CN106822169B

CN106822169B - Application of cordycepin in preparation of medicine for preventing and/or treating radiation injury

Info

Publication number: CN106822169B
Application number: CN201710053823.4A
Authority: CN
Inventors: 史春梦; 王子文
Original assignee: Third Military Medical University TMMU
Current assignee: Third Military Medical University TMMU
Priority date: 2017-01-22
Filing date: 2017-01-22
Publication date: 2020-08-21
Anticipated expiration: 2037-01-22
Also published as: CN106822169A

Abstract

The invention discloses application of cordycepin in preparation of a medicine for preventing and/or treating radiation injury and preparing tumor radiotherapy sensitization. Pharmacodynamic experiments prove that the cordycepin can enhance the oxidation resistance and colony forming capability of cells, reduce DNA damage caused by irradiation, reduce the activity of hydrogen peroxide damaged cells, reduce hematopoietic system damage caused by irradiation, promote the recovery of the hematopoietic system, promote wound healing of wound healing complex, reduce scar formation, reduce skin ulcer and edema caused by irradiation, increase regeneration and repair of dermal cells and epidermal cells, promote repair of local abdominal irradiation intestinal tracts, reduce intestinal tract damage, enhance sensitivity of tumor radiotherapy and protect normal tissue cell functions, and provide a new medicine for preventing and treating radiation damage and enhancing sensitivity of tumor radiotherapy.

Description

Application of cordycepin in preparation of medicine for preventing and/or treating radiation injury

Technical Field

The invention relates to the technical field of medicines, in particular to application of cordycepin in preparing medicines for preventing and/or treating radiation injury and preparing tumor radiotherapy sensitization.

Background

Radiation injury refers to acute, delayed or chronic damage to body tissue caused by ionizing radiation. The large dose of radiation can produce visible physical effects within a few days, and the DNA damage caused by the radiation can cause severe damage to various tissues and organs of the irradiated person, and even genetic defects in the offspring. The clinical evolution and severity of radiation damage depends on the type of radiation source, radiation dose and dose rate, exposure site and extent, etc.

Radiation injury is divided into whole body radiation injury and local radiation injury. Radiation damage is manifested not only in damage to the skin, but also in other organs and tissues. Systemic irradiation damage is mainly manifested by damage to the hematopoietic system; the local radiation injury is mainly manifested by skin and intestinal tract injury. In an accidental irradiation accident, the local irradiation damage is the most part due to the irradiation of high intensity radioactive sources. Local radiation injury conditions evolve more slowly, often days or even weeks later to manifest their clinical symptoms, and while not sufficiently fatal, its delayed effects can lead to more severe injury.

The large-dose radiation injury lacks an ideal treatment means, some anti-radiation medicines have been studied aiming at the whole body radiation injury at present, but the treatment for local tissue organ injury, especially for a large number of irradiated persons, has no standard treatment scheme so far. According to the symptoms, conservative treatment and surgical treatment are mainly adopted. Among these, conservative treatment approaches are mainly pain relief, inflammation reduction, prevention of further infections of the circulatory system, circulation improvement, accelerated healing, wound cleansing and reduction of fibrosis, but conservative treatment is slower. If conservative treatment is ineffective, surgical treatment and reconstructive modeling surgery need to be applied. Although surgical treatment has a certain therapeutic effect, it is technically demanding, complicated to operate, and because delayed effects of irradiation can lead to more serious secondary injury, localized lesions can develop into persistent progressive lesions, and the boundary of the injury is difficult to determine initially by surgical treatment.

Cordycepin (cordycepin), 3 '-deoxyadenosine (3' -deoxyadenosine), is a nucleoside analog. The chemical name of cordycepin is 3' -deoxyadenosine, and its chemical formula is C₁₀H₁₃N₅O₃Is a basic needle-shaped or sheet-shaped crystal with a melting point of 228-231 ℃. Cordycepin has strong hydrophilicity and low solubility in nonpolar solvents. Cordycepin is a special active component in Cordyceps, and has various pharmacological effects of resisting tumor, leukemia, bacteria and virus, regulating immunity, reducing blood sugar, reducing blood lipid and resisting bronchial asthma. In recent years, the potential development and application value of the compound has attracted high attention of the world. However, the effect of cordycepin on preventing and treating radiation damage is not clear, and the influence of cordycepin on radiation damage is not reported in related documents.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of cordycepin in the preparation of a medicament for preventing and/or treating radiation injury. The application provides a new application of cordycepin, and also provides a new medicine for preventing and treating radiation injury.

In order to achieve the above purpose, the invention provides the following technical scheme:

application of cordycepin in preparing medicine for preventing and/or treating radiation injury is provided.

The radiation damage refers to DNA and tissue cell damage caused by irradiation.

The radiation damage refers to DNA damage caused by irradiation or reduction of antioxidant capacity and colony forming capacity of cells.

The radiation damage refers to hydrogen peroxide damage to cell viability.

The radiation damage refers to damage of the hematopoietic system and damage of the hematopoietic system caused by radiation.

The radiation injury refers to the formation of wound surface of the wound and scar.

The radiation injury refers to skin ulcer and edema caused by radiation.

The radiation injury refers to the injury of dermal cells and epidermal cells.

The radiation injury refers to local abdominal irradiation intestinal injury.

Application of cordycepin in preparing tumor radiotherapy sensitizing drugs is provided.

The cordycepin has the following structure:

the invention has the beneficial effects that: the cordycepin is used for preventing and treating radiation injury, and pharmacodynamic experiments prove that the cordycepin can enhance the oxidation resistance and colony forming capability of skin fibroblasts, reduce DNA (deoxyribonucleic acid) injury caused by irradiation and reduce the cell activity damaged by hydrogen peroxide; the composition can relieve the damage of a hematopoietic system caused by irradiation, promote the recovery of the hematopoietic system, promote the healing of a wound surface of a wound and a wound complex and relieve the formation of scars; can relieve skin ulcer and edema caused by large dose irradiation, and increase regeneration and repair of dermal cells and epidermal cells; can promote the repair of local abdominal irradiation intestinal tract and reduce the damage of intestinal tract; enhancing normal cell protection function and sensitizing tumor radiotherapy. Because the cordycepin is a pure natural substance, the cordycepin has the advantages of safer medication and small toxic and side effect, is convenient to use, and provides a new medicine for preventing and treating radiation damage and sensitizing tumor radiotherapy.

Drawings

FIG. 1 shows that cordycepin of example 1 of the present invention can significantly enhance the antioxidant capacity and colony forming capacity of skin fibroblasts; wherein, 1a is the cell pre-cultured by cordycepin, the expression of antioxidant protein (SOD1, SOD2 and GPX-1) is increased, and 1b is the colony forming capability of the cell pre-cultured by cordycepin after non-irradiation or irradiation is obviously stronger than that of the cell not pre-cultured;

FIG. 2 shows that cordycepin of example 2 of the present invention can significantly reduce DNA damage caused by irradiation; wherein 2a is the cells cultured by cordycepin, which can obviously reduce the formation of gamma-H2 AX in the cell nucleus caused by irradiation compared with the cells of a control group, 2b is the cells pre-cultured by cordycepin, and the gamma-H2 AX protein is obviously lower than that of the control group;

FIG. 3 shows that cordycepin of example 3 of the present invention can significantly reduce the hydrogen peroxide damaged cell viability; compared with cells of a control group, the cordycepin cultured cells can obviously reduce H₂O₂Decreased cell viability due to culture; FIG. 4 shows that cordycepin of example 4 of the present invention can reduce irradiation-induced damage to and promote recovery of the hematopoietic system; the white blood cell number of the treatment group and the control group is reduced sharply at day 3, but the reduction degree of the treatment group is not obvious than that of the control group, the white blood cell number of the treatment group is recovered more quickly, and the white blood cell level of the treatment group is basically recovered to be normal at day 20;

FIG. 5 shows that cordycepin of example 5 of the present invention can promote the healing of the wound surface of the trauma complex and reduce the scar formation; wherein, 5a is that compared with a control group, the wound healing speed of the administration group is faster, and 5b and 5c are that compared with the control group, scars formed after the administration group is healed are thinner, which shows that the cordycepin can improve the speed and the quality of wound healing;

FIG. 6 shows that cordycepin of example 6 of the present invention can significantly reduce skin ulcer and edema caused by radiation and increase regeneration and repair of dermal cells and epidermal cells; wherein 6a is compared with a control group, the administration group can obviously reduce skin ulcer and edema caused by radiation, 6b is compared with the control group, the administration group can obviously increase regeneration and repair of dermal cells and epidermal cells, 6c is that the dermal layer fiber tissue of the administration group is compact and has small gaps, the control group shows that the fiber tissue is sparse, scattered and irregular, and 6d is that the muscle fiber density of the control group is obviously reduced, the skeletal muscle structure is loose, and the cell gaps are obviously increased;

fig. 7 shows that cordycepin of example 7 of the present invention promotes intestinal repair by local abdominal irradiation and alleviates intestinal damage, wherein 7a is that the degree of intestinal damage in the administration group is light and intestinal villi is intact, and 7b is that the K i67 positive rate in the prevention group and the treatment group is significantly higher than that in the control group;

fig. 8 shows that cordycepin of example 8 of the present invention has little effect on the viability of human bronchial epithelial-like cells (HBE), and can significantly reduce the viability of tumor a549 and H460 cells, and that cordycepin can increase the colony forming ability of human bronchial epithelial-like cells (HBE) and reduce the colony forming ability of tumor a549 cells. 8a is that cordycepin has little influence on the activity of human bronchial epithelial-like cells (HBE), and simultaneously can obviously reduce the activity of tumor A549 and HBE cells, 8b is that the colony forming capability of the human bronchial epithelial-like cells (HBE) pre-cultured by cordycepin after non-irradiation or irradiation is obviously stronger than that of the cells not pre-cultured, and 8c is that compared with the cells not pre-cultured, the colony forming capability of the tumor A549 cells pre-cultured by cordycepin after non-irradiation or irradiation is obviously less than that of the cells not pre-cultured.

Detailed Description

Reagents and instruments used in the examples:

the cell culture was performed using CO from Thermo Fisher, USA₂Cell culture chamber (model HERAMell 150 i).

Cell viability was measured at a wavelength of 450nm using a Dojindo CCK-8 reagent, Japan, and a full-wavelength microplate reader (Multiskan GO, model 1510), manufactured by Thermo Fisher, USA.

Immunohistochemical experiments the results of the experiments were observed using a fluorescent microscope (model BX51) from Olympus, Japan.

All primary antibodies were purchased from Cell Signaling, and secondary antibodies were purchased from petunia.

The rest of the reagents are commercially available biological products.

The invention will be further described with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

Example 1

Detection of antioxidant proteins

Dissolving cordycepin in low-sugar culture medium to make the concentration of cordycepin in the low-sugar culture medium 200 μ M. Culturing human dermal fibroblasts in a low-sugar medium containing 200 μ M cordycepin for 0, 2, 4, 6, 8, 12, 24, 48, 72 and 96 hours respectively. After the cells are cultured by a low-sugar culture medium containing cordycepin, the cells are collected and added with protein lysate to lyse the cells, the protein concentration is detected by a BCA method, the denatured protein is subjected to electrophoresis, membrane conversion and sealing, and secondary antibody is added for development after the primary antibody is sealed overnight. The antioxidant proteins detected include SOD1, SOD2, and GPX-1. The results showed that the level of antioxidant proteins (SOD1, SOD2, GPX-1) was elevated in the cells pre-cultured with cordycepin (FIG. 1 a).

Colony formation assay

Dissolving cordycepin in low-sugar culture medium to make the concentration of cordycepin in the low-sugar culture medium respectively 0, 100, 200 and 300 μ M. Culturing human dermal fibroblast, human bronchial epithelial-like cell (HBE) and tumor A549 cell with low-sugar culture medium containing 0, 100, 200 and 300 μ M cordycepin for 72h, and replacing with low-sugar culture medium without cordycepin. One group of each cell was irradiated with gamma-rays at 5Gy irradiation dose, the other group was not irradiated, and the cells were re-seeded on a 6-well plate (1X 103/well) and cultured (low-sugar medium without cordycepin) for 14 days until colonies were clearly visible. Single colonies of over 50 cells were counted using 0.5% crystal violet staining after 4% paraformaldehyde fixation. The results show that the colony forming ability of the human dermal fibroblasts (fig. 1b) and human bronchial epithelial-like cells (fig. 8b) pre-cultured with cordycepin is significantly stronger after non-irradiation or irradiation than that of the non-pre-cultured cells, and that the colony forming ability of the tumor a549 cells pre-cultured with cordycepin is significantly less after non-irradiation or irradiation than that of the non-pre-cultured cells (fig. 8 c).

Example 2

Gamma-H2 AX formation test

Dissolving cordycepin in low-sugar culture medium to make the concentration of cordycepin in the low-sugar culture medium 200 μ M. Culturing human dermal fibroblast cells with low-sugar culture medium containing 200 μ M cordycepin for 72h, inoculating the cells on a cover glass after 72h, culturing overnight (low-sugar culture medium without cordycepin), and irradiating with 5Gy gamma ray. Fixing cells with 4% paraformaldehyde at different time (0.5, 2, 6, 12 and 24H) after irradiation, sealing goat serum for 30 minutes after 1% triton solution culture, incubating at 4 ℃ overnight with gamma-H2 AX antibody, incubating at 37 ℃ in dark for 2 hours with goat anti-mouse fluorescent secondary antibody, counterstaining cell nuclei with DAPI, and collecting images with a fluorescence microscope to observe the formation condition of gamma-H2 AX. The results show that cordycepin culture can significantly reduce irradiation-induced formation of γ -H2AX in nuclei compared to control cells (fig. 2 a).

Gamma-H2 AX protein detection

Dissolving cordycepin in low-sugar culture medium to make the concentration of cordycepin in the low-sugar culture medium 200 μ M. Culturing human dermal fibroblast with low-sugar culture medium containing 200 μ M cordycepin for 72 hr, and replacing with low-sugar culture medium without cordycepin. After the culture medium is changed into a low-sugar culture medium without cordycepin, the culture medium is irradiated by gamma rays with the irradiation dose of 5 Gy. Adding proper amount of protein lysate to lyse cells at different time (0, 1, 2, 4, 8, 12h) after irradiation. And detecting the protein concentration by using a BCA method, preparing protein gel (SDS-PAGE gel), carrying out electrophoresis on the denatured protein, transferring a membrane, sealing, and adding a secondary antibody for developing after the primary antibody is sealed overnight. The results show that gamma-H2 AX protein was significantly lower in the cells pre-cultured with cordycepin than in the control group (FIG. 2 b).

Example 3

Cell viability assay

Human dermal fibroblasts were seeded in 96-well plates (1 × 10)⁴/well) overnight (low sugar medium without cordycepin). Adding cordycepin with concentration of 0, 100, 200, 300 μ M into the cells at 37 deg.C and 5% CO₂Culturing for 72h under the condition. With 900 μ M H₂O₂After 12 hours of culture, the relative viability of the cells was determined using the CCK-8 assay. The results show that cordycepin was cultured as compared to the control cellsThe cells can obviously reduce H₂O₂Decreased cell viability upon culture (figure 3).

Human bronchial epithelial-like cells (HBE), tumor A549 and H460 cells were seeded in 96-well plates

Cultured (low-sugar medium without cordycepin) overnight in (1X 104/well). Then, cordycepin with the concentration of 0, 50, 100, 200 and 300 mu M is added into the cells respectively and cultured for 72 hours at the temperature of 37 ℃ and under the condition of 5 percent CO 2. After 72h, the relative viability of the cells was determined using the CCK-8 assay. The results show that cordycepin has small influence on the viability of human bronchial epithelial-like cells (HBE), and can significantly reduce the viability of tumor A549 and H460 cells (FIG. 8 a).

Example 4

Routine blood test

The 6-8 week-old C57 mice were randomly divided into 2 groups, a control group and a treatment group, each of which contained 10 mice. The treatment group was administered 7 days after irradiation, and the daily dose was 60mg/kg (intraperitoneal administration). Blood was collected from the tail vein on

days

3, 7, 10, 14, and 20 after gamma irradiation (4Gy), and routine blood tests were performed. The results showed that the white blood cell counts of the treated group and the control group were sharply reduced at day 3, but the reduction degree of the treated group was not obvious in the control group, the white blood cell counts of the treated group were recovered more rapidly, and the white blood cell levels of the treated group were substantially recovered to normal by day 20 (fig. 4).

Example 5

Wound-releasing compound wound animal test

The 6-8 week-old C57 mice were randomly divided into 3 groups, a control group, a prevention group, and a treatment group, each of which was 12 mice. The medicine is administered for 7 days in a week before the radiation of the prevention group, and is administered for 7 days after the radiation of the treatment group, and the daily dose is 60 mg/kg. After gamma irradiation (4Gy), the rat was constructed at 1cm on its back²And (5) forming an irradiation compound wound animal model by using large and small wound surfaces. And (5) observing the wound healing conditions of animals in different administration groups. The results show that the wound healing speed of the administration group is faster (figure 5a) and the scar formed after healing is thinner (figures 5b and 5c) compared with the control group, and show that the cordycepin can improve the speed and the quality of wound healing.

Example 6

Local soft tissue high dose irradiation animal test

The SD rats of 6-8 weeks old are divided into 2 groups, control group and administration group, wherein the administration group is continuously administered in a dose of 60mg/kg per day before and after irradiation for one week. The right hind leg was exposed to topical gamma irradiation (40Gy), which at high doses aggravated skin erythema, desquamation and ulceration. The material was taken by photographing on day 15 after irradiation. The results show that the administration group can obviously reduce skin ulcer and edema caused by radiation (figure 6a), and increase regeneration and repair of dermal cells and epidermal cells (figure 6b), the dermal layer of the administration group has dense fibrous tissues and small gaps, the control group shows sparse, scattered and irregular fibrous tissues (figure 6c), the muscle fiber density of the control group shows obvious reduction, the skeletal muscle structure shows loose, and the cell gaps are obviously increased (figure 6 d).

Example 7

Topical abdominal irradiation animal test

The 6-8 week-old C57 mice were randomly divided into 3 groups, a control group, a prevention group, and a treatment group, each of which contained 6 mice. The medicine is administered for 7 days in a week before the radiation of the prevention group, and is administered for 3 days after the radiation of the treatment group, and the daily dose is 60 mg/kg. Mice were harvested on day 4 after topical abdominal gamma irradiation (12 Gy). The small intestine damage and repair conditions of different administration groups (treatment and prevention groups) are observed. HE results showed that the degree of damage to the small intestine was less and the villi was intact in the administered group compared to the control group (fig. 7 a). Meanwhile, the cell proliferation index (Ki 67) is detected by immunohistochemistry, and the K i67 positive rate of the prevention group and the treatment group is obviously higher than that of the control group (figure 7 b).

Claims

1. Application of cordycepin as a unique active ingredient in preparing a medicament for preventing and/or treating injury caused by formation of compound wound surfaces and formation of scars due to gamma rays.

2. Application of cordycepin as the only active ingredient in preparing a medicament for preventing and/or treating skin ulcer and edema caused by gamma rays.

3. Application of cordycepin as the only active ingredient in preparing a medicament for preventing and/or treating damage of dermal cells and epidermal cells caused by gamma rays.