CN117838707A - Application of lithocholic acid in the preparation of drugs for preventing and/or treating radiation-induced intestinal injury - Google Patents

Abstract

The invention relates to preparation of a medicine for preventing and/or treating radioactive intestinal injury, in particular to application of lithocholic acid in preparation of a medicine for preventing and/or treating radioactive intestinal injury, and aims to overcome the defect that the existing radioactive protective agent for treating radioactive intestinal injury is lacking.

Description

Application of lithocholic acid in the preparation of drugs for preventing and/or treating radiation-induced intestinal injury

Technical Field

The invention relates to the preparation of a medicine for preventing and/or treating radiation intestinal injury, and in particular to the application of lithocholic acid in the preparation of a medicine for preventing and/or treating radiation intestinal injury.

Background technique

Radiation-induced intestinal injury is the main complication of radiotherapy for patients injured in nuclear radiation accidents and patients with abdominal or pelvic tumors. Currently, more than 50% of cancer patients worldwide receive radiotherapy, which not only kills tumor cells but also often causes damage to normal tissues. Complications that may occur in these patients receiving abdominal radiotherapy include nausea, vomiting, abdominal pain, diarrhea, dehydration, systemic infection, and in extreme cases, septic shock and death. It is reported that 90% of patients receiving abdominal radiotherapy develop radiation-induced intestinal injury within a few weeks, 60% to 80% of patients develop acute clinical and histopathological symptoms, 90% of patients develop permanent changes in bowel habits, and 10% to 15% of patients develop life-threatening complications within 10-20 years after radiotherapy. Radiation-induced intestinal injury seriously affects the treatment of patients with abdominal or pelvic tumors and reduces the quality of life of patients.

At present, there is still a lack of effective treatment for radiation-induced intestinal injury, which is limited to symptomatic supportive treatment such as anti-infection, nutritional support, protection of gastrointestinal mucosa, antidiarrhea, regulation of intestinal flora, and surgical resection of diseased intestinal segments. There is a lack of a targeted radioprotective agent suitable for clinical use. With the increasing use of nuclear-related technologies, more and more people will be exposed to radiation actively or passively. Therefore, there is an urgent need to further clarify the pathogenesis of radiation-induced intestinal injury and determine effective and safe treatment strategies.

Bile acids (BAs) are steroid molecules synthesized from precursor cholesterol in the liver through a series of enzymes. BAs have been shown to be important signaling molecules for maintaining intestinal barrier function and regulating epithelial cell death, survival and proliferation. Lithocholic acid (LCA) is a monohydroxy bile acid produced by intestinal flora. During its first passage through the liver, LCA is rapidly metabolized to less toxic metabolites by sulfation, while other BAs lack this efficient sulfation ability. LCA has been found to be associated with a variety of liver and intestinal diseases, but whether and how LCA is involved in the repair of radiation-induced intestinal damage has not yet been reported.

Summary of the invention

The purpose of the present invention is to solve the deficiency of the existing lack of radioactive protective agents for radiation intestinal injury and to provide an application of lithocholic acid in the preparation of drugs for preventing and/or treating radiation intestinal injury.

To achieve the above purpose, the technical solution provided by the present invention is as follows:

The invention provides an application of lithocholic acid in preparing a medicine for preventing radiation intestinal damage.

And the application of lithocholic acid in the preparation of drugs for treating radiation-induced intestinal injury.

And the use of lithocholic acid in the preparation of drugs for increasing the number of small intestinal crypts after radiation.

And the application of lithocholic acid in the preparation of drugs for promoting the proliferation of small intestinal crypt cells after radiation.

And the application of lithocholic acid in the preparation of drugs to promote the proliferation of intestinal stem cells at the crypt base and "+4" intestinal stem cells after radiation.

And the application of lithocholic acid in the preparation of drugs for increasing the length of intestinal villi after radiation.

And the application of lithocholic acid in the preparation of drugs to promote the proliferation of intestinal stem cells in the villus region of the intestine after radiation.

Beneficial effects of the present invention:

The present invention uses mice with radiation-induced intestinal injury as research objects to explore the preventive and therapeutic effects of lithocholic acid on radiation enteritis, and finds that radiation causes bile acid metabolic spectrum of mice to be disordered, and among the bile acids downregulated by radiation, LCA is the bile acid that is most significantly reduced. The present invention applies lithocholic acid to the prevention and/or treatment of radiation-induced intestinal injury for the first time, and the results show that exogenous supplementation of lithocholic acid can well protect the intestinal villus structure of irradiated mice, increase the number of crypts and the length of the colon of mice, and prolong the survival time of mice, and exogenous supplementation of LCA can promote intestinal crypt regeneration and repair of radiation-induced intestinal injury.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the effect of γ-ray irradiation on bile acid metabolism in mice; wherein A is a flow chart for detection of bile acid metabolic profiles in mice irradiated with γ-rays; B is the relative abundance of primary BAs and secondary BAs in a single mouse; C is the relative abundance of primary BAs and secondary BAs in normal control group and irradiated control group mice; D is the LCA content of normal control group and irradiated control group mice after irradiation; data are expressed as Mean±SD, n=10, ****p<0.0001.

Figure 2 shows the effect of X-ray irradiation on bile acid metabolism in mice; A is the relative abundance of primary BAs and secondary BAs in a single mouse; B is the relative abundance of primary BAs and secondary BAs in normal control group and irradiated control group mice; C is the LCA content of normal control group and irradiated control group mice after irradiation; data are expressed as Mean±SD, n=10, **p<0.01.

Figure 3 shows the radiation intestinal damage of mice caused by γ-ray irradiation after exogenous supplementation of LCA; wherein, A is the survival rate of mice in the four groups; B is the percentage of body weight of mice after the experiment relative to that before the experiment; C is the gross morphology of the mouse colon; D is a representative image of HE staining of mouse small intestinal tissue, Scale bar = 50 μm; E is the quantification of the length of the mouse colon; F is the quantification of the number of small intestinal crypts in the mouse; G is the quantification of the length of the villi in the small intestinal tissue sections of the mouse; data are expressed as Mean±SD, n=10, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 4 shows the changes in radiation-induced intestinal damage in mice caused by X-ray irradiation after exogenous supplementation of LCA; wherein A is the percentage of body weight of mice after the experiment relative to before the experiment; B is the quantification of water intake of mice; C is the quantification of food intake; D is the quantification of colon length; E is the gross morphology of mouse colon; F is a representative image of HE staining of mouse small intestinal tissue, Scale bar = 50 μm; G is the quantification of the number of small intestinal crypts; H is the quantification of villus length in small intestinal tissue sections; data are expressed as Mean ± SD, n = 10, *p < 0.05, ***p < 0.001, ****p < 0.0001.

Figure 5 shows the effect of LCA on the intestine of normal mice; wherein, A is the food intake analysis of normal mice and LCA-treated mice; B is the water intake analysis; C is the percentage of body weight of mice after the experiment relative to that before the experiment; D is the quantification of the length of the mouse colon; E is a photograph of the gross morphology of the mouse colon; F is a representative image of HE staining of mouse small intestinal tissue, Scale bar = 50 μm; G is the quantification of the number of small intestinal crypts in each group of mice; H is the quantification of the length of villi in the small intestinal tissue sections of each group of mice; the data are expressed as Mean ± SD, n = 10.

FIG6 shows the effects of LCA on the organs of normal mice; A is a representative image of HE staining of colon, thymus, and spleen of normal mice and LCA-treated mice, Scale bar = 50 μm; B is a representative image of HE staining of liver, kidney, and lung of normal mice and LCA-treated mice, Scale bar = 50 μm.

Figure 7 shows the effect of LCA on the growth of normal mouse small intestinal organoids; wherein, A is a microscopic image of intestinal crypts isolated from mouse small intestine, showing the growth of organoids when exposed to 10μM, 25μM, 50μM, and 100μM LCA, respectively, Scale bar=400μm (top), Scale bar=50μm (bottom); B is a quantitative statistical analysis of the newly formed crypts; data are expressed as Mean±SD, n=4, **p<0.01.

Figure 8 shows the effect of LCA on the growth of mouse small intestinal organoids after radiation damage; wherein, A is a representative image of small intestinal organoids on the 7th day after 4 Gy irradiation with 10 μM LCA, Scale bar = 400 μm (top), Scale bar = 50 μm (bottom); B is a quantitative statistical analysis of the newly formed crypts after 4 Gy irradiation; C is a representative image of small intestinal organoids on the 7th day after 6 Gy irradiation with 10 μM LCA, Scale bar = 400 μm (top), Scale bar = 50 μm (bottom); D is a quantitative statistical analysis of the newly formed small intestinal crypts after 6 Gy irradiation; data are expressed as Mean±SD, n=4, **p<0.01, ***p<0.001.

Figure 9 shows the effect of LCA on inflammatory factors in the small intestinal tissue of mice with radiation-induced intestinal injury; A is the expression of IL-1β in the small intestinal tissue; B is the expression of IL-6 in the small intestinal tissue; C is the expression of TNF-α in the small intestinal tissue; data are expressed as Mean±SD, n=10, **p<0.01, ***p<0.001.

Figure 10 shows the proliferation of small intestinal crypt cells in mice with radiation-induced intestinal injury after LCA intervention; A is a representative image of immunohistochemical staining of PCNA and Ki-67 in mouse small intestinal tissue, Scale bar = 100 μm; B is a quantitative analysis of immunohistochemical staining of PCNA-positive cells; C is a quantitative analysis of immunohistochemical staining of Ki-67-positive cells; data are expressed as Mean±SD, n=8, *p<0.05, **p<0.01.

Figure 11 shows the effect of LCA on intestinal stem cells at the base of the small intestinal crypts of irradiated mice; wherein, A is the immunofluorescence detection of Lgr5 in mouse small intestinal tissue, Scale bar = 100 μm; B is the qRT-PCR detection of the mRNA level of Lgr5 in mouse small intestinal crypt cells; C is the Western detection of the protein expression level of Lgr5 in small intestinal crypt cells; D is the qRT-PCR detection of the mRNA expression level of the intestinal stem cell marker Sox9 in mouse small intestinal tissue; E is the qRT-PCR detection of the mRNA expression level of the intestinal stem cell marker Ascl2 in mouse small intestinal tissue; F is the qRT-PCR detection of the mRNA expression level of the intestinal stem cell marker Olfm4 in mouse small intestinal tissue; data are expressed as Mean±SD, n=5, *p<0.05, **p<0.01, ***p<0.001.

Figure 12 shows the effect of LCA on "+4" intestinal stem cells of irradiated mice; wherein, A is the mRNA expression level of "+4" intestinal stem cell marker Hopx in the small intestinal tissue of mice detected by qRT-PCR; B is the mRNA expression level of "+4" intestinal stem cell marker krt19 in the small intestinal tissue of mice detected by qRT-PCR; C is the mRNA expression level of "+4" intestinal stem cell marker Lrig1 in the small intestinal tissue of mice detected by qRT-PCR; data are expressed as Mean±SD, n=8, *p<0.05, **p<0.01.

Figure 13 shows the effect of LCA on intestinal stem cell-related markers in the intestinal villus region of irradiated mice; wherein, A is the mRNA expression level of DCLK1 in the small intestinal tissue of mice detected by qRT-PCR; B is the mRNA expression level of chga in the small intestinal tissue of mice detected by qRT-PCR; data are expressed as Mean±SD, n=8, *p<0.05, ***p<0.001, ****p<0.0001.

Figure 14 shows the effect of LCA on the expression of bile acid membrane receptor TGR5 in mice with radiation-induced intestinal injury; wherein, A is a representative image of immunohistochemical staining of TGR5 in mouse small intestine and colon tissue, Scale bar = 100 μm; B is a quantitative analysis of immunohistochemical staining of TGR5-positive cells in mouse small intestine tissue; C is a quantitative analysis of immunohistochemical staining of TGR5-positive cells in mouse colon tissue; D is qRT-PCR detection of the mRNA expression level of TGR5 in mouse small intestinal crypt cells; data are expressed as Mean±SD, n=8, *p<0.05, **p<0.01, ***p<0.001.

Detailed ways

The application of lithocholic acid in the preparation of drugs for preventing and/or treating radiation intestinal injury of the present invention is studied with mice with radiation intestinal injury as the research object. The technical scheme in the embodiment of the present invention is described in detail and completely below. The experimental methods used in the embodiments are conventional methods unless otherwise specified; the materials, reagents, etc. used can be obtained from commercial channels unless otherwise specified.

In the present invention, healthy adult male C57BL/6J mice aged 6 to 8 weeks (20 to 22 g) were used. The mice had free access to water and food under a 12-hour dark/light cycle, were raised under a normal light-dark cycle, and the bedding was changed regularly to ensure a clean feeding environment. Subsequent experiments were performed after one week of adaptive feeding.

1. Experimental Methods

(I) Choice of dosage and method of administration

To determine the optimal dose of LCA for the prevention and treatment of radiation-induced intestinal injury. A radiation-induced intestinal injury model was established by irradiating the abdomen of mice with 12Gy ⁶⁰ Coγ-rays. Two LCA administration groups were designed, with the doses of 15mg/kg for the low-dose group and 30mg/kg for the high-dose group.

(II) Experimental Animal Grouping

A total of 40 SPF C57BL/6J male mice were randomly divided into 4 groups: normal control group, irradiation control group, LCA administration group (15 mg/kg), LCA administration group (30 mg/kg), and the number of mice in each group was n=10 for γ-ray irradiation. The same 40 mice were prepared and divided into 4 groups by the same method for X-ray irradiation.

(III) Method of administration

The mice in the LCA administration group were irradiated once with gamma rays or X-rays, and the drug was administered 1 hour before irradiation and continued for 5 consecutive days after irradiation, once a day. The irradiation control group was given corn oil according to the above dosing time nodes, once a day. The daily dosage of the mice in the LCA administration group was: the drug in the LCA administration group (15 mg/kg) contained 15 mg/kg LCA, and the drug in the LCA administration group (30 mg/kg) contained 30 mg/kg LCA.

4. Drug pretreatment

Dosing configuration: LCA is in powder form. Weigh the corresponding weight of LCA powder according to the weight of the mouse and put it in a test tube with a lid. Dissolve the powder in the corresponding volume of corn oil according to the number of mice. The dosage per gram of mouse is 10 μL corn oil. During the dissolution process, a vortex oscillator, ultrasound and heating are used to assist dissolution.

(V) Construction of a mouse model of radiation-induced intestinal injury

The experiment used ⁶⁰ Coγ-rays, divided into normal control group, control group, LCA administration group (15 mg/kg), LCA administration group (30 mg/kg) of mice abdomen, irradiation range of mice sternum xiphoid process to pubic symphysis, the rest of the body with 5 cm thick lead shielding. ⁶⁰ Coγ-rays were used at 1 Gy/min, irradiation dose of 12 Gy, 5 days after irradiation samples were collected for follow-up experiments.

The X-ray device was a Rad Source RS2000 series X-ray biological irradiator, with an operating voltage of 160 kV, an operating current of 25 mA, and a dose rate of 1 Gy/min (monitored in real time by a radiation dosimeter). The experiment was divided into a normal control group, an irradiation control group, an LCA administration group (15 mg/kg), and an LCA administration group (30 mg/kg). The abdomen of the mice (the irradiation range was from the sternal xiphoid process to the pubic symphysis of the mice), and the rest of the body was shielded with a 5 cm thick lead block. The X-ray was irradiated at 1 Gy/min, and the irradiation dose was 10 Gy. The samples were collected on the 5th day after irradiation and subsequent experiments were performed.

(VI) Sample preparation

The survival rate of mice was observed on the 5th day after irradiation, and the small intestinal tissues of mice were obtained, fixed with 4% paraformaldehyde, and stained with HE. All detection reagents used commercial kits.

(VII) Detection of bile acid metabolic profile in mouse feces

Fresh feces from all mice on day 5 after irradiation were collected in 1.5 mL sterile tubes and stored in liquid nitrogen as samples before analysis. The samples were thawed in an ice bath to reduce sample degradation, and then about 10 mg of fecal sample was mixed with about 25 mg of pre-cooled grinding beads and 200 mL of acetonitrile/methanol (v/v=8:2) mixed solvent containing 10 mL of internal standard. Centrifuged at 13,500 rpm for 20 min at 4°C. 10 μL of the supernatant was diluted with 90 mL of solvent. The solvent was 1:1 acetone (80/20)/methanol (80/20) and ultrapure water. After dilution, the samples were vortexed, centrifuged, and used for injection analysis. The injection volume was 5 mL, and the concentration of BAs in feces was determined by liquid chromatography-mass spectrometry/mass spectrometry (UPLC-MS/MS). The raw data files generated by UPLC-MS/MS were processed using MassLynx software (v 4.1, Waters, Milford, MA, USA) to integrate, construct standard curves, and quantify each bile acid. The data were plotted using Prism 9.2 (GraphPad, San Diego, CA).

(VIII) HE staining

(1) Place the radiation-damaged colon mucosal tissue slices in a 60°C oven for 2 h;

(2) Dewaxing and hydration: Place tissue sections in the following reagents in sequence: biological clearing agent I for 10 min, biological clearing agent II for 10 min, 100% alcohol for 5 min, 90% alcohol for 2 min, 80% alcohol for 2 min, 70% alcohol for 2 min, and ultrapure water for 2 min;

(3) The tissue sections were stained with hematoxylin for 5 min and rinsed with running water for 2 min;

(4) Differentiate the tissue sections with hydrochloric acid alcohol for a few seconds and rinse with running water for 2 minutes;

(5) The tissue sections were stained with eosin for 2 min and washed with distilled water for 1-2 s;

(6) Place tissue sections in the following reagents in sequence: 70% alcohol for 1 min, 95% alcohol for 2 min, 100% alcohol for 2 min, biological clearing agent I for 5 min, and biological clearing agent II for 5 min;

(7) Seal the tissue sections with neutral gum and take photos under a microscope.

(IX) Isolation of mouse small intestinal crypt cells and organoid culture

(1) Place the small intestine in a 10 cm dish, add 10 mL of cold (2-8°C) PBS, and insert a 1 mL pipette tip into one open end of the small intestine to gently rinse the small intestine;

(2) Use small scissors to make a longitudinal incision along the entire length of the intestine, open the intestinal segment, and gently wash it with 1 mL of cold (2-8°C) PBS. Wash it two more times, for a total of 3x 1 mL washes;

(3) Transfer the intestinal segment to a clean 10 cm dish containing 15 mL of fresh cold (2-8°C) PBS. Using forceps, move the segment through the cleaning buffer to rinse thoroughly;

(4) Add 15 mL of cold (2-8°C) PBS to a 50 mL conical tube. Use forceps to fix one end of the cleaned intestine to the test tube. Starting from the bottom of the intestine, use scissors to cut the intestine into 2 mm fragments, allowing the fragments to fall into the buffer in the test tube;

(5) Use a pre-wetted 10 mL serological pipette to wash the fragments by pipetting up and down 5 times. Allow the intestinal pieces to settle under gravity and carefully remove the supernatant. Add 15 mL of cold (2-8°C) PBS. Repeat this washing procedure 15-20 times or until the supernatant is clear;

(6) Remove the supernatant and resuspend in 25 mL of mild cell dissociation reagent at room temperature (15-25°C). Incubate on a shaker at 20 rpm for 15 min at room temperature (15-25°C);

(7) Allow the intestinal slices to settle under gravity for about 30 seconds, then carefully remove the supernatant;

(8) Resuspend the intestinal fragments in 10 mL of cold (2-8°C) PBS + 0.1% BSA and pipette up and down 5 times to allow most of the fragments to settle to the bottom. Remove the supernatant and pass the supernatant through a 70 μm filter into a 50 mL conical tube to obtain fragment 1, which is marked as "1" and placed on ice;

(9) Repeat step (8) 3 times to generate fragments 2-4, and transfer fragments 2-4 to new 15 mL conical tubes labeled with corresponding numbers. Centrifuge fragment 4 at 1280 rpm for 5 min at 2-8°C, carefully remove the supernatant, and leave the precipitate in the test tube;

(10) Resuspend the pellet in 10 mL of cold (2-8°C) PBS + 0.1% BSA and centrifuge at 1000 rpm for 5 min at 2-8°C;

(11) Gently pour off and discard the supernatant; the precipitated crypts will remain in the test tube.

(12) After the small intestinal crypt cells were isolated, they were prepared in a 1:1 (matrigel: culture medium) ratio, mixed with a pipette, and 40 uL was taken from each well and inoculated into a 24-well plate.

(13) After incubation in the incubator for 30 min, add 500 uL of culture medium along the inner wall of the 24-well plate. Change the culture medium every two days, and organoids will be obtained in about one week.

10. Organoid passaging and irradiation

(1) Thaw 500uL of Matrigel on ice (keep the Matrigel on ice after taking it out);

(2) Remove the cultured organoids from the 37°C incubator, discard the culture medium, add 1 mL of cell dissociation reagent to the organoids, and incubate at room temperature for 1 min;

(3) Transfer to a 15 mL centrifuge tube and shake for 10 min to fully digest;

(4) Centrifuge at 1280 rpm for 5 min at 2-8°C, carefully remove the supernatant, and leave the precipitate in the test tube;

(5) Resuspend the pellet in 10 mL of cold (2-8°C) PBS + 0.1% BSA and centrifuge at 1000 rpm for 5 min at 2-8°C;

(6) Gently pour out the supernatant. The precipitated crypt cells will remain at the bottom of the test tube. Mix the matrix gel and culture medium in a 1:1 ratio with a gun and add them to the test tube. Take 40uL and inoculate it in a 24-well plate. After 30 minutes, add 500uL of culture medium along the inner wall of the 24-well plate.

(7) Gamma ray irradiation was performed with a total irradiation amount of 4 Gy or 6 Gy and a dose rate of 1 Gy/min.

11. Enzyme-linked immunosorbent assay

Under pre-cooling conditions, the intestinal tissue was dissolved with PBS and homogenized in a homogenizer (Leica) to extract total protein. According to the instructions, the expression levels of IL-1β, IL-6 and TNF-α in the supernatant of small intestinal tissue were quantitatively determined using an enzyme-linked immunosorbent assay kit (ELISA kit).

12. Immunohistochemical staining

(1) After dewaxing and rehydrating the tissue sections obtained in step (VIII), microwave antigen repair was performed using sodium citrate antigen repair solution, first at medium-high heat for 5 minutes, then slowly put the sections in at high heat for 5 minutes, and then at medium-low heat for 10 minutes;

(2) Rinse the tissue sections with PBS for 3 min × 3 times, add endogenous peroxidase blocker, incubate at room temperature for 10 min; then rinse with PBS for 5 min × 3 times.

(3) Antibody incubation: Add the prepared antibody dropwise onto the tissue section and incubate at 4°C for 10-12 h;

(4) After incubation, the tissue sections were placed at room temperature for 15 min and rinsed with PBS for 5 min × 3 times;

(5) Incubation with secondary antibody: Add reagent secondary antibody and place in a wet box, incubate at room temperature for 30 min;

(6) DAB staining was performed on the tissue sections. After the reaction was completed under a microscope, the sections were placed in PBS solution to terminate the reaction.

(7) Hematoxylin counterstaining: The tissue sections were stained with hematoxylin for 5 min, rinsed with running water for 1 min, differentiated with hydrochloric acid and alcohol for a few seconds, and rinsed with running water for 2 min;

(8) Place tissue sections in 70% alcohol for 1 min, 95% alcohol for 2 min, 100% alcohol for 2 min, biological clearing agent I for 5 min, and biological clearing agent II for 5 min, respectively, seal the sections with neutral gum, and take photos under a microscope.

13. Immunofluorescence staining

(1) Perform antigen repair on the tissue sections prepared in advance, which is the same as the antigen repair in the immunohistochemical staining step (1);

(2) After cooling, the tissue sections were rinsed with PBS for 3 min × 3 times and then blocked with 5% BSA for 1 h;

(3) Antibody incubation: Add the prepared antibody dropwise onto the tissue section and incubate at 4°C for 10-12 h;

(4) The tissue sections were placed at room temperature for 15 min and then rinsed with PBS for 5 min × 3 times;

(5) Incubation with secondary antibody: prepare secondary antibody at a concentration of 1:200 in the dark, add secondary antibody and place in a wet box, incubate at room temperature for 30 min, and keep away from light throughout the process;

(6) Wash the sections with PBS for 5 min × 3 times in a dark environment;

(7) DAPI counterstaining of nuclei: tissue sections were stained with DAPI for 10 min and rinsed with PBS for 5 min × 3 times;

(8) Add anti-fluorescence quenching agent to the tissue section for sealing and take photos under a microscope.

(XIV) Real-time fluorescence quantitative PCR

①Total RNA extraction

(1) Isolate mouse small intestinal crypt cells, add 1 ml of TRIzol, and let stand for 5 min;

(2) Add 200 μL of chloroform, shake vigorously for 15 seconds, let stand for 3 minutes, and centrifuge at 4°C and 12,000 rpm for 15 minutes;

(3) Gently aspirate the upper aqueous phase;

(4) Add 500 μL of isopropanol to the aqueous phase and incubate at room temperature for 10 min;

(5) Centrifugation at 12,000 rpm for 10 min at 4°C;

(6) discarding the waste liquid and adding 75% ethanol to wash the precipitate;

(7) Centrifuge at 7500 rpm for 5 min to remove residual liquid;

(8) After drying for 5 min, add enzyme-free water to obtain total RNA and measure the concentration;

(9) The RNA concentration and purity were determined using NanoDrop 2000. The RNA samples were diluted to the same concentration and reverse transcribed. The resulting cDNA was stored in a −20°C refrigerator.

Among them, reverse transcription is specifically:

A. Prepare the reverse transcription reaction system according to the table below. Perform the preparation process on ice.

Composition Usage amount 5×All-in-One RT MasterMix 4μL Total RNA 2μL RNase-Free ddH ₂ O 14μL

B. The reaction system was reacted at 25℃ for 10 min, 42℃ for 15 min, and 85℃ for 5 min. The cDNA was stored in a -20℃ refrigerator.

② Prepare PCR reaction solution according to the table below

Composition Usage amount 2×Super Real PreMix Plus 10μL Forward primer (Forward) (10μM) 0.5μL Reverse primer (Reverse) (10 μM) 0.5μL cDNA template 1μL RNase-Free ddH ₂ O 8μL

The forward primer and reverse primer are shown in the following table:

③Set the PCR reaction program according to the table below

④ Cycle threshold was used to quantify the mRNA levels in the normal control group, irradiation control group and drug-treated group. GAPDH was used as the internal reference gene, and the 2- ^△△CT method was used to compare the differences between the control group and the experimental group.

(XV) Protein immunoblotting

(1) Small intestinal crypt cell protein extraction and BCA protein quantification

First, add corresponding proportions of protease inhibitors, phosphatase inhibitors and PMSF to RIPA lysis buffer; place on ice for lysis for 30 minutes, and vortex and oscillate every 5 minutes to mix; centrifuge at 4°C, 12000rpm for 15 minutes; gently aspirate the supernatant and use the BCA quantitative method; add 5× Loading Buffer to the supernatant, heat in a metal bath at 100°C for 10 minutes to denature the protein, and obtain the protein sample, which is stored in a -80°C refrigerator for subsequent experiments.

(2) SDS-PAGE electrophoresis: SDS-polyacrylamide gel was prepared according to the molecular weight of the protein, and the sample load was 30 μg. The electrophoresis was first performed at a constant voltage of 80 V. After the protein migrated to the separation gel (about 30 min), the electrophoresis was continued at a constant voltage of 120 V.

(3) Transfer: Pre-activate the 0.22 μm PVDF membrane with methanol for 15 s, and transfer the membrane at a constant current of 200 mA for 120 min. The transfer time is determined according to the specific molecular weight.

(4) Blocking: 5% skim milk powder, block for 2 h at room temperature;

(5) Primary antibody incubation: Incubate with primary antibody at 4°C overnight;

(6) Wash the membrane with TBST for 5 min × 3 times;

(7) Secondary antibody incubation: dilute the corresponding species secondary antibody at 1:5000 and incubate at room temperature for 2 h;

(8) Wash the membrane for 5 min × 3 times;

(9) The HRP-ECL chemiluminescence method was used to analyze the bands.

2. Statistical Analysis

All data in experimental analysis were generated using GraphPad Prism 9.2 software (San Diego, CA, USA). Unpaired t-test or one-way ANOVA was used. All values are expressed as Mean ± SD. p < 0.05 was considered statistically significant.

3. Experimental Results

1. Effects of γ-ray irradiation on bile acid metabolism in mice

C57BL/6J mice were given ⁶⁰ Coγ-ray 12Gy abdominal irradiation to prepare a radiation intestinal injury model (10 mice in the irradiation control group and 10 mice in the normal control group). Fresh feces of mice were collected on the 5th day after irradiation, and the fecal bile acid metabolic profile of mice was detected by ultra high resolution liquid chromatography-mass spectrometry/mass spectrometry (UPLC-MS/MS). The results are shown in Figure 1. In the figure, Control represents the normal control group and 12Gy represents the irradiation control group. It can be seen from Figure 1A that irradiation significantly changed the primary and secondary bile acid profiles, which was manifested by an increase in the relative abundance of primary BAs and a decrease in the relative abundance of secondary BAs. Among the secondary BAs, LCA, isoLCA, dehydroLCA, bHDCA, HDCA and 6-keto-LCA were the most significantly changed bile acids, among which LCA decreased most significantly.

2. Effects of X-ray irradiation on bile acid metabolism in mice

C57BL/6J mice were given 10Gy of X-ray abdominal irradiation to prepare a radiation intestinal injury model (10 mice in the irradiation control group and 10 mice in the normal control group). Fresh feces of mice were collected on the 5th day after irradiation, and the concentration of BAs in feces was measured. The results are shown in Figure 2. Irradiation significantly changed the primary and secondary bile acid profiles, showing an increase in the relative abundance of primary BAs and a decrease in the relative abundance of secondary BAs. Among the bile acids downregulated by radiation, LCA was the most significantly decreased bile acid.

(III) Exogenous LCA supplementation can alleviate radiation-induced intestinal damage in mice

To explore whether LCA has a protective effect on radiation-induced intestinal injury, C57BL/6J mice were irradiated with 12Gy of gamma rays, and different doses of LCA (15mg/kg or 30mg/kg) were given 1 hour before irradiation for 5 consecutive days. As shown in Figure 3, 15mg/kg LCA reduced the mortality of irradiated mice, promoted the weight growth of irradiated mice, significantly increased the number of crypts and the length of intestinal villi, and significantly increased the length of the mouse colon, all of which were better than supplementing 30mg/kg LCA. The results show that supplementing LCA can promote the repair of radiation-induced intestinal injury in mice.

(IV) Exogenous LCA supplementation can also reduce radiation-induced intestinal damage in mice

Under X-ray irradiation conditions, in order to clarify whether LCA also has the effect of regulating the repair of radiation-induced intestinal damage, irradiated mice were supplemented with LCA. The results in Figure 4 show that LCA increased the amount of water and food consumed by irradiated mice, promoted the weight gain of irradiated mice, and significantly increased the length of the colon of mice. Consistent with the results of γ-ray irradiation, supplementation with 15 mg/kg LCA was more effective than supplementation with 30 mg/kg LCA in increasing the length of intestinal villi and the number of crypts.

(V) LCA has no significant effect on the intestines of normal mice

In order to detect whether LCA has an effect on the intestines of normal C57BL/6J mice, normal mice were treated with LCA (15 mg/kg, 30 mg/kg). The results showed that, as shown in Figure 5, LCA had no significant effect on the drinking water and food intake of normal mice, and there was no significant change in the weight of mice after LCA treatment, and no significant change in the length of the colon. HE staining analysis of small intestinal tissues showed that there was no significant change in the length of intestinal villi and the number of crypts after LCA treatment compared with normal mice.

(VI) Effects of LCA on the main organs of normal mice

We also tested whether LCA had any effect on the main organs of normal mice, and performed HE staining on the main organs of mice. As shown in Figure 6, after LCA treatment, the colon tissue structure was clear, and there was no significant change in the depth of the crypts and the number of goblet cells; the thymus capsule was intact, and the cortical-medullary junction was clear; the spleen capsule was intact, and the white pulp and red pulp boundary was clear; the liver tissue capsule was intact; the kidney tissue capsule was intact, the glomerular structure was normal, and the renal tubular structure was normal; the lung tissue bronchial structure was intact and clear. The above results suggest that LCA at 15mg/kg or 30mg/kg has no significant effect on the main organs of normal mice.

(VII) LCA promotes the growth of normal mouse small intestinal organoids

We confirmed the protective effect of LCA on radiation-induced intestinal injury in mice through in vivo experiments. Next, we cultured the small intestinal organoids of normal mice in vitro, treated them with LCA after irradiation, and used the LCA administration group at a dosage of 10 μM, dissolved in DSMO. At the same time, we set up a blank control group and an irradiation control group treated only with DSMO after irradiation. The budding of the organoids was observed under a microscope 7 days later. As shown in Figure 7, 10 μM LCA significantly promoted the regeneration of the small intestinal organoids of normal mice. It is worth noting that low-dose LCA treatment enhanced the budding of the organoids, while high-dose LCA treatment inhibited the growth of the organoids.

(VIII) LCA promotes regeneration of mouse small intestinal organoids after radiation damage

Normal mouse small intestinal crypts were used to culture small intestinal organoids in vitro. To further clarify the effect of LCA on the growth of organoids after radiation damage, 4Gy and 6Gy of γ-ray irradiation were then given. After irradiation, LCA (10μM) and DMSO were given, and the regeneration of small intestinal organoids was observed under a microscope 7 days later. The results are shown in Figure 8. Whether after 4Gy or 6Gy irradiation, LCA can significantly promote the growth of irradiated small intestinal organoids.

(IX) LCA reduces the expression of inflammatory factors in the small intestine of mice with radiation-induced intestinal injury

To detect whether LCA has an anti-inflammatory effect in mice with radiation-induced intestinal injury, the expression levels of IL-1β, IL-6, and TNF-α in small intestinal tissue were detected by ELISA. As shown in Figure 9, the expression levels of IL-1β, IL-6, and TNF-α were upregulated after irradiation, and LCA treatment significantly reduced the expression of these three inflammatory cytokines in small intestinal tissue. These results indicate that LCA can have an anti-inflammatory effect on radiation-induced intestinal mice by reducing the expression of IL-1β, IL-6, and TNF-α.

(X) LCA promotes the proliferation of small intestinal crypt cells in mice with radiation-induced intestinal injury

To investigate whether LCA has a protective effect on the proliferation of crypt cells in mice with radiation-induced intestinal injury, C57BL/6J mice were irradiated with 12Gy of gamma rays, and different doses of LCA were given 1 hour before irradiation for 5 consecutive days. A blank control group and an irradiation control group were also provided. The expression levels of PCNA and Ki-67 in the small intestinal tissue of mice with radiation-induced intestinal injury were detected by immunohistochemistry. As shown in Figure 10, gamma rays significantly reduced the expression levels of PCNA and Ki-67 in the crypt cells of the small intestine of mice, while LCA had a protective effect on the proliferation of crypt cells in mice. After irradiation with 12Gy of gamma rays, the decrease in the expression levels of PCNA and Ki-67 was slowed down, that is, the expression of PCNA and Ki-67 in the crypt cells of the small intestine of mice was promoted, indicating that LCA can promote the proliferation of crypt cells in the small intestine of mice with radiation-induced intestinal injury.

(XI) LCA treatment increases the expression of intestinal stem cells at the base of the small intestinal crypts in irradiated mice

To detect whether LCA affects the loss of ISCs at the base of the crypts caused by irradiation, C57BL/6J mice were irradiated with 12 Gy of gamma rays, and different doses of LCA were given 1 hour before irradiation for 5 consecutive days. A blank control group and an irradiation control group were also provided. Immunofluorescence staining was performed on the small intestinal tissues of mice with radiation-induced intestinal injury to detect the expression of ISCs marker Lgr5 in the small intestinal crypt cells. As shown in Figure 11, irradiation reduced the number of Lgr5-positive cells in the mouse crypt cells, while LCA treatment increased the positive rate of Lgr5 in the mouse crypt cells. qRT-PCR and immunoblotting were further used to detect the mRNA and protein expression levels of Lgr5. It was found that irradiation caused the expression of Lgr5 in the small intestinal tissues of mice to be downregulated, and LCA promoted the expression of Lgr5 in the mouse crypt cells. This indicates that LCA can increase the expression of intestinal stem cell markers Sox9, Ascl2, and Olfm4 at the base of the crypts in mice with radiation-induced intestinal injury. Therefore, LCA promotes intestinal damage repair by increasing the expression of intestinal stem cells at the base of the small intestinal crypts.

(XII) LCA treatment increases the expression of “+4” intestinal stem cells in irradiated mice

In order to detect the effect of LCA on "+4" intestinal stem cells, "+4" intestinal stem cells in mouse intestinal crypt cells were detected. The results are shown in Figure 12. The expression of "+4" intestinal stem cells Hopx, krt19 and Lrig1 was significantly reduced after radiation damage, and their expression was significantly increased after LCA intervention, indicating that LCA treatment increased the expression of "+4" intestinal stem cells in irradiated mice and promoted intestinal damage repair.

(XIII) LCA treatment increases the expression of intestinal stem cells in the intestinal villus region

At the same time, the expression of intestinal stem cell-related markers in the intestinal villus region was also detected. As shown in Figure 13, the expression of DCLK1 and chga was significantly reduced after radiation damage, and their expression was significantly increased after LCA intervention. This suggests that LCA can promote the expression of intestinal stem cells in the intestinal villus region of mice with radiation-induced intestinal damage and promote the repair of radiation-induced intestinal damage.

(XIV) LCA promotes the expression of TGR5 in mice with radiation-induced intestinal injury

The regulatory function of BAs is mainly the result of the activation of intracellular ligand-activated nuclear receptors (NRs), such as farnesoid X receptor (FXR, NR1H4) and cell surface G protein-coupled receptors (GPCRs), especially the G protein-coupled BAs receptor TGR5. TGR5 is a class of BAs receptors, and LCA is its most effective natural agonist. In order to gain a deeper understanding of whether the potential mechanism of LCA promoting intestinal regeneration after irradiation involves TGR5, mice were irradiated abdominally and given LCA. Five days later, the small intestine and colon tissues of mice were taken for immunohistochemistry and qRT-PCR to detect the expression of TGR5. The results are shown in Figure 14. After irradiation, the expression level of TGR5 in the small intestine tissue of mice with radiation-induced intestinal injury was reduced, while LCA could significantly enhance the protein expression level of TGR5 in the small intestine tissue and colon tissue of mice after irradiation and the mRNA expression level in the small intestine tissue of mice.

Analysis

Radiation intestinal injury, also known as radiation enteritis, is a common complication in cancer patients receiving abdominal radiotherapy. This not only limits the use of high-dose ionizing radiation to effectively kill tumor cells, but also brings great pain and even death to patients. Clinical data show that although the advancement of radiotherapy technology has greatly reduced the damage of radiotherapy to tissues outside the target area, 90% of patients receiving radiotherapy will experience abdominal pain, diarrhea, blood in the stool, dehydration, malabsorption, enterobacterial infection, fecal incontinence and weight loss. Severe cases may develop septic shock and death. 50% of patients have digestive tract symptoms that affect their quality of life, and 20% to 40% of patients have moderate to severe symptoms.

The small intestine is the main sensitive site for abdominal radiotherapy. During the radiotherapy of abdominal and pelvic malignancies, the healthy intestine is inevitably exposed to radiation. Although the intestine can initiate the regeneration process after radiation damage, there is also a rapid loss of intestinal stem cells and proliferative progenitor cells. Ionizing radiation can damage basal epithelial cells, hinder their renewal, and lead to histologically detectable changes in IECs, such as reduced height and number of intestinal villi, inflammation, and intestinal wall edema. Although the pathogenesis of radiation-induced intestinal injury is more related to the production of free radicals, the loss of intestinal stem cells, and intestinal microbiota dysbiosis, it is not clear whether metabolic dysfunction is also involved. At present, there is a lack of effective means for the treatment of radiation-induced intestinal injury, which is limited to anti-infection, nutritional support, fecal microbiota transplantation, and traditional Chinese medicine. Therefore, there is an urgent need to find new targets and intervention strategies.

Abnormal regulation of BAs and impaired BAs receptor transduction are associated with intestinal diseases. This experiment was the first to find that irradiation can affect BAs metabolism, leading to a disorder in the bile acid metabolic profile of the mouse intestine, which was manifested by an increase in primary bile acids and a significant decrease in multiple secondary bile acids after irradiation, among which LCA decreased most significantly, indicating that radiation caused a disorder in the bile acid metabolic profile of mice. Among the bile acids downregulated by radiation, LCA was the most significantly reduced bile acid.

In the present invention, mice were supplemented with LCA before irradiation, and samples were taken on the 5th day after irradiation for subsequent experiments. The results showed that the weight loss of mice treated with LCA was reduced, the colon length increased, the intestinal villus length increased, and the number of crypts increased significantly. These results confirmed that supplementing LCA can alleviate the intestinal damage of mice caused by abdominal irradiation. At the same time, normal mice were treated with LCA, and the results showed that LCA had no significant effect on the intestines and major organs of normal mice. By isolating the small intestinal crypt cells of mice for organoid culture, and treating them with different concentrations of LCA, the growth of organoids can be significantly promoted when LCA is 10μM. The cultured organoids were irradiated and treated with 10μM LCA. The results showed that LCA can significantly promote the regeneration of small intestinal organoids after irradiation. The present invention detects the changes in proinflammatory factors after radiation by ELISA. The results show that LCA can effectively reduce the expression levels of IL-1β, IL-6 and TNF-α, promote intestinal mucosal repair, and have anti-inflammatory effects on radiation-damaged mice. The expression of PCNA and Ki-67 was detected by immunohistochemistry, and the results showed that LCA can significantly promote the proliferation of crypt cells in mice with radiation-damaged intestines. This suggests that radiation can affect the metabolism of bile acids, and bile acids, especially LCA, can promote the repair of radiation-induced intestinal damage.

The small intestine is a highly sensitive tissue. When exposed to high doses of radiation, the intestinal crypts lose all surviving cells with regenerative capacity and become "inactive", leading to crypt loss. Intestinal crypts serve as a niche for intestinal stem cells and are the source of intestinal cell regeneration. Crypt loss seriously affects the renewal of intestinal cells. Intestinal crypt loss is an important pathological basis for radiation-induced intestinal injury, and intestinal crypt reconstruction is the key to repairing radiation-induced intestinal injury. In this part of the experiment, LCA treatment increased the expression of intestinal stem cell markers in the crypt cells of the small intestine of irradiated mice, including intestinal stem cells at the base of the crypts, "+4" intestinal stem cells, and intestinal stem cells in the villus region of the small intestine. This suggests that LCA can promote the renewal of intestinal stem cells and promote intestinal cell proliferation.

G protein-coupled receptor 1 (also known as TGR5), as a membrane receptor for BAs, is widely expressed in the gastrointestinal tract, pancreas, liver, gallbladder, and adipose tissue. The expression level of BAs receptors in gastrointestinal tissues depends on the intestinal microbiota, which is significantly reduced during inflammation. TGR5 mediates BAs to regulate intestinal homeostasis and function, including intestinal hormone secretion, gastrointestinal motility, and local immune function. Mice lacking the membrane receptor TGR5 are prone to intestinal inflammation, indicating that receptors activated by BAs are essential for maintaining intestinal homeostasis. LCA is the strongest natural agonist of bile acid receptor TGR5. Our results showed that the expression of TGR5 was reduced in mice with radiation-induced intestinal injury, while LCA could promote the expression of TGR5 in mouse small intestinal tissue and colon tissue. Activating TGR5 has a cytoprotective effect and promotes intestinal damage repair, further indicating that LCA may be a potential radioprotector, providing a new perspective for the clinical treatment of radiation-induced intestinal injury.

At the same time, according to the above experimental results, it was found that the number of PCNA, Ki67 positive cells, the number of crypts, and the height of small intestinal villi in the low-dose group were optimal, while those in the high-dose group were poor. Therefore, from the aspects of PCNA, Ki67 positive cell number, the number of crypts, and the height of small intestinal villi, the 15 mg/kg administration group had better effect.

Claims (7)

1. Application of lithocholic acid in the preparation of drugs for preventing radiation-induced intestinal injury. 2. Application of lithocholic acid in the preparation of drugs for the treatment of radiation-induced intestinal injury. 3. Application of lithocholic acid in the preparation of drugs for increasing the number of small intestinal crypts after radiation. 4. Application of lithocholic acid in the preparation of drugs for promoting proliferation of small intestinal crypt cells after radiation. 5. Application of lithocholic acid in the preparation of drugs to promote the proliferation of intestinal stem cells at the base of crypts and "+4" intestinal stem cells after radiation. 6. Application of lithocholic acid in the preparation of drugs for increasing the length of intestinal villi after radiation. 7. Application of lithocholic acid in the preparation of drugs for promoting the proliferation of intestinal stem cells in the villi region of the intestine after radiation.