Application of TPT-260 in preparation of medicine for treating plateau encephalopathy

Abstract

The invention relates to the field of medicines, and discloses application of TPT-260 in preparation of a medicine for treating plateau encephalopathy, and the research shows that the TPT-260 has a remarkable treatment effect on plateau encephaledema. The TPT-260 can reduce intracellular cholesterol accumulation by promoting outflow of cholesterol in low-oxygen exposed microglial cells, down-regulate expression of aquaporin 4 of low-oxygen exposed astrocytes, reduce cholesterol accumulation in brains of mice exposed in plateau, and improve plateau encephaledema. Compared with the prior art, the TPT-260 serving as the medicine for treating the plateau encephalopathy has the advantages of quick effect, obvious effect and strong targeting property.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to the application of TPT-260 in preparing a medicine for treating high altitude encephalopathy. Background Art

[0002] The geographical and climatic characteristics of low oxygen, low pressure, cold and dryness caused by the rapid rise of people from the plains can cause severe discomfort in the body's tissues and organs, induce acute mountain sickness, and even cause high-altitude cerebral edema in severe cases. High-altitude cerebral edema is a relatively rare but extremely dangerous type of plateau disease. The characteristic manifestations of high-altitude cerebral edema are a significant increase in the water content in the brain, a significant upregulation of aquaporin 4 (AQP-4), and a large number of neuronal deaths. However, the occurrence mechanism of high-altitude cerebral edema is not yet fully understood, resulting in the lack of targeted drugs. The prevention or treatment of high-altitude cerebral edema has always been a difficult point in the prevention and treatment of plateau disease.

[0003] The brain is one of the organs with the highest fat content in the human body. Lipid homeostasis, especially cholesterol homeostasis, is crucial to the maintenance of brain function. Most of the cholesterol in the brain is stored in the myelin sheath, which plays a key role in the growth of the myelin membrane during brain maturation. Cholesterol in neurons, as the main lipid on the cell membrane, participates in the synaptic conduction process. It can not only promote the fusion of synaptic vesicles and presynaptic membranes, but also change the dynamics of postsynaptic membrane receptors, thereby affecting synaptic activity. Mature neurons have a weak ability to synthesize cholesterol on their own, and meet their own needs by taking in apolipoprotein E-cholesterol excreted by glial cells. Excessive accumulation of cholesterol in the brain can induce apoptotic neuronal death, impaired synaptic plasticity, and cause cognitive impairment. The latest research believes that glial cholesterol efflux disorders are an important cause of neurological diseases. In previous reports, there has been no research focusing on the prevention and treatment of acute high-altitude encephalopathy from the perspective of lipid homeostasis. Summary of the invention

[0004] The object of the present invention is to provide the use of TPT-260 in the preparation of a drug for treating high altitude encephalopathy.

[0005] In order to solve the above technical problems, the present invention provides the use of TPT-260 in the preparation of a drug for treating brain diseases, wherein the molecular formula of TPT-260 is C8H 14 Cl2N4S3, CAS number is 2076-91-7, the chemical structure is as follows:

[0006]

[0007] Among them, the high altitude encephalopathy is cerebral edema or cholesterol accumulation caused by high altitude exposure.

[0008] Furthermore, the drug is used to alleviate cerebral edema caused by low pressure and low oxygen exposure at high altitude.

[0009] Furthermore, the drug is used to alleviate cerebral edema caused by acute low-pressure and low-oxygen exposure at high altitude.

[0010] Furthermore, the drug is used to reduce cholesterol accumulation caused by high altitude hypoxia exposure.

[0011] Furthermore, the drug is used to reduce cholesterol accumulation caused by acute hypoxia exposure at high altitude.

[0012] Furthermore, the drug is used to reduce microglial cholesterol accumulation caused by high altitude hypoxia exposure.

[0013] Furthermore, the drug is used to promote cholesterol efflux from microglia under high-altitude hypoxia exposure conditions.

[0014] Furthermore, the drug is used to reduce astrocyte swelling caused by high altitude hypoxia exposure.

[0015] Furthermore, the drug is used to down-regulate the expression level of aquaporin 4 in astrocytes under high altitude hypoxia exposure conditions.

[0016] Furthermore, the drug is administered by injection, and the drug concentration is 50 mg / kg / day.

[0017] Compared with the prior art, the present invention has found through research that the use of TPT-260 can promote the efflux of cholesterol from microglia exposed to hypoxia to reduce cholesterol accumulation in cells, down-regulate the expression of aquaporin 4 (AQP-4) in astrocytes exposed to hypoxia, reduce cholesterol accumulation in the brains of mice exposed to plateau, and improve high-altitude cerebral edema. It has the advantages of rapid effect, obvious effect, and strong targeting. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the analysis diagram of cholesterol content and lipid profile in the brain of mice in the NN group and the HH group in Example 1;

[0019] Figure 2 This is an analysis diagram of the cholesterol content and brain edema in each group of mice in Example 2;

[0020] Figure 3 This is a graph showing the detection and analysis of cholesterol accumulation and cholesterol efflux in microglia in the Nor group, Hyp group, and Hyp+TPT-260 group in Example 3;

[0021] Figure 4 These are the immunoblot images and statistical graphs of the changes in AQP-4 expression levels in astrocytes in the Nor group, TPT-260 group, Hyp group, and Hyp+TPT-26 group in Example 4. DETAILED DESCRIPTION

[0022] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the content specifically described below is illustrative and not restrictive, and should not be used to limit the scope of protection of the present invention. The experimental methods for which specific conditions are not indicated in the embodiments are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0023] The Chinese and English names of the drug used in the invention are both TPT-260, and the molecular formula is C8H 14 Cl2N4S3, molecular weight 333.32, CAS No. 2076-91-7, purchased from Sellleck, product No. S0197. Unless otherwise specified, reagents used in the examples of the present invention can be obtained from sales companies.

[0024] Example 1

[0025] Experiment on brain edema and cholesterol content detection in mice with high altitude cerebral edema induced by hypobaric and hypoxic exposure:

[0026] (1) NN group: 10 male adult mice weighing 25 g.

[0027] (2) HH group: 10 male adult mice weighing 25 g were exposed to a hypobaric hypoxic chamber simulating an altitude of 7000 m for 48 h.

[0028] After the above experiment, 5 mice were randomly selected from each group and injected with Evans blue (EB) dye solution through the tail vein. After 1 hour, the mice were anesthetized and physiological saline was perfused through the heart to remove EB in the circulatory system. After the brain tissue was peeled off, it was homogenized and the residual EB content in the brain tissue was detected using an enzyme marker. For the remaining 5 mice, the mouse cerebral cortex was quickly peeled off and placed in a muffle furnace at 100°C for 24 hours. The wet weight and dry weight of the brain were weighed before and after drying. The calculation formula for brain water content: Brain Water Content (%) = (brain wet weight-brain dry weight) / brain weight loss × 100%. Part of the brain tissue of the mouse was taken, homogenized, and total cholesterol and free cholesterol were detected using the cholesterol detection kit of Solebao. Non-targeted lipid metabolomics detection technology was used to analyze the changes in lipid component content in mouse brain tissue by high performance liquid chromatography.

[0029] Figure 1The following are the cholesterol content and lipid spectrum analysis diagrams of the brains of mice in the NN group and the HH group in this embodiment; wherein, Figure A is a statistical diagram of the water content of the brains of the two groups of mice, Figure B is a diagram of the Evans blue leakage in the brains of the two groups of mice, Figure C is a diagram of the total cholesterol content of the brains of the two groups of mice, Figure D is a diagram of the free cholesterol content of the brains of the two groups of mice, and Figure E is a diagram of lipid spectrum clustering, pink and green are two groups, and the results represent obvious differences between the groups. F represents differential lipid components, and it can be seen that multiple cholesterol esters (CE) are upregulated in the HH group.

[0030] Depend on Figure 1 A shows that the brain water content in the HH group increased significantly, which is consistent with the characteristics of the high altitude cerebral edema model. Figure 1 B shows that the EB residues in the brain of mice in the HH group increased, indicating that the tight junctions of blood vessels in the brain were damaged, which is consistent with the characteristics of the high-altitude cerebral edema model. Figure 1 C and 1D show that the levels of total cholesterol and free cholesterol in the brain of mice in the HH group were significantly increased. Figure 1 As shown in Figures 1E and 1F, the content of cholesterol ester in the brain of mice in the HH group increased significantly (the red box on the right side of Figure 1F indicates upregulation, and the blue box indicates downregulation). Therefore, it is proved that cholesterol accumulates significantly in the brain of mice with HH-induced high-altitude cerebral edema.

[0031] Example 2

[0032] Experiment on TPT-260 alleviating brain cholesterol accumulation and brain edema in high altitude cerebral edema model mice induced by hypobaric hypoxia exposure:

[0033] (1) NN group: 10 male adult mice weighing 25 g.

[0034] (2) TPT-260 group: 10 25 g male adult mice were intraperitoneally injected with 50 mg / kg TPT-260 for 24 hours.

[0035] (3) HH group: 10 male adult mice weighing 25 g were exposed to a hypobaric hypoxic chamber simulating an altitude of 7000 m for 48 h.

[0036] (4) HH+TPT-260 group: Ten male adult mice weighing 25 g were exposed to a hypobaric hypoxic chamber at a simulated altitude of 7000 m for 48 h, and then intraperitoneally injected with 50 mg / kg TPT-260 for 24 h.

[0037] After the above experiment, 5 mice were randomly selected from each group, and part of the brain tissue of the mice was taken. After homogenization, the total cholesterol was detected using the cholesterol detection kit of Solebold. The remaining brain tissue was placed in a muffle furnace and dried at 100°C for 24 hours. The wet weight and dry weight of the brain before and after drying were weighed. The calculation formula of brain water content: Brain Water Content (%) = (brain wet weight-brain dry weight) / brain weight loss × 100%. The remaining 5 mice in each group were injected with Evans blue (EB) dye solution through the tail vein, and the mice were anesthetized after 1 hour, and physiological saline was perfused through the heart to remove EB in the circulatory system. The brain tissue was then fixed with neutral paraformaldehyde, and tissue sections were made along the sagittal plane using frozen section technology, and microscopic imaging was performed using a laser confocal microscope in the near-infrared Ex630 / Em660 to detect EB residues in the brain tissue.

[0038] Figure 2 The following are analysis diagrams of the cholesterol content and brain edema in the brains of mice in each group of mice in this embodiment. Among them, Figure A is a diagram of the total cholesterol content in the brains of mice in the NN group, HH group, and HH+TPT-260 group. Figure B is a statistical diagram of the water content in the brains of mice in the NN group, HH group, TPT-260 group, and HH+TPT-260 group. Figure C is an Evans blue staining diagram of the brains of mice in the NN group, HH group, and HH+TPT-260 group.

[0039] Depend on Figure 2 A shows that compared with the NN group, the total cholesterol in the brain of mice in the HH group was significantly increased; after TPT-260 administration, the total cholesterol in the brain of mice exposed to HH decreased significantly. Figure 2 B shows that compared with the NN group, TPT-260 had no significant effect on the brain water content of mice, and the brain water content of mice in the HH group increased significantly. In the HH+TPT-260 group, the brain water content was significantly reduced compared with the HH group. Figure 2 C shows that the EB residue in the brain of mice in the HH group was higher than that in the NN group; and after TPT-260 administration, the EB residue in the brain of mice exposed to HH was significantly reduced. Therefore, the above results prove that TPT-260 can effectively reduce the cholesterol accumulation in the brain of mice induced by HH and the symptoms of high-altitude cerebral edema.

[0040] Example 3

[0041] TPT-260 reduces cholesterol accumulation and enhances cholesterol efflux in hypoxia-exposed microglia.

[0042] (1) Nor: primary cultured mouse microglia.

[0043] (2) Hyp group: Primary cultured mouse microglia were exposed to 1% O2 in a cell hypoxia workstation for 24 hours.

[0044] (3) Hyp+TPT-260 group: After primary cultured mouse microglia were exposed to 1% O2 in a cell hypoxia workstation for 24 hours, the cells were incubated with 10 μM TPT-260 for 12 hours.

[0045] After the above experiment was completed, Figure 3 The cell treatment conditions of A and B are as follows: the microglia were immediately fixed, the cholesterol in the microglia was stained with Filipin, and the images were taken with a laser confocal microscope and the intensity of the Filipin fluorescence signal in the cells was counted. Figure 3 The cell treatment conditions of C are as follows: cells are incubated with Bodipy-conjugated cholesterol (Bodipy-CE), and fresh medium without dye is replaced after 30 minutes and further incubated for 1 hour; then the cells are fixed with neutral paraformaldehyde and photographed using a laser confocal microscope. The intensity of the Bodipy signal remaining in the cells is negatively correlated with the ability of the cells to efflux cholesterol.

[0046] Figure 3 The above are the cholesterol accumulation and cholesterol efflux detection and analysis diagrams in the microglia of the Nor group, Hyp group, and Hyp+TPT-260 group in Example 3. Figure A is the cholesterol staining diagram of each group of cells. Figure B is the statistical diagram of the cholesterol accumulation degree of each group of cells. Figure C is the cholesterol efflux detection diagram of each group of cells.

[0047] Depend on Figure 3 As shown in A and 3B, the cholesterol accumulation in microglia in the Hyp group was significantly increased compared with that in the Nor group, and TPT-260 significantly reduced the cholesterol accumulation in microglia exposed to Hyp. Figure 3 C shows that cholesterol efflux from microglia in the Hyp group was significantly blocked, and TPT-260 treatment significantly improved the problem of blocked cholesterol efflux from microglia caused by hypoxia exposure, that is, enhanced cholesterol efflux from Hyp-exposed mice. Therefore, the above results prove that TPT-260 can effectively reduce cholesterol accumulation in the brain of mice induced by hypoxia and enhance cholesterol efflux.

[0048] Example 4

[0049] TPT-260 attenuates astrocyte swelling induced by hypoxia exposure.

[0050] (1) Nor: primary cultured mouse astrocytes.

[0051] (2) TPT-260 group: Primary cultured mouse astrocytes were incubated with 10 μM TPT-260 for 12 h.

[0052] (3) Hyp group: Primary cultured mouse astrocytes were exposed to 1% O2 in a cell hypoxia workstation for 24 hours.

[0053] (4) Hyp+TPT-260 group: After primary cultured mouse astrocytes were exposed to 1% O2 in a cell hypoxia workstation for 24 hours, the cells were incubated with 10 μM TPT-260 for 12 hours.

[0054] Brain edema caused by plateau exposure is mainly manifested as astrocyte swelling. The up-regulation of AQP-4 expression in astrocytes is a marker of cell swelling. Therefore, after the above experiment, we extracted cell proteins and detected the expression level of AQP-4 in cells by Western blot and performed statistics. The results are shown in Figure 2. Figure 4 As shown in the figure, the expression level of AQP-4 in astrocytes in the TPT-260 group did not change significantly compared with the Nor group, and the expression level of AQP-4 in the Hyp group was significantly upregulated; the expression level of AQP-4 in the Hyp+TPT-260 group was significantly downregulated compared with the Hyp group. Therefore, it is proved that TPT-260 can effectively reduce the expression of aquaporins in astrocytes induced by hypoxia, suggesting that it has a therapeutic effect on hypoxia-induced astrocyte swelling.

[0055] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended to include all changes within the meaning and scope of the equivalent elements of the claims. Although the embodiments of the present invention have been shown and described, it is understood by those of ordinary skill in the art that various changes, modifications, substitutions and deformations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the attached claims and their equivalents.

Claims

1. Use of TPT-260 in the preparation of a drug for treating high altitude encephalopathy, wherein the molecular formula of TPT-260 is C8H 14 Cl2N4S3, CAS number is 2076-91-7, the chemical structure is as follows: in, The high altitude encephalopathy is cerebral edema or cholesterol accumulation caused by high altitude exposure.

2. The use according to claim 1, characterized in that: The drug is used to reduce cerebral edema caused by low pressure and low oxygen exposure at high altitude.

3. The use according to claim 1, characterized in that: The drug is used to alleviate cholesterol accumulation caused by high altitude hypoxic exposure.

4. The use according to claim 1, characterized in that: The drug is used to alleviate microglial cholesterol accumulation caused by high altitude hypoxia exposure.

5. The use according to claim 1, characterized in that: The drug is used for promoting cholesterol efflux from microglia under high altitude hypoxia exposure conditions.

6. The use according to claim 1, characterized in that: The drug is used to reduce astrocyte swelling caused by high altitude hypoxia exposure.

7. The use according to claim 1, characterized in that: The drug is used to down-regulate the expression level of aquaporin 4 in astrocytes under high altitude hypoxia exposure conditions.

8. The use according to claim 1, characterized in that: The drug is administered by injection, and the drug concentration is 50 mg / kg / day.

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