Application of hydrogen-rich water containing salidroside in preparation of medicine for preventing plateau encephaledema

Abstract

The invention relates to the field of biological medicine, in particular to application of hydrogen-rich water containing salidroside in preparation of a medicine for preventing plateau encephaledema. The salidroside and the hydrogen-rich water are creatively combined for application, and the effect of preventing the plateau encephaledema is achieved. Results of the specific embodiment of the invention show that after salidroside and hydrogen-rich water are combined, the plateau encephaledema can be effectively prevented, which is specifically reflected in improving biochemical indexes caused by the plateau encephaledema, reducing accumulation of brain water content, improving brain tissue pathological damage, enhancing neuronal activity and relieving damage of hypobaric hypoxia to mitochondria.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of hydrogen-rich water containing salidroside in the preparation of a medicine for preventing high-altitude cerebral edema. Background Art

[0002] High altitude cerebral edema (HACE) is a serious disorder of central nervous system function caused by acute hypoxia, and is the terminal stage of acute mountain sickness. HACE is characterized by rapid onset, and clinical manifestations are characterized by severe headache, vomiting, ataxia, and progressive disturbance of consciousness. In severe cases, convulsions, heart failure, shock, pulmonary edema, severe infection, and cerebral hemorrhage may occur. HACE develops rapidly and can often be life-threatening. The pathological changes of the disease mainly include ischemic or hypoxic damage to brain tissue, cerebral circulation disorders, and ultimately brain edema and increased intracranial pressure. If not treated properly, it is often life-threatening. In China, it was previously called high altitude coma, cerebral mountain sickness, acute high altitude encephalopathy, and high altitude cerebral hypoxia syndrome. With the increase in altitude, the oxygen and air pressure in the air decrease, a series of organ tissues change, and with the increase in altitude, the blood oxygen saturation decreases, which eventually leads to HACE, which seriously affects people's health.

[0003] At present, the main method for treating high-altitude cerebral edema is drug therapy. Taking drugs such as dexamethasone, furosemide, and mannitol can reduce intracranial pressure. However, long-term use of such drugs will have many side effects on the human body. Therefore, how to effectively prevent high-altitude cerebral edema is very critical. At present, there are no reports on the use of salidroside and hydrogen-rich water to prevent high-altitude cerebral edema. Summary of the invention

[0004] The purpose of the present invention is to provide an application of hydrogen-rich water containing salidroside in the preparation of a drug for preventing high-altitude cerebral edema, so as to solve the problems existing in the above-mentioned prior art. The present invention creatively combines salidroside and hydrogen-rich water to achieve the effect of preventing high-altitude cerebral edema.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an application of hydrogen-rich water containing salidroside in preparing a medicine for preventing high-altitude cerebral edema. The preparation method of the hydrogen-rich water containing salidroside comprises the steps of injecting hydrogen into a salidroside aqueous solution to obtain the hydrogen-rich water containing salidroside.

[0007] Preferably, the concentration of salidroside in the hydrogen-rich water containing salidroside is 30 mg / kg-100 mg / kg, and the concentration of hydrogen is 4 mg / kg-6 mg / kg.

[0008] Further preferably, the concentration of salidroside in the hydrogen-rich water containing salidroside is 50 mg / kg, and the concentration of hydrogen is 5.35 mg / kg.

[0009] The invention provides a medicine for preventing high-altitude cerebral edema, the medicine comprising hydrogen-rich water containing salidroside; the preparation method of the hydrogen-rich water containing salidroside comprises the step of injecting hydrogen into a salidroside aqueous solution to obtain the hydrogen-rich water containing salidroside.

[0010] Preferably, the concentration of salidroside in the hydrogen-rich water containing salidroside is 30 mg / kg-100 mg / kg, and the concentration of hydrogen is 4 mg / kg-6 mg / kg.

[0011] Further preferably, the concentration of salidroside in the hydrogen-rich water containing salidroside is 50 mg / kg, and the concentration of hydrogen is 5.35 mg / kg.

[0012] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0013] The present invention discloses the following technical effects:

[0014] The present invention creatively combines hydrogen-rich water and salidroside to produce a synergistic effect, thereby achieving better therapeutic effects. The results of the specific embodiments of the present invention show that the combination of salidroside and hydrogen-rich water can effectively prevent high-altitude cerebral edema, which is specifically reflected in: improving biochemical indicators caused by high-altitude cerebral edema, reducing the accumulation of brain water content, improving brain tissue pathological damage, enhancing neuronal activity, and alleviating low-pressure hypoxia damage to mitochondria. The present invention has important clinical significance for strengthening the exploration of the pathogenesis and prevention and treatment of high-altitude cerebral edema. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 The graphs are the changes of biochemical indexes of rats with high altitude cerebral edema in each experimental group; A is the SOD statistical graph; B is the MAD statistical graph; C is the CAT statistical graph; D is the GSH-Px statistical graph; CON is the control group, HH is the model group (denoted as HH), HRW is the hydrogen-rich water group, SAL is the salidroside group, combined is the hydrogen-rich water combined with salidroside administration group, ACZ is the acetazolamide positive control group;

[0017] Figure 2Schematic diagram of the changes in brain water content of rats in each experimental group; CON is the control group, HH is the model group (denoted as HH), HRW is the hydrogen-rich water group, SAL is the salidroside group, HRV+SAL is the hydrogen-rich water combined with salidroside administration group, and ACZ is the acetazolamide positive control group; ## ≤0.01, *p≤0.05, **p≤0.01, ***p≤0.001;

[0018] Figure 3 Transmission electron microscopy of mitochondria and endoplasmic reticulum in the brain of rats in each experimental group; CON is the control group, HH is the model group (denoted as HH), HRW is the hydrogen-rich water group, SAL is the salidroside group, HRV+SAL is the hydrogen-rich water combined with salidroside group, and ACZ is the acetazolamide positive control group;

[0019] Figure 4 HE staining images of the cerebral cortex and hippocampus sections of rats in each experimental group; CON is the control group, HH is the model group (denoted as HH), HRW is the hydrogen-rich water group, SAL is the salidroside group, HRV+SAL is the hydrogen-rich water combined with salidroside group, ACZ is the acetazolamide positive control group; Hippocampus is the hippocampus, CA1 is the CA1 region of the hippocampus, CA2 is the CA2 region of the hippocampus, CA3 is the CA3 region of the hippocampus, DG is the DG region of the hippocampus, and Cortex is the cortex; the scale bar is 100 μm;

[0020] Figure 5 The Nissl staining images of the cerebral cortex and hippocampus of rats in each experimental group; CON is the control group, HH is the model group (denoted as HH), HRW is the hydrogen-rich water group, SAL is the salidroside group, HRV+SAL is the hydrogen-rich water combined with salidroside administration group, ACZ is the acetazolamide positive control group; Hippocampus is the hippocampus, CA1 is the CA1 area of ​​the hippocampus, CA2 is the CA2 area of ​​the hippocampus, CA3 is the CA3 area of ​​the hippocampus, DG is the DG area of ​​the hippocampus, and Cortex is the cortex; the scale bar is 100 μm;

[0021] Figure 6 Protein expression profiles detected by western blot in each experimental group; lane 1 is the CON group, lane 2 is the HH group, lane 3 is the HRW group, lane 4 is the SAL group, lane 5 is the HRV+SAL group, and lane 6 is the ACZ group. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] Unless otherwise specified, the materials used in the present invention are all purchased by those skilled in the art; the methods used in the present invention are all methods well known to those skilled in the art.

[0028] Hydrogen-rich water is composed of molecular hydrogen and physiological saline. Molecular hydrogen is a flammable, colorless, odorless, and non-toxic gas. It can remove a large number of hydroxyl radicals (·OH) in mitochondria and other organelles and convert them into water molecules. Hydrogen-rich water has antioxidant, anti-inflammatory, anti-apoptotic, and autophagy regulating functions. It can selectively remove harmful free radicals such as hydroxyl radicals and nitrite anions, reduce oxidative damage to proteins, DNA, etc.; reduce the production of oxidative stress markers, increase the activity of antioxidant enzymes such as catalase, superoxide dismutase, and glutathione peroxidase, and reduce the activity of oxidases such as myeloperoxidase; reduce the inflammatory response by reducing the release of proinflammatory factors such as TNF-α, IL-1β, IL-6, and HMGB1, and increasing the release of anti-inflammatory factor interleukin-10; hydrogen can inhibit cell apoptosis by reducing the expression of cysteine-containing aspartate protease 3, 8, and 9. Rhodiola rosea L. is a perennial herbaceous plant of the genus Rhodiola in the Crassulaceae family. Its nature and flavor are cold, sweet, and astringent. It belongs to the lung meridian and has the effects of replenishing qi and clearing the lungs, improving intelligence and nourishing the heart, astringing and stopping bleeding, and dispersing blood stasis and reducing swelling. It is mainly used to treat qi deficiency and physical weakness, fear of cold after illness, shortness of breath and fatigue, lung heat cough, hemoptysis, leucorrhea and diarrhea, and traumatic injuries. In the present invention, salidroside can protect central nervous system diseases by regulating inflammation, oxidative stress, apoptosis and autophagy. Both hydrogen-rich water and salidroside can protect against nervous system diseases.

[0029] Example 1 Preparation of Hydrogen-Rich Water Containing Salidroside

[0030] Mixing salidroside with water to obtain a salidroside aqueous solution; the concentration of salidroside in the salidroside aqueous solution is 30 mg / kg-100 mg / kg;

[0031] After injecting 50mL of salidroside aqueous solution into the bag, evacuate the air in the bag, connect the non-needle end of a 20mL sterile empty needle to the hydrogen output end of the hydrogen generator, insert the needle into a physiological saline soft bag, and allow hydrogen to enter the bag. After the soft bag is expanded and filled with hydrogen, pull out the needle, put it into an aluminum bag, and store it at 4°C for 24 hours to obtain hydrogen-rich water containing salidroside. The concentration of hydrogen in the hydrogen-rich water containing salidroside is 4mg / kg-6mg / kg.

[0032] Hydrogen molecules have strong penetrating power. If the container is not selected properly, it will cause the hydrogen molecules to escape. Therefore, vacuum aluminum foil packaging with strong sealing properties is used.

[0033] Example 2 Animal Experiment

[0034] 1. Experimental Animals

[0035] Thirty-six Wistar rats, weighing 180-220 g, were provided by Beijing Huafukang Biotechnology Co., Ltd., with animal production certificate number: SCXK (Beijing) 2019-0008. The experimental animals were raised and managed in accordance with SPF animal management requirements.

[0036] 2. Modeling

[0037] The rats were exposed to a simulated plateau environment animal test chamber for 72 hours, and then ascended to a simulated altitude of 6000m and 47.2kPa at a speed of 10m / s to obtain high-altitude cerebral edema model rats. After the experiment, the rats were anesthetized immediately to obtain brain tissue and blood samples.

[0038] 3. Grouping and intervention

[0039] After one week of adaptive feeding, 36 Wistar rats were randomly divided into a control group (denoted as CON), a model group (denoted as HH), a hydrogen-rich water group (purchased from Shanghai Huimei, denoted as HRW), a salidroside group (50 mg / kg, denoted as SAL), a hydrogen-rich water combined with salidroside administration group (50 mL of hydrogen-rich water containing salidroside prepared by the preparation method provided in Example 1, the concentration of salidroside in the hydrogen-rich water containing salidroside is 50 mg / kg, and the concentration of hydrogen is 10 ... The concentration was 5.35 mg / kg, recorded as combined or HRV+SAL), acetazolamide positive control group (200 mg / kg, recorded as ACZ), a total of 6 groups, 10 rats in each group, and the drug was administered for 7 days before modeling. The control group and the model group were intraperitoneally injected with normal saline (2 mL), and the other drug-dosing groups were intraperitoneally injected with salidroside, hydrogen-rich water, or hydrogen-rich water combined with salidroside. The injection volume was 0.1 mL / 10 g, and the intraperitoneal injection volume was the same. Modeling was performed after administration, and the body weight of the rats was measured every day before and after modeling.

[0040] 4. Detection methods for preventing high altitude cerebral edema in each experimental group

[0041] After the experiment, the rats were anesthetized and killed, and some tissues were taken for the detection of biochemical indicators, including SOD, CAT, GSH-Px, and MDA.

[0042] After the experiment, the rats were anesthetized and killed, and the brain tissue was quickly dissected and separated to obtain 100 mg of the left hemisphere. The wet weight of the tissue was quickly weighed, and then it was placed on filter paper and placed in a constant temperature oven at 80°C for 72 h. The dry weight of the tissue was weighed to ensure that it reached a constant weight (the mass error between the two times was less than 0.0003 g). The brain water content was calculated using the following formula: brain water content / % = (wet weight - dry weight) / wet weight × 100%.

[0043] After the experiment, the rats were anesthetized and killed, and 1mm*1mm*1mm brain tissue was quickly put into electron microscope fixative at 4℃ for 2-4h. Rinse 3 times with 0.1M phosphate buffer (PB, pH 7.4), 15min each time, and then fix, dehydrate, infiltrate, embed, slice, stain, observe under transmission electron microscope, and collect images for analysis.

[0044] After the experiment, the rats were anesthetized and killed, the brain tissues were fixed with paraformaldehyde, the samples were embedded in paraffin, sectioned, stained with hematoxylin-eosin and Nissl, observed under an optical microscope, and photographed.

[0045] Expression of related proteins: Add lysis buffer (RIPA: PMSF = 100: 1), place in 2mL EP tube, add 2 steel beads to each tube, balance, and homogenize at low temperature. Stand at room temperature for 30min, centrifuge at 12000rpm at low temperature (4℃) for 15min, take the supernatant; determine the protein concentration by BCA method, and calculate the loading amount. Take the supernatant after centrifugation and add 4× loading buffer (1 / 3 sample volume), 100℃, 5min, denature the protein, and divide the sample. Prepare gel electrophoresis, load the sample, electrophoresis (80V / 20min, 110V / 40min), transfer to membrane (220mA, 1.5h), and then block the PVDF membrane in 5% skim milk powder for 2h. Primary antibodies (Nrf1 (1: 1000), Nrf2 (1: 1000) and β-actin (1: 10000)) are incubated at 4℃ overnight. The next day, the membrane was washed with 1×TBST buffer for 3 times, 10 min each time; the secondary antibody was incubated for 2 h, and the membrane was washed again for 3 times, 10 min each time. After that, ECL luminescent solution was added to the membrane to make it color and emit light, and the degree of protein luminescence was counted. ImageJ was used for quantification, and the gray value was expressed.

[0046] 5. Statistical methods

[0047] All data were calculated using SPSS 16.0 software. The measurement data were expressed as mean ± standard deviation (Mean ± SD). The independent sample t test was used for comparison between the groups. p ≤ 0.05 was considered statistically significant.

[0048] 6. Experimental results

[0049] Changes of biochemical indexes in rats with high altitude cerebral edema in each experimental group Figure 1 As shown, the results showed that: SOD, CAT, and GSH-Px in the model group showed a downward trend, and the hydrogen-rich water combined with salidroside administration group could increase these three indicators; MDA in the model group increased, and the hydrogen-rich water combined with salidroside administration group decreased, indicating that hydrogen-rich water combined with salidroside improved the biochemical indicators of rats with high-altitude cerebral edema and increased the energy metabolism level of rats.

[0050] Changes of brain water content in rats in each experimental group Figure 2 As shown, brain water content is the gold standard for measuring whether the model is successful. It can be seen that the brain water content of rats in the model group was significantly increased, and the hydrogen-rich water combined with salidroside administration group can reduce the accumulation of brain water content.

[0051] Transmission electron microscopy observation of brain tissue of rats in each experimental group Figure 3 As shown, the green arrow indicates healthy mitochondria, the yellow arrow indicates healthy endoplasmic reticulum, the red arrow indicates swollen mitochondria, and the blue arrow indicates damaged endoplasmic reticulum. In the model group, mitochondrial swelling, mitochondrial cristae damage, and severely dilated and moderately edematous endoplasmic reticulum were observed, while the mitochondrial structure of the hydrogen-rich water combined with salidroside administration group was relatively intact.

[0052] HE staining of brain tissue of rats in each experimental group is shown in Figure 4 As shown, the neurons in the control group had normal morphology and regular arrangement, and no obvious damage to the morphological structure. The model group showed severe pathological damage, with condensation and dark staining of neuronal nuclei, and the formation of vacuoles. The cells were arranged relatively loosely, and the intercellular spaces were enlarged. The group receiving hydrogen-rich water combined with salidroside showed improvement in brain tissue pathological damage.

[0053] Nissle staining of brain tissues of rats in each experimental group is shown in the figure below. Figure 5 As shown, the neurons in the control group were arranged neatly, the cytoplasm was filled with a large number of blue granular Nissl bodies, the nuclear membrane and nucleolus were clear, the neurons in the model group were irregular in morphology, the cell viability was significantly decreased, the nuclear membrane and nucleolus were blurred, and the Nissl bodies were lightly stained. The hydrogen-rich water combined with salidroside administration group significantly reversed these adverse changes and enhanced neuronal activity.

[0054] Protein expression profiles detected by western blot in brain tissue of rats in each experimental group are shown in Figure 6 As shown. The complex biological process of mitochondrial biogenesis is regulated by multiple protein molecules such as PGC-1α, NRF1 / NRF2, and TFAM. In this example, Western Blot was used to evaluate the expression of key proteins in the SIRT1 / PGC-1α pathway. The results showed that when the rat brain was injured, the expression levels of SIRT1, PGC-1α, NRF1, NRF2, and TFAM all decreased, indicating that hypoxic stimulation caused damage to mitochondrial synthesis. The expression of SIRT1, PGC-1α, NRF1, NRF2, and TFAM, which were pre-protected by the hydrogen-rich water combined with salidroside administration group, showed an upward trend, indicating that the hydrogen-rich water combined with salidroside administration group can alleviate the damage of low-pressure hypoxia to mitochondria.

[0055] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. The use of hydrogen-rich water containing salidroside in the preparation of a drug for preventing high-altitude cerebral edema, characterized in that: The method for preparing hydrogen-rich water containing salidroside comprises the step of injecting hydrogen into a salidroside aqueous solution to obtain the hydrogen-rich water containing salidroside.

2. The use according to claim 1, characterized in that: The concentration of salidroside in the hydrogen-rich water containing salidroside is 30 mg / kg-100 mg / kg, and the concentration of hydrogen is 4 mg / kg-6 mg / kg.

3. A drug for preventing high altitude cerebral edema, characterized in that: The medicine comprises hydrogen-rich water containing salidroside; and the preparation method of the hydrogen-rich water containing salidroside comprises the step of injecting hydrogen into a salidroside aqueous solution to obtain the hydrogen-rich water containing salidroside.

4. The drug according to claim 3, characterized in that The concentration of salidroside in the hydrogen-rich water containing salidroside is 30 mg / kg-100 mg / kg, and the concentration of hydrogen is 4 mg / kg-6 mg / kg.

5. The drug according to claim 3, characterized in that The medicine also includes pharmaceutically acceptable auxiliary materials.

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