New application of L-theanine in improving acute plateau hypoxia tolerance (viability)

Abstract

The invention provides a new application of L-theanine in improvement of acute plateau hypoxia tolerance (viability), and finds and verifies that L-theanine can significantly improve the survival rate of mice in an acute hypobaric hypoxia environment and also can relieve brain tissue injury and body oxidative stress injury induced by plateau hypobaric hypoxia for the first time. And the generation of brain energy is promoted. In addition, in an in-vitro cell model, the L-theanine can also effectively improve hypoxia injury of the cobalt chloride induced hippocampal neuron cell line HT22. The invention provides a natural and safe active substance capable of improving the plateau hypoxia tolerance of an organism, provides a scientific basis for research and development of a series of acute physiological and pathological reactions caused by plateau hypoxia, and has a significant application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically involving a new use of L-theanine to improve acute high-altitude hypoxia tolerance (survival ability). Background Technology

[0002] Hypoxic hypoxia in high-altitude environments is a key factor affecting normal bodily functions. Acute or chronic exposure to high-altitude hypoxia often triggers a series of complex physiological and pathological reactions. When people are suddenly exposed to high-altitude hypoxia without acclimatization, it often causes a combined stress response in the nervous, respiratory, and circulatory systems, mainly manifested as dizziness, loss of appetite, nausea, palpitations, and decreased physical strength. If the condition worsens, it may lead to central nervous system dysfunction and cardiopulmonary dysfunction, resulting in acute mountain sickness such as high-altitude cerebral edema and high-altitude pulmonary edema, and even death—the extreme form of altitude sickness. The brain, due to its high demand for metabolic oxygen, is the most sensitive organ to hypoxia. Acute exposure to high-altitude hypoxia easily leads to hypoxic brain damage, resulting in cognitive impairment. Currently, there are few drugs available clinically to alleviate adverse reactions induced by acute high-altitude hypoxia, mainly including acetazolamide and dexamethasone, but these drugs all have various side effects. Therefore, developing new drugs that can improve tolerance to hypoxia at high altitudes is of great significance and will help meet the health needs of people such as workers, travelers and military personnel in high-altitude areas.

[0003] L-theanine (LTA) is a non-protein amino acid unique to tea and a major source of its flavor. It is widely used as a beverage ingredient and dietary supplement in the modern food industry. Recent studies have shown that L-theanine possesses bioactivities such as antioxidant, anti-inflammatory, anti-cancer, mood-regulating, and cognitive-enhancing effects, as well as improving sleep quality. Patent CN108114045A discloses a traditional Chinese medicine composition with L-theanine as the main component for treating hypoxia-induced insomnia, revealing its sedative and sleep-aiding effects. Furthermore, literature reports that theanine has a protective effect against ischemic brain injury. Ischemic brain injury is a type of brain tissue damage caused by insufficient blood supply to the brain due to cerebral vascular stenosis or occlusion. Its main causes include arteriosclerosis, cerebral artery stenosis, cerebral aneurysm, or embolism. The key to prevention and treatment is improving blood supply, such as through thrombolysis. High-altitude hypoxia-induced brain injury is caused by environmental factors such as low pressure and hypoxia at high altitudes, and is usually accompanied by increased cerebral blood flow. The key to prevention and treatment is to increase oxygen supply, such as through supplemental oxygen therapy. Therefore, ischemic brain injury and high-altitude hypoxia-induced brain injury differ significantly in etiology and prevention strategies. Currently, there are no research reports on L-theanine's ability to enhance high-altitude hypoxia tolerance or improve the biological function of brain tissue damage induced by acute high-altitude hypoxia. Summary of the Invention

[0004] In view of this, the present invention aims to propose a novel pharmaceutical use for L-theanine. The novel pharmaceutical use of L-theanine provided by the present invention is its application in the preparation of drugs to improve tolerance to acute high-altitude hypoxia. Experiments have shown that L-theanine can significantly improve the survival rate of mice under acute low-pressure hypoxia, reduce brain tissue damage caused by high-altitude hypoxia, promote brain energy production, alleviate oxidative stress damage, and effectively improve the cell viability of HT22 hippocampal neurons in a cobalt chloride-induced chemical hypoxia model in mice.

[0005] To achieve the above objectives, the technical solution of the present invention is implemented as follows: In a first aspect, the present invention provides the application of L-theanine in the preparation of drugs to improve tolerance to acute high-altitude hypoxia.

[0006] Furthermore, the structural formula of the L-theanine (CAS: 3081-61-6) is as follows:

[0007] Furthermore, the application includes one of the following: (1) Application in the preparation of drugs to improve survival ability in extreme high-altitude hypoxic environments; (2) Application in the preparation of drugs to improve hypoxic brain injury at high altitudes; (3) Application in the preparation of drugs to alleviate oxidative stress damage under high-altitude hypoxia conditions; (4) Application in the preparation of drugs that promote brain energy generation under high-altitude hypoxia conditions; In the application described, the drug is made with L-theanine as the active component, using pharmaceutically or physiologically acceptable excipients and conventional pharmaceutical preparation processes.

[0008] The drug may be an oral preparation or an injectable preparation.

[0009] Furthermore, the anti-altitude hypoxia activity of L-theanine is manifested in the following ways: (1) The results of the acute hypoxia experiment showed that L-theanine can effectively improve the survival rate of mice in an environment at an altitude of 10,000 meters within a certain period of time, and has the potential to enhance the tolerance to acute high altitude hypoxia. (2) Through the analysis of pathological changes and related biochemical indicators of mouse brain tissue at an altitude of 8,000 meters, it was found that L-theanine can significantly reduce the pathological damage of the cerebral cortex and hippocampus of mice, promote the generation of brain energy, and inhibit the occurrence of oxidative stress in the body. (3) In vitro cell experiments have shown that the intervention of L-theanine can effectively improve the cell viability of HT22 hippocampal neurons in mice in a chemical hypoxia model induced by cobalt chloride.

[0010] Furthermore, after one week of use, it can alleviate brain pathological damage caused by high-altitude hypoxia and improve the body's tolerance to low-pressure hypoxia.

[0011] Compared with existing technologies, the application of L-theanine described in this invention in the preparation of drugs to improve tolerance to acute high-altitude hypoxia has the following advantages: This invention is the first to discover and verify that L-theanine can alleviate brain tissue damage induced by acute high-altitude hypoxia, improve brain energy metabolism disorders, and reduce the body's oxidative stress level. It provides a natural, safe active ingredient that can improve the body's tolerance to high-altitude hypoxia, providing a scientific basis for the research and development of drugs to alleviate the negative effects induced by acute high-altitude hypoxia in clinical practice, and has significant application value. Attached Figure Description

[0012] Figure 1 The effect of L-theanine intervention for one week on the survival rate of mice at an altitude of 10,000 meters in Example 1 of this invention; Figure 2 This is a graph showing the H&E staining results and corresponding pathological scores of mouse brain tissue from each group after one week of L-theanine intervention in Example 2 of this invention, at an altitude of 8000 meters (scale bar: 100 μm); where * p <0.05;** p <0.01.

[0013] Figure 3 This is a graph showing the Nissl staining and corresponding pathological scores of mouse brain tissue from each group after one week of L-theanine intervention in Example 2 of this invention, at an altitude of 8000 meters (scale bar: 100 μm); where * p <0.05;** p <0.01.

[0014] Figure 4 This is a graph showing the levels of adenosine triphosphate (ATP) in the brains of mice in each group after one week of L-theanine intervention in Example 2 of this invention, at an altitude of 8000 meters; where * p <0.05;** p <0.01; **** p <0.0001.

[0015] Figure 5This is a graph showing the effects of L-theanine intervention for one week on oxidative stress indicators such as malondialdehyde (MDA), superoxide dismutase (SOD), reduced glutathione / oxidized glutathione (GSH / GSSG), total antioxidant capacity (T-AOC), and catalase (CAT) levels in mice at an altitude of 8000 meters in Example 2 of this invention; where * p <0.05;** p <0.01; **** p <0.0001.

[0016] Figure 6 This is a graph showing the effect of L-theanine on HT22 cell viability in the cobalt chloride-induced in vitro hypoxia model of the present invention, as shown in Example 3 of this invention. * p <0.05; *** p <0.001; **** p <0.0001. Detailed Implementation

[0017] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0018] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0019] Example 1: Effect of L-theanine supplementation on the tolerance of mice to acute hypoxia Animal grouping: 20 male C57BL / 6J mice, 6 weeks old, were randomly divided into 2 groups of 10 mice each, namely the control group (Con) and the L-theanine group (LTA).

[0020] Experimental Methods: Mice were administered L-theanine (LTA) via gavage after acclimatization. The L-theanine group was then administered 200 mg / kg LTA daily via gavage, while the control group received an equal volume of distilled water. This gavage regimen lasted for one week. Two hours after the final gavage, all mice were placed in a hypobaric chamber to simulate a high-altitude hypoxic environment. The simulated altitude was uniformly increased to 10,000 meters at a rate of 20 m / s. Survival was then observed for 20 minutes. After the experiment, all surviving mice were euthanized.

[0021] like Figure 1As shown, compared with the Con group, the survival rate of mice in the LTA group was significantly increased by 40%, indicating that L-theanine has the potential to improve the survival ability of mice in acute high-altitude hypoxia.

[0022] Example 2: Protective effect of L-theanine pretreatment on brain injury and oxidative stress injury induced by acute high-altitude hypoxia. Animal grouping: 40 male C57BL / 6J mice, 6 weeks old, were randomly divided into 4 groups of 10 mice each: normoxic control group (Con), hypobaric hypoxia group (HH), low-dose L-theanine group (LTA100), and high-dose L-theanine group (LTA200).

[0023] Experimental Methods: Mice were administered L-theanine via gavage. After acclimatization, the low-dose and high-dose groups were administered L-theanine at 100 mg / kg and 200 mg / kg daily, respectively. The normoxic control group and the hypoxic-hypoxic group were administered the same volume of distilled water daily. The gavage cycle lasted one week. Two hours after the last gavage, the normoxic control group continued to be housed in a normoxic environment, while the other groups were placed in a hypoxic-hypoxic chamber to simulate a high-altitude hypoxic environment. The simulated altitude was initially increased at a constant rate of 5 m / s to 4500 meters, held for 30 minutes, and then increased at a constant rate of 10 m / s to 8000 meters, maintained for 24 hours. After the experiment, blood samples were collected from the mice via ocular sampling. All mice were then euthanized, and brain tissue was immediately collected. Rinse the surface of the brain tissue with pre-cooled saline solution to remove bloodstains. After blotting with filter paper, divide the brain into two halves, left and right. Fix one half in neutral formalin solution to assess brain pathological changes; store the other half in a pre-cooled cryovial at -80°C for later use.

[0024] H&E staining: Fresh brain tissue was fixed in neutral formalin solution for more than 24 hours, and then sequentially placed in a dehydrator, embedding machine, and microtome for graded alcohol dehydration, embedding, and coronal sectioning. Paraffin sections were then dewaxed to water and stained with hematoxylin and eosin. Hematoxylin stained the cell nuclei, and eosin stained the cytoplasm. After staining, the sections were dehydrated and mounted with neutral resin.

[0025] Nissl staining: Fresh brain tissue was fixed in neutral formalin solution for more than 24 hours, and then subjected to gradient alcohol dehydration, embedding, and coronal sectioning in a dehydrator, embedding machine, and microtome. The paraffin sections were then dewaxed to water and stained with toluidine blue. Nissl bodies appeared purple, and cell nuclei appeared pale purple. After staining, the sections were dehydrated and mounted with neutral resin.

[0026] Brain ATP content measurement: Brain ATP levels were measured using an ATP assay kit (chemiluminescence method).

[0027] Oxidative stress markers were measured: serum levels of MDA, SOD, GSH / GSSG, T-AOC, and CAT were determined according to the instructions of the corresponding commercial reagent kits.

[0028] like Figure 2 As shown, compared to the Con group, the HH group mice exhibited abnormal brain tissue structure. Numerous neuronal degeneration, pyknosis, and deep staining of the nuclei were observed in the CA1, CA3, and DG regions of the hippocampus. A small number of neurons showed edema, and the arrangement of neurons in the cerebral cortex was disordered, with a significantly increased number of degenerated neurons. The LTA100 and LTA200 groups showed improvement in brain tissue damage, specifically with more orderly arrangement of neurons in the cortical region and a gradual decrease in the number of degenerated neurons in the hippocampus. The LTA200 group showed better improvement in the pathological damage to the hippocampus and cortex. Scoring results showed that the pathological scores of the hippocampus (P<0.01) and cortex (P<0.05) were significantly higher in the HH group compared to the Con group. The pathological scores of both the LTA100 and LTA200 groups were lower than those of the HH group, with the LTA200 group showing a significantly lower score (P<0.05).

[0029] like Figure 3 As shown, compared to the Con group, the brain tissue structure of mice in the HH group was more abnormal. Neurons in the CA1, CA3, and DG regions of the hippocampus exhibited irregular morphology, impaired Nissl body structure integrity, blurred nuclear membranes and nucleoli, and disordered neuronal cell arrangement in the cerebral cortex, with a significantly increased number of degenerated neurons. These pathological changes were reversed to varying degrees in both the LTA100 and LTA200 groups. Based on Nissl staining pathological scoring calibration, the results showed that the pathological scores of the hippocampus (P<0.05) and cortical regions (P<0.01) in the HH group were significantly lower than those in the Con group; the pathological scores in the LTA100 and LTA200 groups were higher than those in the HH group.

[0030] The above results indicate that L-theanine can alleviate pathological damage to the cerebral cortex and hippocampus induced by acute high-altitude hypoxia.

[0031] like Figure 4 As shown, the brain ATP levels in the HH group were significantly lower than those in the Con group, while the ATP levels in the LTA100 and LTA200 groups increased by 29.76% (P<0.05) and 48.28% (P<0.01), respectively, compared with the HH group. This indicates that L-theanine has a potential role in promoting brain energy production under acute high-altitude hypoxia.

[0032] Multiple studies have shown that hypoxia can disrupt the homeostasis of the body's redox system. For example... Figure 5 As shown, by measuring oxidative stress-related indicators in mouse serum, it was found that the MDA level in the HH group was significantly higher than that in the Con group (P<0.05), while the levels of SOD, GSH / GSSG, T-AOC, and CAT in the serum were significantly lower than those in the Con group. Both LTA100 and LTA200 groups effectively alleviated oxidative stress damage caused by acute low-pressure hypoxia. These results further demonstrate that L-theanine has anti-acute high-altitude hypoxia effects.

[0033] Example 3: Protective effect of L-theanine against hypoxic injury induced by cobalt chloride in HT22 cells in vitro. Experimental method: In vitro, HT22 cells were pretreated with 1000 μM and 2000 μM L-theanine for 8 hours, respectively. After removing the culture medium, cobalt chloride (250 μM) or cobalt chloride (250 μM) combined with L-theanine (1000 μM and 2000 μM) was added to the fresh culture medium, respectively. The cells were cultured for another 24 hours, and then the cell viability was detected by crystal violet staining.

[0034] like Figure 6 As shown, cobalt chloride treatment induced approximately 30% inhibition in HT22 cells (P<0.0001), while L-theanine significantly alleviated cobalt chloride-induced hypoxic cell damage, increasing cell survival rates by 9.48% (P<0.05) and 13.71% (P<0.001), respectively. These results indicate that L-theanine possesses potential anti-hypoxic effects in the in vitro HT22 cell hypoxia model, and this effect is dose-dependent.

[0035] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Use of L-theanine in the preparation of a drug for improving acute high altitude hypoxia tolerance.

2. Use according to claim 1, characterized in that, The use is the use of L-theanine in the preparation of a drug for improving the survival ability in an extreme high altitude hypoxia environment.

3. Use according to claim 1, characterized in that, The use is the use of L-theanine in the preparation of a drug for improving acute high altitude hypoxia brain injury.

4. Use according to claim 1, characterized in that, The use is the use of L-theanine in the preparation of a drug for reducing the oxidative stress injury of the body under high altitude hypoxia conditions.

5. The use according to claim 1, characterized in that, The use is the use of L-theanine in the preparation of a drug for improving brain energy supply under high altitude hypoxia conditions.

6. Use according to claim 1, characterized in that, The use is the use of L-theanine in the preparation of a drug for improving the viability of hippocampal neuron cells under cobalt chloride-induced hypoxic environment.

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