Application of schizandrin B in preparation of medicine for preventing or treating intestinal radiation injury

Abstract

The invention relates to the field of medicines, in particular to application of schisandrin B in preparation of medicines for preventing or treating intestinal radiation injury. The invention finds the protective effect of the schisandrin B on the irradiated intestinal tract, and experiments prove that the schisandrin B can reduce the pathological injury degree of the irradiated intestinal tract, including increasing the villus length of the intestinal tract and the number of crypts, maintaining the stability of the intestinal mucosal barrier, reducing the inflammatory factor content of the intestinal tract tissue, and improving the intestinal tract tissue. The activity of the irradiated intestinal epithelial cells is recovered, and the apoptosis of the intestinal epithelial cells is inhibited. Meanwhile, schisandrin B can relieve ferroptosis caused by irradiation. Therefore, the schizandrin B has a wide application prospect in treatment of the radioactive intestinal injury and drug development as a potential radiation protective agent.

Description

Technical Field

[0001] The invention relates to the field of medicine, in particular to the application of schisandrae alcohol B, an effective ingredient in schisandrae chinensis, in the preparation of medicine for preventing or treating intestinal radiation damage. Background Art

[0002] Radiation-induced intestinal injury (RIII) is an important cause of death in nuclear radiation accidents and a common complication of radiotherapy for cancer patients. RIII is mainly manifested by necrosis and shedding of the small intestinal mucosal epithelium, apoptosis and necrosis of crypt cells, reduction in the number of crypts, mucosal inflammation, and intestinal flora ectopic. Currently, most drugs for the prevention and treatment of radiation-induced intestinal injury are in the research stage. Most drugs face problems such as unclear mechanisms and high toxicity. In addition, the only anti-radiation drug approved by the FDA, amifostine (WR-2721), has problems such as high toxicity and short half-life, which greatly limits its clinical use. So far, the treatment for intestinal damage caused by ionizing radiation has not been met, and the medical needs are urgent.

[0003] Schisandrol B is one of the main active ingredients of Schisandra chinensis. Schisandrol B has a certain protective effect on the heart, lungs and kidneys. Studies have shown that Schisandrol B can alleviate acetaminophen-induced acute liver injury, promote liver regeneration after partial hepatectomy, increase hepatocyte size and promote liver regeneration. However, there has been no systematic study on the prevention and treatment of radiation-induced intestinal injury by Schisandrol B. Summary of the invention

[0004] The purpose of the present invention is to provide a new use of Schisandrin B, namely, its use in preventing or treating radiation-induced intestinal injury, so as to achieve effective treatment of radiation-induced intestinal injury.

[0005] In order to achieve the above-mentioned object, the first aspect of the present invention provides the use of Schisandrin B in the preparation of a drug for preventing or treating intestinal radiation damage.

[0006] Furthermore, the drug for preventing or treating intestinal radiation damage is a drug composition with schisandrae alcohol B as the only active monomer component, or schisandrae alcohol B and at least one pharmaceutically permitted excipient.

[0007] Furthermore, the purity of Schisandrin B in the drug for preventing or treating intestinal radiation damage is not less than 99.9%.

[0008] Furthermore, the dosage of Schisandrin B in the drug for preventing or treating intestinal radiation damage is 25 mg / kg.

[0009] Furthermore, the drug for preventing or treating intestinal radiation damage is an intravenous injection.

[0010] Furthermore, the site of intestinal radiation damage is selected from at least one of the small intestine, colon, and rectum.

[0011] Furthermore, the radiation in the intestinal radiation damage is selected from X-rays.

[0012] Furthermore, the drug for preventing or treating intestinal radiation damage is a drug that increases the length of intestinal villi and the number of crypts, maintains the stability of the intestinal mucosal barrier, reduces the content of inflammatory factors in intestinal tissue, restores the vitality of intestinal epithelial cells after irradiation, inhibits their apoptosis, and reduces ferroptosis caused by radiation.

[0013] The second aspect of the present invention provides a drug for preventing or treating intestinal radiation damage, wherein the drug for preventing or treating intestinal radiation damage comprises schisandra chinensis alcohol B as the only active monomer component, or schisandra chinensis alcohol B and at least one pharmaceutically permitted excipient constitute a pharmaceutical composition.

[0014] Schisandrin B, as one of the important active ingredients of Schisandra chinensis, has been proven to have biological functions such as antioxidant, anti-stress, and anti-inflammatory.

[0015] The invention discloses an application of schisandrin B in the preparation of a drug for preventing or treating radiation-induced intestinal injury. By establishing animal and cell models of radiation-induced intestinal injury, the protective effect of schisandrin B on the irradiated intestine and its possible mechanism are explored. After ionizing irradiation, schisandrin B can increase the expression of intestinal tight junction proteins, protect the intestinal barrier, reduce DNA damage, reduce inflammatory factors in intestinal tissue, inhibit apoptosis of intestinal cells, and improve the vitality of intestinal epithelial cells after irradiation. The invention has potential application value in drugs for preventing and treating radiation-induced intestinal injury.

[0016] The present invention has the advantages that:

[0017] The present invention has found the protective effect of Schisandrin B on intestinal tissue after irradiation, and confirmed that it can reduce the degree of intestinal pathological damage after irradiation, maintain the stability of membrane barrier, reduce DNA damage, reduce the content of inflammatory factors (IL-1β, TNF-α) in intestinal tissue, restore the vitality of intestinal epithelial cells after irradiation, and inhibit their apoptosis. At the same time, Schisandrin B can also reduce the iron death of intestinal cells caused by radiation. Therefore, Schisandrin B has a wide range of application prospects in the treatment of radiation-induced intestinal injury and drug development as a potential radiation protector. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the results of the radiation protective effect of schisandrin B on intestinal cell lines.

[0019] Figure 2 This is a schematic diagram of the results of the radiation protective effect of schisandra chinensis alcohol B on intestinal tissue.

[0020] Figure 3 This is a schematic diagram of the protective effect of schisandrin B on the intestinal mucosal barrier after irradiation.

[0021] Figure 4 This is a schematic diagram of the effects of Schisandrin B on intestinal inflammation after irradiation.

[0022] Figure 5 This is a schematic diagram of the results showing that the protective effect of schisandra chinensis alcohol B on radiation-induced intestinal injury involves the Nrf2 pathway.

[0023] Figure 6 This is a schematic diagram of the inhibitory effect of schisandrin B on radiation-induced ferroptosis. DETAILED DESCRIPTION

[0024] The specific implementation methods provided by the present invention are described in detail below in conjunction with examples.

[0025] The schisandrae alcohol B used in the experiment in the embodiment of the present invention was purchased from MCE. The male C57BL / 6J mice used in the experiment were 6-8 weeks old, weighing about 20±3g, SPF-level management, and purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The room temperature was about 20-25°C, and distilled water and standard mouse food were freely available. The experimental process and related operations were approved by the Experimental Animal Ethics Center of the Academy of Military Medical Sciences (IACUC-DWZX-2021-557).

[0026] Experimental Example 1: Radioprotective effect of Schisandrin B on intestinal cell lines

[0027] 1. Experimental Grouping and Drug Administration

[0028] Cell experiment: divided into normal group Control, irradiation group IR, irradiation + schisandrae B group IR + Sol B. Normal group: no treatment of cells; irradiation group: irradiation of cells by X-rays; irradiation + schisandrae B group: cells were stimulated with schisandrae B 12h before irradiation.

[0029] 2. Experimental Methods

[0030] (1) In vitro culture of NCM460 and IEC-6 cells

[0031] The cells were cultured in DMEM medium supplemented with 10% (v / v) fetal bovine serum, 100μ / mL penicillin and 100μ / mL streptomycin. The cells were passaged at 37°C and 5% CO2 until they were 80% to 90% confluent. The culture medium in the culture vessel was removed by pouring out, the cells were washed once with PBS, the PBS was discarded, an appropriate amount of 0.1% trypsin solution was added according to the culture area for digestion, the cells were placed at 37°C for 1 min, and observed under an inverted phase contrast microscope. When most of the cells became round and suspended, the trypsin was discarded and the culture medium was added to terminate the digestion. The cells were dispersed into clusters by blowing and aspirating several times, and the cells were passaged at a ratio of 1:3. The cells after passage were changed once after 24 hours.

[0032] (2) CCK8 detection of cell viability

[0033] Cells were seeded into 96-well plates, with 5 × 10 cells per well. 3 12h before irradiation, schisandrin B (2.5μM, 5μM, 10μM, 20μM) was given to stimulate. Then the cells were irradiated with 4Gy, placed in the incubator for 24h, and then 10μL CCK8 detection solution was added. The culture was continued for 2.5h, and finally the OD value was detected by an enzyme marker at a wavelength of 450-490nm. The cell survival rate was calculated as follows:

[0034] Cell survival rate = [(As-Ab)] / [(Ac-Ab)]×100%

[0035] As experimental wells: culture medium containing cells, CCK8, and schisandrin B;

[0036] Ac control wells: medium containing cells, CCK8, and no schisandrin B;

[0037] Ab blank wells: medium without cells and Schisandrin B, CCK8.

[0038] (3) Calcein AM / PI live-dead double staining to observe cell live-dead

[0039] Cells were seeded into 6-well plates and stimulated with 10 μM and 20 μM schisandrin B 12 h before irradiation. 24 h after 4 Gy irradiation, cells were stained with Calcein AM and PI. Cells were then observed using an Agilent BioTekCytation 5 according to the manufacturer's instructions.

[0040] (4) Flow cytometry to detect cell viability

[0041] Cells were seeded into 6-well plates and stimulated with 20 μM schisandrin B 12 h before irradiation. 24 h after 4 Gy irradiation, cells were stained with Calcein AM and PI. Cells were then analyzed using a flow cytometer (FACSAria III) according to the manufacturer's instructions.

[0042] 3. Experimental results:

[0043] like Figure 1 As shown, Figure 1 This is a schematic diagram of the radiation protective effect of schisandrin B on intestinal cell lines.

[0044] The CCK8 method was used to detect the effects of different concentrations of schisandrin B on cell viability under a 4Gy irradiation dose. Figure 1 A and B are schematic diagrams of using CCK-8 method to detect the effect of Schisandrin B on the viability of irradiated NCM460 and IEC-6 cells. Figure 1 As can be seen in A, 2.5, 5, 10, and 20 μM concentrations of Schisandrin B can promote the viability of NCM460 cells after 4 Gy irradiation. Figure 1 As can be seen in B, schisandrin B in the concentration range of 10 and 20 μM can promote the viability of IEC-6 cells after 4 Gy irradiation.

[0045] Calcein AM / PI live-dead double staining was used to detect the protective effect of Schisandrin B on irradiated NCM460 and IEC-6 cells. Figure 1 C, D Schematic diagram of the effect of Schisandrin B on NCM460 and IEC-6 cells using Calcein AM / PI live-dead double staining. Figure 1 As can be seen from Figure C, 20 μM concentration of Schisandrin B can significantly inhibit the cell death of NCM460 cells after 4 Gy irradiation. Figure 1 As can be seen in D, 20 μM concentration of schisandrin B can significantly inhibit the death of IEC-6 cells after 4 Gy irradiation.

[0046] Flow cytometry was used to detect the protective effect of schisandrin B on irradiated NCM460 and IEC-6 cells. Figure 1 E, F, G, H Schematic diagram of flow cytometry to detect the effect of Schisandrin B on cell survival. Figure 1 As can be seen from E and F, 20 μM concentration of schisandrin B can significantly inhibit the death of NCM460 cells after 4 Gy irradiation. Figure 1 As can be seen from G and H, 20 μM concentration of schisandrin B can significantly inhibit the death of IEC-6 cells after 4 Gy irradiation.

[0047] Example 2: Radioprotective effect of Schisandrin B on intestinal tissue

[0048] 1. Experimental Grouping and Drug Administration

[0049] Animal experiment: divided into normal group, irradiation group, irradiation + IR + Sol B. Normal group: 200 μL normal saline was injected into the abdomen of mice every day; irradiation group: irradiated by X-ray after 3 consecutive days of normal saline abdominal injection; irradiation + IR + Sol B group: irradiated by X-ray after 3 consecutive days of IR + Sol B abdominal injection.

[0050] 2. Experimental Methods

[0051] Extraction of intestinal tissue: 6-8 week old C57BL / 6J mice were killed by cervical dislocation, and the abdominal cavity of the mice was cut open with high temperature sterilized ophthalmic scissors. The stomach and 3 cm small intestine and colon below the cecum of the mice were taken and fixed in 4% paraformaldehyde.

[0052] 3. Experimental results

[0053] like Figure 2 As shown, Figure 2 This is a schematic diagram of the results of the radiation protective effect of schisandra chinensis alcohol B on intestinal tissue. Figure 2 In the figure, A and B are schematic diagrams showing the effect of schisandrin B on apoptosis of the small intestine after irradiation. C and D are schematic diagrams showing the effect of schisandrin B on apoptosis of the colon after irradiation. E and F are schematic diagrams showing the effect of schisandrin B on DNA damage of the small intestine after irradiation. G and H are schematic diagrams showing the effect of schisandrin B on DNA damage of the colon after irradiation.

[0054] Figure 1 A and B show that schisandrin B can inhibit the apoptosis of small intestinal tissue after irradiation. Figure 1 C and D show that schisandrin B can inhibit the apoptosis of colon tissue after irradiation. Figure 1 E and F show that schisandrin B can improve DNA damage in small intestine tissue after irradiation. Figure 1 G and H showed that schisandrin B could improve DNA damage in colon tissue after irradiation.

[0055] Example 3: Protective effect of schisandrae alcohol B on intestinal mucosal barrier after irradiation

[0056] 1. Experimental Grouping and Drug Administration

[0057] Animal experiment: divided into normal group, irradiation group, irradiation + IR + Sol B. Normal group: 200 μL normal saline was injected into the abdomen of mice every day; irradiation group: irradiated by X-ray after 3 consecutive days of normal saline abdominal injection; irradiation + IR + Sol B group: irradiated by X-ray after 3 consecutive days of IR + Sol B abdominal injection.

[0058] 2. Experimental Methods

[0059] Immunofluorescence experiments:

[0060] 1) Take out the glass-bottomed culture dish, discard the culture medium and wash three times with sterile PBS (be careful to keep the matrix gel intact), then fix with 4% paraformaldehyde for 30 minutes;

[0061] 2) Aspirate the fixative, wash three times with PBS (be careful not to aspirate the small intestinal organoids), add 0.5% Triton for 20 minutes, and then block with 1% bovine serum albumin (BSA, prepared in PBS) for 90 minutes;

[0062] 3) After discarding BSA, wash with PBS three times, add primary antibody (anti-TLR4, diluted with 1% BSA, 1:250), and incubate the antibody at 4°C overnight;

[0063] 4) Aspirate and discard the primary antibody, wash three times with PBS, add fluorescent secondary antibody (diluted with 1% BSA, 1:1000), incubate for 90 minutes, wash three times with PBS, add DAPI staining solution and incubate for 30 minutes, then drop glycerol into the slides, and then observe the staining under a fluorescence microscope and take fluorescent pictures;

[0064] 3. Experimental results:

[0065] Figure 3 This is a schematic diagram of the effects of schisandrin B on intestinal tight junction proteins. Figure 3 In the figure, A, B, C, and D are schematic diagrams of detecting the expression of ZO-1 protein in the intestinal tissue of mice by immunofluorescence. E, F, G, and H are schematic diagrams of detecting the expression of ZO-1 protein in the intestinal tissue of mice by immunofluorescence. I and J are schematic diagrams of detecting the LPS content in the serum of mice by ELISA.

[0066] The intestinal mucosal barrier is an important defense against the invasion of pathogenic bacteria, and irradiation greatly damages the intestinal mucosal barrier. ZO-1 and Occludin are two intestinal tight junction proteins. The immunofluorescence method of ZO-1 and Occludin was used to evaluate the effect of Schisandrin B on the intestinal mucosal barrier. Figure 3 As shown in A, B, C, and D, the expression of Occludin in the IR+Sol B group was significantly higher than that in the IR group; Figure 3 As shown in Figures E, F, G, and H, the expression of ZO-1 in the IR+Sol B group was significantly higher than that in the IR group, which means that schisandrin B can alleviate the damage of the intestinal mucosal barrier after irradiation. Figure 3 As shown in I and J, the serum LPS level in the IR group was significantly higher than that in the IR+Sol B group, which indirectly indicated that Schisandrin B could alleviate the damage of the intestinal mucosal barrier after irradiation.

[0067] Example 4: Schisandrin B reduces intestinal inflammation after irradiation

[0068] 1. Experimental Grouping and Drug Administration

[0069] Animal experiment: divided into normal group, irradiation group, irradiation + IR + Sol B. Normal group: 200 μL normal saline was injected into the abdomen of mice every day; irradiation group: irradiated by X-ray after 3 consecutive days of normal saline abdominal injection; irradiation + IR + Sol B group: irradiated by X-ray after 3 consecutive days of IR + Sol B abdominal injection.

[0070] 2. Experimental Methods

[0071] ELISA test method:

[0072] 1) Add the antigen or antibody solution to be detected to the microplate, and form a layer of fixed antigen or antibody on the surface of the microplate.

[0073] 2) Add blocking agents to reduce nonspecific binding and lower background signals.

[0074] 3) The sample to be tested is added to the microplate, and the target antigen or antibody in the sample interacts with the antibody or antigen fixed on the well plate.

[0075] 4) Remove unbound material by repeatedly washing the plate.

[0076] 5) Add an enzyme-labeled secondary antibody (or enzyme-labeled antigen) that specifically binds to the object to be detected, so that it binds to the target object in the sample.

[0077] 6) Wash the plate again to remove unbound enzyme markers.

[0078] 7) Add a substrate that interacts with the enzyme label to produce a measurable signal.

[0079] 8) Add a stop agent to stop the reaction of the substrate and prevent further increase in the signal.

[0080] 9) Use a spectrometer or fluorescence meter to measure the signal generated by the substrate reaction, and determine the concentration of the target object in the sample based on the intensity of the signal.

[0081] 3. Experimental results:

[0082] Figure 4 This is a schematic diagram of the results of schisandrae alcohol B alleviating intestinal inflammation after irradiation. Figure 4 In the figure, A, B, C, and D are schematic diagrams of the contents of pro-inflammatory factors IL-1β, IL-18, IL-6, and TNF-ɑ in the small intestine and colon 12 h and 24 h after irradiation, respectively.

[0083] like Figure 4A, B, C, D, 12h and 24h after irradiation, the pro-inflammatory factors IL-1β, IL-18, IL-6, and TNF-ɑ in the small intestine and colon decreased significantly, indicating that schisandrin B can significantly reduce intestinal inflammation after irradiation.

[0084] Example 5: The protective effect of Schisandrin B on radiation-induced intestinal injury involves the Nrf2 pathway

[0085] 1. Experimental Grouping and Drug Administration

[0086] Animal experiment: divided into normal group, irradiation group, irradiation + IR + Sol B. Normal group: 200 μL normal saline was injected into the abdomen of mice every day; irradiation group: irradiated by X-ray after 3 consecutive days of normal saline abdominal injection; irradiation + IR + Sol B group: irradiated by X-ray after 3 consecutive days of IR + Sol B abdominal injection.

[0087] Cell experiment: divided into normal group Control, irradiation group IR, irradiation IR+ML385 group, irradiation+Schisandrin B group IR+Sol B, irradiation+Schisandrin B group IR+Sol B+ML385 group. Normal group: no treatment was done on the cells; irradiation group: the cells were irradiated by X-rays; irradiation IR+ML385 group: ML385 was used for stimulation 12h before irradiation. Irradiation+Schisandrin B group: Schisandrin B was used for stimulation 12h before irradiation. Irradiation+Schisandrin B group IR+Sol B+ML385 group: Schisandrin B and ML385 were used for stimulation 12h before irradiation.

[0088] 2. Experimental Methods

[0089] (1) Immunofluorescence experiment:

[0090] 1) Add the antigen or antibody solution to be detected to the microplate, and form a layer of fixed antigen or antibody on the surface of the microplate.

[0091] 2) Add blocking agents to reduce nonspecific binding and lower background signals.

[0092] 3) The sample to be tested is added to the microplate, and the target antigen or antibody in the sample interacts with the antibody or antigen fixed on the well plate.

[0093] 4) Remove unbound material by repeatedly washing the plate.

[0094] 5) Add an enzyme-labeled secondary antibody (or enzyme-labeled antigen) that specifically binds to the object to be detected, so that it binds to the target object in the sample.

[0095] 6) Wash the plate again to remove unbound enzyme markers.

[0096] 7) Add a substrate that interacts with the enzyme label to produce a measurable signal.

[0097] 8) Add a stop agent to stop the reaction of the substrate and prevent further increase in the signal.

[0098] 9) Use a spectrometer or fluorescence meter to measure the signal generated by the substrate reaction, and determine the concentration of the target object in the sample based on the intensity of the signal.

[0099] (2) Calcein AM / PI live-dead double staining to observe cell live-dead

[0100] Cells were seeded into 6-well plates and stained with Calcein AM and PI. Cells were then visualized using an Agilent BioTek Cytation 5 according to the manufacturer's instructions.

[0101] 3. Experimental results:

[0102] Figure 5 This is a schematic diagram of the results that the protective effect of schisandra chinensis B on radiation-induced intestinal injury involves the Nrf2 pathway. Figure 4 In the figure, A, B, C, and D are schematic diagrams of Nrf2 expression in the small intestine and colon 24 hours after irradiation. E and F are schematic diagrams of Calcein AM / PI live-dead double staining results of NCM46 and IEC-6, respectively.

[0103] like Figure 5 A, B, C, D, 24 hours after irradiation, the expression of Nrf2 in the small intestine and colon increased significantly. Figure 5 E, F, compared with the irradiation + schisandrin B group, the cell viability of the IR + Sol B + ML385 group was significantly decreased, indicating that the intestinal radiation protection effect of schisandrin B may involve the Nrf2 pathway.

[0104] Example 6: Inhibitory effect of schisandrae alcohol B on irradiation-induced ferroptosis

[0105] 1. Experimental Grouping and Drug Administration

[0106] Animal experiment: divided into normal group, irradiation group, irradiation + Schisandrin B IR + Sol B group, irradiation + Schisandrin B IR + Sol B + ML385 group. Normal group: 200 μL normal saline was injected into the abdomen of mice every day; irradiation group: irradiation was performed by X-ray after 3 consecutive days of abdominal injection of normal saline; irradiation + Schisandrin B group: irradiation was performed by X-ray after 3 consecutive days of abdominal injection of Schisandrin B; irradiation + Schisandrin B IR + Sol B + ML385 group: irradiation was performed by X-ray after 3 consecutive days of abdominal injection of Schisandrin B + ML385.

[0107] Cell experiment: divided into normal group Control, irradiation group IR, irradiation IR+ML385 group, irradiation+Schisandrin B group IR+Sol B, irradiation+Schisandrin B group IR+Sol B+ML385 group. Normal group: no treatment was done on the cells; irradiation group: the cells were irradiated by X-rays; irradiation IR+ML385 group: ML385 was used for stimulation 12h before irradiation. Irradiation+Schisandrin B group: Schisandrin B was used for stimulation 12h before irradiation. Irradiation+Schisandrin B group IR+Sol B+ML385 group: Schisandrin B and ML385 were used for stimulation 12h before irradiation.

[0108] 2. Experimental Methods

[0109] (1) Small intestinal tissue immunohistochemistry

[0110] 1) The small intestinal tissue was fixed with 4% paraformaldehyde for 1 day, then replaced with 70% ethanol to preserve the tissue, and then the tissue was trimmed into 5 mm tissue blocks and placed in an embedding box;

[0111] 2) The tissue was dehydrated by soaking in 70% ethanol, 85% ethanol, 90% ethanol, 95% ethanol and anhydrous ethanol for 60 min, then treated with dewaxing solution I, dewaxing solution II and dewaxing solution III for 30 min, and then treated with paraffin I, paraffin II and paraffin III for 60 min;

[0112] 3) After the tissue was embedded with an embedding machine, the paraffin sections were cut into 3 μm, and then the paraffin sections were dewaxed. The process was as follows: baking (60°C, 120 min) → soaking in dewaxing solution for 5 min (xylene II, xylene I, alcohol + xylene, 100% alcohol II, 100% alcohol I, 95% alcohol, 90% alcohol, 85% alcohol, 70% alcohol) → washing with distilled water twice;

[0113] 4) Antigen retrieval was performed using EDTA (pH 9.0) at 121°C (treatment for 2 min), and the sections were washed twice with PBS after natural cooling, and then blocked with hydrogen peroxide and 1% BSA in sequence (washed twice with PBS in between), and then incubated with primary antibody (prepared with 1% BSA) at 4°C overnight;

[0114] 5) After incubation with primary antibody overnight, the primary antibody was discarded and the slides were washed three times with PBS. Then, the slides were incubated with horseradish peroxidase-labeled secondary antibody for 90 min and washed three times with PBS. Then, the slides were developed with 3-diaminobenzidine (DAB), counterstained with hematoxylin, and washed to blue. Finally, the slides were dehydrated and sealed with neutral gum.

[0115] (2)Ferrorange dyeing

[0116] Cells were seeded into 6-well plates and stained with Ferrorange. Cells were then visualized using an Agilent BioTek Cytation 5 according to the manufacturer's instructions.

[0117] (3) LPO detection

[0118] Cells were seeded into 6-well plates and stained with Lipid Peroxidation Probe-BDP 581 / 591C11. Cells were then visualized using Agilent BioTek Cytation5 according to the manufacturer's instructions.

[0119] (4) Calcein AM / PI live-dead double staining to observe cell live-dead

[0120] Cells were seeded into 6-well plates and stained with Calcein AM and PI. Cells were then visualized using an Agilent BioTek Cytation 5 according to the manufacturer's instructions.

[0121] 3. Experimental results:

[0122] Figure 6 This is a schematic diagram of the results of schisandrin B inhibiting GPX4-mediated ferroptosis through the Nrf2 pathway. Figure 6 In the figure, A, B, are schematic diagrams of GPX4 expression in the small intestine and colon 24 hours after irradiation. C and D are schematic diagrams of iron ion fluorescence in NCM460 and IEC-6 cells after irradiation, E and F are schematic diagrams of lipid peroxidation in NCM460 and IEC-6 cells, and G and H are schematic diagrams of Calcein AM / PI live-dead double staining results in NCM460 and IEC-6 cells.

[0123] like Figure 6 A, B, 24 h after irradiation, the expression of GPX4 in the small intestine and colon increased significantly, and after administration of ML385, the expression of GPX4 decreased. Figure 6 C, D, the fluorescence of iron ions in the irradiation + schisandrin B group (IR + Sol B) + ML385 group increased significantly compared with the irradiation + schisandrin B group. Figure 6 Compared with the irradiation + schisandrin B group, the degree of lipid peroxidation in the irradiation + schisandrin B group and the IR + Sol B + ML385 group was significantly increased, as shown in Figures G and H. Compared with the irradiation + schisandrin B group, the cell viability in the irradiation + schisandrin B group and the IR + Sol B + ML385 group was significantly decreased, indicating that schisandrin B may inhibit GPX4-mediated ferroptosis through the Nrf2 pathway.

[0124] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Application of Schisandrin B in the preparation of drugs for preventing or treating intestinal radiation damage.

2. The use of Schisandrin B according to claim 1 in the preparation of a drug for preventing or treating intestinal radiation damage, characterized in that: The drug for preventing or treating intestinal radiation damage uses schisandra chinensis alcohol B as the only active monomer component, or schisandra chinensis alcohol B and at least one pharmaceutically permitted auxiliary material to form a pharmaceutical composition.

3. The use of Schisandrin B according to claim 1 in the preparation of a drug for preventing or treating intestinal radiation damage, characterized in that: The purity of schisandra chinensis alcohol B in the medicine for preventing or treating intestinal radiation damage is not less than 99.9%.

4. The use of Schisandrin B according to claim 1 in the preparation of a drug for preventing or treating intestinal radiation damage, characterized in that: The dosage of Schisandrin B in the medicine for preventing or treating intestinal radiation damage is 25 mg / kg.

5. The use of Schisandrin B according to claim 1 in the preparation of a drug for preventing or treating intestinal radiation damage, characterized in that: The medicine for preventing or treating intestinal radiation damage is an intravenous injection.

6. The use of Schisandrin B according to claim 1 in the preparation of a drug for preventing or treating intestinal radiation damage, characterized in that: The site of intestinal radiation damage is selected from at least one of the small intestine, colon and rectum.

7. The use of Schisandrin B according to claim 1 in the preparation of a drug for preventing or treating intestinal radiation damage, characterized in that: The radiation in the intestinal radiation damage is selected from X-rays.

8. The use of Schisandrin B according to claim 1 in the preparation of a drug for preventing or treating intestinal radiation damage, characterized in that: The drug for preventing or treating intestinal radiation damage is a drug that increases the length of intestinal villi and the number of crypts, maintains the stability of the intestinal mucosal barrier, reduces the content of inflammatory factors in intestinal tissue, restores the vitality of intestinal epithelial cells after irradiation, inhibits their apoptosis, and reduces ferroptosis caused by radiation.

9. A drug for preventing or treating intestinal radiation damage, characterized in that: The drug for preventing or treating intestinal radiation damage uses schisandrae alcohol B as the only active monomer component, or schisandrae alcohol B and at least one pharmaceutically permitted auxiliary material to form a drug composition.

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