VIP of the OPLS-DA model ≥ 1, ② |Fold change| ≥ 1.2 or ≤ 0.83, ③ p-value < 0.

05.

9. The screening method according to claim 5, characterized in that In step (4), the metabolic pathways significantly associated with radiation proctitis include: tryptophan metabolism, glyoxylate and dicarboxylate metabolism, citric acid cycle, pyruvate metabolism, lipoic acid metabolism, arginine and proline metabolism, β-alanine metabolism, histidine metabolism, cysteine ​​and methionine metabolism, inositol phosphate metabolism and galactose metabolism.

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