CN107296815A - Application of the zymosan in protection acute radiation bone marrow injury medicine is prepared - Google Patents

Abstract

The present invention relates to biomedicine field, specifically application of the zymosan (Zymosan A) or derivatives thereof in protection acute radiation bone marrow injury medicine is prepared.The present invention passes through mouse experiment in vivo, it was demonstrated that zymosan can substantially mitigate the destructiveness of medulla hematopoietic system caused by ionising radiation, promotes the recovery of hemopoietic system, suppresses peripheral white blood cells caused by ionising radiation and declines；Tested by cell in vitro, zymosan can be reduced according to rear apoptosis rate, improved and shone rear cell proliferation activity；Prove that zymosan plays Study On The Radioprotective by targeted activation TLR2 signal paths by TLR2 knock-out mices.Illustrate to use zymosan to mitigate mouse bone marrow cells hemopoietic system radioactive damage by activating TLR2 signal paths according to preceding, protect peripheral white blood cells, new foundation is provided for zymosan protection acute radiation bone marrow injury.

Description

Application of zymosan in preparation of medicine for protecting acute radiation-induced bone marrow injury

technical field

The invention relates to the field of biomedicine, specifically, the application of zymosan or its derivatives in the preparation of medicines for protecting acute radiation bone marrow injury, and the pharmaceutical composition containing zymosan or its derivatives.

Background technique

At present, the nuclear industry, nuclear energy and nuclear radiation technology have been widely used in the national economy, military and medical fields. The wide application of nuclear and radiation has greatly increased the chances of radiation workers and the public being injured by nuclear radiation. Nuclear radiation damage and protection It has also attracted more and more attention from academia and the public. After receiving large doses of ionizing radiation, exposed personnel may develop acute radiation sickness, among which acute radiation-induced bone marrow injury (also known as bone marrow-type acute radiation sickness) has attracted increasing attention due to its high incidence and difficulty in prevention.

The mechanism of acute radiation-induced bone marrow injury is mainly as follows: the ionization of high-energy rays acts on intracellular biological macromolecules such as DNA and protein through direct and indirect effects, leading to bleeding in the hematopoietic system of the bone marrow, destruction of sinusoids, and death of bone marrow cells, which in turn leads to Clinical manifestations such as leukopenia, infection, and bleeding.

At present, the protective drugs for acute radiation-induced bone marrow injury are mainly oxygen free radical scavengers (such as W2721), anti-inflammatory drugs (such as glucocorticoids), and cytokines (such as G-CSF). However, the side effects and lack of specificity of these drugs limit their wide application in clinical practice. Therefore, finding a new effective, less toxic and side effect, and highly specific nuclear radiation protection drug is the focus and difficulty of current radiation biology and radiation medicine research.

Zymosan (Zymosan-A, CAS No.: 58856-93-2) is extracted from the cell wall of yeast fungi, and studies have proved that it is a TLR2 (Toll-like receptor 2) ligand (see literature: Li JT, Wang WQ, Wang L, Liu NN, Zhao YL, Zhu XS, Liu QQ, Gao CF, Yang AG, Jia LT: Subanesthetic isoflurane relieves zymosan-induced neutrophil inflammatory response by targeting NMDA glutamate receptor and Toll-like receptor 2 signaling. Oncotarget 2016; 7:31792-317). The research on zymosan mainly focuses on its ability to activate the body's immune system and enhance the body's immunity. Previous studies have found that activation of the TLR2 signaling pathway can promote nuclear translocation of NF-κB, upregulate a variety of cytokines, and exert its protection against acute radiation-induced bone marrow injury (see literature: Gao F, Zhang C, Zhou C, Sun W, Liu X, Zhang P, Han J, Xian L, BaiD, Liu H, Cheng Y, Li B, Cui J, Cai J, Liu C: A critical role of toll-like receptor2(TLR2) and its'in vivo ligands in radio -resistance. Sci Rep 2015;5:13004.)

So far, there is no literature report that zymosan plays a protective role in acute radiation-induced bone marrow injury by activating TLR2 signaling pathway.

Contents of the invention

The object of the present invention is to provide a new medical application of zymosan or derivatives thereof, and a pharmaceutical composition containing zymosan or derivatives thereof.

The first aspect of the present invention provides the application of zymosan or its derivatives in the preparation of medicines for protecting acute radiation-induced bone marrow injury.

Further, the medicine for protecting acute radiation-induced bone marrow injury is a medicine for reducing the degree of damage to the bone marrow hematopoietic system caused by ionizing radiation, reducing the apoptosis rate of cells after irradiation, and improving the proliferation activity of cells after irradiation. Furthermore, the drug for reducing the degree of destruction of the bone marrow hematopoietic system caused by ionizing radiation is a drug for reducing hemorrhage of the bone marrow hematopoietic system and destruction of bone marrow structure.

Further, the drug for protecting acute radiation-induced bone marrow injury is a drug targeting activation of TLR2. It can be seen from the above that the activation of the TLR2 signaling pathway can promote the nuclear translocation of NF-κB and up-regulate various cytokines to play a role in radiation protection.

The zymosan and its derivatives in the present invention refer to zymosan compounds capable of exerting pharmacological effects, which are glucan polymers linked by β-1,3 glycosidic bonds.

In order to verify the protective effect of zymosan on acute radiation-induced bone marrow injury, and whether it plays a role in radiation protection through the TLR2 signaling pathway, the present invention verified the effect of zymosan on mouse bone marrow through in vivo experiments on mice and in vitro cytology experiments. Radiation protection of hematopoietic system and human lymphoid B cells AHH-1, human small intestinal epithelial cells HIEC.

For the in vivo experiments in mice, C57/BL6 male wild-type mice aged 8 weeks were selected for experiments, and the mice were intraperitoneally injected with zymosan (25 mg/kg/time) 24 hours before irradiation and 2 hours before irradiation. The irradiation method for protecting mice was a single whole body irradiation, the irradiation dose was 7.0Gy, the dose rate was 1Gy/min, and the irradiation source was ⁶⁰ Coγ-rays; TLR2 knockout mice were given small doses 24 hours before irradiation and 2 hours before irradiation. Rats were intraperitoneally injected with zymosan (25mg/kg/time). The irradiation method for protecting mice is a single whole body irradiation, the irradiation dose is 6.0Gy, the dose rate is 1Gy/min, and the irradiation source is ⁶⁰ Co gamma rays; for cytological in vitro experiments, 12 hours before irradiation and 2 hours before irradiation, 40ug /ml zymosan treated HIEC and AHH-1 cells, the protected HIEC and AHH-1 cells received irradiation dose gradients of 4.0Gy and 8.0Gy, the dose rate was 1Gy/min, and the irradiation source was ⁶⁰ Coγ-rays.

Through experiments, it was found that administration of mice before irradiation can significantly reduce the bone marrow hemorrhage and blood sinus damage in wild-type mice caused by irradiation, protect the number of bone marrow nucleated cells, reduce the apoptosis of bone marrow nucleated cells, and accelerate the repair of bone marrow damage; Experiments on TLR2-knockout mice confirmed that zymosan plays a radioprotective role by targeting and activating TLR2; experiments on HIEC and AHH-1 showed that irradiation can induce cell apoptosis and inhibit cell proliferation, while pre-administration can reduce cell proliferation. After irradiation, the apoptosis rate was improved, and the inhibition of proliferation was improved. It shows that the use of zymosan before exposure can alleviate acute radiation-induced bone marrow injury in mice, protect nucleated cells in bone marrow, and accelerate the recovery of bone marrow hematopoietic system after exposure.

Further, the medicine for protecting acute radiation bone marrow injury is zymosan or its derivatives as the only active ingredient or a pharmaceutical composition containing zymosan or its derivatives.

The medicine or pharmaceutical composition can be made into any dosage form with pharmaceutically commonly used auxiliary materials, for example, it can be decoction, powder, pill, wine, lozenge, gel, plaster, tea, koji, cake , lotion, stick, thread, strip, nail, moxibustion iron, ointment, elixir, microcapsule, intravenous emulsion, liposome preparation, aerosol, prodrug preparation, injection, mixture, oral Ampoules, tablets, capsules, dripping pills, emulsions, ointments, rubber plasters, films, sponges, iontophoretic agents, or transdermal absorption agents.

The second aspect of the present invention provides a pharmaceutical composition for protecting acute radiation-induced bone marrow injury, which consists of zymosan or its derivatives and pharmaceutically acceptable auxiliary materials.

The zymosan or its derivatives of the present invention is used to prevent acute radiation-induced bone marrow injury, and is especially suitable for acute radiation-induced bone marrow injury associated with nuclear wars, nuclear accidents, radiation workers, tumor radiotherapy patients, and the like. The zymosan or derivatives thereof of the present invention are especially useful for preventing acute radiation-induced bone marrow injury, that is, administering it to people who may suffer from radiation-induced bone marrow injury in advance, and the administration method is not limited to oral administration, injection and the like.

The beneficial effects of the present invention are:

In the present invention, through experiments in mice, zymosan can significantly reduce the degree of damage to the bone marrow hematopoietic system caused by ionizing radiation, promote the recovery of the hematopoietic system, and inhibit the decrease in peripheral blood leukocytes caused by ionizing radiation; through in vitro cytology experiments, zymosan can reduce The apoptosis rate of cells after irradiation increases the proliferation activity of cells after irradiation; it is confirmed by TLR2 knockout mice that zymosan plays a role in radiation protection by targeting and activating TLR2; through in vitro cytology experiments, zymosan can reduce the apoptosis rate of cells after irradiation , to improve proliferation inhibition, indicating that the use of zymosan before exposure can alleviate acute radiation-induced bone marrow injury in mice by targeting TLR2, protect bone marrow nucleated cells, protect peripheral blood leukocytes, and reduce the sensitivity of the mouse bone marrow hematopoietic system to radiation. Therefore, the present invention provides a new basis for the protection of zymosan or its derivatives from acute radiation-induced bone marrow injury.

Current studies have confirmed that zymosan has the functions of adsorbing pathogenic bacteria, anti-tumor and enhancing immune function, and the present invention provides a new indication of zymosan, and provides a new drug for protecting damage caused by nuclear radiation.

Description of drawings

Figure 1 is a graph showing the effect of zymosan on bone marrow injury in mice after irradiation. Among them, A is the result graph of zymosan alleviating bone marrow damage in mice after irradiation, and B is the result graph of mouse bone marrow nucleated cells, which is used to measure the bone marrow damage.

Fig. 2 is a graph showing the effect of zymosan on peripheral blood leukocytes of mice induced by irradiation.

Fig. 3 is a graph showing the effect of zymosan on the spleen coefficient (spleen weight/body weight) of mice.

Fig. 4 is a graph showing the effect of zymosan on the apoptosis of AHH-1 cells in vitro. Among them, A is the result of the AnnexinV/PI kit detected by flow cytometry; B is the quantitative statistical result graph of the effect of zymosan on the apoptosis rate.

Fig. 5 is a graph showing the results of zymosan alleviating the inhibitory effect of HIEC proliferation.

Fig. 6 is a graph showing the effect of zymosan on the survival period of TLR2 wild-type and TLR2 knockout mice.

detailed description

The specific implementation modes provided by the present invention will be described in detail below in conjunction with the embodiments and the accompanying drawings.

Example 1: Zymosan can reduce bone marrow injury in mice after irradiation

1. Experimental method

C57/BL6, 8-week-old male mice were selected for the experiment, and the mice were divided into a zymosan group and a control group. 24 hours before irradiation and 2 hours before irradiation, mice were given intraperitoneal injection of zymosan (25mg/kg/time). Since zymosan was dissolved in sterile saline, the mice in the control group were given intraperitoneal injection of the same dose of sterile saline.

The mice in the zymosan group and the control group were irradiated by a single whole-body irradiation with a dose of 7.0Gy and a dose rate of 1Gy/min, and the irradiation source was ⁶⁰ Coγ-rays.

The bone marrow of the mice was harvested on the 1st, 5th, 10th, 15th, and 30th day after irradiation for pathological examination. The pathological examination was HE staining, showing the bone marrow tissue structure and lesions. In addition to pathological examination, we also measured bone marrow nucleated cell count, peripheral blood white blood cell count, and spleen index. The spleen index is the ratio of the newly removed spleen tissue to body weight. This ratio can be used to indicate the damage of the peripheral blood system, thus indirectly inferring The degree of damage to the bone marrow hematopoietic system. Generally, the more serious the hematopoietic system injury, the lower the spleen index.

2. Experimental results

The above experimental results are shown in Figures 1-3:

(1) HE staining of bone marrow sections indicated that the bone marrow tissues of mice in the control group and the administration group were not significantly different on the first day after irradiation; on the fifth and tenth days after irradiation, the damage in the control group was significantly worse than that in the administration group, The number of nucleated cells in the bone marrow decreased significantly; on the 20th day after irradiation, the hematopoietic system began to recover, and the treatment group recovered faster than the control group (Figure 1A). The number of nucleated cells in the bone marrow of the mice detected by flow cytometry also indicated that the injury in the treatment group was significantly less than that in the control group (Fig. 1B). Thus, zymosan attenuated bone marrow damage in mice after irradiation.

(2) Peripheral blood leukocyte count showed that the administration group significantly increased the peripheral blood leukocyte count (Fig. 2).

(3) Spleen coefficient results showed that irradiation caused a decrease in spleen coefficient, while zymosan could increase spleen coefficient, indicating that zymosan had a protective effect on the hematopoietic system (Figure 3).

Example 2: Zymosan reduces the apoptosis of AHH-1 after irradiation

1. Experimental method:

In order to verify the effect of zymosan on apoptosis after irradiation, apoptosis detection was performed on AHH-1 after irradiation.

Apoptosis was detected by AnnexinV/PI double-staining (purchased from Invitrogen) flow cytometry method, the principle of which is: apoptosis is a process of programmed initiation, which can be divided into two processes: early apoptosis and late apoptosis. During early apoptosis, phosphatidylserine (PS) can change from the original state inside the cell phospholipid membrane to outward. At this time, Annexin V can combine with the everted PS as a detection mark of early apoptosis. In the late stage of apoptosis, the permeability of the cell membrane increases. At this time, propidium iodide (PI), as a nucleic acid dye, can enter the interior of the cell and bind to the nucleic acid, and the nucleus is stained red. Annexin V is labeled with fluorescein, and when used in combination with PI, the level of apoptosis (including early apoptosis and late apoptosis) can be identified through flow cytometry analysis.

The specific operation steps are: AHH-1 (purchased from ATCC) cells were given zymosan (40ug/ml) or sterile saline 12 hours before irradiation and 2 hours before irradiation, and then the cells were irradiated at a dose of 4.0Gy and 8.0Gy, the dose rate is 1Gy/min. Cells were digested 24 hours after irradiation, stained with Annexin V/PI, and analyzed by flow cytometry 20 minutes later.

2. Experimental results

As shown in Figure 4, the apoptosis rate in the irradiation + zymosan group was significantly lower than that in the irradiation + NS group, manifested by a decrease in the proportion of cells in the upper right and lower right quadrants (Figure 4A). The cell apoptosis rate is the sum of the cell proportions in the upper right quadrant plus the lower right quadrant of the coordinate graph. Statistical quantification shows that zymosan significantly reduces the apoptosis rate of cells after irradiation ( FIG. 4B ).

Example 3: Zymosan reduces the proliferation inhibitory effect of irradiation on HIEC

1. Experimental method:

The detection of cell proliferation ability adopts CCK-8 experiment, the principle is: WST-8 in CCK-8 reagent can be reduced to highly water-soluble yellow formazan product by dehydrogenase in cell mitochondria, and the formed formazan product The number is proportional to the number of viable cells. The light absorption value was measured at a wavelength of 450nm by an enzyme-linked immunosorbent assay instrument, which can indirectly reflect the number of living cells.

The specific operation steps are as follows: 24 hours before irradiation, plate HIEC cells on a 96-well plate (5000 cells/well). /ml) and NS. The irradiation dose was 8.0Gy, and the dose rate was 1Gy/min. After the irradiation, the culture medium was replaced and the culture was continued. The light absorption value of the cells was measured with an enzyme-linked immunoassay instrument at a wavelength of 450nm 24 hours after the irradiation.

2. Experimental results

As shown in Figure 5, compared with the control group, the absorbance of cells in the administration group increased significantly, indicating that zymosan alleviated the inhibitory effect of irradiation on cells and promoted the proliferation of cells after irradiation.

Example 4: TLR2 knockout reverses the radioprotective effect of zymosan on mice

1. Test method

TLR2 knockout type, 8-week-old male mice were selected for the experiment, and the mice were divided into two groups: a zymosan group and a control group. 24 hours before irradiation and 2 hours before irradiation, mice were given intraperitoneal injection of zymosan (25mg/kg/time). Since zymosan was dissolved in sterile saline, the mice in the control group were given intraperitoneal injection of the same dose of sterile saline.

The mice in the zymosan group and the control group were irradiated by a single whole body irradiation, the dose was 6.0Gy, the dose rate was 1Gy/min, and the irradiation source was ⁶⁰ Coγ-rays.

The average survival time and survival period of mice were observed after irradiation.

2. Experimental results

As shown in Figure 6, there was no significant difference in the survival period between the administration group and the control group, and zymosan had no radioprotective effect on TLR2 knockout mice, proving that zymosan plays a radioprotective role mainly through the TLR2 signaling pathway.

The above-mentioned examples took zymosan as an example to explore the effect of zymosan on the acute bone marrow injury after irradiation in mice, the apoptosis of AHH-1 after irradiation, and the inhibition of proliferation of HIEC. However, the present invention is not limited to zymosan and can play a role in Zymosan derivatives with the same pharmacological effects also belong to the protection scope of the present invention.

The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments, and those skilled in the art can also make various equivalents without violating the spirit of the present invention. These equivalent modifications or replacements are all included within the scope defined by the claims of the present application.

Claims (7)

1. The application of zymosan or its derivatives in the preparation of medicines for protecting acute radiation-induced bone marrow injury. 2. The application of zymosan or its derivatives according to claim 1 in the preparation of medicines for protecting acute radiation-induced bone marrow injury, characterized in that, the medicine for protecting acute radiation-induced bone marrow injury is to reduce the destruction of bone marrow hematopoietic system caused by ionizing radiation A drug that reduces the rate of cell apoptosis after irradiation and improves the viability of cell proliferation after irradiation. 3. The application of zymosan or its derivatives according to claim 2 in the preparation of medicines for protecting against acute radiation-induced bone marrow injury, characterized in that the medicine for reducing the degree of damage to the bone marrow hematopoietic system caused by ionizing radiation is to reduce bone marrow hematopoiesis Drugs for systemic bleeding, bone marrow structural damage. 4. The use of zymosan or its derivatives according to claim 1 in the preparation of a drug for protecting acute radiation-induced bone marrow injury, characterized in that the drug for protecting acute radiation-induced bone marrow injury is a drug that targets and activates TLR2. 5. The application of zymosan or its derivatives according to any one of claims 1-4 in the preparation of medicines for protecting acute radiation-induced bone marrow injury, characterized in that, the medicine for protecting acute radiation-induced bone marrow injuries is zymosan or derivatives thereof as the only active ingredient or as a pharmaceutical composition containing zymosan or its derivatives. 6. The application of zymosan or its derivatives according to claim 1 in the preparation of medicines for protecting acute radiation-induced bone marrow injury, characterized in that, the medicine for protecting acute radiation-induced bone marrow injury is decoction, powder, pill, wine , lozenge, gel, plaster, tea, koji, cake, dew, stick, thread, strip, nail, moxibustion, ointment, elixir, microcapsule, intravenous emulsion, lipid Body preparations, aerosols, prodrug preparations, injections, mixtures, oral ampoules, tablets, capsules, dripping pills, emulsions, ointments, rubber plasters, films, sponges, iontophoresis, or penetrants Skin absorbent. 7. A pharmaceutical composition for protecting acute radiation-induced bone marrow injury, characterized in that it consists of zymosan or its derivatives and pharmaceutically acceptable auxiliary materials.