Application of butylphthalide in prevention and treatment of acute plateau hypoxic ischemic brain injury

Abstract

The invention relates to the technical field of medicine application, in particular to application of butylphthalide in prevention and treatment of acute plateau hypoxic-ischemic brain injury, and the action mechanism of butylphthalide in protection of hypoxic-ischemic brain injury is as follows: butylphthalide can reduce brain water content so as to reduce edema; by improving oxygen free radical balance of hypoxic ischemic brain injury, oxidative stress injury is relieved; by reducing the number of leukocytes of a body, ischemic injury of brain tissues is relieved, and expression of inflammatory factors such as VEGF (vascular endothelial growth factor), TNF-alpha, IL-1beta and IL-6 in brain tissue homogenate is improved, namely butylphthalide can effectively prevent and treat acute plateau hypoxia ischemic brain injury.

Description

Technical Field

[0001] The invention relates to the technical field of drug application, and in particular to application of butylphthalide in preventing and treating acute high altitude hypoxic-ischemic brain damage. Background Art

[0002] Plateau generally refers to areas above 3000m above sea level. Its special natural environment, such as thin oxygen, cold climate and low oxygen partial pressure, will have serious effects on the pathology and physiology of the body. High altitude cerebral edema (HACE) is the most serious stage in the development of acute mountain sickness (AMS). The main clinical manifestations of HACE are headache, loss of coordination, weakness, and decreased level of consciousness (including disorientation, memory loss, hallucinations and psychotic behavior). HACE is considered to be the last stage of AMS. The hallmark symptoms of AMS progressing to HACE are the occurrence of ataxia or changes in consciousness, and occasionally focal neurological deficits. Studies have found that HACE is generally more common in unacclimated individuals who rise to an altitude of more than 3000m. Although the incidence of HACE is low (the incidence of HACE at 4000-5000m is about 3.4%), the mortality rate is extremely high. It is reported that currently there are drugs or related health products that can prevent and treat altitude sickness, such as Rhodiola rosea, Gaoyuanning, American ginseng, Danshen pills, Baifuning, etc. However, these drugs or foods have defects such as slow effect and many side effects, especially the poor effect on the treatment of acute altitude sickness.

[0003] There is no unified standard for the study of the pathogenesis of plateau hypoxic-ischemic brain damage. The main research focuses on two aspects: oxidative stress and inflammatory signaling pathways. Brain tissue is rich in unsaturated fatty acids, has a fast metabolic rate, and is extremely sensitive to hypoxic environments. Therefore, oxygen free radicals play an important role in the formation of high-altitude cerebral edema and are an important factor in the increased permeability of the blood-brain barrier under plateau conditions. Botaro et al. found that after rats were exposed to an altitude of 8,000 meters for 24 hours, the water content of the rat brain increased, the MDA level in the brain tissue increased, and the antioxidant capacity (SOD and GSH) decreased, indicating that the occurrence of HACE is closely related to oxidative stress in brain tissue. At the same time, Jing et al. first proposed that cold stress may promote the formation of HACE by aggravating brain damage caused by continuous hypoxia exposure, and provided an effective and reliable HACE rat experimental model by combining continuous hypoxia with temperature fluctuations. A large number of other studies have shown that hypoxia can activate nuclear transcription factor-κB (NF-κB), which plays a pivotal role in regulating the immune system and inflammation. Neuroinflammation caused by hypoxia has been identified as one of the triggering factors of HACE. Wang et al. reported that hypoxia triggered an inflammatory response, during which astrocyte aquaporin 4 could be upregulated by microglia releasing tumor necrosis factor-α and interleukin-6, and excessive expression of AQP4 would aggravate hypoxic brain damage. Ren Xiaoxia, Ma Junyi, Zheng Yidan et al. found that hypoxia-induced brain edema could significantly upregulate IL-1β, IL-6, VEGF and TNF-α.

[0004] At present, drugs or related health products such as Rhodiola rosea, Gaoyuanning, American ginseng, Danshen pills, Baifuning, and hyperbaric oxygen therapy that are effective in preventing and treating acute high-altitude brain injury include. However, these drugs or foods have defects such as slow effect and many side effects, especially for the treatment of acute mountain sickness. Hyperbaric oxygen can increase tissue oxygen pressure, stimulate the production of endothelial growth factor, and activate cell and vascular repair mechanisms. However, improper use will lead to some unsafe factors, such as flammability, explosiveness, and the occurrence of complications such as barotrauma.

[0005] For example, application number CN1565441A discloses the use of L-butylphthalide in preventing and treating dementia, application number CN1863523A discloses the use of L-butylphthalide in preparing drugs for preventing and treating cerebral infarction, and application number CN106963757A discloses the use of butylphthalide or its derivatives in treating radiation-induced brain damage. However, the above applications do not disclose the use of butylphthalide in preventing high altitude hypoxia.

[0006] Therefore, those skilled in the art are committed to developing an application of butylphthalide in preventing and treating acute high-altitude hypoxic-ischemic brain damage, clarifying the mechanism of action of butylphthalide in preventing and treating acute high-altitude hypoxic-ischemic brain damage, and overcoming the problem that there is currently no optional drug for preventing acute high-altitude hypoxic-ischemic brain damage. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide an application of butylphthalide in preventing and treating acute high-altitude hypoxic-ischemic brain damage, to clarify the mechanism of action of butylphthalide in preventing and treating acute high-altitude hypoxic-ischemic brain damage, and to overcome the problem that there is currently no optional drug for preventing acute high-altitude hypoxic-ischemic brain damage.

[0008] The technical solution of the present invention to solve the above technical problems is as follows:

[0009] Application of butylphthalide in drugs for reducing water content in hypoxic-ischemic brain tissue.

[0010] Application of butylphthalide in improving oxidative stress injury and inflammation in hypoxic-ischemic brain damage.

[0011] Application of butylphthalide in improving the ultrastructure of hypoxic-ischemic brain tissue.

[0012] In the above application, butylphthalide is racemic-3-n-butylphthalide.

[0013] The butylphthalide of the present invention can reduce brain water content, thereby reducing edema; by improving the oxygen free radical balance of hypoxic-ischemic brain damage, oxidative stress damage is reduced; by reducing the number of leukocytes in the body, ischemic damage to brain tissue is reduced, and the expression of inflammatory factors such as VEGF, TNF-α, IL-1β and IL-6 in brain tissue homogenate is improved, that is, butylphthalide can effectively prevent and treat acute high-altitude hypoxic-ischemic brain damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The molecular structure diagram of butylphenol in the embodiment of the present invention;

[0015] Figure 2 Schematic diagram of the effect of preventive administration of butylphthalide on oxidative stress indicators and inflammatory factors in high altitude hypoxic-ischemic rats in an embodiment of the present invention. DETAILED DESCRIPTION

[0016] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0017] The drug for preventive administration in the embodiment is a butylphthalide solution prepared by using the butylphthalide raw material and 2% Tween 80 solvent, and is administered to rats by oral gavage.

[0018] like Figure 1 As shown, the molecular formula of butylphthalide used in the embodiment is: C 12 H 14 O2, molecular weight: 190.24, purchased from a pharmaceutical company.

[0019] All rats used in the experiment were male Sprague-Dawley rats, weighing 200±20g, purchased from the Experimental Animal Center of a Medical University. Before the experiment, the rats were kept in the animal room of a military general hospital for 1 week to adapt to the environment, kept under constant conditions (temperature 20±2℃, humidity 40-60%, light / dark cycle 12h), and fed with standard feed and water.

[0020] Embodiment 1

[0021] Testing the effect of preventive administration of butylphthalide on brain water content in high altitude hypoxic-ischemic rats includes the following steps:

[0022] Step S100. 30 rats were randomly divided into 3 groups, namely, a blank control group, a hypoxia-ischemia group, and a hypoxia-ischemia+butylphthalide group, with 10 rats in each group;

[0023] Step S200: Control group: Rats were intragastrically administered with a blank solvent (2% Tween 80) once a day for 7 days and were raised in a normal environment.

[0024] Step S300 Hypoxia-ischemia group: Rats were first gavaged with blank solvent (2% Tween 80) once a day for 4 days. The right common carotid artery was surgically ligated for 2 hours before entering the chamber. Then, the rats were placed in a special artificial oxygen chamber in the northwest to simulate the 6000m plateau environment. The artificial oxygen chamber was opened for 1 hour every day to add food and water to the animals, and blank solvent was gavaged at the same time. The rats were treated in the oxygen chamber for a total of 3 days.

[0025] Step S400 Hypoxia-ischemia + butylphthalide group: Rats were intragastrically administered (butylphthalide dosage 120 mg / kg), once a day for a total of 4 days. The right common carotid artery was surgically ligated for 2 hours before entering the chamber, and then placed in a special artificial oxygen chamber in the northwest to simulate the 6000m plateau environment. The artificial oxygen chamber was opened for 1 hour every day to add food and water to the animals, and butylphthalide solution was administered intragastrically at the same time. The rats were treated in the artificial oxygen chamber for a total of 3 days.

[0026] Step S500: 3 days after leaving the cabin, take the rat brain tissue, number the brain tissue and weigh the wet weight, put it in a vacuum drying oven at 80°C for 72 hours, weigh the dry weight after drying, and then calculate the brain water content = (wet weight-dry weight) / wet weight×100%.

[0027] Table 1. Changes in water content of rat brain tissue under hypoxic conditions

[0028]

[0029] Note: Compared with the blank group, ** P<0.01, compared with the hypoxia-ischemia group, a P<0.05.

[0030] As shown in Table 1, after being treated for 3 days under high altitude hypoxia-ischemia conditions, rats developed obvious cerebral edema, that is, the brain water content increased significantly (P<0.01) compared with the control group; when butylphthalide was used for preventive and therapeutic administration, the brain water content of rats decreased significantly (P<0.05), indicating that the preventive and therapeutic administration of butylphthalide significantly inhibited the brain edema of rats caused by high altitude hypoxia-ischemia conditions.

[0031] Embodiment 2

[0032] To test the effects of preventive administration of butylphthalide on oxidative stress indicators and inflammatory factors in high altitude hypoxic-ischemic rats:

[0033] Step S100: 30 rats were randomly divided into 3 groups, namely a blank control group, a hypoxia-ischemia group, and a hypoxia-ischemia+butylphthalide group, with 10 rats in each group.

[0034] Step S200: Control group: Rats were intragastrically administered with a blank solvent (2% Tween 80) once a day for 7 days and were raised in a normal environment.

[0035] Step S300: Hypoxia-ischemia group: Rats were first gavaged with blank solvent (2% Tween 80) once a day for 4 days. The right common carotid artery was surgically ligated for 2 hours before entering the chamber. Then, the rats were placed in a special artificial oxygen chamber in the northwest to simulate the 6000m plateau environment. The artificial oxygen chamber was opened for 1 hour every day to add food and water to the animals, and blank solvent was gavaged at the same time. The rats were treated in the oxygen chamber for a total of 3 days.

[0036] Step S400: Hypoxia-ischemia + butylphthalide group: Rats were intragastrically administered (butylphthalide dosage 120 mg / kg), once a day for a total of 4 days. The right common carotid artery was surgically ligated for 2 hours before entering the chamber, and then placed in a special artificial oxygen chamber in the northwest to simulate the 6000m plateau environment. The artificial oxygen chamber was opened for 1 hour every day to add food and water to the animals, and butylphthalide solution was administered intragastrically at the same time. The rats were treated in the artificial oxygen chamber for a total of 3 days.

[0037] Step S500: 3 days after leaving the cabin, take the rat brain tissue, rinse the brain tissue with pre-cooled PBS (0.01M, pH=7.4), remove residual blood, weigh and cut the tissue into pieces, add the cut tissue and the corresponding volume of PBS (generally at a weight-to-volume ratio of 1:9, such as 1g tissue sample plus 9mL PBS) into a homogenizer, grind it thoroughly on ice, and continue grinding after repeated freezing and thawing to further lyse tissue cells. Finally, centrifuge the homogenate at 13000r / min for 5-10 minutes, and take the supernatant for detection. Superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), vascular endothelial growth factor (VEGF), tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were detected respectively.

[0038] like Figure 2 As shown in the data, after rats were treated with high altitude hypoxia-ischemia for 3 days, compared with the control group, the SOD content in the hypoxia-ischemia group was significantly decreased (P<0.05), the MDA content was significantly increased (P<0.01), and the GSH-Px content was significantly decreased (P<0.01); VEGF, TNF-α, IL-1β and IL-6 were significantly increased (P<0.01), while compared with the hypoxia-ischemia group, the SOD and GSH-Px contents in the butylphthalide preventive administration group were significantly increased (P<0.01), and MDA, VEGF, TNF-α, IL-1β, and IL-6 were significantly decreased (P<0.01), indicating that butylphthalide has a good effect in alleviating oxygen free radical damage and inflammatory damage caused by high altitude hypoxia-ischemia.

[0039] Embodiment 3

[0040] The effect of butylphthalide on the prevention and treatment of high altitude hypoxia-ischemia rat brain tissue hematoxylin-eosin (HE) staining was tested:

[0041] Step S100: 30 rats were randomly divided into 3 groups, namely a blank control group, a hypoxia-ischemia group, and a hypoxia-ischemia+butylphthalide group, with 10 rats in each group.

[0042] Step S200: Control group: Rats were intragastrically administered with a blank solvent (2% Tween 80) once a day for 7 days and were raised in a normal environment.

[0043] Step S300: Hypoxia-ischemia group: Rats were first gavaged with blank solvent (2% Tween 80) once a day for 4 days. The right common carotid artery was surgically ligated for 2 hours before entering the chamber. Then, the rats were placed in a special artificial oxygen chamber in the northwest to simulate the 6000m plateau environment. The artificial oxygen chamber was opened for 1 hour every day to add food and water to the animals, and blank solvent was gavaged at the same time. The rats were treated in the oxygen chamber for a total of 3 days.

[0044] Step S400: Hypoxia-ischemia + butylphthalide group: Rats were intragastrically administered (butylphthalide dosage 120 mg / kg), once a day for a total of 4 days. The right common carotid artery was surgically ligated for 2 hours before entering the chamber, and then placed in a special artificial oxygen chamber in the northwest to simulate the 6000m plateau environment. The artificial oxygen chamber was opened for 1 hour every day to add food and water to the animals, and butylphthalide solution was administered intragastrically at the same time. The rats were treated in the artificial oxygen chamber for a total of 3 days.

[0045] Step S500: 3 days after leaving the cabin, anesthetize and dissect quickly to completely remove the brain, immediately wash it with 0.9% saline, and fix it with 10% neutral formaldehyde solution. After fixation for 2 days, cut brain slices from behind the optic chiasm as samples, and continue to fix them. The samples are paraffin-embedded, tissue sliced, and stained with hematoxylin-eosin, and then observed and photographed with an optical microscope.

[0046] SPSS26.0 software package was used for statistical analysis. All data were expressed as mean ± standard deviation (X ± S). One-way analysis of variance was used for comparison among groups. P < 0.05 was considered statistically significant.

[0047] like Figure 2 As shown in the figure, the effect of preventive and therapeutic administration of butylphthalide on the hematoxylin-eosin (HE) staining of brain tissue of rats with high altitude hypoxia-ischemia was observed. After the rats were treated with high altitude hypoxia-ischemia conditions for 3 days, the brain tissue structure of the rats in the blank group was intact, the neuronal cell morphology was normal, the hippocampal cells were neatly arranged, and the perivascular space was small. The neuronal cells in the brain tissue of the rats in the hypoxia-ischemia group showed nuclear condensation, the pericellular space and perivascular space began to increase, and the arrangement of cells in the hippocampus was disordered. After the intervention of butylphthalide, the number of swollen brain tissue cells decreased, and the perivascular space was also significantly reduced.

[0048] In summary, the results of Examples 1 to 3 of the present invention show that butylphthalide has a good preventive and therapeutic effect on the animal model of hypoxic-ischemic brain damage, successfully expands the alternative drugs for hypoxic-ischemic brain damage, provides a reference for the selection of clinical therapeutic drugs, and can enable personnel working in the plateau to quickly adapt to the adverse environment of low pressure and low oxygen in the plateau to reduce brain damage (edema, oxygen free radical damage and inflammatory damage) in the body, and at the same time enable them to still maintain considerable work ability and strong physical strength on the plateau, which is more meaningful for special people who are stationed in the plateau for training.

[0049] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of butylphthalide in drugs for reducing water content in hypoxic-ischemic brain tissue.

2. Application of butylphthalide in improving oxidative stress injury and inflammatory drugs in hypoxic-ischemic brain damage.

3. Application of butylphthalide in improving the ultrastructure of hypoxic-ischemic brain tissue.

4. The use according to any one of claims 1 to 3, characterized in that: Butylphthalide is racemic-3-n-butylphthalide.

Images