Application of hydrogen-rich water containing salidroside in preparation of medicine for preventing high altitude cerebral edema

Abstract

The present application relates to the field of biological medicine, and particularly relates to application of hydrogen-rich water containing salidroside in preparation of a medicine for preventing high altitude cerebral edema. The present application creatively combines salidroside and hydrogen-rich water, and achieves the effect of preventing high altitude cerebral edema. The results of the specific embodiments of the present application show that the combination of salidroside and hydrogen-rich water can effectively prevent high altitude cerebral edema, and specifically can improve biochemical indexes caused by high altitude cerebral edema, reduce accumulation of brain water content, improve brain tissue pathological damage, enhance neuron activity, and relieve damage of mitochondria caused by low pressure and hypoxia.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of hydrogen-rich water containing rhodioloside in the preparation of drugs for the prevention of high-altitude cerebral edema. Background Technology

[0002] High altitude cerebral edema is a severe impairment of central nervous system function caused by acute hypoxia, representing the terminal stage of acute mountain sickness. It is characterized by a rapid onset, with clinical manifestations including severe headache, vomiting, ataxia, and progressive loss of consciousness. In severe cases, it can lead to seizures, heart failure, shock, pulmonary edema, severe infection, and cerebral hemorrhage. High altitude cerebral edema has a rapid onset and can often be life-threatening. The pathological changes primarily involve ischemic or hypoxic damage to brain tissue, cerebral circulatory disturbances, ultimately resulting in cerebral edema and increased intracranial pressure. Improper treatment can be life-threatening. In China, it has previously been referred to as high-altitude coma, cerebral mountain sickness, acute mountain sickness encephalopathy, and high-altitude hypoxic-cold syndrome. As altitude increases, the oxygen and air pressure in the air decrease, causing changes in a series of organs and tissues. Furthermore, with increasing altitude, blood oxygen saturation decreases, ultimately leading to high altitude cerebral edema, a disease that seriously affects people's health.

[0003] Currently, the main treatment for high-altitude cerebral edema is medication, which involves taking drugs such as dexamethasone, furosemide, and mannitol to reduce intracranial pressure. However, long-term use of these drugs can produce many side effects. Therefore, effective prevention of high-altitude cerebral edema is crucial. Currently, there are no reports on the use of rhodioloside and hydrogen-rich water for the prevention of high-altitude cerebral edema. Summary of the Invention

[0004] The purpose of this invention is to provide the application of hydrogen-rich water containing rhodioloside in the preparation of drugs for preventing high-altitude cerebral edema, thereby solving the problems existing in the prior art. This invention creatively combines rhodioloside and hydrogen-rich water to achieve the effect of preventing high-altitude cerebral edema.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides the application of hydrogen-rich water containing rhodioloside in the preparation of drugs for preventing high-altitude cerebral edema. The preparation method of the hydrogen-rich water containing rhodioloside includes the step of injecting hydrogen gas into an aqueous solution of rhodioloside to obtain the hydrogen-rich water containing rhodioloside.

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

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

[0009] This invention provides a drug for preventing high-altitude cerebral edema, the drug comprising hydrogen-rich water containing rhodioloside; the method for preparing the hydrogen-rich water containing rhodioloside comprises injecting hydrogen gas into an aqueous solution of rhodioloside to obtain the hydrogen-rich water containing rhodioloside.

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

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

[0012] Preferably, the drug also includes pharmaceutically acceptable excipients.

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

[0014] This invention creatively combines hydrogen-rich water and rhodioloside to produce a synergistic effect, thereby achieving better therapeutic efficacy. Results from specific embodiments of this invention show that the combination of rhodioloside and hydrogen-rich water can effectively prevent high-altitude cerebral edema, specifically by: improving biochemical indicators caused by high-altitude cerebral edema, reducing brain water accumulation, improving pathological damage to brain tissue, enhancing neuronal activity, and alleviating mitochondrial damage caused by hypobaric hypoxia. This invention has significant clinical implications for further exploring the pathogenesis and prevention of high-altitude cerebral edema. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The graphs show the changes in biochemical indicators of rats with high-altitude cerebral edema in each experimental group; where 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 rhodioloside group; combined is the hydrogen-rich water combined with rhodioloside administration group; and ACZ is the acetazolamide positive control group.

[0017] Figure 2The diagram shows the changes in brain water content in rats in each experimental group; where CON is the control group, HH is the model group (denoted as HH), HRW is the hydrogen-rich water group, SAL is the rhodioloside group, HRV+SAL is the hydrogen-rich water combined with rhodioloside 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 brains of rats in each experimental group; CON was the control group, HH was the model group (denoted as HH), HRW was the hydrogen-rich water group, SAL was the rhodioloside group, HRV+SAL was the hydrogen-rich water combined with rhodioloside administration group, and ACZ was the acetazolamide positive control group.

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

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

[0021] Figure 6 The images show the protein expression profiles of each experimental group as detected by Western blot. 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 Implementation

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

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0027] Unless otherwise specified, all materials used in this invention are those that are routinely purchased by those skilled in the art; all methods used in this invention are those that are 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 that can remove large amounts of hydroxyl radicals (·OH) from mitochondria and other organelles, converting 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, reducing oxidative damage to proteins and DNA; reduce the production of oxidative stress markers, increase the activity of antioxidant enzymes such as catalase, superoxide dismutase, and glutathione peroxidase, and decrease the activity of oxidative enzymes such as myeloperoxidase; reduce the release of pro-inflammatory factors such as TNF-α, IL-1β, IL-6, and HMGB1, and increase the release of the anti-inflammatory factor interleukin-10 to alleviate inflammatory responses; hydrogen can inhibit apoptosis by reducing the expression of cysteine-containing aspartate proteases 3, 8, and 9. Rhodiola rosea L. is a perennial herb belonging to the genus Rhodiola in the family Crassulaceae. Its properties are cold, sweet, and astringent; it enters the lung meridian and has the effects of tonifying qi and clearing the lungs, benefiting 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 weakness, post-illness aversion to cold, shortness of breath and fatigue, lung heat cough, hemoptysis, leukorrhea and diarrhea, and traumatic injuries. In this invention, rhodioloside can protect against central nervous system diseases by regulating inflammation, oxidative stress, apoptosis, and autophagy. Both hydrogen-rich water and rhodioloside have protective effects against nervous system diseases.

[0029] Example 1: Preparation of hydrogen-rich water containing rhodioloside

[0030] Rhodioloside was mixed with water to obtain an aqueous solution of rhodioloside; the concentration of rhodioloside in the aqueous solution was 30 mg / kg-100 mg / kg.

[0031] After injecting 50 mL of rhodioloside aqueous solution into the bag, the air inside the bag is evacuated. The non-needle end of a 20 mL sterile syringe is connected to the hydrogen output end of a hydrogen generator. The needle is inserted into the physiological saline soft bag, and hydrogen enters the bag. After the soft bag expands and is filled with hydrogen, the needle is removed, and the bag is placed in an aluminum bag and stored at 4°C for 24 hours to obtain hydrogen-rich water containing rhodioloside. The hydrogen concentration in the hydrogen-rich water containing rhodioloside is 4 mg / kg-6 mg / kg.

[0032] Hydrogen molecules have strong penetrating power, and if the container is not chosen properly, hydrogen molecules will escape. Therefore, vacuum aluminum foil packaging with strong sealing properties is used.

[0033] Example 2 Animal Experiment

[0034] 1. Laboratory animals

[0035] 36 Wistar rats, weighing 180 - 220 g, were provided by Beijing Huafukang Biotechnology Co., Ltd., with the animal production license number: SCXK(Beijing)2019 - 0008. The experimental animals were raised and managed according to the requirements of SPF - level animal management.

[0036] 2. Model establishment

[0037] The rats were exposed to a simulated high - altitude environment animal test chamber for 72 h, ascending to a simulated altitude of 6000 m (47.2 kPa) at a speed of 10 m / s to obtain rats with high - altitude cerebral edema model. Immediately after the experiment, the rats were anesthetized and brain tissue and blood samples were taken.

[0038] 3. Grouping and intervention

[0039] After one - week adaptive feeding of the 36 Wistar rats, they 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 this hydrogen - rich water containing salidroside was 50 mg / kg, and the concentration of hydrogen was 5.35 mg / kg, denoted as combined or HRV + SAL), and an acetazolamide positive control group (200 mg / kg, denoted as ACZ), a total of 6 groups, with 10 rats in each group. Seven days before model establishment, the control group and the model group were intraperitoneally injected with normal saline (2 mL), and the remaining administration groups were intraperitoneally injected with salidroside, hydrogen - rich water or hydrogen - rich water combined with salidroside, with an injection volume of 0.1 mL / 10 g. The intraperitoneal injection volumes were the same. After administration, model establishment was carried out, and the body weights of the rats were measured every day before and after model establishment.

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

[0041] After the experiment, the rats were anesthetized and sacrificed, and part of the tissue was used to detect biochemical indexes, including SOD, CAT, GSH - Px, and MDA.

[0042] After the experiment, the rats were anesthetized and sacrificed. The brain was quickly dissected and separated, and 100 mg of the left half - brain was taken. The wet weight of the tissue was quickly weighed, and then it was placed on filter paper and put into an 80°C constant - temperature oven for 72 h. The dry weight of the tissue was weighed to ensure constant weight (the mass error between the two times was less than 0.0003 g), and the brain water content was calculated. The calculation formula for brain water content is: brain water content / %=(wet weight−dry weight) / wet weight×100%.

[0043] After the experiment, the rats were anesthetized and euthanized. Brain tissue samples (1mm x 1mm x 1mm) were quickly immersed in electron microscopy fixative at 4°C for 2-4 hours. The samples were then rinsed three times with 0.1M phosphate buffer (PB, pH 7.4) for 15 minutes each time. After fixation, dehydration, infiltration, embedding, sectioning, and staining, the samples were observed under a transmission electron microscope, and images were acquired and analyzed.

[0044] After the experiment, the rats were anesthetized and euthanized. The brain tissue was fixed with paraformaldehyde, the samples were embedded in paraffin, sectioned, stained with hematoxylin and eosin and Nissl stain, and observed and photographed under an optical microscope.

[0045] Expression of relevant proteins: Add lysis buffer (RIPA:PMSF = 100:1), place in 2mL EP tubes, add 2 steel balls to each tube, balance, and homogenize at low temperature. Incubate at room temperature for 30 min, centrifuge at 12000 rpm at low temperature (4℃) for 15 min, collect the supernatant; determine protein concentration using the BCA method, and calculate the loading amount. Add 4× loading buffer (1 / 3 sample volume) to the supernatant after centrifugation, incubate at 100℃ for 5 min to denature the protein, and aliquot the sample. Prepare gel electrophoresis, load the sample, perform electrophoresis (80V / 20 min, 110V / 40 min), transfer to membrane (220mA, 1.5 h), and then block the PVDF membrane in 5% skim milk powder for 2 h. Incubate with primary antibodies (Nrf1 (1:1000), Nrf2 (1:1000), and β-actin (1:10000)) at 4℃ overnight. The following day, the membrane was washed three times with 1×TBST buffer, 10 min each time; it was then incubated with secondary antibody for 2 h, followed by three more washes, 10 min each time. After incubation, ECL luminescent solution was added to the membrane to induce color development and luminescence, and the degree of protein luminescence was statistically analyzed. Quantification was performed using ImageJ, and the results were expressed as grayscale values.

[0046] 5. Statistical methods

[0047] All data were calculated using SPSS 16.0 software. Quantitative data were expressed as mean ± standard deviation (Mean ± SD). Independent samples t-tests were used for comparisons between groups, and p ≤ 0.05 was considered statistically significant.

[0048] 6. Experimental Results

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

[0050] Changes in brain water content in rats of each experimental group are as follows: Figure 2 As shown, brain water content is the gold standard for measuring the success of modeling. It can be seen that the brain water content of rats in the model group is significantly increased, while the group treated with hydrogen-rich water combined with rhodioloside can reduce the accumulation of brain water.

[0051] Transmission electron microscopy images of rat brain tissue from each experimental group are shown below. Figure 3 As shown, green arrows represent healthy mitochondria, yellow arrows represent healthy endoplasmic reticulum, red arrows represent swollen mitochondria, and blue arrows represent damaged endoplasmic reticulum. In the model group, mitochondrial swelling, mitochondrial cristae damage, and severely dilated and moderately edematous endoplasmic reticulum were observed. In the group receiving hydrogen-rich water combined with rhodioloside, the mitochondrial structure remained relatively intact.

[0052] HE staining images of rat brain tissue from each experimental group are shown below. Figure 4 As shown, the control group showed normal neuronal morphology and regular arrangement with no obvious damage to morphology and structure, while the model group showed severe pathological damage, with condensed and deeply stained neuronal nuclei forming vacuoles, loosely arranged cells, and increased intercellular spaces. The group treated with hydrogen-rich water combined with rhodioloside showed improvement in the pathological damage of brain tissue.

[0053] Nissle staining images of rat brain tissue from each experimental group are shown below. Figure 5 As shown, neurons in the control group were neatly arranged, with a large number of blue granular Nissl bodies in the cytoplasm, and clear nuclear membranes and nucleoli. Neurons in the model group had irregular morphology, significantly reduced cell viability, blurred nuclear membranes and nucleoli, and lightly stained Nissl bodies. The group treated with hydrogen-rich water combined with rhodioloside significantly reversed these adverse changes and enhanced neuronal activity.

[0054] Protein expression profiles of rat brain tissue detected by Western blot in each experimental group are as follows: Figure 6 As shown, the complex biological processes of mitochondrial biogenesis are regulated by multiple protein molecules, including PGC-1α, NRF1 / NRF2, and TFAM. This example uses Western blotting to assess the expression of key proteins in the SIRT1 / PGC-1α pathway. The results show that in rat brain injury, the expression levels of SIRT1, PGC-1α, NRF1, NRF2, and TFAM all decreased, suggesting that hypoxia stimulation leads to impaired mitochondrial synthesis. However, in the hydrogen-rich water combined with rhodioloside administration group, the expression of SIRT1, PGC-1α, NRF1, NRF2, and TFAM showed an increasing trend under pre-protection, indicating that the hydrogen-rich water combined with rhodioloside administration group can alleviate mitochondrial damage caused by hypobaric hypoxia.

[0055] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of hydrogen-rich water containing rhodioloside in the preparation of drugs for preventing high-altitude cerebral edema, characterized in that, The method for preparing the hydrogen-rich water containing rhodioloside includes the step of injecting hydrogen gas into an aqueous solution of rhodioloside to obtain the hydrogen-rich water containing rhodioloside; the concentration of rhodioloside in the hydrogen-rich water containing rhodioloside is 30 mg / kg-100 mg / kg, and the concentration of hydrogen gas is 4 mg / kg-6 mg / kg.

2. A drug for preventing high-altitude cerebral edema, characterized in that, The drug includes hydrogen-rich water containing rhodioloside; the preparation method of the hydrogen-rich water containing rhodioloside includes the step of injecting hydrogen gas into an aqueous solution of rhodioloside to obtain the hydrogen-rich water containing rhodioloside; the concentration of rhodioloside in the hydrogen-rich water containing rhodioloside is 30 mg / kg-100 mg / kg, and the concentration of hydrogen gas is 4 mg / kg-6 mg / kg.

3. The drug according to claim 2, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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