Application of nano hydrogen bubble water in preparation of medicine for preventing or treating radiation

Abstract

The invention discloses an application of nano hydrogen bubble water in preparation of a medicine for preventing or treating radiation, and discovers and verifies that the nano hydrogen bubble water has a new anti-radiation application for the first time, and can obviously prolong the lifetime of a mouse after radiation. And the compound has a more prominent effect in the aspect of treating sensitive tissue and organ injuries caused by radiation.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to an application of nano hydrogen bubble water in the preparation of a medicine for preventing or treating radiation. Background Art

[0002] Ionizing radiation has always been closely related to a variety of biological damage effects. The main mechanism of ionizing radiation damage is that radiation can activate water molecules to produce a large number of free radicals. These bursts of free radicals can cause damage to biological macromolecules such as DNA and proteins, leading to cell death and damage to sensitive tissues and organs. Therefore, removing free radicals caused by irradiation is an effective strategy for radiation protection.

[0003] In recent years, many studies have found that hydrogen can selectively scavenge oxidative free radicals and has a certain protective effect against radiation damage ([1]Terasaki Y,Terasaki M,Shimizu A.Protective Effects of Hydrogen against Irradiation.Curr Pharm Des.2021;27(5):679-686.[2]Qiu X,Dong K,Guan J,He J.Hydrogen attenuates radiation-induced intestinal damage by reducingoxidative stress and inflammatory response.Int Immunopharmacol.2020Jul;84:106517.[3]Yin Z,Xu W,Ling J,Ma L,Zhang H,Wang P.Hydrogen-rich solutionalleviates acute radiation pneumonitis by regulating oxidative stress andmacrophages polarization.J Radiat Res.2024May 23;65(3):291-302.). However, hydrogen is poorly soluble in water, which reduces its absorption efficiency in the body and severely limits its application in radiation protection.

[0004] To this end, cavitation technology is used to prepare hydrogen into nanoscale hydrogen bubbles, which significantly increases its solubility in water. This not only improves the storage efficiency of hydrogen, but also finds that hydrogen in the form of nanobubbles can increase the scavenging effect on free radicals, and the small bubbles can be quickly absorbed into cells, improving the cell's utilization of hydrogen. These indicate that nano hydrogen bubbles are very different from hydrogen in both physical characteristics and biological effects. Therefore, nano hydrogen bubbles can exert a radiation protection effect that is superior to hydrogen, and this method can be used to achieve the preparation of large-scale hydrogen-rich solutions, which is of great significance to the development of hydrogen medicine and hydrogen agriculture. Summary of the invention

[0005] The purpose of the present invention is to provide an application of nano hydrogen bubble water in preparing medicine for preventing or treating radiation.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides a use of nano hydrogen bubble water in the preparation of a medicine for preventing or treating radiation.

[0008] The diameter of the hydrogen bubbles in the nano hydrogen bubble water is between 50 and 500 nm, the average particle size is 205.5 nm, the concentration is 1.5 ppm, and 70% of the hydrogen bubbles still exist after being stored for 24 weeks.

[0009] The nano hydrogen bubble water refers to hydrogen water in the form of nano bubbles (Hydrogen Nanobubble), which differs from the traditional hydrogen-rich water prepared by pressurizing hydrogen in terms of physical properties: Nano hydrogen bubbles increase the stability of hydrogen by 3.5 times, making it easy to store and transport for a long time; Nano hydrogen bubbles have a higher free radical scavenging efficiency than hydrogen. Biological characteristics include: Nano hydrogen bubbles are easier to enter cells, and the radiation protection effect on cells is better than ordinary hydrogen.

[0010] The radiation is selected from alpha rays, beta rays, gamma rays or X-ray radiation.

[0011] The radiation dose rate was 1 Gy / min.

[0012] The invention provides an application of the nano hydrogen bubble water in the preparation of a medicine for preventing or treating radiation, wherein the nano hydrogen bubble water is used as the only active ingredient.

[0013] The second aspect of the present invention provides a pharmaceutical composition, which is made of an active component and a pharmaceutically acceptable excipient; wherein the active component is the above-mentioned nano hydrogen bubble water.

[0014] Furthermore, the pharmaceutical composition of the present invention is used in combination with other drugs for preventing or treating radiation.

[0015] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0016] The present invention discovers and confirms for the first time that nano hydrogen bubble water has a new anti-radiation use, can significantly improve the survival time of mice after radiation, and has a more prominent effect in the treatment of sensitive tissue and organ damage caused by radiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the performance characterization results of nano hydrogen bubble water.

[0018] Figure 2 Schematic diagram of the uptake of nano hydrogen bubble water and hydrogen-rich water by radiation-sensitive cells (HUVEC).

[0019] Figure 3 Schematic diagram of the results of measuring the free radical scavenging ability of nano hydrogen bubble water by electron spin resonance (ESR).

[0020] Figure 4 This is a quantitative statistical graph of the ROS fluorescent probe in radiation-sensitive cells (HUVEC).

[0021] Figure 5 Schematic diagram of quantitative statistics of apoptosis of radiation-sensitive cells (HUVEC).

[0022] Figure 6 This is a quantitative statistical graph of Edu fluorescence staining of radiation-sensitive cells (HUVEC).

[0023] Figure 7 This is a line diagram showing the survival rate of mice 30 days after γ-ray irradiation.

[0024] Figure 8 Schematic diagram of H&E pathological test results of mouse small intestine.

[0025] Fig. 9 This is a quantitative statistical graph of fluorescent staining of Lgr5+ in mouse small intestinal stem cells.

[0026] Fig.10 Schematic diagram of the statistical results of peripheral blood leukocyte count in mice.

[0027] Fig.11 Schematic diagram of H&E pathological test results of mouse bone marrow. DETAILED DESCRIPTION

[0028] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0029] Example 1

[0030] Nano hydrogen bubble water was prepared using a high-concentration and high-stability micro-nano hydrogen bubble water generating device prepared in Example 1 of the patent application with publication number CN115138279A.

[0031] Nano hydrogen bubble water was prepared according to Example 2 in the patent application with publication number CN115138279A:

[0032] Step 1: The plunger pump fills water to supply water to the electrolysis device;

[0033] Step 2: Electrolyze water through the electrolysis device, and send the hydrogen generated by the negative electrode of the electrolysis device into the transmission channel, while the gas collection bag starts to collect hydrogen;

[0034] Step 3: The plunger pump fills water to supply water to the venturi tube;

[0035] Step 4: The first interface of the venturi tube receives hydrogen generated by the electrolysis device, and the second interface receives water provided by the plunger pump. The received hydrogen and water are quickly mixed inside the venturi tube to form a gas-liquid two-phase flow;

[0036] Step 5: The third interface of the ceramic membrane tube receives the hydrogen output from the gas collection bag and stores it between the shell and the ceramic membrane. The ceramic membrane and the stainless steel rod receive the hydrogen-water mixture from the venturi tube. When the hydrogen pressure between the shell and the ceramic membrane is greater than the critical value, the hydrogen will be squeezed into the ceramic membrane. The squeezed hydrogen will be sheared by the hydrogen-water mixture to generate small bubbles of hydrogen.

[0037] Step 6: The hydrogen-water mixture output by the ceramic membrane tube is input into the microfluidic shearer, and after being diverted, sheared and squeezed through the microchannel, high-concentration and high-stability micro-nano hydrogen bubble water is formed;

[0038] Step 7: The micro-nano hydrogen bubble water produced by the microfluidic shearing device is sent to the water storage device.

[0039] Among them, the specific working process of the microfluidic shearer in step 6 is as follows: the liquid inlet area of ​​the microfluidic shearer receives the mixed liquid transmitted from the ceramic membrane tube, the microchannel diverts the hydrogen-water mixed liquid in the liquid inlet area, and the hydrogen bubbles in the mixed liquid after diversion are broken into micro-nano hydrogen bubbles under the shearing and extrusion of the static shear rod to enhance the stability of hydrogen in water; since the length of the high-speed shear zone is shorter than that of the pre-shear zone and the output zone, the flow rate of the mixed liquid in the high-speed shear zone is relatively slow, and placing the high-speed shear zone in the middle of the shear chamber can improve the extrusion and shearing efficiency of the mixed liquid; finally, the stable micro-nano hydrogen bubble water converges in the liquid outlet area and is sent to the hydrogen water storage device.

[0040] Alternatively, the nano hydrogen bubble water can be prepared by using the Venturi ultrasonic multi-scale bubble generator in the patent application with publication number CN111266027A.

[0041] Specifically as follows: The Venturi ultrasonic multi-scale bubble generator provided by the present invention comprises a first-stage Venturi tube assembly 1, an air inlet plug 2, a second-stage Venturi tube assembly 8, a third-stage Venturi tube assembly 14, a first ultrasonic generator 6, a second ultrasonic generator 7, a third ultrasonic generator 12, a fourth ultrasonic generator 13, a fifth ultrasonic generator, a sixth ultrasonic generator, a first-stage tee 3, a second-stage tee 10, a first outlet valve 4 of the device, a second-stage Venturi tube inlet valve 5, a second outlet valve 9 of the device, a third-stage Venturi tube inlet valve 11 and a third outlet valve 15 of the device; the three-stage Venturi tubes are coaxially installed, Each level of the venturi tubes is flange-connected by a tee with a valve, and the internal parts of the first-level venturi tube assembly 1, the second-level venturi tube assembly 8, and the third-level venturi tube assembly 14 are bonded by glue; the first-level venturi main pipe 103 has an opening 10 mm away from the starting area of ​​the throat, and the air inlet plug 2 is connected to the venturi tube by threading at the opening, and the second-level venturi main pipe 804 is symmetrically arranged along the circumference at 10 mm away from the starting area of ​​the throat. The third-level venturi main pipe 1402 is stacked and arranged along the circumference at 10 mm away from the starting area of ​​the throat. The first-level venturi main pipe 103, the second-level venturi main pipe 804, and the third-level venturi main pipe 1402 have the same pipe diameter change structure, and each level of the venturi main pipe includes a connected inlet straight pipe section, a pipe diameter contraction section, a throat section, a pipe diameter expansion section, and an outlet straight pipe section. The length of the inlet straight pipe section is 50 mm, and the length of the outlet straight pipe section is 80 mm. The inner wall diameter of the inlet straight pipe section of each level of the venturi main pipe is 50 mm, the outer wall diameter is 55 mm, the angle between the pipe wall of the pipe diameter contraction section and the center of the pipeline is 20° to 25°, the throat length of the minimum cross-section area of ​​the pipe is 20 to 30 mm, the throat aspect ratio is equal to 1, and the angle between the pipe wall of the pipe diameter expansion section and the center of the pipeline is 10° to 15°. The contraction angle is preferably 22.5°, the diffusion angle is preferably 12.5°, the throat section length is preferably 20 mm, and the throat diameter is preferably 20 mm. The first ultrasonic generator 6, the second ultrasonic generator 7, the third ultrasonic generator 12, the fourth ultrasonic generator 13, the fifth ultrasonic generator, and the sixth ultrasonic generator in the present invention all include ultrasonic probes.

[0042] An optical compensation water tank is arranged outside each level of the Venturi main pipe, and the optical compensation water tank includes a water tank with an opening composed of a bottom plate and a plurality of side plates; the main pipe runs through the bottom plate, and the plurality of side plates surround the outer circumference of the main pipe. A mounting hole is arranged on the side of the optical compensation water tank, and the ultrasonic probe support frame is mounted on the outer wall of the throat section of the second-level Venturi main pipe and the third-level Venturi main pipe through the mounting hole, and the ultrasonic probe is mounted on the ultrasonic probe support frame. The throat section of the second-level Venturi main pipe and the third-level Venturi main pipe is provided with a plurality of straight holes along the circumference, and the straight holes are coaxially arranged with the ultrasonic probe support frame arranged on the outer side of the second-level Venturi main pipe and the third-level Venturi main pipe. The probe of the ultrasonic generator extends into the straight hole through the ultrasonic probe support frame.

[0043] The first ultrasonic generator 6, the second ultrasonic generator 7, the third ultrasonic generator 12, the fourth ultrasonic generator 13, the fifth ultrasonic generator, and the sixth ultrasonic generator are respectively connected to the first ultrasonic support frame 803, the second ultrasonic support frame 805, the third ultrasonic support frame 1405, the fifth ultrasonic support frame 1407, the fourth ultrasonic support frame 1406, and the sixth ultrasonic support frame 1408 through flanges, and the structure is a rod type, and ultrasonic vibration is generated at the front end surface of the ultrasonic probe. The millimeter-level bubbles are broken into micron-level (average diameter 500 microns) and submicron-level (average diameter 800 nanometers) through the ultrasonic action in the second-stage venturi tube and the third-stage venturi tube, and the working gas content can reach up to 30%. The frequency of the sound generator is stable at 28±0.2 kHz; the power range is from 50 watts to 400 watts.

[0044] The first-stage Venturi main pipe 103, the second-stage Venturi main pipe 804, the third-stage Venturi main pipe 1402 and the optical compensation box of the present invention are all made of acrylic material.

[0045] When the multi-scale bubble generator of the present invention is working, the liquid phase working medium flows into the device from the inlet of the device of the present invention, i.e., the inlet of the first-stage venturi tube. The millimeter-level bubble flow can be obtained at the first outlet of the device by closing the inlet valve 5 of the second-stage venturi tube and opening the first outlet valve 4 of the device; the first outlet valve 4 of the device is closed, the inlet valve 5 of the first-stage venturi tube is opened, the first ultrasonic generator 6 and the second ultrasonic generator 7 at the throat of the second-stage venturi tube are opened, the second outlet valve 9 of the device is opened, and the inlet valve 11 of the third-stage venturi tube is closed, and micron-level bubbles broken by ultrasonic cavitation at the throat of the second-stage venturi tube can be obtained at the second outlet of the device; the second outlet valve 9 of the device is closed, the outlet valve 15 of the third-stage venturi tube is opened, the third ultrasonic generator 12, the fifth ultrasonic generator, the fourth ultrasonic generator 13, and the sixth ultrasonic generator are opened, and the micron-scale bubble flow can be broken for the third time in the third-stage venturi tube, and finally a bubble flow containing submicron-level bubbles can be obtained at the third outlet of the device.

[0046] The multi-scale bubble generator of the present invention reduces the diameter of the tube, increases the flow rate, and reduces the pressure in the throat area of ​​the first-stage Venturi tube. The air is sucked into the Venturi tube through the air inlet plug and mixed with the liquid phase, and enters the first-stage Venturi tube diffusion section in the form of a bubbly flow with centimeter-scale bubbles. In the diffusion section of the first-stage Venturi tube, the centimeter-scale bubbles are broken into millimeter-scale bubbles through turbulence modulation and turbulent shear stress. At the throat of the second-stage Venturi tube, the width of the throat flow area is smaller than a single wavelength at this frequency, and it is in the proximal area where ultrasound and fluid act. The bubble vibration is severe and the crushing phenomenon is obvious, and micron-scale bubbles can be obtained. The ultrasonic vibration at the throat of the third-stage Venturi tube is more intense, and a bubbly flow containing smaller-scale bubbles can be obtained at the third outlet of the device, thereby obtaining three bubbly flows containing bubbles of different scales in a single device. The multi-scale bubble generator of the present invention uses the bubble turbulence breakup theory and the ultrasonic cavitation bubble vibration theory, and adopts turbulence modulation and turbulent shearing action in the diffusion section of the Venturi tube to perform primary bubble breakage. At the same time, a two-stage ultrasonic generating device is added to increase the millimeter-level bubble breakage into a new type of micron-level bubble breakage method. By adjusting the outlet valve of the invented device and the power of the ultrasonic probe, the same device can produce multi-scale bubble-like flows.

[0047] The diameter of hydrogen bubbles in the prepared nano hydrogen bubble water is between 50 and 500 nm, the average particle size is 205.5 nm, the concentration is 1.5 ppm, and 70% of the hydrogen bubbles still exist after being stored for 24 weeks.

[0048] Figure 1 The figure is a schematic diagram of the performance characterization results of nano hydrogen bubble water; wherein A represents the schematic diagram of the preparation and formation process of nano hydrogen bubbles. First, deionized water is injected, and hydrogen is injected at low pressure to form hydrogen bubbles (gas flow rate: 0.12m 3 / h). Then, the large hydrogen bubbles are broken into nano-sized hydrogen bubbles by cavitation, and nano hydrogen bubble water can be obtained by circulating for 20 minutes. B is a schematic diagram of the cavitation process. Large hydrogen bubbles are broken into nano-sized hydrogen bubbles under the cavitation of high temperature and pressure. C is a process of hydrogen bubbles gradually turning into nano hydrogen bubbles over time, which is photographed by a high-speed camera. D is a schematic diagram of nano bubbles observed by high-speed imaging and laser scattering at 10000×, and E is a schematic diagram of the characterization of nano bubbles, from left to right, the average particle size of nano bubbles, surface potential, and the number of nano bubbles per liter of water. The results show that the average particle size of hydrogen nano bubbles is 205.5 nanometers, the surface potential is -16.5 millivolts, and there are 14 billion stable nano bubbles per liter of water.

[0049] The hydrodynamic diameter of the nanoparticles in the nanohydrogen bubble water was measured using a nanoparticle tracking analyzer (Particle Metrix, Zetaview TWIN, Germany): the average particle size was 205.5 nm ( Figure 1 The particle concentration is 14 billion stable nanobubbles per liter of water ( Figure 1 E) and a surface charge (Zeta potential) of -16.5 mV ( Figure 1 (shown in E). Figure 1 D and E in the figure are the results of the measurement of the generated nano hydrogen bubble water using high-speed imaging and laser scattering methods. The results show that the average particle size of the nano hydrogen bubbles is 205.5 nanometers, the surface charge, i.e., the Zeta potential, is -16.5 millivolts, and the number density is 14×10 10 / L.

[0050] The morphology and size of the nano hydrogen bubbles in the nano hydrogen bubble water were characterized using a Bruker Nanoscope Icon atomic force microscope (AFM) in ScanAsyst mode at room temperature. The hydrodynamic size of the nano hydrogen bubble water was measured using dynamic light scattering (Malvern Zetasizer Nano). Figure 1 (shown in D) and apparent zeta potential.

[0051] Figure 1 In F, the hydrogen concentration in the nano hydrogen bubble water and hydrogen-rich water prepared by the present invention is continuously monitored with a hydrogen electrode under full exposure (the hydrogen-rich water is prepared using a hydrogen-rich cup (brand CROOZER), and the saturation state is reached after 15 minutes of preparation, i.e., 1.5ppm). The results show that the hydrogen concentration of the hydrogen-rich water before the test is 1.5ppm, which drops to 20% after 3 hours and 19 minutes, while the hydrogen concentration of the nano hydrogen bubble water prepared by the present invention is 1.5ppm before the test, and after 3 hours and 19 minutes, the hydrogen concentration in the nano hydrogen bubble water is 70%, and the stability is improved by 3.5 times. The present invention can adjust the content of nano hydrogen bubbles and the size of nano hydrogen bubbles in nano hydrogen bubble water.

[0052] Example 2

[0053] Detection of the cell uptake ability of the nano hydrogen bubble water prepared in Example 1

[0054] Specific testing method: deionized water was injected to prepare nano hydrogen bubble water with an osmotic pressure of 300mOsm / kg, and hydrogen-rich water was prepared using a purchased hydrogen water cup (brand CROOZER). The prepared solution was mixed with the culture medium in a mass ratio of 1:2, and then radiation-sensitive cells (HUVEC) were cultured. The holographic label-free three-dimensional living cell imaging microscope was used to observe and record the cell's uptake of nano hydrogen bubble water and hydrogen-rich water.

[0055] Figure 2The figure is a schematic diagram of the uptake of nano hydrogen bubble water and hydrogen-rich water by radiation-sensitive cells (HUVEC). It can be seen from the figure that nano hydrogen bubbles can quickly enter the cells within 30 seconds and exist stably in the cells. However, since hydrogen-rich water releases hydrogen, it can freely enter and exit the cells and cannot exist stably in the cells.

[0056] Example 3

[0057] Test the antioxidant activity of the nano hydrogen bubble water prepared in Example 1

[0058] Specific method: Electron spin resonance (ESR) was used to measure the scavenging capacity of hydroxyl radicals (·OH) and superoxide radical anions (O 2 ·- ) ability.

[0059] Measurement of the scavenging rate of hydroxyl radicals (·OH) (Liu Guanghong [1], Wang Xiaoxuan [1], Hu Shaogang [1], et al. Determination of hydroxyl radicals by electron spin resonance spectrometry [J]. College Laboratory Science and Technology, 2019 (1): 2.) 2+ / 200μM H 2 O 2 ) produces hydroxyl radicals, which, upon contact with H 2 O 2 ESR spectra were recorded starting 1 min after the generation of -OH. ·OH was trapped by DMPO in the form of the spin adduct DMPO / ·OH, and the amount of ·OH was quantitatively estimated by the peak-to-peak height of the second line of the ESR spectrum.

[0060] Measurement of superoxide radical anion (O 2 ·- ) clearance rate (MisakA, Brezova V, Chovanec M, etal.EPR Study ofKO2 as a Source ofSuperoxide and BMPO-OH / OOH Radical ThatCleaves Plasmid DNA and Detects Radical Interaction with H 2 S and Se-Derivatives[J].Multidisciplinary Digital Publishing Institute,2021(8).): During ESR measurement, spin trap BMPO was used to identify superoxide anions. Enzymatic xanthine / xanthine oxidase (Xan / XOD, XOD catalyzes the oxidation of Xan to uric acid to produce O 2 ·-) System verification of nano hydrogen bubbles to remove O 2 ·- The reaction was initiated by adding XOD.

[0061] Grouping:

[0062] Control group: no hydrogen-rich water, nano hydrogen bubble water or nano air;

[0063] H 2 Group: Hydrogen-rich water made using a hydrogen water cup and titrated water;

[0064] Nano H 2 Group: nano hydrogen bubble water prepared in Example 1;

[0065] Nano air group: using the nano air bubbles prepared in Example 1.

[0066] In a quartz capillary with an inner diameter of 0.9 mm, 50 μl of the sample was placed in each of the above groups. Unless otherwise specified, all ESR measurements were performed at ambient temperature using a Bruker EMX ESR spectrometer (Billerica, MA) with a microwave power of 20 mW and a field modulation of 1G.

[0067] The results are as follows Figure 3 As shown, Figure 3 The figure is a schematic diagram of the results of measuring the free radical scavenging ability of nano hydrogen bubble water by electron spin resonance (ESR); A represents the content of hydroxyl radicals measured by electron spin resonance (ESR), the upper figure is the signal intensity of ESR, and the lower figure is the density of hydroxyl radicals contained in each group. The stronger the ESR signal, the more ·OH is captured by DMPO in the form of spin adduct DMPO / ·OH. As can be seen from Figure A, H 2 Group, Nano H 2 The ESR signal intensity was significantly reduced in the Nano H 2 The signal intensity of the Nano H 2 The nano hydrogen bubble water contained in the group is higher than H 2 The hydrogen-rich water contained in the group has higher reducing power. B represents the content of superoxide anions measured by electron spin resonance (ESR). That is, the superoxide anion scavenging ability of nano hydrogen bubble water was studied using the enzyme superoxide anion generation system. Among them, the upper figure is the signal intensity of ESR, and the lower figure is the density of superoxide anions contained in each sample. The typical four-line ESR spectrum shown in the upper figure (relative intensity is 1:1:1:1:1) shows that O 2 ·- A free radical adduct is formed between the nanostructured H and the spin-trap BMPO. 2The results of the group suggest that when nano hydrogen bubble water is added, the ESR signal intensity of free radicals is significantly reduced. 2 The results of the two groups were compared and it was found that in the presence of nano hydrogen bubble water, O 2 ·- The decomposition of hydroxyl radicals is more effective than in the presence of hydrogen-rich water. In summary, nano hydrogen bubble water has a stronger ability to remove hydroxyl radicals and superoxide anions. Free radicals are important mediators of radiation damage, so this result suggests that nano hydrogen bubble water has better free radical elimination ability under in vitro conditions.

[0068] Grouping:

[0069] Non-IR group: no radiation;

[0070] IR group: radiation alone;

[0071] IR+H 2 Group: Add hydrogen-rich water prepared using a hydrogen water cup before irradiation;

[0072] IR+Nano H 2 Group: Nano hydrogen bubble water prepared in Example 1 was added before irradiation.

[0073] Radiation-sensitive HUVEC cells were seeded in 6-well plates, with 1×10 5 DMEM (containing 10% FBS) was added and incubated for 24 h. 2 Group, IR+Nano H 2 The groups were treated with culture medium, culture medium, 10% hydrogen-rich water, and nano hydrogen bubble water, respectively; 30 minutes later, the IR group, IR+H 2 Group, IR+Nano H 2 The group was irradiated with γ-rays, with a total dose of 6 Gy and a dose rate of 1 Gy / min, while the Non-IR group was not irradiated as a control. After irradiation, the cells were incubated for another 6 h, the culture medium was removed, 1 mL of 5 μM 2,7-dichlorodihydrofluorescein (DCFH-DA) was added, incubated for 20 min, and washed 3 times with PBS. The cells were collected, and the fluorescence intensity under 488 nm excitation was recorded by flow cytometry, and the cell images were taken by fluorescence microscopy.

[0074] The results are as follows Figure 4 As shown, Figure 4This is a quantitative statistical diagram of the ROS fluorescent probe of radiation-sensitive cells (HUVEC); it can be seen from the figure that nano hydrogen bubble water can reduce the amount of reactive oxygen species (ROS) in HUVEC cells after irradiation: DCFH-DA is a ROS fluorescent probe that emits green fluorescence after being oxidized by ROS. The more fluorescence it emits, the more ROS content it has. It can be seen that bright green fluorescence signals can be observed in cells treated with γ rays, and IR+H 2 The fluorescence signal of cells treated with hydrogen-rich water in the group was significantly weakened, while that of cells treated with IR+Nano H 2 The fluorescence signal of cells treated with nano hydrogen bubble water was extremely weak. Therefore, the antioxidant effect of nano hydrogen bubble water in cells is stronger than that of hydrogen-rich water. It can be used as an effective antioxidant to better promote the removal of free radicals in cells, thereby protecting cells from oxidative stress.

[0075] Example 4

[0076] Example 1 Preventive and therapeutic effects of nano hydrogen bubble water on radiation-induced cell damage

[0077] Grouping:

[0078] Non-IR group: no radiation;

[0079] IR group: radiation alone;

[0080] IR+H 2 Group: Add hydrogen-rich water prepared using a hydrogen water cup before irradiation;

[0081] IR+Nano H 2 Group: Nano hydrogen bubble water prepared in Example 1 was added before irradiation.

[0082] Radiation-sensitive cells (HUVEC) were collected and digested from target tissues or cell samples using trypsin containing EDTA. The collected cell samples were then prepared into single-cell suspensions, and unbound antibodies and other impurities were removed by centrifugation and washing. Apoptotic cells were labeled with the Annexin V-FITC / PI apoptosis detection kit (TransGen Biotech Crop. Ltd, Beijing, China). After incubation at room temperature for a period of time, the stained single-cell suspension was placed in a flow cytometer for analysis.

[0083] Figure 5 The quantitative statistical diagram of apoptosis of radiation-sensitive cells (HUVEC) is shown in the figure. 2The nano hydrogen bubble water used in the group can reduce cell apoptosis after irradiation: Annexin V-FITC / PI cell apoptosis is a method for detecting cell apoptosis. Annexin-V labeled with fluorescein (FITC, Alexa Fluor488, etc.) is used as a probe, which can bind with high affinity to phosphatidylserine (PS) in early apoptotic cells that flip from the inside of the cell membrane to the surface. Propidium iodide (PI) is a nucleic acid dye that cannot penetrate the intact cell membrane, but in cells in the middle and late stages of apoptosis and necrotic cells, PI can penetrate the cell membrane and dye the cell nucleus red. Therefore, by combining Annexin-V with PI, early and late apoptotic cells in the cell population can be detected. It can be seen that the cell apoptosis rate increased significantly after radiation, while IR+H 2 The hydrogen-rich water and IR+Nano H 2 The nano hydrogen bubble water used in both groups could significantly reduce the cell apoptosis rate, among which IR+Nano H 2 The effect of the nano hydrogen bubble water used in the group was better.

[0084] Grouping:

[0085] Non-IR group: no radiation;

[0086] IR group: radiation alone;

[0087] IR+H 2 Group: Add hydrogen-rich water prepared using a hydrogen water cup before irradiation;

[0088] IR+Nano H 2 Group: Nano hydrogen bubble water prepared in Example 1 was added before irradiation.

[0089] Radiation-sensitive cells (HUVEC) in the logarithmic growth phase were digested and resuspended in complete medium to a density of 1×10^6 / ml. They were then inoculated into a 96-well plate with 1×10^6 cells per well. 4 The cells were placed in a 37°C constant temperature incubator for 24 hours to allow the cells to adhere to the wall. After irradiation, the cell supernatant was taken and incubated with Edu preparation solution (BeyoClick TM Edu-594 cell Proliferation Kit with Alexa Fluor 594), and the cell nucleus was stained with DAPI. After staining for 15 minutes, the positive rate of Edu (5-ethynyl-2'-deoxyuridine) was observed and detected using a fluorescence microscope.

[0090] Figure 6This is a quantitative statistical diagram of Edu fluorescence staining of radiation-sensitive cells (HUVEC); it can be seen from the figure that nano hydrogen bubble water can reduce the proliferation inhibition effect caused by radiation: Edu fluorescence staining is an important method to evaluate cell proliferation ability, and the more fluorescence it has, the stronger the proliferation ability. As can be seen from the figure, after irradiation, the number of fluorescent cells decreased significantly, indicating that epithelial cell proliferation was weakened; while IR+H 2 Group and IR+Nano H 2 After the two groups were treated with hydrogen-rich water or nano hydrogen bubble water, the number of fluorescence increased significantly, and IR+Nano H 2 Group ratio IR+H 2 This indicates that both nano-bubble hydrogen water and hydrogen-rich water can alleviate the proliferation inhibition caused by radiation, but the effect of nano-bubble hydrogen water is significantly better than that of hydrogen-rich water.

[0091] Example 5

[0092] Example 1 Preventive and therapeutic effects of nano hydrogen bubble water on radiation-induced damage to sensitive tissues and organs

[0093] Specific methods:

[0094] Male wild-type C57BL / 6 mice (6-8 weeks, weighing 17-20 g) were purchased from Jihui Experimental Animal Breeding Co., Ltd. (Shanghai). All mice were housed in the animal room of the Radiological Medicine Unit of the Naval Medical Department of the Chinese People's Liberation Army Naval Medical University (Shanghai). They were kept on a 12h-12h circadian rhythm and given adequate food and water. All animal surgeries were in accordance with the requirements of the Ethics Committee of the Naval Medical University.

[0095] Grouping:

[0096] Non-IR group: no radiation;

[0097] IR group: radiation alone;

[0098] IR+H 2 Group: Add hydrogen-rich water prepared using a hydrogen water cup before irradiation;

[0099] IR+Nano H 2 Group: Nano hydrogen bubble water prepared in Example 1 was added before irradiation.

[0100] The mice were randomly divided into the above four groups. Twelve hours before irradiation, the mice drank hydrogen-rich water (IR+H 2 group) or nano hydrogen bubble water (IR+Nano H 2 group), then IR group, IR+H 2 Group, IR+Nano H 2The mice in the two groups received whole-body γ-ray radiation with a total dose of 9.5 Gy and a radiation rate of 1 Gy / min. After radiation, they continued to drink hydrogen-rich water or nano hydrogen bubble water for 1 week.

[0101] To evaluate the damage to sensitive tissues and organs of mice, the following tests were performed after irradiation:

[0102] (1) Survival rate of mice 30 days after irradiation;

[0103] (2) H&E pathological examination of mouse small intestine 5 days after irradiation;

[0104] (3) Status of mouse intestinal stem cells 5 days after irradiation (Lgr5+ staining);

[0105] (4) Peripheral blood leukocyte counts of mice 1, 3, and 7 days after irradiation;

[0106] (5) H&E pathological examination of mouse bone marrow 7 days after irradiation.

[0107] The results are as follows Figures 7 to 11 As shown:

[0108] Figure 7 The survival rate of mice after γ-ray irradiation for 30 days is shown in the figure. It can be seen from the figure that IR+Nano H 2 The nano hydrogen bubble water prepared in Example 1 used in the IR group can enhance the survival rate of mice after 30 days of irradiation: IR group: all mice died 8 days after irradiation; IR+H 2 Group and IR+Nano H 2 The survival rate of mice in the IR+Nano H 2 The survival rate of the group was higher, which shows that the effect of nano hydrogen bubble water is more significant than that of hydrogen-rich water.

[0109] Figure 8 This is a schematic diagram of the H&E pathological test results of the mouse small intestine; it can be seen from the figure that nano hydrogen bubble water can reduce radiation-induced intestinal damage in mice: Non-IR group: The mouse intestine contains a large number of villi and crypts. IR group: The intestinal villi and crypts are reduced. IR+H 2 Group: The intestinal villi and crypts of mice increased significantly compared with the IR group. 2 Group (Figure shows IR+NBH 2 ):The number of intestinal villi and crypts in mice increased significantly, and the increase was greater than that in IR+H 2 Group more.

[0110] In summary, nano hydrogen bubble water has a stronger preventive and therapeutic effect on intestinal tissue damage caused by radiation than hydrogen-rich water.

[0111] Fig. 9This is a quantitative statistical chart of the fluorescence staining of Lgr5+ in the small intestine stem cells of mice; it can be seen from the figure that nano hydrogen bubble water can reduce the damage of stem cells in the small intestine of mice after irradiation: the intestinal tissue of mice was taken for Lgr5+ fluorescence labeling on the third day after irradiation. Lgr5+ is a molecular marker specific to small intestinal stem cells, and the more its number, the more stem cells there are.

[0112] Non-IR group: The positive expression rate of Lgr5+ fluorescence was about 30%.

[0113] IR group: The positive expression rate of Lgr5+ fluorescence was significantly reduced to about 8%.

[0114] IR+H 2 Group: The positive expression rate of Lgr5+ fluorescence was about 14% which was higher than that of IR group.

[0115] IR+Nano H 2 Group (Figure shows IR+NB H 2 ): The positive expression rate of Lgr5+ fluorescence was significantly higher than that of IR group, which was about 21%. 2 Group more.

[0116] Compared with the Non-IR group, the number of fluorescent cells in the IR group decreased significantly after 9.5 Gy irradiation, indicating that stem cell damage increased; while the IR+NB H 2 Group and IR+H 2 The fluorescence quantity of the IR group increased significantly compared with that of the IR group, indicating that the damage of stem cells was alleviated. 2 Group ratio IR+H 2 This suggests that nano hydrogen bubble water is more effective than hydrogen-rich water in alleviating the damage of stem cells in the small intestine of mice after irradiation.

[0117] Fig.10 This is a schematic diagram of the statistical results of peripheral blood leukocyte counts in mice. The leukocytes in the peripheral blood of mice were counted 1, 3, and 7 days after irradiation.

[0118] Non-IR group: The mean number of peripheral blood leukocytes on days 1, 3, and 7 after irradiation was 6.2×10 7 about.

[0119] IR group: The white blood cell count decreased sharply, about half of that in the Non-IR group.

[0120] IR+H 2 Group: The mean white blood cell count on the first day after irradiation was 4.6×10 7 The average white blood cell count on the third day was 4.2×10 7 The average white blood cell count on day 7 was 4.0×107 The number of white blood cells showed a slowly decreasing trend in the three counts.

[0121] IR+Nano H 2 Group: The white blood cell count on the 3rd day after irradiation was significantly higher than that on the 1st day after irradiation. The white blood cell count on the 3rd and 7th days was significantly higher than that on the IR+H 2 group, significantly increased.

[0122] It can be seen from the figure that IR+Nano H 2 The nano hydrogen bubble water prepared in Example 1 used in the IR group can significantly increase the number of peripheral blood leukocytes in mice after irradiation: the number of leukocytes in the IR group decreased significantly. 2 Group and IR+Nano H 2 There was no significant difference in the content of peripheral blood leukocytes between the two groups, but on the 3rd and 7th days after irradiation, the IR+Nano H 2 The peripheral blood leukocyte count in the IR+H group was significantly higher than that in the 2 Therefore, nano hydrogen bubble water has a stronger protective effect on peripheral blood leukocytes, can reduce the damage of peripheral blood leukocytes caused by radiation, and promote their recovery.

[0123] Fig.11 Schematic diagram of H&E pathological test results of mouse bone marrow.

[0124] Non-IR group: The bone marrow cavity was relatively full, containing many trabeculae, bone marrow, cells and other structures.

[0125] IR group: The structures of bone trabeculae, bone marrow, cells, etc. in the bone marrow cavity were sharply reduced, and contained a small amount of new hematopoietic foci in the bone marrow.

[0126] IR+H 2 Group: The bone trabeculae, bone marrow, cells and other structures in the bone marrow cavity decreased, the degree of which was between the IR group and the IR+Nano H 2 There are more new hematopoietic foci and cells in the bone marrow between the two groups.

[0127] IR+Nano H 2 Group (Figure shows IR+NB H 2 ): The structures of trabeculae, bone marrow, cells, etc. in the bone marrow cavity are reduced, but relatively mild, and contain a large number of new hematopoietic foci and cells in the bone marrow.

[0128] As can be seen from the figure, nano hydrogen bubble water can reduce radiation-induced bone marrow damage in mice. Bone marrow is one of the radiation-sensitive tissues and organs. The trabeculae, bone marrow, cells and other structures in the bone marrow cavity of the IR group were sharply reduced. 2 and IR+H 2The bone marrow damage in the IR+Nano H 2 The group contained more new hematopoietic foci and cells in the bone marrow, which indicated that IR+Nano H 2 The nano hydrogen bubble water prepared in Example 1 used in the group has a stronger protective effect on the bone marrow and the ability to promote bone marrow regeneration and recovery after irradiation damage, and the effect is better than that of IR+H 2 The effect of hydrogen-rich water in the group was more significant.

[0129] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. Application of nano hydrogen bubble water in the preparation of medicine for preventing or treating radiation.

2. The use of the nano hydrogen bubble water according to claim 1 in preparing a medicine for preventing or treating radiation, characterized in that: The diameter of the hydrogen bubbles in the nano hydrogen bubble water is between 50 and 500 nm, the average particle size is 205.5 nm, the concentration is 1.5 ppm, and 70% of the hydrogen bubbles still exist after being stored for 24 weeks.

3. The use of the nano hydrogen bubble water according to claim 1 in preparing a medicine for preventing or treating radiation, characterized in that: The radiation is selected from alpha rays, beta rays, gamma rays or X-ray radiation.

4. The use of the nano hydrogen bubble water according to claim 1 in preparing a medicine for preventing or treating radiation, characterized in that: The radiation dose rate was 1 Gy / min.

5. The use of the nano hydrogen bubble water according to claim 1 in preparing a drug for preventing or treating radiation, characterized in that: In the application, nano hydrogen bubble water is used as the only active ingredient.

6. A pharmaceutical composition, characterized in that The invention is made of active components and pharmaceutically acceptable excipients; wherein the active component is nano hydrogen bubble water.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition is used in combination with other drugs for preventing or treating radiation.

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