CN112891364A - Medicine for preventing and treating cell damage caused by gamma ray and neutron nuclear radiation - Google Patents
Medicine for preventing and treating cell damage caused by gamma ray and neutron nuclear radiation Download PDFInfo
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Abstract
The invention discloses a medicine for preventing and treating cells from being damaged by gamma rays and neutron nuclear radiation, which comprises NMN. The medicine has the advantages of low cost, convenient storage, stable property, capability of effectively preventing and treating the damage of cells caused by gamma rays and neutron nuclear radiation and the like.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a medicine for preventing and treating cells from being damaged by gamma rays and neutron nuclear radiation.
Background
Nuclear weapon radiation damage, civil nuclear power station and other nuclear facilities leakage accidents under the war condition make people face nuclear radiation risks. Existing research has shown that nuclear warfare and nuclear leakage accidents are dominated by radiation damage emitted in the form of rays or particles, with gamma rays and neutrons being the primary rays and particles of nuclear radiation damage. The gamma ray and neutron nuclear radiation can cause cell DNA damage, so that normal cells of a human body are subjected to apoptosis, and physiological functions of the human body are disordered and damaged.
In order to prevent cells from being damaged by radiation and treat radiation-damaged diseases, a series of researches are carried out, chemical drugs, Chinese herbal medicines, biological agents and the like are adopted to prevent and treat ultraviolet rays, and certain results are obtained on x-ray calculation. In order to find a safer, lower-cost and easily-stored drug for preventing and treating cell radiation injury, a series of researches are carried out:
for example, in patent document of invention publication No. CN1270034A, the use of a coenzyme for the prevention and treatment of histocytochemistry and radiation damage is disclosed, which discloses the use of NADH for the prevention and treatment of histocytochemistry and radiation damage;
the research of the above documents shows that NADH can down-regulate the expression of apoptosis-related proteins p53, p21, p16 and bax, and inhibit apoptosis; NADH can up-regulate the expression of death inhibitory molecule bcl-2, bcl-2 can inhibit the generation of active oxygen and cell apoptosis by changing the permeability of mitochondrial membrane; NADH can inhibit the release of cytochrome C from mitochondria and can inhibit radiation-induced normal apoptosis through a Caspase pathway.
The research better solves the problems of high cost and insufficient safety of the medicine for preventing and treating the cell radiation damage, but the NADH is easy to be oxidized and is limited in preparation, storage, administration and other aspects; meanwhile, NADH is easy to be damaged by gastric acid environment and easy to be oxidized under the influence of temperature and PH, so that the NADH is limited in manufacturing, storage and application.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide the medicine which is convenient to store, high in absorption efficiency and low in cost and is used for preventing and treating the cells from being damaged by gamma rays and neutron nuclear radiation.
In order to achieve the purpose, the invention adopts the following technical scheme:
a medicament for the prevention and treatment of cell damage from gamma radiation and neutron nuclear radiation, said medicament comprising NMN.
In the present invention, it is further preferable that the drug is an injection.
In the invention, a further preferable scheme is that the medicament contains NMN and physiological saline, and the concentration of the NMN in the physiological saline is 20-400 mug/mL.
In the present invention, it is further preferable that the concentration of NMN in physiological saline is 50 to 400. mu.g/mL.
In the present invention, it is further preferable that the drug is one of a tablet, a capsule, a granule, and a powder.
In the invention, a further preferable scheme is that the medicament contains NMN and pharmaceutically acceptable auxiliary materials, and the mass percentage of the NMN in the medicament is 20-90%.
In the invention, a further preferable scheme is that the mass percentage of the NMN in the medicine is 90%.
In the invention, a further preferable scheme is that the medicament is an intestinal tract sustained-release agent.
Compared with the prior art, the invention has the beneficial effects that: the medicament for preventing and treating the cell damage caused by gamma rays and neutron nuclear radiation selects NMN to replace NADH, so that the medicament is more stable in the preparation, storage and administration processes, the cost of the medicament can be further reduced, and the popularization and the application are more facilitated; meanwhile, the medicine can effectively prevent and treat the damage of cells caused by gamma rays and neutron nuclear radiation.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the contents in the embodiments of the present invention, and it is obvious that the described embodiments are some, not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A medicament for the prevention and treatment of radiation damage to cells, the medicament comprising NMN. NMN, abbreviation for nicotinamide mononucleotide, CAS No.: 214-136-5 can be metabolized in human cells to form NADH, so that the expression of apoptosis-related proteins p53, p21, p16 and bax can be reduced, the expression of death inhibition molecules bcl-2 can be up-regulated, the release of cytochrome C from mitochondria can be inhibited, normal apoptosis can be induced by radiation through a Caspase pathway, and the cells can be well prevented and treated from being damaged by radiation. By adopting the precursor NMN of NADH, the property of NMN is more stable than NADH, and the NMN is more stable than NADH in the preparation, storage and administration processes, thereby further saving the costs of production, storage and the like.
The medicine for preventing and treating cell radiation injury can be prepared into different dosage forms according to requirements, such as liquid preparations, solid preparations and the like, specifically, the liquid preparations can be injection, oral liquid and the like, and the solid preparations can be tablets, capsules, granules, powder and the like.
For injection, the injection can be prepared by selecting corresponding pharmaceutically acceptable liquid preparation auxiliary materials and NMN according to the requirements, for example, physiological saline is adopted, and the concentration of the NMN in the physiological saline is 20-400 mu g/mL; in order to ensure that the effect of preventing and treating the cell radiation injury is better, the concentration of the NMN in the physiological saline is preferably 50-400 mu g/mL.
For a solid preparation, pharmaceutically acceptable auxiliary materials can be selected according to different requirements to be mixed with NMN for preparation, wherein the mass percentage of the NMN in the medicine is 20-90% (namely, the mass of the NMN contained in each 1g of the medicine is 200-900 mg); in order to obtain better prevention and treatment effect on cell radiation injury, the mass percentage of the NMN in the medicine is 90%.
NMN is absorbed through the intestinal in the human body, and the efficiency that current NMN absorbs in the intestinal is not high, in order to promote NMN's absorption efficiency in the intestinal, can make into the intestinal sustained-release agent, can promote NMN's release and dwell time in the intestinal like this, and then increase medicine absorption efficiency, avoid because waste and the too high cost that absorption efficiency is low and cause, also need not increase consumer's misery through the injection administration, make the use also more convenient, the cost is lower.
Example 1
A medicine for preventing and treating cell damage caused by gamma rays and neutron nuclear radiation is an injection, and comprises NMN and physiological saline, wherein the concentration of the NMN in the physiological saline is 20 mu g/mL.
Example 2
A medicine for preventing and treating cell damage caused by gamma rays and neutron nuclear radiation is an injection, and comprises NMN and physiological saline, wherein the concentration of the NMN in the physiological saline is 50 mu g/mL.
Example 3
A medicine for preventing and treating cells from being damaged by gamma rays and neutron nuclear radiation is an injection, and comprises NMN and physiological saline, wherein the concentration of the NMN in the physiological saline is 400 mu g/mL.
Experimental example 1
Selecting 30 healthy Kunming mice, half of which are male and female, and the weight of which is 18-22g and the age of which is 6-8 weeks; dividing 30 Kunming mice into three groups (10 mice in each group, five mice in each group), wherein 10 mice are used as normal control groups, and no injection or radiation is applied; another 10 of the cells were irradiated with radiation as a control group; the last 10, radiation was applied and the drug of example 2 was injected once a day 3 days before radiation application (injection amount was 50mg/kg in NMN injection amount to mouse body weight); irradiation conditions were as follows: gamma rays (irradiation dose is 6Gy, dose rate is 500cGy/min, irradiation source is 100cm away from the center of the target) are adopted.
Peripheral hemogram changes
On days 1, 7 and 15 after irradiation, the mouse eyeballs were bled and the number of leukocytes was measured using a SYSMEX KX-21 hemacytometer in Japan, and the data are shown in Table 1 below.
Mouse bone marrow cell nucleated cell number detection
After 24h of irradiation, the mice are decapped and killed, the complete femur is taken, the femoral head and the tibia end are cut off, the RPMI-1640 buffer solution is absorbed by a No. 6 needle syringe to flush out all bone marrow in the femur, then a No. 4 needle is used for filtering to prepare single cell suspension, 20 mu L of the single cell suspension is taken, 0.38ml of leukocyte diluent is added, the mixture is uniformly mixed and then added into a cell counting plate for counting, and the specific measurement data refer to Table 2.
Detection of mouse bone marrow cell apoptosis rate
After 24h of irradiation, the mouse is removed from the neck and killed, the complete femur is taken, the femoral head and the tibia end are cut off, the RPMI-1640 buffer solution is absorbed by a No. 6 needle syringe to flush out all bone marrow in the femur, and then a No. 4 needle is used for filtering to prepare single cell suspension; centrifuging 100 μ L of single cell suspension for 5min under the condition of 500-; collecting the precipitated cells, then resuspending the cells, incubating the cells at room temperature in the dark for 10-15min, centrifuging the cells at 1000r/min for 5min, washing the cells with an incubation buffer solution for 1 time, adding a fluorescence (SA-FLOUS) solution, incubating the cells at 4 ℃ for 20min, and vibrating the cells at intervals in the dark; then analyzing by a flow cytometer, exciting light with the wavelength of 488nm, detecting FITC fluorescence by a band-pass filter with the wavelength of 515nm, detecting PI by a filter with the wavelength of more than 560nm, adding 5 mu L Annexin V-FITC and 2 mu L PI, placing for 15min at room temperature in a dark condition, adding 0.4ml PBS to suspend cells, measuring cell fluorescence by the flow cytometer, calculating the percentage of apoptotic cells, and referring to the specific measurement data in Table 2.
Effect of NMN on Caspase-3 Activity of irradiated mouse bone marrow cells
After 24h of irradiation, the mouse is removed from the neck and killed, the complete femur is taken, the femoral head and the tibia end are cut off, the RPMI-1640 buffer solution is absorbed by a No. 6 needle syringe to flush out all bone marrow in the femur, and then a No. 4 needle is used for filtering to prepare single cell suspension; washing with PBS for 2 times, centrifuging at 2000r/min for 5min, removing supernatant, and collecting cell precipitate; then adding 50 μ L ice-cold lysine Buffer into the cell precipitate, and placing on ice to crack for 20min, wherein the vortex oscillation is carried out for 4 times, 10s each time; centrifuging at 4 deg.C at 10000r/min for 1min, determining supernatant protein concentration in cell lysate, and adjusting protein concentration to 1.8 μ g/μ L; using a 96-well plate, 50. mu.L of the supernatant of the cell lysate after adjustment of the protein concentration was aspirated from each well, 50. mu.L of lysine Buffer was used as a blank, 0.5. mu.L of DTT was added to each 50. mu.L of 2 × Reaction Buffer, 50. mu.L of 2 × Reaction Buffer was aspirated from each well, 5. mu.L of Caspase-3Subst rate was added thereto, incubation was carried out at 37 ℃ in the dark for 4 hours, and then A405 was measured by a microplate reader, and the specific measurement data are shown in Table 2.
The measured data were processed with SPSS statistical software and analyzed for differences, see tables 1-2.
A. Effect of NMN on mouse peripheral blood leukocytes
Table 1: data sheet for NMN effect on mouse peripheral blood leukocytes
F and P statistics for major effect and F and P statistics for # interactive effect.
As can be seen from the data in Table 1 above, the peripheral blood leukocytes of the smaller mice in each time point group of the normal control group have no statistical significance; the peripheral blood leucocyte of the mouse at each time point of irradiating the control group has statistical significance; the peripheral blood leucocyte of the mouse at each time point of the NMN irradiation group has statistical significance; after 15 days of irradiation, the leukocytes of the NMN-irradiated group had returned to 92% of the time before irradiation, while the leukocytes of the irradiated control group were returned to only 73% of the original time, which indicates that administration of NMN after irradiation significantly promoted the recovery of leukocytes (WBCs) in the irradiated mice.
B. Effect of NMN on mouse bone marrow apoptosis Rate, Nuclear cell count, Caspase-3 Activity
Table 2: data sheet of the effects of NMN on mouse bone marrow apoptosis rate, nucleated cell count, Caspase-3 activity
Major component (P) is less than 0.001 compared with normal component;
as can be seen from the data in table 2 above, the numbers of nucleated cells in the femoral bone marrow of the irradiation control group are all lower than those of the normal control group and the NMN irradiation group, and the numbers of nucleated cells in the femoral bone marrow of the NMN irradiation group are both lower than those of the normal control group but higher than those of the irradiation control group;
the irradiation control group has a higher femoral bone marrow apoptosis rate than the normal control group and the NMN irradiation group, and the NMN irradiation group has a higher femoral bone marrow apoptosis rate than the normal control group but a lower femoral bone marrow apoptosis rate than the irradiation control group, which indicates that the NMN can inhibit the bone marrow apoptosis;
the activity of the femoral bone marrow cell Caspase-3 of the irradiation control group is higher than that of the normal control group and the NMN irradiation group, and the activity of the femoral bone marrow cell Caspase-3 of the NMN irradiation group is higher than that of the normal control group but lower than that of the irradiation control group, which indicates that the NMN can inhibit the activity of Caspase-3 activity, thereby inhibiting the apoptosis of bone marrow cells;
in conclusion, the medicine of the embodiment 1 can effectively prevent and treat the cells from being damaged by the gamma-ray radiation.
Experimental example 2
The normal human liver cell line L02 was purchased from Shanghai cell institute of Chinese academy of medical sciences and cultured in RPMI 1640 medium containing 10% fetal bovine serum. The experiment is divided into three groups, namely an experiment group I which is a control group without radiation, an experiment example II which is a control group with radiation and an experiment example III which is an experiment group (the medicine of the embodiment 3 of the invention) which is applied with NMN after radiation; irradiation conditions were as follows: adopting gamma rays (the irradiation dose is 6Gy, the dose rate is 500cGy/min, the irradiation source is 100cm away from the center of the target), the field size is 10 multiplied by 10cm, and a wax plate with the thickness of 1.5cm is placed on a culture bottle during irradiation;
l02 cells were 2X 105The cells were inoculated in a 60mm tissue culture plate at 5 ml/well, and after the cells were attached, the supernatant was removed and 0.01mol/L PBS (pH 7.4),5 ml/well, was added. Three experimental groups were irradiated immediately after addition of 5 ml/well of RPMI 1640 complete medium at a final concentration of 400. mu.g/ml at 37 ℃ with 5% CO2Culturing in incubator for 24 hr, digesting with pancreatin, washing with PBS, fixing with 0.5% paraformaldehyde, washing for 2 times, washing with 0.1% Triton-100 and 1% sodium citrate at 0.O1 mol/L PBS for 45min at 4 deg.C, washing for 2 times, and culturing the cells at 2.5 × 105Mu.l of the mixture was divided into tubes, washed, centrifuged, and the supernatant was discarded, and mouse anti-human Bcl-2, Bax and rabbit anti-human Fas monoclonal antibodies (purchased from Beijing Zhongshan Co.) were added to each tube. Mixing, incubating at 37 deg.C for 1 hr, washing, centrifuging, adding 50 μ l FITC-labeled anti-mouse and anti-rabbit secondary antibody into each tube, incubating at 37 deg.C for 30 min, washing, centrifuging, shaking, suspending in 500 μ l PBS solution,the detection was carried out by a flow cytometer FACScan (Beckman COULTER, USA) whose emission light was an argon ion laser and whose emission wavelength was 488 nm. 10000 cells were measured per tube, isotype control was set, processing data was analyzed by lysine II software, and percent positive cells were recorded, and the data for the specific measurements are shown in Table 3 below.
Table 3: expression results of Fas, Bax and Bcl-2 proteins in L02 cells
Variance analysis compared with experiment group I and III, P is less than 0.05
With reference to table 3, it can be seen that the expression of Bcl-2 protein is down-regulated and the expression of Fas and Bax protein is up-regulated after irradiation of L02 hepatocytes in experiment group II, which have significant difference (P <0.05) compared with experiment group III and experiment group i, indicating that the positive percentage of Bcl-2 protein expression is down-regulated and the expression of Fas and Bax is up-regulated after irradiation; NMN can up-regulate Bcl-2 and down-regulate Fas and Bax protein expression.
Observing the L02 cells of experiment examples II and III under an electron microscope, and observing most of apoptosis, nucleus disintegration, a plurality of apoptotic bodies, shrinkage of partial cell bodies, cytoplasm condensation and chromatin condensation of the L02 cells under the electron microscope after the L02 cells are radiated by X rays in the experiment example II, so that the apoptotic bodies contained in two membranes can be seen; in the experimental group III, only a small part of cells are subjected to apoptosis by adding NMN after radiation, most of cell nuclei and cytoplasm are not obviously concentrated, the cell membrane is complete, the chromatin condensation is less, and the radiation damage of the cells can be effectively treated by the NMN.
Testing the influence of different doses of NMN on the radiation apoptosis damage of the L02 liver cell strain: after irradiation, the L02 cells are added with NMN concentration of 0, 100, 200, 300, 400, 500 and 600 mug/ml respectively, and the apoptosis rate is detected in 6, 12 and 24 hours; see table 4 below for specific test data:
table 4: l02 test data table for relation between apoptosis rate and NMN dosage at different time after liver cell lean radiation
As can be seen from the above table 4, the apoptosis rate is low at 6 hours after the cells are irradiated, the apoptosis rate is high at 24 hours, the apoptosis rate is gradually reduced along with the increase of NMN concentration, and a platform is reached when the NMN concentration is 400-600 mug/ml.
Experimental example 3
The performance of the medicament for preventing and treating the neutron radiation damage of the cells is tested by adopting the method of the embodiment 1-2, and the test result shows that the medicament has good effect on preventing and treating the neutron radiation of the cells.
While the invention has been described with reference to specific embodiments, the invention is not limited thereto, and various equivalent modifications and substitutions can be easily made by those skilled in the art within the technical scope of the invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (8)
1. A medicament for the prevention and treatment of cell damage from gamma radiation and neutron nuclear radiation, said medicament comprising NMN.
2. The medicament of claim 1, wherein the medicament is an injection.
3. The medicament according to claim 2, wherein the medicament comprises NMN and physiological saline, and the concentration of the NMN in the physiological saline is 20-400 μ g/mL.
4. The medicament according to claim 3, wherein the NMN is present in physiological saline at a concentration of 50-400 μ g/mL.
5. The medicament of claim 1, wherein the medicament is one of a tablet, a capsule, a granule and a powder.
6. The drug according to claim 5, wherein the drug comprises NMN and pharmaceutically acceptable excipients, and the mass percentage of the NMN in the drug is 20-90%.
7. The medicament according to claim 6, wherein the mass percentage of NMN in the medicament is 20-90%.
8. The medicament of claim 1, wherein the medicament is an enteric sustained release formulation.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1270034A (en) * | 1999-11-04 | 2000-10-18 | 第一军医大学珠江医院 | application of coenzyme in histocyte chemistry and prevention and cure of radiation injury |
| CN108025187A (en) * | 2015-04-28 | 2018-05-11 | 新南创新私人有限公司 | Targeting NAD+ is to treat cognitive impairment, neuropathy that chemotherapy and radiotherapy trigger and inactive |
| CN109674808A (en) * | 2019-01-30 | 2019-04-26 | 四川大学 | β-nicotinamide mononucleotide or its precursor are preparing the purposes delayed in lung senescence drug |
| US20190350960A1 (en) * | 2017-01-04 | 2019-11-21 | President And Fellows Of Harvard College | Modulating nudix homology domain (nhd) with nicotinamide mononucleotide analogs and derivatives of same |
-
2021
- 2021-03-15 CN CN202110273385.9A patent/CN112891364A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1270034A (en) * | 1999-11-04 | 2000-10-18 | 第一军医大学珠江医院 | application of coenzyme in histocyte chemistry and prevention and cure of radiation injury |
| CN108025187A (en) * | 2015-04-28 | 2018-05-11 | 新南创新私人有限公司 | Targeting NAD+ is to treat cognitive impairment, neuropathy that chemotherapy and radiotherapy trigger and inactive |
| US20190350960A1 (en) * | 2017-01-04 | 2019-11-21 | President And Fellows Of Harvard College | Modulating nudix homology domain (nhd) with nicotinamide mononucleotide analogs and derivatives of same |
| CN109674808A (en) * | 2019-01-30 | 2019-04-26 | 四川大学 | β-nicotinamide mononucleotide or its precursor are preparing the purposes delayed in lung senescence drug |
Non-Patent Citations (1)
| Title |
|---|
| JIE SONG等: "Nicotinamide mononucleotide promotes osteogenesis and reduces adipogenesis by regulating mesenchymal stromal cells via the SIRT1 pathway in aged bone marrow", 《CELL DEATH & DISEASE》, vol. 10, no. 5, 18 April 2019 (2019-04-18) * |
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