CN115364093A - Radiation sensitizer containing schisandrin B and application thereof - Google Patents

Radiation sensitizer containing schisandrin B and application thereof Download PDF

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CN115364093A
CN115364093A CN202210908547.6A CN202210908547A CN115364093A CN 115364093 A CN115364093 A CN 115364093A CN 202210908547 A CN202210908547 A CN 202210908547A CN 115364093 A CN115364093 A CN 115364093A
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schisandrin
cancer
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王若雨
梁珊珊
于炜婷
张玲玲
房艳华
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Dalian University
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    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract

The invention discloses a radiation sensitizer containing schisandrin B and application thereof, belonging to the technical field of medicines. The radiosensitizer disclosed by the invention contains schisandrin B, and also contains one or more of gomisin, deoxyschizandrin, schisandrin C, schisandrin A, schisandrin B, schisantherin A, schisantherin B and schisanhenol.

Description

Radiation sensitizer containing schisandrin B and application thereof
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a schisandrin B-containing radiosensitizer and application thereof in preparation of tumor radiosensitizers.
Background
Malignant tumors are one of the important causes affecting human health. Radiotherapy is one of the effective methods for local treatment of malignant tumors, can relieve more than 80% of malignant tumors to different degrees, and is also an effective means for preventing recurrence after tumor operation. The efficacy of radiotherapy depends primarily on the radiosensitivity of the tumor, which is related to the type, location and volume of the tumor. Highly sensitive cancer cells such as leukemia, germ cell tumor, most lymphomas and the like can be rapidly killed by medium dose of rays, and radiation-resistant tumors such as breast cancer, melanoma and the like can not be cured by safe dose in clinical practice. In addition, radiation therapy resistance is also a major cause of failure of radiation therapy for some tumors.
Radiotherapy is one of the important means for treating malignant tumors, and the treatment failure is caused by the fact that the normal tissues are limited in tolerance dose so that the tumor cannot be given enough radiation dose, and how to improve the sensitivity of the tumor to radiation is a prominent problem in clinical tumor radiotherapy. The radiosensitizer is used as a medicine for improving the radiosensitivity of tumors and enhancing the curative effect of radiotherapy, and achieves the radiosensitization effect by radiating induced oxygen free radicals and DNA damage and regulating and controlling radiotherapy key molecular targets. The radiotherapy sensitizer commonly used in clinic at present, such as platinum, alkylating agent, fluorouracil and the like, has toxic and side effects on the whole body. Therefore, it is important to develop a radiosensitizer that can increase the radiosensitivity of cancer, and the treatment method with the radiosensitizer has lower radiation dose, specificity of potential target and clinically acceptable toxicity.
Schisandra chinensis fruit of Schisandra of Magnoliaceae is a famous traditional nourishing traditional Chinese medicine, and has the functions of invigorating qi, promoting fluid production, astringing, invigorating kidney, calming heart, protecting brain nerve cells, protecting liver, resisting inflammation, resisting oxidation, resisting tumor, etc.It has good therapeutic effect on chronic diarrhea, chronic cough, asthma, short breath, deficiency of pulse, palpitation, and insomnia. The main active ingredients of the schisandra chinensis alcohol extract are lignans, and the lignans mainly comprise: schizandrin A, schizandrin B, schizandrin C, schizandrol A, schizandrol B, schisantherin A, schisantherin B, schisanhenol, gomisin, etc. Numerous studies have found that schisandrin B has various pharmacological effects including anti-inflammatory, antioxidant, anti-fibrosis, lipid regulation, etc., and has little toxic and side effects on normal cells. Schisandrin B has high safety in animal experiments and low toxicity to cells, and low, medium and high dosage (1.67, 5.00 and 15.00 mu g/mL) of Schisandrin B is proved by research -1 ) Has no adverse effect on cell growth and metabolism, and indicates that schisandrin B has no cytotoxic effect. However, no literature report and patent discloses a radiation sensitizer containing schisandrin B and application thereof in preparing tumor radiation sensitizing drugs.
Disclosure of Invention
In view of the above, the invention aims to provide a schisandrin B-containing radiosensitizer and application thereof in preparing tumor radiosensitizing drugs.
The purpose of the invention is realized by the following modes:
the invention provides a radiosensitizer, which contains schisandrin B.
Based on the technical scheme, the radiosensitizer further comprises one or more than two of gomisin, deoxyschizandrin, schisandrin A, schisandrin B, schisantherin A, schisantherin B and schisanhenol.
The invention also provides the application of the radiosensitizer in the preparation of tumor radiosensitizing drugs.
Based on the technical scheme, the tumor is breast cancer, nasopharyngeal carcinoma, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, cervical cancer, gastric cancer, colorectal cancer, glioma, hepatobiliary cancer, leukemia, melanoma, esophageal cancer and thymoma.
Based on the technical scheme, further, the cell line of the breast cancer is MDA-MB-231 or MCF-7, and the cell line of the nasopharyngeal carcinoma is CNE1.
Based on the technical scheme, furthermore, the content of schisandrin B in the medicine is 0.6-90wt%; the content of other components of the schisandra chinensis is 0-19.4wt%.
Based on the technical scheme, the medicine further comprises a pharmaceutically acceptable carrier and/or an auxiliary material, and the content of the carrier and/or the auxiliary material is 10-80wt%.
Based on the technical scheme, the administration route of the medicine further comprises oral administration, intravenous administration, intramuscular administration, subcutaneous injection, nasal administration, intraperitoneal injection, sublingual administration, transdermal administration and anal administration.
The radiosensitizer can be administered in one or more doses, adjusted according to the route of administration, the age, weight, body surface area, type and severity of the disease of the patient.
Based on the technical scheme, the medicine is further applied to the radiation therapy (external irradiation therapy) process and the radionuclide therapy (internal irradiation therapy) process.
Based on the technical scheme, the medicine is further applied before a radiotherapy (external irradiation treatment) process and a radionuclide treatment (internal irradiation treatment) process.
Based on the technical scheme, the radiotherapy dose in the radiotherapy process is further used according to 25% -50% of the calculated radiotherapy dose under the condition of not using the tumor radiosensitizer.
The invention has the beneficial effects that:
the radiosensitizer provided by the invention has the characteristics of increasing the curative effect of radiotherapy, reducing the radiation dose, potential target specificity and clinically acceptable toxicity, wherein the radiation dose can be reduced, the radiotherapy effect is unchanged or even better, and the influence on surrounding normal tissues is reduced, so that the complications caused by radiation toxic and side effects are reduced.
Drawings
In order to more clearly illustrate the embodiments of the present invention, the drawings to which the embodiments relate will be briefly described below.
FIG. 1 shows the effect of Schizandrin B on the cell cycle of breast cancer cells (A) and nasopharyngeal cancer cells (B);
FIG. 2 shows the effect of Schisandrin B on breast cancer radiosensitivity (A-B), lung cancer cell radiosensitivity (C-D) and nasopharyngeal cancer cell radiosensitivity (E);
FIG. 3 shows the results of qualitative (A) and quantitative (B) experiments of schisandrin B-enhanced breast cancer cell radiation damage;
FIG. 4 shows the results of qualitative (A) and quantitative (B) experiments of using pentamer B to delay the repair of radiation damage in breast cancer cells;
FIG. 5 shows the expression results of Schisandrin B radiosensitizing core target in various tumor cancers (A) and tissues adjacent to the cancer (B);
FIG. 6 shows the effect of Schizandrin B on the cell activities of normal mammary epithelial cells MCF-10A (A) and human fibroblast HFF-1 (B).
Detailed Description
The present invention is described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto, and it is obvious that the examples in the following description are only some examples of the present invention, and it is obvious for those skilled in the art to obtain other similar examples without inventive exercise and falling into the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1 flow cytometry to examine the cycle Effect of Schizandrin B on breast cancer cells MCF-7 and on nasopharyngeal carcinoma cells CNE-1
MCF-7 cells are divided into a control group (NC) and a schisandrin B group (10, 20, 40 and 60 mu m), CNE-1 cells are divided into a control group (NC) and a schisandrin B group (10, 20, 30 and 40 mu m), schisandrin B acts for 48h, then the cells are digested and centrifuged, 500ul PI/RNase staining buffer is added to suspend the cells, then the cells are incubated for 15min at room temperature, and after sieving, the cells are analyzed by a flow cytometer.
As shown in FIG. 1, it can be seen that the ratio of G1 phase in MCF-7 cells and CNE-1 cells gradually increases and the ratio of S phase gradually decreases as the concentration of schisandrin B increases; the result shows that the schisandrin B blocks the cell cycle of breast cancer and nasopharyngeal carcinoma from the G1 stage which is relatively sensitive to radiation, and reduces the proportion of S stage of radiation resistance.
Example 2 plate cloning experiment to examine the Effect of Schizandrin B on radiation sensitivity of breast cancer and nasopharyngeal carcinoma
Inoculating 5000 cells per well of MCF-7 cells in logarithmic growth phase to a 6-well plate, dividing the cells into X-ray irradiation groups (NC, 2Gy, 4Gy, 6Gy, 8 Gy), and irradiating with 6Mv energy and 600cGy/min dose rate; treating Schizandrin B group (20 μm, 40 μm) for 48 hr; subjecting Schisandrin B to X-ray irradiation (20 μm +2Gy, 20 μm +4Gy, 20 μm +6Gy, 20 μm +8Gy, 40 μm +2Gy, 40 μm +4Gy, 40 μm +6Gy, 40 μm +8 Gy), adding Schisandrin B (20, 40 μm) after cell adhesion, pretreating for 4h, irradiating with 6Mv energy and 600cGy/min dose rate, replacing culture medium after 48h, culturing in culture box for 7 days, fixing with 4% paraformaldehyde for 15min when monoclonal antibody is observed under the mirror, washing with clear water twice, staining with 0.01% crystal violet for 30min, washing with clear water to remove residual crystal violet, and air drying. Clones containing more than 50 cells were recorded as one valid clone and the relative colony formation rate was calculated. Relative clone formation rate = (number of cell clones/number of seeded cells)/(number of control clones/number of seeded cells), and the results are shown in table 1.
Inoculating 2000 cells per well of H1299 cells in logarithmic growth phase to a 6-well plate, dividing the cells into X-ray irradiation groups (NC, 2Gy, 4Gy, 6Gy and 8 Gy), and irradiating with 6Mv energy and 600cGy/min dosage rate; treating Schizandrin B group (20, 30 μm) for 48 hr; schisandrin B is irradiated by combined X-ray (20 mu m +2Gy, 20 mu m +4Gy, 20 mu m +6Gy, 20 mu m +8Gy, 30 mu m +2Gy, 30 mu m +4Gy, 30 mu m +6Gy and 30 mu m +8 Gy), schisandrin B (20 and 30 mu m) is added for pretreatment for 4 hours after the cells are attached to the wall, the cells are irradiated at 6Mv energy and 600cGy/min dosage rate, the culture medium is replaced after 48 hours, and then the cells are cultured in an incubator for 14 days, and the rest is subjected to the experiment process of plate clone of the breast cancer, and the result is shown in a table 2.
400 cells per well in the logarithmic growth phase CNE-1 are inoculated in a 6-well plate, the cells are divided into a control group (NC), a schisandrin B group (10 mu m), an X-ray irradiation group (4 Gy) and a schisandrin B combined X-ray irradiation group (10 mu m Sch B +4 Gy), schisandrin B (10 mu m) is added for pretreatment for 6h after the cells are attached to the wall, X-ray 4Gy dosage, 6Mv energy and 600cGy/min dosage rate irradiation are carried out, and the cells are cultured for 5d in an incubator, and the rest is the same as the breast cancer plate cloning experiment process, and the result is shown in figure 2.
TABLE 1 Effect of SchB on radiation sensitivity of breast cancer cells MCF-7
Figure BDA0003773174510000051
Note: d0, average lethal dose, namely the radiation dose required by the remaining 37 percent of cells after irradiation; dq, subthreshold dose, representing the width of the viable shoulder, also called wasted radiation dose; SF2:2Gy survival score, SF2=2Gy group colony formation rate/Control group colony formation rate; SER: radiosensitization ratio, SER = D0 (control group)/D0 (experimental group).
TABLE 2 influence of SchB on the radiosensitivity of breast cancer cells H1299
Figure BDA0003773174510000052
Note: d0, average lethal dose, namely the radiation dose required by the remaining 37 percent of cells after irradiation; dq, subthreshold dose, representing the width of the viable shoulder, also known as wasted radiation dose; SF2:2Gy survival score, SF2=2Gy group colony formation rate/Control group colony formation rate; SER radiosensitization ratio, SER = D0 (control group)/D0 (experimental group).
According to the results of table 1, table 2 and fig. 2, it can be seen that schizandrin B combined irradiation reduces the cell colony forming ability of breast cancer (a), lung cancer (B) and nasopharyngeal carcinoma (C), and the SchB has synergistic effect with radiotherapy by the numerical value of radiosensitization ratio.
Example 3 immunofluorescence detection of the Effect of Schisandrin B in combination with radiation therapy on DNA Damage to Breast cancer cells MCF-7 cells 1x10 5 The cells were separated by plating in 12-well plates with round slidesBlank group, control group, schisandrin B group (60 mu m, 2H), X-ray group (8Gy, 2h) and Sch B (60 mu m) are pre-acted for 1H and combined with the X-ray group (8 Gy treated for 2H), old culture medium is discarded, PBS buffer solution is washed for 2 times, 4% paraformaldehyde is fixed for 15min, the washing is carried out for 1H at normal temperature by using confining liquid after washing, rabbit anti-human IgG primary antibody (gamma-H2 AX) is incubated overnight at 4 ℃, PBS is washed for 3 times, goat anti-rabbit IgG secondary antibody (488 Dylight) is incubated for 1H at normal temperature, PBS is washed for 3 times, DAPI is stained for 15min and then is mounted, and gamma-H2 AX focus is observed by 1000 times under a confocal microscope.
The result is shown in FIG. 3, which shows that the simple Schisandrin B action has no DNA double strand break damage; after the X-ray irradiation is carried out for 2 hours, partial DNA double-strand damage occurs in the X-ray group; compared with the X-ray group, the schisandrin B is combined with the X-ray after pre-acting for 1h (treating for 2h in 8 Gy), so that the DNA double-strand damage is enhanced; the result shows that the schisandrin B has the function of enhancing the damage of X-ray to the DNA double strand of the breast cancer cells.
Example 4 immunofluorescence assay for the Effect of Schizandrin B in combination with radiation therapy on the repair of DNA Damage in Breast cancer cells
MCF-7 cells 1x10 5 The cells were separated into Blank, control, sch B (60. Mu. M, 13 h), X-ray (8 Gy, 12h) and Sch B (60. Mu. M) by pre-treatment for 1h in combination with X-ray (8 Gy for 12 h), and the other experiments were carried out in the same manner as in example 3, in 12-well plates equipped with circular slides.
The result is shown in figure 4, the Schisandrin B still has no DNA double-strand damage after being acted for 13 hours; compared with the X-ray group, sch B (60 mu m) is acted for 1h in advance and combined with the X-ray (8 Gy treated for 12 h), DNA double-strand damage still exists, and the result shows that the schisandrin B has the effect of delaying the repair of the DNA double-strand damage of the breast cancer cells.
Example 5GEPIA database verifies the expression of Schisandrin B radiosensitizing core target in each tumor
Schisandrin B core target was imported via GEPIA database (http:// GEPIA. Cancer-pku. Cn /), FIG. 5A shows the expression of tumor tissue core target, and FIG. 5B shows the expression of cancer-adjacent tissue core target.
The results are shown in fig. 5, and the schisandrin B radiosensitization core target is highly expressed in liver cancer, gastric cancer, ovarian cancer, pancreatic cancer, cervical cancer, gastric cancer, colorectal cancer, glioma, hepatobiliary cancer, leukemia, melanoma, esophageal cancer and thymoma; the results show that the schisandrin B has radiosensitization effect in the tumors through the core target.
Example 6CCK8 experiment to examine the Effect of Schisandrin B on Normal cell Activity
The experimental cells are selected from normal mammary epithelial cells MCF-10A and human fibroblasts HFF-1, MCF-10A and HFF-1 are respectively inoculated on a 96-well plate at 3000/well and 3500/well, each group is provided with 5 multiple wells, schisandrin B (with the final concentration of 0, 20, 40, 60, 80, 100 and 120 mu m) is respectively added after 15h adherent culture, then the cells are placed in an incubator for continuous culture, the culture is respectively terminated at 24h, 48h and 72h, 110 mu l of serum-free culture medium containing 10 CCK-8 is added into each well, and the cells are placed in the incubator for culture for 2h. Measuring the light absorption value A value of 450nm wavelength of each hole by a multifunctional enzyme-labeling instrument, and determining the cell survival rate (%) = (A) test -A blank ) /(A control -A blank )×100％。
The results are shown in figure 6, with the increase of schisandrin B concentration and the prolonging of action time, the effect on MCF-10A cell activity is small, and the concentration less than 20 μm has the effect of promoting cell proliferation; with the increase of schisandrin B concentration and the prolongation of action time, no cytotoxic action is caused to HFF-1 cells; the result shows that the schisandrin B as a radiosensitizer has low toxicity to normal tissues.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. A radiosensitizer is characterized in that the radiosensitizer contains schisandrin B.
2. The radiosensitizer according to claim 1, wherein the radiosensitizer comprises one or more of gomisin, deoxyschizandrin, schisandrin c, schizandrol a, schizandrol b, schisantherin a, schisantherin b, and schisanhenol.
3. Use of a radiosensitizer according to claim 1 or 2 in the preparation of a tumor radiosensitizing drug.
4. The use of claim 3, wherein the tumor is breast cancer, nasopharyngeal carcinoma, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, cervical cancer, gastric cancer, colorectal cancer, glioma, hepatobiliary cancer, leukemia, melanoma, esophageal cancer, thymoma.
5. The use of claim 3, wherein the medicament contains schisandrin B in an amount of 0.6-90wt%; the total content of one or more of gomisin, deoxyschizandrin, schisandrin C, schisandrin A, schisandrin B, schisantherin A, schisantherin B and schisanhenol is 0-19.4wt%.
6. The use according to claim 3, wherein the medicament comprises a pharmaceutically acceptable carrier and/or adjuvant in an amount of 10-80wt%.
7. The use of claim 3, wherein the route of administration of said medicament comprises oral, intravenous, intramuscular, subcutaneous, nasal, intraperitoneal, sublingual, transdermal and anal administration.
8. The use according to claim 3, wherein the medicament is used in radiotherapy and radionuclide therapy.
9. The use of claim 3, wherein said medicament is used prior to a radiation therapy procedure and prior to a radionuclide therapy procedure.
10. The use of claim 8, wherein the radiation dose of the radiation therapy session is used in the range of 25% to 50% of the radiation dose calculated without the use of the tumor radiosensitizing drug.
CN202210908547.6A 2022-07-29 2022-07-29 Radiation sensitizer containing schisandrin B and application thereof Pending CN115364093A (en)

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Citations (1)

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Publication number Priority date Publication date Assignee Title
CN102068496A (en) * 2009-11-20 2011-05-25 益生生物科技有限公司 Radiosensitizer compositions comprising schisandra chinensis(turcz.)baill

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102068496A (en) * 2009-11-20 2011-05-25 益生生物科技有限公司 Radiosensitizer compositions comprising schisandra chinensis(turcz.)baill

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Application publication date: 20221122