WO2022134306A1

WO2022134306A1 - Use of 3-hydroxybutyric acid or derivative thereof or bacterial composition capable of producing same for treating radiation intestinal injury

Info

Publication number: WO2022134306A1
Application number: PCT/CN2021/078878
Authority: WO
Inventors: 秦启元; 黄斌杰; 王辉; 黄小艳; 吕金艳; 司徒卫; 余柳松
Original assignee: 中山大学附属第六医院; 珠海麦得发生物科技股份有限公司
Priority date: 2020-12-22
Filing date: 2021-03-03
Publication date: 2022-06-30
Also published as: CN112826814A

Abstract

Use of 3-hydroxybutyric acid or a derivative thereof or a bacterial composition capable of producing same for treating radiation intestinal injury. Constructing a radiation intestinal injury animal model verifies that 3-hydroxybutyric acid has the effects of anti-fibrosis, reducing inflammatory cell infiltration, promoting intestinal repair, etc. and can effectively treat radiation intestinal injury. 3-Hydroxybutyric acid, the derivative thereof and the composition thereof can be made into products such as medicaments and functional formulations.

Description

Use of 3-hydroxybutyric acid or its derivative or bacterial composition capable of producing the substance in the treatment of radiation-induced intestinal injury

technical field

The present invention relates to the field of biomedicine, more particularly, to the application of 3-hydroxybutyric acid or a derivative thereof or a bacterial composition capable of producing 3-hydroxybutyric acid or a derivative thereof in the treatment of radiation-induced intestinal injury.

Background technique

Radiotherapy is one of the most effective means of treating pelvic malignancies. About 35%-61% of patients with pelvic malignancies have received pelvic radiotherapy. According to reports, the number of new cases of malignant pelvic tumors in China alone in 2015 is estimated to exceed 500,000. . Although radiotherapy significantly prolongs the survival time of patients, its physical effects on normal tissues can lead to damage to the pelvic and abdominal organs. Radiation Intestinal Injury (RII) refers to intestinal damage caused by pelvic malignancies such as cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, rectal cancer, bladder cancer, etc. the large intestine. According to the time of onset and changes in the course of the disease, it can be divided into acute radiation intestinal injury (ARII) and chronic radiation intestinal injury (CRII), usually 3 months as the boundary. ARII occurs in more than 75% of patients who receive pelvic radiotherapy, and about 5% to 20% of patients develop CRII. In fact, the incidence of CRII is very likely to be underestimated. It is reported that 81% of pelvic radiotherapy patients have gastrointestinal symptoms, but only 55% of the patients seek professional medical attention.

The main pathological changes of CRII include abnormal mucosal vascular proliferation, submucosal fibrosis, obstructive endarteritis, and inflammatory cell infiltration. Symptoms of CRII patients are repeated, protracted and difficult to heal, and show a tendency of progressive aggravation. The main clinical symptoms in the early stage are blood in the stool, anemia, diarrhea, abdominal cramps, anal pain, tenesmus, incontinence, chronic malabsorption, etc. In the late stage, serious complications such as gastrointestinal bleeding, abscess, perforation, intestinal fistula, rectal stricture, and obstruction are prone to occur. Severe anemia, etc., and even cause death. Clinical diagnosis and treatment are difficult, patients suffer, and their physical and mental health is seriously affected. According to statistics, about 90% of CRII patients can have permanent changes in bowel habits, and 50% of patients say that their quality of life is affected by various gastrointestinal symptoms. In addition, CRII patients can be combined with radiation-induced small bowel injury, radiation-induced colorectal injury, radiation-induced bladder injury, radiation-induced pelvic injury, and primary tumor recurrence and metastasis, which brings great difficulties and challenges to the formulation of patient diagnosis and treatment plans. At present, CRII lacks fundamental prevention and treatment methods, and its treatment has become a major problem for medical workers and patients.

At present, domestic and foreign drugs are mainly used for mild to moderate CRII (such as topical sucralfate, steroids, sulfasalazine, metronidazole, rebamipide and short-chain fatty acids such as sodium butyrate, etc.), topical formalin Lin therapy, endoscopic argon coagulation (APC), laser therapy and hyperbaric oxygen therapy, etc. The main routes of drug treatment include oral and retention enemas. At present, the effect of oral treatment with conventional drugs is very limited. Although retention enema has a certain effect on mild CRII, the operation is cumbersome and patient compliance is poor. Topical formalin therapy requires special caution, is mainly performed in the operating room, and carries risks of anal ulcers, rectal strictures, anal incontinence, and anal pain. Endoscopic treatment may also carry the risk of anal bulge, pain, worsening ulcers, and even rectal fistulas. After taking the above treatments, the patient's symptoms are repeated or progressively aggravated, and it is easy to gradually progress to severe CRII, which requires surgical treatment.

Surgical treatment mainly includes stoma bypass and diseased bowel resection. Ostomy bypass alone does not remove damaged tissue, and residual bowel disease still leads to intractable symptoms, putting patients at risk of ongoing complications. At the same time, long-term stoma is prone to stoma complications, such as stoma prolapse, parastomal hernia, etc., clinical care is cumbersome, and some patients are forced to choose diseased bowel resection due to intractable pain, perforation and other complications . Resection of the diseased bowel is difficult, high-risk, and perioperative complications are common.

In the treatment decision of CRII, the clinical symptoms and endoscopic manifestations should be integrated, and the main symptoms should be relieved by non-surgical treatment as much as possible to avoid the occurrence of serious complications. Drug therapy is favored by patients and medical workers because of its simple treatment method and high patient compliance. However, at present, there are very few drugs for CRII treatment with little effect, while the symptoms of CRII are stubborn, the lesions are irreversible, and it is easy to gradually progress to serious complications, which requires surgical treatment. Therefore, there is an urgent need to develop a more effective, safe and highly compliant CRII therapeutic drug or functional preparation.

SUMMARY OF THE INVENTION

In view of this, the present invention provides 3-hydroxybutyric acid or a derivative thereof or a bacterial composition capable of producing 3-hydroxybutyric acid or a derivative thereof in the treatment of radioactive intestinal The application of injury can solve the technical problem that surgical treatment is prone to complications and CRII treatment drugs are very few and have little effect.

In order to solve the above-mentioned technical problems, the present invention adopts the following technical solutions:

Use of 3-hydroxybutyric acid (3-hydroxybutyric acid or 3-HB) or a derivative thereof or a bacterial composition capable of producing the substance in the treatment of radiation-induced intestinal injury.

The structural formula of 3-hydroxybutyric acid (3-HB) or its derivatives is as follows:

R ₁ in formula I and formula II are both H, straight-chain or branched-chain alkyl groups of different chain lengths (such as C1-C20, C1-C10, C1-C6 or C1-C3 straight-chain or branched chain alkyl groups) , Linear or branched alkoxy with different chain lengths (such as C1-C20, C1-C10, C1-C6 or C1-C3 linear or branched alkoxy), cycloalkyl (such as C3-C6 cycloalkyl) or aryl;

R in _formula I is H, straight or branched alkyl of different chain lengths (such as C1-C20, C1-C10, C1-C6 or C1-C3 straight or branched alkyl), cycloalkane group (such as C3-C6 cycloalkyl) or aryl;

R ₂ in formula II is a non-toxic metal ion (such as sodium, potassium or calcium, etc.), and the value of n in formula II is determined according to the valence state of the metal ion.

The 3-hydroxybutyric acid or its derivatives can be either D-type or L-type, or a mixture of D-type and L-type.

Specifically, it can be: 3-hydroxybutyric acid (3-HB) methyl ester, 3-hydroxybutyric acid (3-HB) ethyl ester, 3-hydroxybutyric acid (3-HB) or its salts (including Its sodium salt, potassium salt, calcium salt, etc.), 3-hydroxyhexanoic acid (3-hydroxyhexanoic acid or 3-HHx) methyl ester, 3-hydroxyhexanoic acid (3-HHx) ethyl ester, 3-hydroxyhexanoic acid (3-hydroxyhexanoic acid) -HHx) or its salts (including its sodium salts, potassium salts, calcium salts, etc.).

The 3-hydroxybutyric acid and its derivatives described in the present invention can be obtained by various methods such as hydrolysis and alcoholysis of various polyhydroxy fatty acid PHAs. After purification by distillation, it is confirmed by GC analysis that the purity is extremely high, and there is no harm such as double bonds. By-products of cell growth (Chen GQ, Wu Q. Microbial Production and Applications of Chiral Hydroxyalkanoates. Appl Microbiol Biotechnol, 67(2005) 592-599).

The 3-hydroxybutyric acid or its derivatives or the bacterial composition that can produce 3-hydroxybutyric acid or its derivatives can be made into medicines, health products or food functional preparations, preferably oral preparations, more preferably made into Sustained and controlled release targeted formulations, and the formulation methods used in the preparation of the present invention are known to those skilled in the art.

The 3-hydroxybutyric acid or its derivatives of the present invention or the bacterial composition capable of producing 3-hydroxybutyric acid or its derivatives can be prepared by oral administration, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, Intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration.

The 3-hydroxybutyric acid or its derivatives of the present invention or the bacterial composition that can produce 3-hydroxybutyric acid or its derivatives can be prepared into preparations including injections, tablets, pills, powders, granules, capsules, oral liquids , suppository or film and other dosage forms, the medicines of the above-mentioned various dosage forms can be prepared according to the conventional methods in the field of pharmacy.

The present invention provides that 3-HB or a derivative or a composition thereof has the effect of reducing excessive fibrosis, can treat excessive fibrosis of intestinal wall formed by radiation-induced intestinal injury, and can also treat diseases involving fibrotic manifestations.

The present invention provides that 3-HB or a derivative or a composition thereof has the effect of reducing inflammatory response, can treat radiation enteritis, and can also treat diseases involving the manifestation of intestinal inflammatory response.

Treatment of radiation-induced intestinal injury can be carried out in two ways:

①Relieve the excessive fibrosis of the intestinal wall after irradiation: The present invention found that 3-HB can effectively reduce the intestinal wall fibrosis of mice after pelvic irradiation.

②Reduce the infiltration of inflammatory cells in the intestinal wall after irradiation: The present invention found that 3-HB can improve the infiltration of inflammatory cells in the intestinal wall of mice after pelvic irradiation, and significantly reduce the secretion of pro-inflammatory factors.

Compared with the prior art, the present invention has the following beneficial effects: the present invention is verified by constructing an animal model of radiation intestinal injury, and it is found for the first time that 3-hydroxybutyric acid has obvious anti-excessive fibrosis, relieves intestinal wall inflammatory response, promotes intestinal repair, etc. It can effectively treat radiation-induced intestinal injury, and provides a new direction for non-surgical treatment of radiation-induced intestinal injury. 3-HB) or its derivatives or bacterial compositions that can produce 3-hydroxybutyric acid or its derivatives, its application as a drug for the treatment of radiation-induced intestinal injury makes the treatment of CRII more effective and safe, with higher compliance, and has a wide range of applications. application prospects.

Description of drawings

Figure 1 is a schematic diagram of C57BL/6J mice undergoing 25Gy pelvic external irradiation.

Figure 2 is a line chart of changes in body weight of mice in blank control group, simple irradiation group, low-dose 3-HB administration group, and high-dose 3-HB administration group 8 weeks after irradiation.

Figure 3 shows the 6-month survival curves of mice in the blank control group, the simple irradiation group, and the high-dose irradiation + 3-HB administration group.

Figure 4 shows the screenshots of the colonoscopy examination with the anus up 1-2 cm after 8 weeks of irradiation of the mice in the blank control group, the simple irradiation group, the low-dose 3-HB administration group, and the high-dose 3-HB administration group. Scoring under the mirror.

Figure 5 shows the results of HE staining and MASSON staining of intestinal tissue and radiation damage score of mice in blank control group, simple irradiation group, low-dose 3-HB administration group, and high-dose 3-HB administration group after 8 weeks of irradiation.

Figure 6 shows the intestinal fibrosis markers fibronectin, T 1collagen and T 3collagen of mice in the blank control group, simple irradiation group, low-dose 3-HB administration group, and high-dose 3-HB administration group after 8 weeks of irradiation Immunohistochemical staining and the proportion of submucosa in the full thickness of the intestinal wall.

Figure 7 shows the immunohistochemical staining of the intestinal wall macrophage marker F4/80 in the blank control group, the simple irradiation group, the mice in the low-dose 3-HB administration group, and the mice in the high-dose 3-HB administration group after 8 weeks of irradiation and intestinal wall infiltrating macrophage count.

Figure 8 shows the mRNA expression of inflammatory factors in the intestinal wall tissue of mice in the blank control group, the simple irradiation group, the mice in the low-dose 3-HB administration group, and the mice in the high-dose 3-HB administration group after 8 weeks of irradiation.

Detailed ways

The present invention will be described below through specific examples, but the present invention is not limited thereto.

The experimental methods used in the following examples are conventional methods unless otherwise specified; the reagents, biological materials, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1: 3-Hydroxybutyric acid alleviates the symptoms of radiation injury in the RII mouse model.

80 healthy female SPF C57BL/6J mice, weighing 18-20g (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.), were randomly divided into 4 groups with 20 mice in each group: blank control group, The mice in the simple irradiation group, the low-dose 3-HB administration group and the high-dose 3-HB administration group. Among them, the blank control group was routinely reared without any other treatment; the simple irradiation group was routinely reared and received a single 25Gy pelvic irradiation, and the irradiation range was a rectangular area with a length of 1 cm from the anus to the upper side (Figure 1); the low-dose 3-HB administration group and The high-dose 3-HB administration group was given 75 mg/kg and 150 mg/kg body weight of 3-hydroxybutyric acid in their drinking water, respectively, and the mice took water freely throughout the experiment, and the rest of the treatments were the same as those in the simple irradiation group. Among the 20 mice in each group, 5 mice recorded their body weight on the day before irradiation and 8 weeks after irradiation, and were sacrificed at the 8th week after irradiation to collect intestinal tissue 1-2 cm above the anus for subsequent experiments; 5 mice Endoscopy was performed and scored at the 8th week after irradiation; another 10 mice were used to observe and record the survival curve half a year after irradiation.

Statistics and data analysis: The percentage change of body weight was displayed by mean±SEM, and the statistical comparison was performed by Student's-t test; the semi-quantitative score of proctoscopy and semi-quantitative intestinal radiation damage score of mice with radiation-induced intestinal injury were analyzed by Mann-Whitney test. Comparison; survival curves were statistically compared using log rank test; P values less than 0.05 were considered significant differences.

Figure 2 shows the percentage change in body weight of the mice in the blank control group, the simple irradiation group, the low-dose 3-HB administration group, and the high-dose 3-HB administration group 8 weeks after irradiation compared with the body weight on the day before irradiation.

It can be seen from Figure 2 that the body weight of mice after 25Gy pelvic irradiation was significantly lower than that of mice in the blank control group, but administration of high doses of 3-HB could effectively inhibit the weight loss of mice after irradiation. obvious.

The experimental results showed that the drug significantly improved the weight loss of animals induced by 25Gy pelvic irradiation.

Figure 3 shows the survival curves of mice in the blank control group, the simple irradiation group, and the high-dose 3-HB administration group within half a year after irradiation.

It can be seen from Figure 3 that the mice will die from 2 months to 6 months after 25Gy pelvic irradiation, and high-dose 3-HB can significantly prolong the survival time of mice after 25Gy pelvic irradiation.

The experimental results show that the drug can prolong the survival time of mice after 25Gy pelvic irradiation.

Fig. 4 shows the screenshots and internal examinations of the intestinal endoscopy with the anus up 1-2 cm after 8 weeks of irradiation in the blank control group, the simple irradiation group, the mice in the low-dose 3-HB administration group, and the mice in the high-dose 3-HB administration group. Scoring under the mirror.

Semiquantitative scoring of proctoscopy for radiation-induced intestinal injury in mice

Table 1 Semi-quantitative scores of mice with radiation-induced intestinal injury proctoscopy

Table 2 Semi-quantitative score of proctoscopy with radiation-induced intestinal injury in mice

Note: This score was independently scored by an endoscopist using a single-blind method.

It can be seen from Figure 4 that after 25Gy pelvic irradiation, the mucosa of the irradiated intestinal segment of mice appeared pale edema, erosion, ulceration and even perforation, and different doses of drug treatment can effectively alleviate the above endoscopic symptoms, and the effect of high-dose treatment group The lower dose group was better.

The experimental results showed that the drug significantly improved the intestinal mucosal injury caused by 25Gy pelvic irradiation under endoscopy.

CONCLUSION: 3-Hydroxybutyric acid can significantly reduce the symptoms of radiation-induced intestinal injury in mice, mainly by inhibiting the weight loss after irradiation, prolonging the survival time, and improving the intestinal mucosal endoscopic injury after irradiation.

Example 2: 3-Hydroxybutyric acid relieves intestinal semi-quantitative pathological scoring in RII mouse model.

(1) Experimental materials

HE staining: Harris hematoxylin, eosin staining solution.

MASSON staining: hematoxylin, anhydrous ethanol, anhydrous ferric chloride, acid fuchsin, phosphomolybdic acid, glacial acetic acid, aniline blue concentrated hydrochloric acid.

MASSON staining main reagent preparation:

Weigent's hematoxylin: Solution A: add 1g of hematoxylin to 100ml of absolute ethanol to dissolve with slight heat, and store at room temperature; solution B: mix 4ml of 30% anhydrous ferric chloride solution, 1ml of concentrated hydrochloric acid and 100ml of distilled water, and store at room temperature. When using, mix A and B in equal amounts, and generally use them now.

Masson Ponceau Acid Fusin Solution: Mix and dissolve 0.7 g of Ponceau red, 0.3 g of acid fuchsin, 99 ml of distilled water, and 1 ml of glacial acetic acid, and store at room temperature.

1% glacial acetic acid aqueous solution: add 1 ml of glacial acetic acid to 100 ml of water and store at room temperature.

1% aqueous solution of phosphomolybdic acid: dissolve 1 g of phosphomolybdic acid in 100 ml of water and store at room temperature.

Aqueous solution of aniline blue: dissolve 2g of aniline blue in 98ml of distilled water and 2ml of glacial acetic acid, and store at room temperature.

1% hydrochloric acid alcohol: add 1 ml of concentrated hydrochloric acid to 1000 ml of absolute ethanol and store at room temperature.

(2) Experimental method

HE staining:

Mouse intestinal tissue for HE (eosin staining method hematoxylin-eosin staining) was placed in 10% neutral formalin for 24 hours, washed with running water, dehydrated, transparent, and embedded in paraffin to prepare 4 μm thick Serial paraffin sections. Paraffin sections were routinely dewaxed to water, stained with hematoxylin at room temperature for 10 min, rinsed with tap water for 30-60 s; differentiated with 1% hydrochloric acid alcohol for 1 s, rinsed with tap water for 1 min; stained with eosin at room temperature for 5-10 min; dehydrated with gradient alcohol; transparent in xylene; neutral gum cover sheet.

MASSON staining:

The mouse intestinal tissue used for MASSON staining was placed in 10% neutral formalin for 24 hours, washed with running water, dehydrated, transparent, and embedded in paraffin to prepare serial paraffin sections with a thickness of 4 μm. Paraffin sections were routinely dewaxed to water, stained with Weigent's hematoxylin at room temperature for 5 min, and rinsed with tap water for 3 min; differentiated in 1% hydrochloric acid alcohol for 1 s, rinsed with tap water for 1 min, and immersed in distilled water for 30 s; stained with acid fuchsin at room temperature for 5 min, and rinsed with distilled water for 30 s. 1% phosphomolybdic acid, observed under the microscope until the muscle fibers were red, and the submucosa was pale pink, directly stained with aniline blue at room temperature for 3 minutes without washing, and immersed in 1% glacial acetic acid for 30-60s; gradient alcohol dehydration; xylene transparent; neutral gum seal.

Intestinal Radiation Injury Semiquantitative Scale

Table 3 Intestinal radiation injury semi-quantitative scoring table

Note: The score is independently scored by a pathologist using a single-blind method. Each score ranges from 0-1, 0-2, or 0-3. The sum of the individual scores of each tissue layer for each pathological section is the Radiation damage score.

It can be seen from Figure 5 that different doses of drugs can improve the degree of pathological damage of the irradiated intestinal segment in mice after 25Gy pelvic irradiation, and reduce the semi-quantitative score of intestinal radiation damage.

Experimental conclusion: Drugs can improve intestinal radiation injury in mice at the level of intestinal pathology.

Example 3: 3-Hydroxybutyric acid attenuates excessive fibrosis of the intestinal wall in the RII mouse model.

(1) Experimental materials

Fibronectin antibody, Type I collagen antibody, Type III collagen antibody, antigen retrieval solution, DAB chromogenic solution (Zhongshan Jinqiao).

(2) Experimental method

The mouse intestinal tissue used for immunohistochemical staining of fiber markers was placed in 10% neutral formalin for 24 hours, washed with running water, dehydrated, transparent, and embedded in paraffin to prepare 4 μm thick serial paraffin sections. Paraffin sections were routinely dewaxed to water, immersed in sodium citrate antigen retrieval solution (10mM, pH 6.0) for high pressure renaturation, slowly cooled after steaming for 3 minutes, and washed with PBS twice after cooling, 5 minutes/time; rabbit serum The blocking solution was blocked at room temperature for 15 minutes; the diluted primary antibodies of fibronectin, Type I collagen, and Type III collagen were directly added dropwise (dilutions were 1:400, 1:50, and 1:100), 4°C for 12 hours, and PBS soaked for 2 times, 5 min/time; add biotinylated secondary antibody dropwise, 40 min at room temperature, wash twice with PBS, 5 min/time; add tertiary antibody dropwise, 40 min at room temperature, wash twice with PBS, 5 min/time; DAB color development, mirror Under observation, terminated in time, rinsed with tap water for 5 min; counterstained with hematoxylin, room temperature for 30 s, differentiated with 1% hydrochloric acid alcohol for 1 s, rinsed with tap water for 5 min; dehydrated with gradient alcohol; transparent in xylene; mounted with neutral gum.

The intestinal pathological sections of mice were observed under an Olympus upright electron microscope, and the intestinal segment with the most obvious intestinal wall thickening was selected under a 10x eyepiece and a 10x objective lens, and the full-thickness thickness of the intestinal wall was measured using the electron microscope's own software. Submucosa thickness (try to keep the two measurement lines parallel and overlapping), and calculate the ratio of submucosa thickness/full thickness of intestinal wall.

Figure 6 shows the intestinal fibrosis markers fibronectin (Fibr) and Type I of mice in the blank control group, simple irradiation group, low-dose 3-HB administration group, and high-dose 3-HB administration group after 8 weeks of irradiation Immunohistochemical staining of collagen (COL1) and Type III collagen (COL3) and the proportion of submucosa in the full thickness of the intestinal wall.

It can be seen from Figure 6 that the coloration of intestinal wall fiber markers in the mice in the administration group is less, the thickness of the intestinal wall is lighter, and the thickness of the submucosa accounts for less of the total thickness of the intestinal wall.

Experimental conclusion: 3-Hydroxybutyric acid can reduce excessive fibrosis of intestinal submucosa in mice irradiated with 25Gy pelvic cavity.

Example 4: 3-Hydroxybutyric acid attenuates intestinal wall inflammation in RII mouse model.

(1) Experimental materials

Immunohistochemical staining:

F4/80 antibody, antigen retrieval solution, DAB chromogenic solution (Zhongshan Jinqiao).

Fluorescence quantitative PCR:

Primers, Trizol, SYBR Green dye, PCR reverse transcription kit.

(2) Experimental method

Immunohistochemical staining:

The operation process is basically the same as that of Example 3, and the dilution of F4/80 primary antibody is 1:50.

Macrophage infiltration score:

The intestinal pathological sections of mice were observed under an Olympus upright electron microscope, and the intestinal segment with the most obvious thickening of the intestinal wall was selected under a 10x eyepiece and a 10x objective lens, and then the objective lens magnification was adjusted as needed, and the macroscopic examination was carried out according to the actual findings. Phagocytosis infiltration score. The specific scoring criteria for the macrophage infiltration score are as follows: 1. Scattered macrophages in the lamina propria, no macrophages in the submucosa and muscularis propria; 2. A small number of macrophages in the mucosal crypts (<10/crypt) , there are no macrophages in the submucosa and the muscularis propria; 3. A large number of macrophages (>10/crypt) in the mucosal crypts, macrophages in the submucosa and the muscularis propria; There are a lot of macrophages. Scores were independently scored by a pathologist using a single-blind method.

Fluorescence quantitative PCR:

1) Extraction of RNA from animal tissues

1. Put the prepared mouse intestinal tissue or human intestinal tissue into a 2ml EP tube, and add 1ml TRIzol;

2. Add 2 autoclaved steel balls with a diameter of 2 mm and 1 steel ball with a diameter of 5 mm into the EP tube, seal the mouth of the tube with sealant, and place it in a shaker at 60 Hz to shake until the tissue is completely fragmented;

3. Take out the steel ball and place it at room temperature for 5 minutes;

4. Add 0.2ml of chloroform to each EP tube, close the tube cap tightly, and shake on the shaker for 10s;

5. Let stand at room temperature until the liquid in the tube is stratified, and centrifuge at 12000rpm at 4°C for 15min;

6. Carefully take out the EP tube. At this time, the sample is divided into three layers: the upper layer is the colorless aqueous phase layer containing RNA, the middle layer is DNA, lipids and proteins, etc., and the lower layer is the red phenol-chloroform layer. Take the clear supernatant solution into a new EP tube, the volume of the aqueous layer that can be absorbed is about 400-500 μl;

7. Add 0.5ml of isopropanol to each new EP tube, shake for 10s on a shaker, place at room temperature for 20min, centrifuge at 12000rpm at 4°C for 10min, carefully remove the supernatant, leaving a white precipitate;

8. Add 1ml of freshly prepared 75% alcohol to the pellet, invert 5 times and mix well, then centrifuge at 12000rpm and 4°C for 5min;

9. Carefully suck off the supernatant, put the tube upside down on the absorbent paper and suck up the solution at the mouth of the tube, open the mouth of the tube and air-dry in a fume hood for 10 minutes;

10. Dissolve the RNA with 20 μl of DEPC water at room temperature for 5 min and measure the concentration.

2) RNA quantification

Quantitation was detected using a Nanodrp 2000 microquantitator. Open the lid of the machine and drop 1 μl of DEPC water at the quantitative point, press "blank" to deduct the effect of volume. Then wipe off the DEPC water with lens tissue, drop 1 μl of the RNA sample to be tested, and perform the “measure” operation to obtain the absolute concentration of RNA. Generally, the RNA concentration of all samples will be adjusted to 1000 μg/ml with DEPC water.

3) Synthesize cDNA by reverse transcription

1. Referring to TOYOBO's RT-PCR kit instructions, the first step is to add samples according to the following reaction system:

Table 4

The reaction conditions were 37°C for 5 min.

2. In the second step, add samples to the reaction system:

table 5

The reaction conditions were 37°C for 15 min, 98°C for 5 min, and storage at 4°C.

4) Real-Time PCR amplification

1. Primer design:

Primer premier 5.0 software was used to design the corresponding primer sequences, sequence similarity query system (BLAST) was used to analyze the homology of primers, and primers were carefully selected to span exons, and the thermodynamic characteristics of primers were analyzed by Oligo 6 software. All primers were synthesized by Guangzhou Aike Biotechnology Co., Ltd. The specific primer sequences required for the experiment are as follows:

Table 6

2. Referring to the SYBR Green Master Mix reagent instructions, add samples according to the following reaction system:

Table 7

3. PCR amplification reaction

Briefly centrifuge and mix well, then put the reaction tube into the PCR machine, and perform the Real-Time PCR reaction according to the following parameter settings: 95°C, 7min; 95°C, 10s; 60°C, 30s, cycle 45 times; 60°C, 30s; Dissolution curve 60℃-98℃; 20℃ for 10s. Three replicate wells were set for each group of samples to ensure the validity and repeatability of the experimental results.

Figure 7 shows that high concentrations of 3-hydroxybutyric acid can significantly reduce intestinal wall macrophage infiltration after irradiation.

Figure 8 shows that 3-hydroxybutyric acid can reduce the transcription of pro-inflammatory chemokines such as CCL2 and CCL7.

Conclusion: 3-Hydroxybutyric acid can reduce and inhibit the inflammatory response of irradiated intestinal segment.

Claims

Use of 3-hydroxybutyric acid or a derivative thereof or a bacterial composition capable of producing the substance in the treatment of radiation-induced intestinal injury.
The application according to claim 1, characterized in that, the 3-hydroxybutyric acid or its derivative is D-form or L-form, or a mixture of D-form and L-form.
The application according to claim 2, wherein the 3-hydroxybutyric acid or its derivatives comprise methyl 3-hydroxybutyrate, ethyl 3-hydroxybutyrate, 3-hydroxybutyric acid or a salt thereof, Methyl 3-hydroxyhexanoate, ethyl 3-hydroxyhexanoate, or 3-hydroxyhexanoic acid or a salt thereof.
The application according to claim 1, wherein the 3-hydroxybutyric acid or its derivative or the bacterial composition capable of producing 3-hydroxybutyric acid or its derivative is made into a medicine, a health product or a food function preparation.
The application according to claim 4, wherein the preparation is an oral preparation.
The application according to claim 5, wherein the preparation is a sustained and controlled release targeted preparation.
The application according to claim 6, wherein the preparation is administered orally, intravenously, intramuscularly, intraarterially, intramedullary, intrathecally, intraventricularly, transdermally, subcutaneously, intraperitoneally, intranasally, enterally, Topical, sublingual or rectal route of administration.
The application according to claim 6, wherein the preparation comprises injection, tablet, pill, powder, granule, capsule, oral liquid, suppository or film.