Application of glucomannan in preparation of product for preventing or treating radioactive intestinal injury

Abstract

The invention relates to an application of glucomannan in preparation of a product for preventing or treating radioactive intestinal injury. The relative molecular weight of the glucomannan disclosed by the invention is 10 to 1,000 kD; monosaccharide components of the glucomannan comprise mannose and glucose; the glucomannan is formed by connecting mannose and glucose through a beta-1, 4-glucosidic bond, and has a glucose branched chain structure and acetyl modification. The glucomannan disclosed by the invention can be used for treating radioactive intestinal injury, directly protecting an organism from small intestine DNA injury and small intestine crypt stem cell injury caused by ionizing radiation, and inhibiting oxidative stress, inhibiting mitochondrial apoptotic cell apoptosis regulatory factors and regulating expression of inflammatory factors in intestinal tissues by regulating an in-vivo SOD (superoxide dismutase) value; and the intestinal flora is kept stable under the influence of ionizing radiation.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular to the application of glucomannan in the preparation of a product for preventing or treating radiation-induced intestinal injury. Background Art

[0002] In the field of modern cancer treatment, radiotherapy is one of the extremely important treatment means, and 65-80% of cancer patients need to receive radiotherapy, which is widely clinically used in the treatment of pelvic, abdominal and retroperitoneal malignant tumors. However, the intestine is inevitably irradiated during radiotherapy, thereby causing radiation-induced intestinal injury (RII). Common damaged sites include the rectum, sigmoid colon, small intestine in the pelvic group and the ileocecal region. Among them, acute RII usually occurs within several days to several weeks after the start of radiotherapy, mainly manifested as acute inflammatory reactions of the intestinal mucosa, such as abdominal pain, diarrhea, nausea, vomiting, etc., with an incidence rate of more than about 75% and having a certain self-limiting property; chronic RII appears 6 months to 2-5 years after the end of radiotherapy, with an incidence rate of about 2.5%-20%, showing a progressive development process and seriously affecting the quality of life and long-term prognosis of patients. ] Clinically, the current treatment means for RII mainly include supportive treatment, symptomatic drug treatment and surgical treatment. Commonly used drugs include anti-inflammatory drugs, antibiotics, antioxidants, antidiarrheal drugs, mucosal protectants, etc. However, the existing drugs have a single effect and large toxic and side effects, and cannot fundamentally solve the problems of intestinal injury and high incidence of RII. There is a lack of an effective preventive drug treatment plan for RII. In recent years, China has issued a number of guidelines such as the "Chinese Expert Consensus on the Integrated Diagnosis and Treatment of Radiation-Induced Intestinal Injury (2023 Edition)", which has promoted the development of the integrated diagnosis and treatment of RII.

[0003] In the scope of traditional Chinese medicine's understanding of etiological factors, radiation belongs to the exogenous "fiery toxin pathogen". RII belongs to the category of "intestinal dysentery" in traditional Chinese medicine. The pathogenesis is characterized by deficiency in the root and excess in the tip, with a mixture of deficiency and excess being the basic pathogenesis. The disease location is in the large intestine, and the main organ involved is the spleen. Heat toxin accumulates, damaging the spleen and stomach, resulting in the loss of the functions of the large intestine in receiving and digesting food, separating the clear from the turbid, and conducting. Among patients, the damp-heat constitution, yang-deficiency constitution, and stasis constitution are more common. The main syndrome type of ARII in traditional Chinese medicine syndrome differentiation is intestinal damp-heat syndrome. The treatment principle is to clear heat and remove dampness, cool the blood and stop bleeding. Examples of prescriptions include Gegen Qinlian Decoction, Qinghua Yichang Decoction, Shaoyao Decoction, or Huangqin Decoction combined with Huaihua Pills with modifications. For retention enema, Bai Tou Weng Decoction, Qingre Liangxue Jiedu Decoction, etc. are selected; the main syndrome type of CRII is spleen deficiency with dampness accumulation syndrome. The treatment principle is to strengthen the spleen and boost qi, astringe and secure the prolapse. Prescriptions such as modified Shenling Baizhu Powder and Cibai Capsule are used. For retention enema, Jiawei Muxiang Shunqi Decoction, etc. are selected. Discovering effective substances with antioxidant and anti-inflammatory effects from heat-clearing and detoxifying traditional Chinese medicines to prevent and treat ARII has excellent R & D prospects (Wang Chuangchuang, Research progress on the effects and mechanisms of traditional Chinese medicine monomers in preventing and treating radiation-induced intestinal injury. China Journal of Chinese Materia Medica, 2023, 48, 3743 - 3752.). The existing expert consensus believes that based on the correlation between traditional Chinese medicine constitution and diseases, starting from the patient's basic constitution type, rationally using body-regulating drugs, health preservation with medicated foods and homologous substances, improving living habits, and personalized body differentiation and nursing, etc., to improve the patient's biased constitution and achieve the goals of preventing disease before it occurs, preventing the progression of the disease during illness, and preventing recurrence after recovery.

[0004] Analyzing from the mechanism of action, RII refers to the physical damage of the intestinal tract near the tumor caused by high-dose radiation exposure. Ionizing radiation can generate a large number of reactive oxygen free radicals, which can then damage the double-stranded DNA structure, change proteins, lipids, carbohydrates, and other complex molecules, activate multiple signaling pathways, cause lipid peroxidation, resulting in cell death, and lead to mucosal damage and subsequent inflammation. The specific pathogenesis of RII is extremely complex and involves multiple molecular mechanism levels. Recent studies have found that intestinal microbiota imbalance plays a key role in the occurrence and development of RII. Radiotherapy can disrupt the balance of the intestinal microbiota, leading to a decrease in the number of beneficial bacteria and overgrowth of harmful bacteria, which in turn affects the intestinal barrier function and immune regulation ability. In addition, apoptosis, autophagy, and abnormal activation of signaling pathways have also been confirmed to be closely related to RII.

[0005] Bletilla striata polysaccharide is the main active ingredient in the traditional Chinese medicine Bletilla striata, with functions such as astringency, hemostasis, tonifying the lung, and immune regulation. In the inventor's previous research, it was found that Bletilla striata polysaccharide is a glucomannan (GM), and this type of polysaccharide can also be prepared from plants of the genus Dendrobium and Amorphophallus konjac. Given that this type of ingredient has a good effect on repairing gastrointestinal mucosal damage, it may have the effect of preventing and treating RII. Through systematic and innovative research, the present invention has been completed.

[0006] Glucomannan within a certain molecular weight range has been discovered, and there is no literature on the specific structure of Bletilla striata polysaccharide and the structure-activity relationship research on anti-intestinal radiation injury. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides an application of glucomannan in the preparation of a product for preventing or treating radiation-induced intestinal injury. The glucomannan of the present invention can treat radiation-induced intestinal injury, can directly protect the body from small intestine DNA damage and small intestine crypt stem cell damage caused by ionizing radiation, inhibit oxidative stress by regulating the SOD value in the body, inhibit mitochondrial apoptosis cell apoptosis regulators, regulate the expression of inflammatory factors in intestinal tissues, and maintain the stability of intestinal flora under the influence of ionizing radiation.

[0008] The present invention is achieved through the following technical solutions:

[0009] The object of the present invention is to provide an application of glucomannan in the preparation of a product for preventing or treating radiation-induced intestinal injury, wherein the relative molecular weight of the glucomannan is 10-1000 kD; the monosaccharide composition of the glucomannan includes mannose and glucose; the glucomannan is obtained by connecting mannose and glucose through β-1,4-glycosidic bonds, and has a glucose branched chain structure and an acetyl group modification.

[0010] In one embodiment of the present invention, the relative molecular weight of the glucomannan is 20-500 kD;

[0011] and / or, the composition ratio of mannose and glucose is 1.4:1-9.0:1;

[0012] and / or, the purity of the glucomannan is greater than 70%.

[0013] In one embodiment of the present invention, the glucomannan is derived from one or more of Amorphophallus konjac, Bletilla striata, and Dendrobium plants. It is obtained through extraction and separation, and if necessary, can be appropriately hydrolyzed to obtain a hydrolysis product with a specific required molecular weight.

[0014] In one embodiment of the present invention, the mass percentage of glucomannan in the product is 10%-100%.

[0015] In one embodiment of the present invention, the dosage form of the product is selected from one of oral liquid preparations, granules, tablets, capsules, injections, and freeze-dried powder injections.

[0016] In one embodiment of the present invention, the product includes one or more of drugs, foods, health foods, and special medical foods.

[0017] In one embodiment of the present invention, the administration method of the drug is selected from oral administration, intravenous injection, or enema.

[0018] In one embodiment of the present invention, the oral dosage calculated according to mouse animals is in the range of 50-200 mg / kg.

[0019] In one embodiment of the present invention, the oral administration frequency is 2 - 3 times a day, and the dosage is 50 - 200 mg each time.

[0020] In one embodiment of the present invention, the oral administration course is synchronous use during the period from 7 days before the start of radiotherapy to 7 days after the end of radiotherapy.

[0021] In one embodiment of the present invention, the drug further comprises a pharmaceutically or pharmacologically acceptable carrier and / or salt.

[0022] In one embodiment of the present invention, the site of intestinal injury is selected from one or more of the small intestine, colon, and rectum.

[0023] In one embodiment of the present invention, the radiation source in the radiation-induced intestinal injury is selected from x-rays and / or γ-rays.

[0024] The above technical solutions of the present invention have the following advantages compared with the prior art:

[0025] (1) The glucomannan of the present invention is a plant polysaccharide of natural origin, with a relative molecular weight range of 10 - 1000 kD; its monosaccharide composition includes mannose and glucose; the mannose and glucose are connected by β-1,4-glycosidic bonds; the glucomannan has a branched structure and acetyl group modification. This type of polysaccharide is mainly distributed in plants of the genera Bletilla and Dendrobium in the Orchidaceae family, as well as plants such as Amorphophallus konjac and Aloe vera. Bletilla striata and Dendrobium officinale are well-known traditional Chinese medicines with mucosal protective effects, and glucomannan is their main active ingredient. The present invention discovers for the first time that glucomannan has the effect of preventing or treating radiation-induced intestinal injury; preferably, glucomannan with a molecular weight range of 20 - 500 kD has a better effect.

[0026] (2) The present invention provides an application of glucomannan in the preparation of a product for preventing or treating radiation-induced intestinal injury. It inhibits the apoptosis of small intestinal epithelial cells by regulating the Bax / Bcl-2 / Caspase-3 cell apoptosis signaling pathway.

[0027] (3) It regulates the expression of inflammatory factors IL-6, IL-10, and TNF-α through the NF-KB pathway, reduces the release of pro-inflammatory factors IL-6 and TNF-α, and increases the release of anti-inflammatory factor IL-10.

[0028] (4) It maintains the stability of the intestinal flora, regulates the abundances of Firmicutes and Bacteroidetes close to the normal values, and reduces the abundance of Proteobacteria.

[0029] (5) It inhibits oxidative stress, regulates the SOD value in serum and intestinal tissues. It reduces the damage of ionizing radiation to DNA. It adjusts ZO-1, protects intestinal villi and crypt stem cells, and improves intestinal cell damage.

[0030] (6) Through cell screening and animal experiment research, it is found that the glucomannan extract of the present invention has the effect of preventing and treating RII, and has no toxic and side effects. Therefore, it can improve and enhance the intestinal RII symptoms and quality of life of cancer patients during radiotherapy.

[0031] (7) The present invention first discovers that glucomannan has the effect of preventing and treating radiation-induced intestinal injury, and preliminarily explores the related mechanisms and pathways, providing a theoretical basis for the development of glucomannan into a drug for preventing and treating radiation-induced intestinal injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to the specific embodiments of the present invention and in combination with the drawings, wherein:

[0033] Figure 1 It is the effect of polysaccharide from Bletilla striata on apoptosis of IEC-6 cells damaged by 4 Gy radiation in the test example of the present invention; wherein, A: The result graph of detecting the apoptosis level by flow cytometry; B: The changes of the percentages of early apoptotic, late apoptotic and total apoptotic cells in IEC-6 cells (n = 3; compared with the Control group, P < 0.001, compared with the IR group, **P < 0.01, ***P < 0.001);

[0034] Figure 2 It is the research on the protein pathway of the anti-apoptotic effect of polysaccharide from Bletilla striata on IEC-6 cells damaged by 4 Gy radiation in the test example of the present invention; wherein, A: The protein expressions of Bax, Bcl-2, Caspase-8, Cleaved-Caspase-3, and GAPDH in IEC-6 cells; B: Semi-quantitative analysis of Bax expression; C. Semi-quantitative analysis of Bcl-2 expression;

[0035] D. Semi-quantitative analysis of Caspase-8 expression; E: Semi-quantitative analysis of Cleaved-Caspase-3 expression (n = 3; compared with the Control group, #P < 0.05, P < 0.001, compared with the IR group, *P < 0.05, **P < 0.01);

[0036] Figure 3 It is the H&E staining of mouse small intestine tissue in the test example of the present invention; A: Representative H&E staining of each group (scale = 200 μm); B: Statistics of the length of small intestine villi (n = 3, compared with the Control group, #P < 0.05, P < 0.001; compared with the IR group, *P < 0.05, ***P < 0.001);

[0037] Figure 4It is the immunohistochemistry of Ki67 and Lgr5 in the intestinal tissue of mice in the test example of the present invention; A: Representative immunohistochemical staining diagrams of Ki67 and Lgr5 in the small intestinal tissue of each group (scale bar = 200 μm); B: Statistical analysis of the number of Ki67 + cells; C: Statistical analysis of the number of Lgr5 + cells (n = 3, compared with the Control group, #P < 0.05; compared with the IR group, *P < 0.05, **P < 0.01, ***P < 0.001);

[0038] Figure 5 It is the immunohistochemistry of γ-H2AX in the intestinal tissue of mice in the test example of the present invention; A: Immunohistochemistry of γ-H2AX in the small intestinal tissue (scale bar = 200 μm); B: Statistical analysis of the number of γ-H2AX + cells (n = 3, compared with the Control group, ##P < 0.01, P < 0.001; compared with IR, *P < 0.05, ***P < 0.001);

[0039] Figure 6 It is the change of the NF-κB pathway and the concentration of inflammatory factors in the small intestine in the test example of the present invention; A: Results of Western Blot experiments of p65 and p-p65 in the NF-κB pathway in the small intestinal tissue; B: Relative content of p-p65 in the small intestinal tissue (n = 3, compared with the Control group, P < 0.001; compared with the IR group, **P < 0.01, ***P < 0.001); C: Contents of inflammatory factors IL-6, TNF-α, and IL-10 in the small intestine (n = 4, compared with the Control group, P < 0.001; compared with the IR group, ***P < 0.001);

[0040] Figure 7 It is the concentration of SOD in the small intestinal tissue and serum in the test example of the present invention (n = 3, compared with the Control group, ##P < 0.01, P < 0.001; compared with the IR group, *P < 0.05, **P < 0.01, ***P < 0.001);

[0041] Figure 8 It is the species analysis of the flora at the phylum level in the test example of the present invention; A. Distribution bar chart; B. Relative abundance of Firmicutes; C. Relative abundance of Bacteroidetes; D. Relative abundance of Proteobacteria (n = 3, compared with the Control group, #P < 0.05; compared with IR, *P < 0.05, ***P < 0.001);

[0042] Figure 9 It is the distribution bar chart of the flora at the genus level in the test example of the present invention;

[0043] Figure 10It is the distribution heat map of the flora at the genus level in the test example of the present invention. Detailed implementation manners

[0044] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the exemplified embodiments are not intended to limit the present invention.

[0045] In the embodiment of the present invention, the uniform polysaccharide means that the chromatographic peak of the polysaccharide sample in the HPGPC (high performance gel chromatography) analysis spectrum is a Gaussian curve with a normal distribution, and the relative molecular weight of the glucomannan is the average weight average molecular weight (Mw) determined by using dextran as a reference substance and analyzed by HPGPC.

[0046] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods, and the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0047] The detection methods used in the following embodiments are as follows:

[0048] The molecular weight and purity of the polysaccharide were analyzed by the HPGPC method. Chromatographic conditions: chromatographic column TSKPW4000 column (7.8 mm × 300 mm); mobile phase water, flow rate 0.6 mL·min -1 ; detected by RI / MALS (Wyatt company) or ELSD (Aotai company); column temperature 40 °C; concentration of the test solution 10 mg / mL, injection volume 10 - 20 μL; analysis time 30 min. Determination was carried out using a series of molecular weight specifications of dextran (Mw 10 - 2000 KD) as a reference substance.

[0049] Example 1: Preparation of Bletilla striata polysaccharide BT300

[0050] Take 1.0 kg of dried Bletilla striata medicinal materials, add 10 L of distilled water and soak overnight, then supplement distilled water to a total volume of 50 L. Reflux extraction was carried out at 100 °C for 2 times, and the extraction time for each time was 1 h. The solutions obtained from the two extractions were filtered through gauze, and the supernatant was concentrated. While stirring the concentrated solution, 75% ethanol with a volume three times that of the concentrated solution was slowly added until obvious turbidity appeared. The solution was allowed to stand, and the white flocculent solid was filtered out and dried at 50 °C for 1.5 h to constant weight to obtain 133.2 g of white solid, namely Bletilla striata polysaccharide BT300.

[0051] The molecular weight of Bletilla striata polysaccharide was obtained as 331 kDa by HPGPC method, and its main component was a single polysaccharide with a purity of 93%. The monosaccharide composition of Bletilla striata polysaccharide was determined by acid hydrolysis-derivatization method to be mainly glucose and mannose. Calculated according to the peak area ratio, the ratio of glucose to mannose was approximately 1:2.99. Through comprehensive structural analysis, the main chain of Bletilla striata polysaccharide was composed of mannose and glucose connected by β-1,4 glycosidic bonds, with partial glucose branches and acetyl group modifications; the molar substitution degree of acetyl group was 0.15-0.22.

[0052] In addition, commercial Bletilla striata medicinal materials from various producing areas were collected, and Bletilla striata polysaccharide was prepared by the method of Example 1 and analyzed by HPGPC. Most of the obtained Bletilla striata polysaccharides were homogeneous polysaccharides with a relative molecular weight of 86.2-344.5 kD and a purity greater than 80% (calculated based on the dried product). It can be seen that the molecular weights of Bletilla striata medicinal materials from different producing areas and sources are different.

[0053] Example 2: Preparation of Bletilla striata polysaccharides BT20-BT110

[0054] Take about 100 g of Bletilla striata polysaccharide obtained in Example 1, dissolve it with 0.01 mol / L HCl to prepare a 2.0% concentration, hydrolyze it in a constant temperature water bath at 75-95 °C for 1.5-4 h, neutralize it to pH 6-7, add 0.1 g of papain in a bath at 50-60 °C and enzymolyze for 6 h, then add 0.5 g of activated carbon and boil for 30 min, centrifuge, filter, filter the filtrate through a 200 nm microfiltration membrane, and freeze-dry to obtain the corresponding hydrolyzed Bletilla striata polysaccharide (60-80 g). The molecular weights of three trial products were analyzed by HPGPC method, which were BT110 (Mw 104 kD), BT50 (Mw 46 kD), and BT20 (Mw 18 kD) respectively, all of which were homogeneous polysaccharides with a purity greater than 95%; the monosaccharide composition ratio of mannose to glucose was 4.9-3.0:1.

[0055] Example 3: Preparation of konjac polysaccharide

[0056] Preparation of Konjac Glucomannan KGM800: The crude konjac glucomannan was a commercially available product, and the konjac glucomannan contained therein was a homogeneous polysaccharide with an Mw of 856 kD analyzed by HPGPC. 50 g of the crude konjac glucomannan was dissolved in 10 L of water to prepare a 0.5% solution, 5 g of papain was added, and the enzymatic hydrolysis was carried out at 50 - 60 °C for 12 h. Then 10 g of activated carbon was added and boiled for 30 min, followed by centrifugation and filtration. The filtrate was filtered through a 1000 nm microfiltration membrane, concentrated under reduced pressure to 5 L, 1.5 times the volume of 95% ethanol was added to cause precipitation, suction filtration was carried out, and drying was carried out under reduced pressure below 60 °C to obtain konjac glucomannan KGM800 (38 g). Through comprehensive structural analysis of KGM, the main chain was composed of mannose and glucose linked by β-1,4 glycosidic bonds, with partial glucose branches and acetyl group modifications; the molar ratio of mannose to glucose monosaccharides was 1.4 - 1.8:1, and the molar substitution degree of acetyl groups was about 0.05 - 0.10.

[0057] Preparation of Konjac Glucomannan Hydrolysate: It was carried out by referring to the method of Example 2. 5 g of konjac glucomannan KGM was weighed and dissolved in 0.01 mol / L HCl to prepare a 0.5% concentration, and hydrolysis was carried out in a constant temperature water bath at 75 - 95 °C for 1.5 - 4 h, neutralized to pH 6 - 7, 0.5 g of activated carbon was added and boiled for 30 min, followed by centrifugation and filtration. The filtrate was filtered through a 200 nm microfiltration membrane and freeze-dried to obtain the corresponding hydrolyzed konjac glucomannan (3.8 - 4.3 g). The molecular weights of three trial products were analyzed by HPGPC method, which were KGM200 (Mw 214 kD), KGM100 (Mw 92 kD), and KGM50 (Mw 48 kD), all of which were homogeneous polysaccharides with a purity greater than 82%.

[0058] Example 4: Preparation of Dendrobium officinale Polysaccharide

[0059] 100 g of Dendrobium officinale medicinal materials were taken, and 1.0 kg of water was added for extraction twice, each time for 1.5 h. The extraction solutions were combined and concentrated under reduced pressure to 1.0 L, filtered. 1.0 g of papain was added to the filtrate and enzymatic hydrolysis was carried out at 50 - 60 °C for 12 h, boiled for 30 min, centrifuged, filtered. 4 times the volume of 95% ethanol was added to the filtrate to cause precipitation, suction filtration was carried out, and drying was carried out under reduced pressure below 60 °C to obtain Dendrobium officinale polysaccharide (1.8 - 4.5 g).

[0060] The molecular weight and purity of polysaccharides from Dendrobium officinale (DOP) were analyzed by HPGPC method. DOP930, DOP150, DOP40, etc. obtained from commercial Dendrobium officinale herbs from various producing areas such as Zhejiang and Yunnan were measured. The relative molecular weight range was 30 - 1415 kD, and the purity was greater than 70% (calculated based on the dried product). DOP from herbs of different sources could be homogeneous polysaccharides or mixed polysaccharides. Through comprehensive structural analysis of DOP (Wen-hua Chen, International Journal of Biological Macromolecules, 2021, 184, 1000), the main chain was composed of mannose and glucose linked by β-1,4 glycosidic bonds, with partial glucose branches and acetyl group modifications; the composition ratio of mannose to glucose monosaccharides was 2.0 - 9.0:1.

[0061] Test Example 1: Radiation protection effect of glucomannan on IEC-6 cells

[0062] To explore the radiation protection effect of glucomannan (GM, Example 1, Mw 331 kDa) on rat small intestinal crypt epithelial cells (IEC-6; purchased from Shanghai Fuheng Biotechnology Co., Ltd.), four groups of cells, namely blank, GM, IR, and IR+GM, were designed. GM and IR+GM were administered (100 μg / mL GM), and IR and IR+GM were irradiated with 4 Gy. After 24 h of culture, all cells were collected and stained, and then sent to a flow cytometer for detection. The GM used in this test example included BT300, BT110, BT50, BT20, KGM800, KGM200, KGM100, KGM50, DOP930, DOP150, DOP40; the results showed that the total number of apoptotic cells in the IR+GM group pretreated with GM (molecular weight range BT20 - DOP930) was lower than that in the IR group (P < 0.05), mainly concentrated in the reduction of late apoptotic cells (P < 0.01).

[0063] Among them, the results of polysaccharides from Bletilla striata BT300 are specifically as Figure 1 shown; Figure 1 A in Figure 1 is the result of detecting the apoptosis level of cells by a flow cytometer, and the percentages of early apoptotic and late apoptotic IEC-6 cells in each group can be obtained (such as

[0064] Test Example 2: Glucomannan exerts an anti-apoptotic effect by inhibiting the mitochondrial apoptosis-related pathway

[0065] In this test example, four groups of IEC-6 cells, namely blank, BSP, IR, and IR + BSP, were designed. 4 hours before irradiation, 100 μg / mL of BSP (BT300) was added to the cells in the BSP and IR + BSP groups. After treatment, the cells in the IR and IR + BSP groups were irradiated with 4 Gy of X-rays, and the cells were collected 24 hours after irradiation for protein quantification analysis. The Western Blot results of each group of cells are as Figure 2 shown. It can be seen that BSP inhibits apoptosis by blocking the increase of pro-apoptotic Bax protein and the decrease of anti-apoptotic Bcl-2 protein, resulting in the down-regulation of the protein expression of Caspase-8 and Cleaved-Caspase-3, thereby alleviating the damage caused by radiation to cells.

[0066] Test Example 3:

[0067] C57BL / 6J mice aged 6 - 8 weeks (purchased from Suzhou Xinuose Biotechnology Co., Ltd.) with an equal number of males and females were randomly divided into 6 groups, with 12 males and females in each group: blank group (Control), simple irradiation group (IR), which were intragastrically administered 200 mg / kg of normal saline according to body weight every day for 7 days before irradiation; low-dose drug irradiation group (IR + LD), medium-dose drug irradiation group (IR + MD), high-dose drug irradiation group (IR + HD), which were intragastrically administered 100, 200, and 400 mg / kg of BSP respectively according to body weight every day for 7 days before irradiation; positive drug irradiation group (IR + Y, the positive drug is the commercially available Tremella fuciformis spore sugar capsule TP), which was intragastrically administered 600 mg / kg of TP according to body weight every day for 7 days before irradiation. The body weight changes of the mice were observed and recorded. On the 7th day, the whole abdomen of the mice was irradiated with 10 Gy, and the mice were sacrificed 3.5 days later. The small intestine, liver, and spleen were taken. The body weight changes, liver index, and spleen index of the mice were statistically analyzed. Pathological section staining analysis of the small intestine of the mice, immunohistochemical analysis of Ki67, Lgr5, γ-H2AX, and immunofluorescence analysis of ZO-1 were performed. The expression of proteins related to the inflammatory pathway and the content of inflammatory factors in the mice were analyzed by Western Blot experiment and ELISA experiment. The feces of the above-mentioned male mice were subjected to 16S rRNA intestinal flora sequencing to analyze the relationship between inflammatory factors and intestinal flora dysregulation.

[0068] (1) H&E pathological section staining was performed on the intestinal tissues of the mice in each group to confirm whether BSP has a protective effect on the small intestinal villi of RII model mice. The results are as Figure 3 shown. According to Figure 3The H&E staining results of A in [context] were compared between the blank group and the IR group. After induction with 10 Gy, the intestinal villi in the small intestine tissue were shortened and the crypts were lost. The positive drug TP could provide good protection for the intestinal villi and crypts. BSP also had a protective effect on the damage of intestinal villi and crypts induced by RII. BSP in the IR+LD and IR+MD groups could maintain the length of the small intestinal villi and retain the crypt structure, and the effect of IR+MD was close to that of the positive drug IR+Y group. The shortening of the intestinal villi in the IR+HD group of mice was more severe and the crypt loss was serious.

[0069] According to the H&E staining results, the length of the small intestinal villi was quantitatively counted ( Figure 3 as shown in B of [context]. After radiation injury, the length of the intestinal villi in both male and female mice was significantly shortened (P < 0.05), and the shortening was more obvious in male mice (P < 0.001). The positive drug TP had a protective effect on the intestinal villi of mice of different genders (P < 0.05). The villus length of mice in the IR+LD and IR+MD groups was extended to a certain extent compared with the IR group, and the effect of IR+MD on male mice was the best (P < 0.05). In addition, the H&E staining results were also consistent with the macroscopic morphology of the small intestinal specimens. The visible permeability of the small intestine in the IR group of mice increased, the intestinal wall became thinner and the contents could even be observed with the naked eye, while the visible permeability decreased in the blank group, IR+MD group and IR+Y group.

[0070] (2) To explore the effects of BSP on RII-induced intestinal epithelial cells and intestinal stem cells, immunohistochemical staining of Ki67 and Lgr5 was performed on the intestinal tissues of mice in each group, and the results were as Figure 4 shown in A of [context]. As Figure 4 shown in B of [context], after irradiation with 10 Gy, the number of Ki67+ in the small intestinal tissue decreased significantly (P < 0.05). Compared with the IR group, the number of Ki67+ in the IR+LD and IR+MD groups of male mice was maintained, and the IR+MD group had statistical significance (P < 0.01). Low and medium doses of BSP could also maintain the number of Ki67 + in female mice (P < 0.001), while BSP in the IR+HD group did not show a good radiation protection effect. By analyzing Figure 4 C of [context], it was found that after irradiation with 10 Gy, the number of Lgr5 + in the small intestinal tissue also decreased significantly (P < 0.05). The number of Lgr5 + in the IR+LD and IR+MD groups of mice of different genders was maintained (P < 0.001). It should be noted that for female mice, high-dose BSP could also protect against the damage of intestinal stem cells induced by RIII (P < 0.001).

[0071] Based on the results of combined H&E staining and immunohistochemistry, it can be concluded that BSP can reduce radiation-induced intestinal villus damage and stem cell damage in mice and maintain cell regeneration ability.

[0072] (3) To explore whether BSP has a protective effect on the DNA of RII mice induced by 10 Gy of ionizing radiation, an immunohistochemistry experiment of γ-H2AX was designed, and the results are as Figure 5 shown. By comparing the Control group and the IR group of mice of different genders, it can be found that the number of γ-H2AX + increased after ionizing radiation treatment (P < 0.01), indicating that ionizing radiation caused relatively severe damage to the DNA of mouse intestinal tissue cells. Different concentrations of BSP can reduce the DNA damage induced by ionizing radiation in mice. For male mice, the IR+LD group (P < 0.001) and the IR+MD group (P < 0.05) were statistically significant. For female mice, both the IR+LD group and the IR+MD group were more significantly statistically significant (P < 0.001). It can be concluded that BSP has a protective effect on the DNA damage of mice caused by ionizing radiation.

[0073] (4) A Western Blot experiment was designed to explore the signal transduction of the NF-KB pathway, and ELISA was used to measure the concentrations of three inflammatory factors, IL-6, IL-10, and TNF-α, in the intestinal tissues of mice in each group, as well as the SOD values in the intestinal tissues and mouse sera. The results are as Figure 6 shown. After irradiation, the content of p-p65 protein in mice increased (P < 0.001), and different concentrations of BSP could inhibit the expression of p-p65 protein (P < 0.001). By comparing the levels of relevant inflammatory factors in the Control group and the IR group, it can be known that after 10 Gy of irradiation, the levels of two inflammatory factors, IL-6 and TNF-α, in the intestinal tissues of mice increased (P < 0.001), while IL-10, as an immunosuppressive factor, decreased (P < 0.001), indicating that ionizing radiation can cause inflammation in the intestines of mice. The three BSP administration groups showed excellent anti-inflammatory effects on the intestinal tissues of male and female mice (P < 0.001). It should be noted that the expression trend of inflammatory factors in the BSP administration group was different from that of p-p65 protein, and the possible reason is that the regulation of inflammatory factor expression involves multiple pathways.

[0074] SOD is a classic endogenous antioxidant enzyme that can regulate the inflammatory response by scavenging free radicals. By comparing the SOD in the sera and intestinal tissues of the blank group and the IR group of mice ( Figure 7) It was found that ionizing radiation could lead to a decrease in the SOD value (P < 0.01), thereby affecting the ability of mouse intestinal tissue to scavenge free radicals and further causing the occurrence of inflammatory reactions in mouse intestinal tissue. The relevant results of the BSP administration group proved that BSP was helpful for the recovery of the SOD value in mice. Good results were obtained with medium and high doses of BSP in the intestinal tissues of both male and female mice (P < 0.001), and the SOD value in the serum of mice in the medium-dose group was also much higher than that in the IR group (P < 0.001).

[0075] In summary, BSP can reduce the expression of pro-inflammatory factors IL-6 and TNF-α in intestinal tissue induced by RIII by regulating the NF-KB pathway, increase IL-10, and its mechanism of action is also related to factors such as regulating the SOD value in the body.

[0076] (5) Extract the fecal DNA of each group of rats, construct a library and sequence, and this process is carried out by Hangzhou Lianchuan Biotechnology Co., Ltd. As Figure 8 shown in A, 30 species classifications with the highest abundances at the phylum level were selected, and the Firmicutes, Bacteroidetes, and Proteobacteria among them were quantitatively statistically compared, and the results are shown in Figure 8 B-D. It can be seen that BSP can inhibit the number of Proteobacteria, and BSP at different administration doses can play a good inhibitory role.

[0077] Figure 9 shows the species distribution at the genus level. Compared with the blank group, the number of Akkermansia bacteria in the intestinal flora of mice in the IR group treated with ionizing radiation increased. Lachnospiraceae_NK4A136_group is a potentially beneficial bacterium, and its abundance decreased after radiation. The number of Akkermansia bacteria in mice in the IR+MD and IR+HD groups was significantly inhibited, and at the same time, the abundance of Lachnospiraceae bacterium was restored, indicating that BSP has a significant impact on these two types of bacteria in this genus.

[0078] From the analysis of the relative abundance heat map ( Figure 10 ), the flora abundance of the medium-dose administration group was closest to that of the Control group, and intestinal symbiotic bacteria such as Duncaniella, Acetatifactor, and kineothrix were well restored compared with the model group and the high- and low-dose administration groups; while the positive drug group showed a trend completely opposite to that of the blank group. In summary, from the perspective of relative abundance, the medium-dose group had the most significant effect, and the intestinal flora of mice with radioactive intestinal injury could be restored to a state close to that of normal mice.

[0079] Obviously, the above embodiments are merely examples for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. Use of glucomannan in the preparation of a product for preventing or treating radiation-induced intestinal injury, characterized in that, The relative molecular weight of the glucomannan is 10-1000 kD; the monosaccharide composition of the glucomannan includes mannose and glucose; the glucomannan is composed of mannose and glucose linked by β-1,4-glycosidic bonds, with a glucose branched-chain structure and acetyl group modification.

2. The application according to claim 1, wherein The relative molecular weight of the glucomannan is 20-500 kD; and / or, the composition ratio of mannose and glucose is 1.4:1-9.0:1; and / or, the purity of the glucomannan is greater than 70%; 3. The application according to claim 1, wherein The glucomannan is derived from one or more of konjac, Bletilla striata and plants of the genus Dendrobium.

4. The application according to claim 1, characterized in that, The mass percentage of glucomannan in the product is 10%-100%.

5. The application according to claim 1, characterized in that, The dosage form of the product is selected from one of oral liquid preparations, granules, tablets, capsules, injections and freeze-dried powder injections.

6. The application according to claim 1, characterized in that, The product includes one or more of drugs, foods, health foods and special medical foods.

7. The application according to claim 6, wherein The administration route of the drug is selected from oral administration, intravenous injection or enema.

8. The application according to claim 6, characterized in that, The drug also includes a pharmaceutically or pharmacologically acceptable carrier and / or salt.

9. The application according to claim 1, wherein The site of intestinal injury is selected from one or more of the small intestine, colon and rectum.

10. The application according to claim 1, characterized in that, The radiation source in the radiation-induced intestinal injury is selected from x-rays and / or γ-rays.

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