Use of schisandrol B in the preparation of a medicine for preventing or treating radiation damage of the intestinal tract

Abstract

The present application relates to the field of medicine, and particularly to the application of schisantherin B in the preparation of a medicine for preventing or treating radiation damage of the intestinal tract. The present application finds that schisantherin B has a protective effect on the intestinal tract after irradiation, and experiments prove that schisantherin B can reduce the pathological damage degree of the intestinal tract after irradiation, including increasing the length of intestinal villi and the number of crypts, maintaining the stability of the intestinal mucosal barrier, reducing the content of inflammatory factors in the intestinal tissue, restoring the activity of the intestinal epithelial cells after irradiation, and inhibiting the apoptosis. Meanwhile, schisantherin B can reduce the iron death caused by irradiation. Therefore, schisantherin B has a wide application prospect as a potential radiation protection agent in the treatment of radiation-induced intestinal injury and drug development.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, specifically to the application of schisandrin ethyl, an active ingredient in Schisandra chinensis, in the preparation of drugs for the prevention or treatment of intestinal radiation damage. Background Technology

[0002] Radiation-induced intestinal injury (RIII) is a significant cause of death in nuclear radiation accidents and a common complication of radiotherapy for cancer patients. RIII is primarily characterized by necrosis and sloughing of the small intestinal mucosa epithelium, crypt cell apoptosis and necrosis, decreased crypt number, mucosal inflammation, and intestinal flora heterotosis. Currently, most drugs for the prevention and treatment of radiation-induced intestinal injury are still in the research stage. Many drugs face challenges such as unclear mechanisms of action and high toxicity. Furthermore, the only FDA-approved anti-radiation drug, amifostine (WR-2721), suffers from high toxicity and a short half-life, significantly limiting its clinical use. To date, treatment options for intestinal injury caused by ionizing radiation remain unmet, highlighting an urgent medical need.

[0003] Schisandrol B is one of the main active components of Schisandra chinensis. Schisandrol B has certain protective effects 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 further promote liver regeneration. However, to date, there are no systematic research reports on the prevention and treatment of radiation-induced intestinal injury with schisandrol B. Summary of the Invention

[0004] The purpose of this invention is to provide a new use for schisandrin ethyl, namely, in the prevention or treatment of radiation-induced intestinal injury, so as to achieve effective treatment of radiation-induced intestinal injury.

[0005] To achieve the above objectives, a first aspect of the present invention provides the use of schisandrin ethyl in the preparation of a medicament for the prevention or treatment of intestinal radiation damage.

[0006] Furthermore, the drug for preventing or treating intestinal radiation damage is a pharmaceutical composition consisting of schisandrol ethyl as the sole active monomeric component, or schisandrol ethyl and at least one pharmaceutically permissible excipient.

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

[0008] Furthermore, the dosage of schisandrin ethyl 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 the intestinal radiation injury is selected from at least one of the small intestine, colon, and rectum.

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

[0012] Furthermore, the drug for preventing or treating intestinal radiation damage is one 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 irradiation-induced ferroptosis.

[0013] In a second aspect, the present invention provides a drug for preventing or treating intestinal radiation damage, wherein the drug for preventing or treating intestinal radiation damage is a pharmaceutical composition consisting of schisandrol ethyl as the sole active monomeric component, or schisandrol ethyl and at least one pharmaceutically permissible excipient.

[0014] Schisandrin ethyl, one of the important active ingredients of Schisandra chinensis, has been verified to have biological functions such as anti-oxidation, anti-stress, and anti-inflammation.

[0015] This invention relates to the application of schisandrol ethyl in the preparation of drugs for the prevention or treatment of radiation-induced intestinal injury. By establishing animal and cell models of radiation-induced intestinal injury, the protective effect and possible mechanism of schisandrol ethyl on the irradiated intestine are explored. After ionizing irradiation, schisandrol ethyl can increase the expression of intestinal tight junction proteins, protect the intestinal barrier, reduce DNA damage, decrease inflammatory factors in intestinal tissue, inhibit apoptosis of intestinal cells, and improve the vitality of intestinal epithelial cells after irradiation. It has potential application value in drugs for the prevention and treatment of radiation-induced intestinal injury.

[0016] The advantages of this invention are:

[0017] This invention has discovered the protective effect of schisandrin ethyl on irradiated intestinal tissue, confirming that it can reduce the degree of pathological damage to the intestine after irradiation, maintain the stability of the membrane barrier, reduce DNA damage, decrease the content of inflammatory factors (IL-1β, TNF-α) in intestinal tissue, restore the vitality of irradiated intestinal epithelial cells, and inhibit their apoptosis. Simultaneously, schisandrin ethyl can also reduce radiation-induced ferroptosis in intestinal cells. Therefore, schisandrin ethyl has broad application prospects as a potential radiation protectant in the treatment and drug development of radiation-induced intestinal injury. Attached Figure Description

[0018] Figure 1 This is a schematic diagram showing the radiation protection effect of schisandrin ethyl on intestinal cell lines.

[0019] Figure 2 This is a schematic diagram showing the radiation protection effect of schisandrin ethyl on intestinal tissue.

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

[0021] Figure 4 This is a schematic diagram showing the effect of schisandrin ethyl on intestinal inflammation after irradiation.

[0022] Figure 5 This is a schematic diagram showing the protective effect of schisandrin ethyl against radiation-induced intestinal injury involving the Nrf2 pathway.

[0023] Figure 6 This is a schematic diagram showing the inhibitory effect of schisandrin ethyl on irradiated ferrodeogenesis. Detailed Implementation

[0024] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0025] Schisandrin ethyl used in the experiments of this invention was purchased from MCE. Male C57BL / 6J mice, 6-8 weeks old, weighing approximately 20±3g, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., under SPF management. The mice were kept at room temperature of approximately 20-25℃ and provided with free access to distilled water and standard mouse feed. All experimental procedures and related operations were approved by the Laboratory Animal Ethics Center of the Academy of Military Medical Sciences (IACUC-DWZX-2021-557).

[0026] Experimental Example 1: Radiation Protection Effect of Schisandrin B on Intestinal Cell Lines

[0027] 1. Experimental grouping and drug administration

[0028] Cell experiments were divided into three groups: a control group (normal), an irradiation-only group (IR), and an irradiation-plus-schisandrol group (IR+Sol B). The control group received no treatment; the irradiation-only group was irradiated with X-rays; and the irradiation-plus-schisandrol group was stimulated with schisandrol for 12 hours prior to irradiation.

[0029] 2. Experimental Methods

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

[0031] Cells were cultured in DMEM medium supplemented with 10% (v / v) fetal bovine serum, 100 μL / mL penicillin, and 100 μL / mL streptomycin. When the cells reached 80%–90% confluence at 37°C and 5% CO2, they were passaged. The culture medium was discarded from the culture dish, and the cells were washed once with PBS. The PBS was discarded, and an appropriate amount of 0.1% trypsin solution was added according to the culture area for digestion. The cells were incubated at 37°C for 1 min and observed under an inverted phase-contrast microscope. Once most cells became rounded and suspended, the trypsin was discarded, and culture medium was added to stop the digestion. The cells were separated by aspiration and respiration several times. Cells were passaged at a 1:3 ratio, and the medium was changed once every 24 hours after passage.

[0032] (2) CCK8 assay for cell viability

[0033] Cells were seeded into 96-well plates, with 5 × 10⁶ cells per well. 3 Cells were stimulated with schisandrin ethyl alcohol (2.5 μM, 5 μM, 10 μM, 20 μM) 12 h before irradiation. Then, each cell was irradiated with 4 Gy and cultured for 24 h. Afterward, 10 μL of CCK8 assay solution was added, and the cells were cultured for another 2.5 h. Finally, the OD value was measured using a microplate reader at a wavelength of 450–490 nm. The cell viability was calculated using the following formula:

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

[0035] As experimental wells: culture medium containing cells, CCK8, schisandrol ethyl;

[0036] Ac control wells: Cell-containing culture medium, CCK8, excluding schisandrol ethyl;

[0037] Ab blank wells: Cell-free and schisandrin-free culture medium, CCK8.

[0038] (3) Observe cell viability using Calcein AM / PI dual staining for cell viability and mortality.

[0039] Cells were seeded into 6-well plates and stimulated with 10 μM and 20 μM schisandrin ethyl alcohol 12 h before irradiation. Twenty-four h after 4 Gy irradiation, cells were stained with Calcein AM and PI. Cells were then observed using an Agilent BioTek Cytation 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 ethyl 12 h before irradiation. Twenty-four h after 4 Gy irradiation, cells were stained with Calcein AM and PI. Cells were then analyzed using a flow cytometer (FACSAriaIII) according to the manufacturer's instructions.

[0042] 3. Experimental Results:

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

[0044] The effect of different concentrations of schisandrin ethyl on cell viability under 4 Gy irradiation was detected using the CCK8 assay. Figure 1 A and B are schematic diagrams illustrating the effect of schisandrin ethyl on the viability of irradiated NCM460 and IEC-6 cells using the CCK-8 assay. Figure 1 As shown in Figure A, schisandrin ethyl alcohol at concentrations of 2.5, 5, 10, and 20 μM can promote the viability of NCM460 cells after 4 Gy irradiation. Figure 1 As shown in Figure B, schisandrin ethyl alcohol at concentrations of 10 and 20 μM can promote the viability of IEC-6 cells after 4 Gy irradiation.

[0045] The protective effect of schisandrin ethyl on irradiated NCM460 and IEC-6 cells was detected using Calcein AM / PI live / dead cell double staining. Figure 1 Schematic diagrams C and D illustrating the effects of Calcein AM / PI live / dead double staining on NCM460 and IEC-6 cells using schisandrin ethyl acetate. From... Figure 1 As shown in Figure C, 20 μM schisandrin significantly inhibited the death of NCM460 cells after 4 Gy irradiation. Figure 1 As shown in Figure D, 20 μM schisandrin ethyl significantly inhibited the death of IEC-6 cells after 4 Gy irradiation.

[0046] The protective effect of schisandrin ethyl on irradiated NCM460 and IEC-6 cells was detected by flow cytometry. Figure 1 E, F, G, and H are schematic diagrams illustrating the effect of schisandrin ethyl on cell viability and mortality using flow cytometry. From... Figure 1 As shown in E and F, 20 μM schisandrol ethyl can significantly inhibit the death of NCM460 cells after 4 Gy irradiation. Figure 1 As can be seen from G and H, 20 μM schisandrin ethyl can significantly inhibit the death of IEC-6 cells after 4 Gy irradiation.

[0047] Example 2: Radiation protection effect of schisandrin ethyl on intestinal tissue

[0048] 1. Experimental grouping and drug administration

[0049] Animal experiments: The mice were divided into a normal group, an irradiation group, and an irradiation group plus schisandrin ethyl acetate (IR) plus Sol B. The normal group received daily abdominal injections of 200 μL of physiological saline; the irradiation group received abdominal injections of physiological saline for 3 consecutive days followed by X-ray irradiation; the irradiation group plus schisandrin ethyl acetate group received abdominal injections of schisandrin ethyl acetate for 3 consecutive days followed by X-ray irradiation.

[0050] 2. Experimental Methods

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

[0052] 3. Experimental Results

[0053] like Figure 2 As shown, Figure 2 This is a schematic diagram showing the radiation protection effect of schisandrin ethyl on intestinal tissue. Figure 2 In the diagram, A and B represent the effects of schisandrol ethyl on small intestinal apoptosis after irradiation. C and D represent the effects of schisandrol ethyl on colonic apoptosis after irradiation. E and F represent the effects of schisandrol ethyl on small intestinal DNA damage after irradiation. G and H represent the effects of schisandrol ethyl on colonic DNA damage after irradiation.

[0054] Figure 2 A and B indicate that schisandrin ethyl can inhibit the degree of apoptosis in the small intestine tissue after irradiation. Figure 2 C and D indicate that schisandrin ethyl can inhibit the degree of apoptosis in colonic tissue after irradiation. Figure 2 E and F indicate that schisandrin ethyl can improve DNA damage in small intestinal tissue after irradiation. Figure 2 G and H indicate that schisandrin ethyl can improve DNA damage in colon tissue after irradiation.

[0055] Example 3: The protective effect of schisandrin ethyl on the intestinal mucosal barrier after irradiation.

[0056] 1. Experimental grouping and drug administration

[0057] Animal experiments: The mice were divided into a normal group, an irradiation group, and an irradiation group plus schisandrin ethyl acetate (IR) plus Sol B. The normal group received daily abdominal injections of 200 μL of physiological saline; the irradiation group received abdominal injections of physiological saline for 3 consecutive days followed by X-ray irradiation; the irradiation group plus schisandrin ethyl acetate group received abdominal injections of schisandrin ethyl acetate for 3 consecutive days followed by X-ray irradiation.

[0058] 2. Experimental Methods

[0059] Immunofluorescence assay:

[0060] 1) Remove 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 min;

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

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

[0063] 4) Discard the primary antibody, wash three times with PBS, add the fluorescent secondary antibody (diluted with 1% BSA, 1:1000), incubate for 90 min, wash three times with PBS, add DAPI staining solution and incubate for 30 min, then add glycerol to mount the slide, and then observe the staining situation and take fluorescence pictures in a fluorescence microscope.

[0064] 3. Experimental Results:

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

[0066] The intestinal mucosal barrier is a crucial defense against pathogenic bacteria, and irradiation significantly damages it. ZO-1 and Occludin are two intestinal tight junction proteins. The effects of schisandrin ethyl on the intestinal mucosal barrier were assessed using immunofluorescence assays for ZO-1 and Occludin. Figure 3 As shown in Figures 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; this suggests that schisandrin ethyl can alleviate damage to the intestinal mucosal barrier after irradiation. Figure 3 As shown in Figures I and J, serum LPS levels in the IR group were significantly higher than those in the IR+Sol B group, indirectly indicating that schisandrin ethyl can alleviate damage to the intestinal mucosal barrier after irradiation.

[0067] Example 4: Schisandrin Ethylene Reduces Post-Irradiation Intestinal Inflammation

[0068] 1. Experimental grouping and drug administration

[0069] Animal experiments: The mice were divided into a normal group, an irradiation group, and an irradiation group plus schisandrin ethyl acetate (IR) plus Sol B. The normal group received daily abdominal injections of 200 μL of physiological saline; the irradiation group received abdominal injections of physiological saline for 3 consecutive days followed by X-ray irradiation; the irradiation group plus schisandrin ethyl acetate group received abdominal injections of schisandrin ethyl acetate for 3 consecutive days followed by X-ray irradiation.

[0070] 2. Experimental Methods

[0071] ELISA detection method:

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

[0073] 2) Add an inhibitor to reduce nonspecific binding and lower background signal.

[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 immobilized on the microplate.

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

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

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

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

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

[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 showing the effect of schisandrin ethyl on reducing intestinal inflammation after irradiation. Figure 4 In the diagram, A, B, C, and D represent the levels of pro-inflammatory factors IL-1β, IL-18, IL-6, and TNF-α in the small intestine and colon 12 hours and 24 hours after irradiation, respectively.

[0083] like Figure 4A, B, C, and D showed a significant decrease in pro-inflammatory factors IL-1β, IL-18, IL-6, and TNF-α in the small intestine and colon at 12 and 24 hours after irradiation, indicating that schisandrin ethyl can significantly reduce intestinal inflammation after irradiation.

[0084] Example 5: The protective effect of schisandrin ethyl against radiation-induced intestinal injury involves the Nrf2 pathway.

[0085] 1. Experimental grouping and drug administration

[0086] Animal experiments: The mice were divided into a normal group, an irradiation group, and an irradiation group plus schisandrin ethyl acetate (IR) plus Sol B. The normal group received daily abdominal injections of 200 μL of physiological saline. The irradiation group received abdominal injections of physiological saline for three consecutive days, followed by X-ray irradiation. The irradiation group plus schisandrin ethyl acetate group received abdominal injections of schisandrin ethyl acetate for three consecutive days, followed by X-ray irradiation.

[0087] Cell experiments were conducted in several groups: a control group (normal), an irradiation-only group (IR), an irradiation-IR+ML385 group, an irradiation-+schisandrol group (IR+Sol B), and an irradiation-+schisandrol group (IR+Sol B+ML385). The control group received no treatment. The irradiation-only group was irradiated with X-rays. The irradiation-IR+ML385 group was stimulated with ML385 for 12 hours prior to irradiation. The irradiation-+schisandrol group was stimulated with schisandrol for 12 hours prior to irradiation. The irradiation-+schisandrol group (IR+Sol B+ML385) was stimulated with both schisandrol and ML385 for 12 hours prior to irradiation.

[0088] 2. Experimental Methods

[0089] (1) Immunofluorescence assay:

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

[0091] 2) Add an inhibitor to reduce nonspecific binding and lower background signal.

[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 immobilized on the microplate.

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

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

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

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

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

[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) Observation of cell viability using Calcein AM / PI dual staining for cell viability and mortality

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

[0101] 3. Experimental Results:

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

[0103] like Figure 5 In samples A, B, C, and D, 24 hours after irradiation, Nrf2 expression in the small intestine and colon showed a significant increase. Figure 5 In E and F, the cell viability of the irradiated + schisandrol group IR + Sol B + ML385 group was significantly lower than that of the irradiated + schisandrol group, indicating that the intestinal radiation protection effect of schisandrol may involve the Nrf2 pathway.

[0104] Example 6: Inhibitory effect of schisandrin ethyl on radiation-induced ferroptosis

[0105] 1. Experimental grouping and drug administration

[0106] Animal experiments: Mice were divided into a normal group, an irradiation group, an irradiation group + schisandrol ethyl IR + Sol B group, and an irradiation group + schisandrol ethyl IR + Sol B + ML385 group. Normal group: Mice were injected abdominally with 200 μL of physiological saline daily; Irradiation group: Mice were treated with abdominal injection of physiological saline for 3 consecutive days followed by X-ray irradiation; Irradiation group + schisandrol ethyl IR + Sol B + ML385 group: Mice were treated with abdominal injection of schisandrol ethyl + ML385 for 3 consecutive days followed by X-ray irradiation.

[0107] Cell experiments were conducted in several groups: a control group (normal), an irradiation-only group (IR), an irradiation-IR+ML385 group, an irradiation-+schisandrol group (IR+Sol B), and an irradiation-+schisandrol group (IR+Sol B+ML385). The control group received no treatment. The irradiation-only group was irradiated with X-rays. The irradiation-IR+ML385 group was stimulated with ML385 for 12 hours prior to irradiation. The irradiation-+schisandrol group was stimulated with schisandrol for 12 hours prior to irradiation. The irradiation-+schisandrol group (IR+Sol B+ML385) was stimulated with both schisandrol and ML385 for 12 hours prior to irradiation.

[0108] 2. Experimental Methods

[0109] (1) Immunohistochemical experiment of small intestine tissue

[0110] 1) After fixing the small intestinal tissue with 4% paraformaldehyde for 1 day, it was replaced with 70% ethanol for preservation. The tissue was then trimmed into 5mm tissue blocks and placed in an embedding cassette.

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

[0112] 3) After embedding the tissue with an embedding machine, cut the paraffin sections to 3μm, and then dewax the paraffin sections. The process is as follows: baking the sections (60℃, 120min) → soaking in dewaxing solutions for 5min each (xylene II, xylene I, alcohol + xylene, 100% alcohol II, 100% alcohol I, 95% alcohol, 90% alcohol, 85% alcohol, 70% alcohol) → washing twice with distilled water;

[0113] 4) Antigen retrieval was performed using EDTA (pH 9.0) at 121℃ (for 2 min). After natural cooling, the antigen was washed twice with PBS, then blocked sequentially with hydrogen peroxide and 1% BSA (washed twice with PBS in between), and then incubated overnight at 4℃ with primary antibody (prepared with 1% BSA).

[0114] 5) After incubating with the primary antibody overnight, wash away the primary antibody and wash three times with PBS. Then, incubate with horseradish peroxidase-labeled secondary antibody for 90 min and wash three times with PBS. Next, develop the color with 3-diaminobenzidine (DAB), then counterstain with hematoxylin and rinse to return to blue. Finally, dehydrate and mount with neutral resin.

[0115] (2)Ferrorange dyeing

[0116] Cells were seeded into 6-well plates and stained with Ferrorange. Cells were then observed 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 observed using Agilent BioTek Cytation5 according to the manufacturer's instructions.

[0119] (4) Observe cell viability using Calcein AM / PI double staining for cell viability and mortality.

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

[0121] 3. Experimental Results:

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

[0123] like Figure 6 In cases A and B, 24 hours after irradiation, GPX4 expression in the small intestine and colon showed a significant increase, and GPX4 expression decreased after administration of ML385. Figure 6 Compared to the irradiated + schisandrol B group (IR + Sol B) + ML385 group, the iron ion fluorescence of groups C and D was significantly increased. Figure 6 Compared with the irradiated + schisandrol group, the lipid peroxidation level of the E and F irradiated + schisandrol group was significantly increased, as shown in Figures G and H. Compared with the irradiated + schisandrol group, the cell viability of the irradiated + schisandrol group was significantly decreased, indicating that schisandrol may inhibit GPX4-mediated ferroptosis through the Nrf2 pathway.

[0124] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. The application of schisandrin ethyl in the preparation of drugs for the prevention or treatment of intestinal radiation injury; wherein the radiation in the intestinal radiation injury is selected from X-rays.

2. The application of schisandrin ethyl as described in claim 1 in the preparation of drugs for the prevention or treatment of intestinal radiation damage, characterized in that, The aforementioned drug for preventing or treating intestinal radiation damage uses schisandrol ethyl as the sole active monomer component, or schisandrol ethyl combined with at least one pharmaceutically permissible excipient to form a pharmaceutical composition.

3. The application of schisandrin ethyl as described in claim 1 in the preparation of drugs for the prevention or treatment of intestinal radiation damage, characterized in that, The purity of schisandrin ethyl in the drug for preventing or treating intestinal radiation damage is not less than 99.9%.

4. The application of schisandrin ethyl as described in claim 1 in the preparation of drugs for the prevention or treatment of intestinal radiation damage, characterized in that, The dosage of schisandrin ethyl in the drug for preventing or treating intestinal radiation damage is 25 mg / kg.

5. The application of schisandrin ethyl as described in claim 1 in the preparation of drugs for the prevention or treatment of intestinal radiation damage, characterized in that, The aforementioned drug for preventing or treating intestinal radiation damage is an intravenous injection.

6. The use of schisandrin ethyl as described in claim 1 in the preparation of drugs for the prevention or treatment of intestinal radiation damage, characterized in that, The site of the intestinal radiation injury is selected from at least one of the small intestine, colon, and rectum.

7. The use of schisandrin ethyl as described in claim 1 in the preparation of drugs for the prevention or treatment of intestinal radiation damage, characterized in that, The drugs mentioned above for preventing or treating intestinal radiation damage increase the length of intestinal villi and the number of crypts, maintain the stability of the intestinal mucosal barrier, reduce the content of inflammatory factors in intestinal tissue, restore the vitality of intestinal epithelial cells after irradiation, inhibit their apoptosis, and reduce irradiation-induced ferroptosis.

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