Application of zanthoxylum amide compound in preparation of medicine for preventing and / or treating nervous system diseases related to hypoxia neuroinflammation

Abstract

The invention provides an application of a zanthoxylum amide compound in preparation of a medicine for preventing and / or treating nervous system diseases related to hypoxia neuroinflammation. The zanthoxylum amide compound disclosed by the invention has the following structure.

Description

Technical Field

[0001] This invention belongs to the fields of natural product chemistry and pharmaceutical technology, specifically relating to the use of a zanthoxylate compound CT-6 in the preparation of a drug for the prevention and / or treatment of neurological diseases related to hypoxia-induced neuroinflammation, particularly high-altitude cerebral edema. Background Technology

[0002] High-altitude cerebral edema (HACE) is a severe central nervous system disorder induced by the low-pressure, low-oxygen environment of high altitudes. It primarily affects individuals who rapidly ascend to high altitudes and are poorly acclimatized. It is a critical and severe form of acute mountain sickness, characterized by brain swelling and increased intracranial pressure. The pathological mechanisms involve hypoxia-induced mitochondrial dysfunction, inflammation and oxidative stress, abnormal sodium-potassium pump function, and blood-brain barrier damage, resulting in cytotoxic edema, ionogenic edema, and vasogenic edema. Currently, acetazolamide, dexamethasone, mannitol, and aminophylline are commonly used in clinical practice to prevent and treat HACE. Natural products derived from unique plants have shown unique advantages and significant potential in the prevention and treatment of HACE. Traditional Chinese medicine preparations such as gastrodin injection, ginseng and deer antler extract tablets, and multi-blood-activating agents have significantly improved the clinical manifestations and prognosis of patients with HACE by reducing hypoxic neurological damage. Due to their novel skeletons, diverse pharmacological activities, and unique mechanisms of action, natural products are an important source of innovative drugs for mountain sickness. Front. Pharmacol. 2024, 15: 1393209. ).

[0003] Amide compounds are from the genus Zanthoxylum (Zanthoxylum). Zanthoxylum L.) plant characteristic secondary metabolites are important components of the "numbing and spicy" sensation of Sichuan pepper. Currently, more than 120 amide compounds have been isolated from plants of the genus Zanthoxylum. These compounds exert neuroprotective, anti-inflammatory, analgesic, and antibacterial effects through biological mechanisms such as regulating voltage-gated sodium / calcium channels, inhibiting neuroinflammatory responses, scavenging free radicals, and activating antioxidant pathways such as Nrf2 / HO-1 (Chinese Journal of Traditional Chinese Medicine, 2023, 48(9):2406-2418.). Qian-Nv Ye et al. from Zanthoxylum armatum Fifteen alkylamide compounds were isolated and identified from the pericarp of DC. Among them, compounds 2, 3, and 4 significantly enhanced H2O2-induced SH-SY5Y cell viability and reduced oxidative stress, thus exhibiting neuroprotective activity. Phytochemistry, 2023, 211: 113704.) Furthermore, the hydroxy-α-sanshool, a sanshoolamine compound, improves the morphological changes in D-galactose / AlCl3-induced Alzheimer's disease-like mice through the Nrf2 / HO-1 signaling pathway, enhances the activity of antioxidant enzymes, and reduces oxidative stress damage, providing a candidate drug for the early prevention and treatment of Alzheimer's disease. Eur. J. Pharmacol., 2022, 914: 174691.However, there are currently no reports on the use of amide compounds from the Zanthoxylum bungeanum for the prevention and treatment of neurological diseases related to hypoxia-induced neuroinflammation, such as high-altitude cerebral edema. Summary of the Invention

[0004] The inventors' research group previously obtained information from Sichuan pepper ( Zanthoxylum bungeanum An amide compound was isolated and prepared from Maxim. CT-6 effectively inhibits the attachment of barnacle larvae and algae, and can be applied to marine antifouling (Chinese Patent No. ZL 202210996685). This invention is based on the surprising discovery that the aforementioned compound CT-6 has a protective effect against lipopolysaccharide (LPS)-induced hypoxic HMC3 microglia, and can be developed into a drug for the prevention and / or treatment of neurological diseases associated with hypoxic neuroinflammation, particularly high-altitude cerebral edema.

[0005] The purpose of this invention is to provide a zanthoxylate compound or its salt as a pharmaceutical active ingredient in the preparation of a medicine for the prevention and / or treatment of neurological diseases related to hypoxic neuroinflammation, especially high-altitude cerebral edema.

[0006] Therefore, the present invention provides the use of a zanthoxylate compound or a salt thereof in the preparation of a medicament for the prevention and / or treatment of neurological diseases associated with hypoxic neuroinflammation, particularly high-altitude cerebral edema, wherein the zanthoxylate compound has the following structure: .

[0007] In this invention, the neurological diseases related to hypoxic neuroinflammation include high-altitude cerebral edema, ischemic stroke, hemorrhagic stroke, hypoxic-ischemic brain injury, traumatic brain injury, Alzheimer's disease, Parkinson's disease, Huntington's disease, epilepsy, post-traumatic stress disorder, etc.

[0008] In one embodiment, the neurological disease associated with hypoxic neuroinflammation is high-altitude cerebral edema.

[0009] In some embodiments of the present invention, the zanthoxylate compounds can inhibit the production of inflammatory factors (including NO, TNF-α and IL-1β) in a hypoxic neuroinflammation model of human microglia HMC3 cells induced by LPS+hypoxia (HY).

[0010] In some embodiments, in the application of the present invention, the zanthoxylate compounds can promote cell growth in a hypoxic neuroinflammation model of human microglia HMC3 cells induced by LPS+hypoxia (HY).

[0011] In some embodiments, in the application of the present invention, the zanthoxylate compounds can inhibit weight loss, increased brain water content (BWC), production of inflammatory factors (including TNF-α and IL-6), histopathological changes, and expression of brain edema-related proteins (including AQP4 and MMP-9) in an animal model of LPS+hypoxia (HY) combined induced high-altitude cerebral edema.

[0012] This invention provides a lead compound of the pepper amide class for the prevention and treatment of high-altitude cerebral edema. The model group (Model) showed significantly higher BWC levels compared to the control group (Control), exhibiting abnormal symptoms such as reduced activity, depression, decreased food intake, and erect hair on the back, as well as significant weight loss. Brain tissue showed disordered neuronal arrangement, cell swelling, nuclear pyknosis, and nuclear fragmentation, with localized cell lysis and vacuolation, accompanied by widened intercellular spaces, microvascular dilation, and inflammatory cell infiltration. Inflammatory factors (TNF-α and IL-6) were significantly increased in brain tissue, while superoxide dismutase (SOD) activity was significantly decreased and malondialdehyde (MDA) levels were elevated. The expression of blood-brain barrier-related proteins (AQP4 and MMP-9) was increased. The CT-6 treatment group showed the following significant effects in a concentration-dependent manner: improved pathophysiology, significantly decreased brain tissue water content, alleviated weight loss trend, enhanced antioxidant capacity (increased SOD activity), reduced lipid peroxidation damage (decreased MDA level), decreased expression of inflammatory factors (TNF-α and IL-6), and downregulation of blood-brain barrier-related proteins (AQP4 and MMP-9). These results indicate that CT-6 can significantly alleviate oxidative stress and inflammatory response induced by hypobaric hypoxia, reduce vascular permeability, and protect blood-brain barrier permeability, thereby preventing the occurrence of HACE. Attached Figure Description

[0013] Figure 1 The effects of different concentrations of CT-6 on LPS+HY-induced inflammatory factors (A: NO; B: TNF-α; C: IL-1β) and cell viability (D) in HMC3 cells were shown.

[0014] Figure 2 The effects of different concentrations of CT-6 on the rate of weight loss (A) and changes in brain water content (B) in HACE model mice are shown.

[0015] Figure 3 The effects of different concentrations of CT-6 on oxidative stress factors (A: SOD; B: MDA) in the brain tissue of HACE model mice were shown.

[0016] Figure 4 The effects of different concentrations of CT-6 on inflammatory factors (A: TNF-α; B: IL-6) in the brain tissue of HACE model mice were shown.

[0017] Figure 5 The pathological analysis of the hippocampal region of brain tissue in HACE model mice with different concentrations of CT-6 is shown.

[0018] Figure 6 The effects of different concentrations of CT-6 on AQP4 and MMP-9 proteins in the brain tissue of HACE model mice are shown by Western blot analysis. In the figures: A shows the effect of different concentrations of CT-6 on AQP4 protein expression; B shows the grayscale analysis of AQP4 protein expression; C shows the effect of different concentrations of CT-6 on MMP-9 protein expression; and D shows the grayscale analysis of MMP-9 protein expression.

[0019] Figure 7 Immunofluorescence staining analysis of MMP-9 (A) and AQP4 (B) proteins in brain tissue of HACE model mice with different concentrations of CT-6 is shown.

[0020] In the image above, compared to the Control group ## P<0.01, ### P < 0.001; compared with the Model group, P > 0.05. P<0.05, P<0.01, P<0.001. Detailed Implementation

[0021] The present invention will be specifically described below through embodiments, but the present invention is not limited to any of the embodiments.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0023] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0024] CT-6 was prepared according to Example 1 in the published patent CN115160176B.

[0025] Example 1: Effects of different concentrations of CT-6 on LPS+HY-induced inflammatory factors and cell viability in HMC3 cells Experimental Methods: A double-antibody sandwich immunoassay was used to detect the production of inflammatory factors in lipopolysaccharide (LPS) + hypoxia (HY)-induced HMC3 cells by CT-6. HMC3 cells (BNCC342264) were seeded in 48-well plates, 100 μL (10 × 10⁻⁶) per well. 4 / mL). Incubate in an incubator until adherence; prepare different concentrations of CT-6 solutions (5 μM, 25 μM, 50 μM) containing 0.5% DMSO using EMEM medium (BNCC364130), add 500 μL to each of 48 wells, and pre-treat in a normal incubator for 4 hours. Without aspirating the original drug-treated medium from the 48-well plate, add 2 µL of LPS (SIGMA-Aldrich, Escherichia coli O55:B5) stock solution (5 μg / mL) prepared in EMEM medium to each well according to the calculated group, gently shake to mix evenly, and then treat in a 0.3% O2 ​​hypoxia workstation for 24 hours. Collect the cell supernatant and centrifuge at 12000 rpm for 10 minutes. The levels of inflammatory factors NO (Beyotime S0021M), TNF-α (E-EL-H0109), and IL-1β (Beyotime PI301) were measured according to the steps in the ELISA kit instructions.

[0026] To assess the effect of CT-6 on the proliferative activity of HMC3 microglia, the effect of LPS on HMC3 cell viability was determined using a CCK-8 assay kit (BioSharp BS350E). HMC3 cells in logarithmic growth phase were inoculated at 3 × 10⁻⁶ cells per cell line. 4 HMC3 cells were seeded in 96-well plates at concentrations of CT-6 (1, 5, 10, 25, 50, 100, 200, 500 µM) for 24 h under hypoxic (1% oxygen in a hypoxic workstation, HY) and normoxic (NOR) conditions, respectively. After treatment, 10 µL / well of CCK-8 reagent was added to each well, and the plates were incubated for 1 h. Absorbance was measured at 450 nm using a microplate reader, cell viability was calculated, and statistical analysis was performed. The cell viability calculation method is shown in the following formula:

[0027] Data are expressed as mean ± SEM (n≥3). Statistical analysis was performed using GraphPadPrism 8.0.2. First, the Kolmogorov-Smirnov or Shapiro-Wilk test was used to test the normality of the data, and the F-test was used to confirm the equality of variances. Then, one-way ANOVA was performed on multiple groups of data, and Tukey's test was conducted; p < 0.05 was considered statistically significant.

[0028] HMC3 cells were treated with different concentrations of CT-6 (5, 25, 50 µM) for 24 h. The release levels of inflammatory factors in the culture supernatant were measured by ELISA. The experimental results are as follows: Figure 1 China A Figure 1B, Figure 1 As shown in Figure C, compared with the control group, the levels of NO, TNF-α, and IL-1β in the LPS group were significantly increased (compared with the Control group). ## P<0.01, ### P < 0.001; compared with the Model group, P > 0.05. P<0.05, P<0.01, (P<0.001), while the CT-6 treatment group significantly inhibited the production of inflammatory factors.

[0029] The effect of CT-6 on HMC3 cell viability was detected using the CCK-8 assay. The experimental results are as follows: Figure 1 As shown in Figure D, under normoxic (NOR) and hypoxic (HY) conditions, CT-6 at a concentration ≤ 100 µM did not significantly inhibit the viability of HMC3 cells.

[0030] Example 2: Effects of different concentrations of CT-6 on changes in body weight and brain water content in HACE model mice Experimental methods: An acute HACE model induced by LPS+HY was constructed using an animal experiment hypobaric oxygen chamber. The efficacy of different concentrations of CT-6 in preventing and treating HACE in mice was evaluated, and changes in mouse body weight and brain water content were studied.

[0031] (1) Drug preparation: Accurately weigh 200 mg and 400 mg of CT-6 and 100 mg of the positive control drug dexamethasone (DEX), dissolve them in a system of 10% DMSO + 40% polyethylene glycol 300 (Peg300) + 5% Tween 80 + 45% physiological saline, and prepare drug solutions with concentrations of 2 mg / mL CT-6, 4 mg / mL CT-6 and 1 mg / mL DEX, respectively, with a volume of 100 mL; (2) Grouping and administration: Sixty C57BL / 6 mice (20 ± 2 g) (purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, license number SCXK(Gan)2020-0002) were raised in a light-dark cycle environment with a temperature of 21-24℃, humidity of 50%-60% and a 12:12 h interval between light and dark, with free access to food and water, and were acclimatized for one week. The subjects were randomly divided into six groups, with 10 subjects in each group, as follows: Control group: administered an equal volume of normal saline by gavage daily for 7 consecutive days; HACE model group: administered an equal volume of normal saline by gavage daily for 7 consecutive days; High-dose CT-6 group (H): administered CT-6 (80 mg / kg) by gavage daily for 7 consecutive days; Medium-dose CT-6 group (M): administered CT-6 (40 mg / kg) by gavage daily for 7 consecutive days; Low-dose CT-6 group (L): administered CT-6 (20 mg / kg) by gavage daily for 7 consecutive days; Positive control group (DEX): administered DEX (10 mg / kg) by gavage daily for 7 consecutive days. (3) HACE model construction: Pretreatment: C57BL / 6 mice in the experimental group and model group were intraperitoneally injected with 0.5 mg / kg LPS for 30 min to ensure free access to food and water; Hypoxia exposure: The mice were placed in the animal experiment hypoxia chamber, the chamber door was sealed and the altitude was increased at the set rate to maintain the target altitude conditions for continuous exposure. The equipment parameters were set as follows: altitude 7000 meters (corresponding pressure 41.0 kPa, oxygen partial pressure 8.3 kPa), increase rate 15 m / s, light cycle 12 h light / 12 h dark, hypoxia treatment for 24 h; Recovery treatment: After 24 h of exposure, the altitude was reduced to the local altitude of Lanzhou (1500 m, 84.0 kPa) at a controllable rate, the chamber door was opened, the animals were taken out, and blood and major organ tissues were collected immediately for the determination of various biochemical indicators, drug efficacy and mechanism of action studies.

[0032] (4) Measurement of mouse body weight and brain water content. Body weight data were measured and recorded when the animals entered and left the hypobaric chamber. Six experimental animals were randomly selected from each group. They were euthanized by decapitation at a predetermined time. The intact brain tissue was immediately removed and placed on ice for operation. The surface blood residue was rinsed with 4°C pre-cooled physiological saline, and excess liquid was absorbed with absorbent paper. Weight measurement: The weight of the culture dish was weighed, the weight of the wet brain tissue was measured, and the total wet weight was recorded. Drying treatment and calculation: The samples were placed in a low-temperature drying oven and dried until constant weight was achieved. The total dry weight was measured, and the brain water content was calculated according to the following Elliot formula:

[0033] Experimental results are as follows Figure 2As shown in Figure A, after 24 hours of acute hypobaric hypoxia treatment, the experimental animals exhibited significant symptoms of typical altitude sickness, such as decreased appetite and rapid breathing, with an average weight loss rate of 11.771 ± 0.72%. Pharmacodynamic evaluation showed that pretreatment with a high dose of CT-6 (80 mg / kg) significantly improved these symptoms, reducing the weight loss rate to 9.091 ± 0.813%. Statistical analysis indicated a highly significant difference in the high-dose group (CT-6-H). P<0.001), while no significant protective effect was observed in the medium and low dose groups (CT-6-M and CT-6-L). In the HACE model evaluation, as shown... Figure 2 As shown in Figure B, both CT-6 pretreatment and the positive control drug dexamethasone (10 mg / kg) effectively reduced the increase in BWC induced by LPS combined with hypobaric hypoxia, and the intervention effect was highly statistically significant. P<0.01 to P<0.001).

[0034] Example 3: Effects of different concentrations of CT-6 on oxidative stress factors in brain tissue of HACE model mice Experimental Methods: Two experimental animals that successfully underwent modeling according to the method in Example 2 were randomly selected from each group. They were euthanized by rapid decapitation on ice, and the brain tissue was completely removed. Residual blood was rinsed off the surface with PBS buffer pre-cooled to 4°C. Tissue homogenate preparation: The wet weight of the brain tissue was accurately weighed and added at a ratio of 1:9 (m / v). For SOD (Beyotime S0109), a special sample preparation solution was added; for MDA (Beyotime S0131S), pre-cooled PBS was added. Tissue homogenization parameters: Single run duration 30 s, linear velocity 6.50 m / s, 4 cycles. Centrifugation and aliquoting: Centrifuged at 12000 r for 15 min at 4°C. The supernatant was carefully transferred to labeled EP tubes to prepare a 10% concentration tissue homogenate suspension. Protein quantification and preservation: Total protein concentration was determined using the BCA method (Beyotime P0012). After aliquoting, the tissue was immediately stored at -80°C. Oxidative stress indicators were detected, including MDA content and SOD enzyme activity. Detection method: Strictly follow the operating specifications of the Beyotime reagent kit, set up a standard curve and quality control samples, and read the absorbance values ​​using an ELISA reader.

[0035] Experimental results are as follows Figure 3 China A Figure 3 As shown in Figure B, the results indicate that 24-hour low-pressure hypoxia treatment significantly affects the redox balance of the mouse cerebral cortex: compared with the normal control group, SOD activity was significantly decreased ( ### P<0.001), while the content of lipid peroxidation product MDA increased significantly ( ##P<0.01. After CT-6 pretreatment, all dose groups showed certain antioxidant protective effects, with low-dose CT-6 showing particularly outstanding effects in enhancing SOD activity. (P<0.001). Although all doses of CT-6 reduced MDA levels, the differences between groups were not statistically significant. The positive control drug dexamethasone (DEX) also showed a significant antioxidant effect, effectively regulating SOD activity and MDA content.

[0036] Example 4: Effects of different concentrations of CT-6 on inflammatory factors in the brain tissue of HACE model mice Experimental Methods: Two experimental animals that successfully underwent modeling according to the method in Example 2 were randomly selected from each group. After rapid decapitation, whole brain tissue was extracted and processed on ice. Surface blood was rinsed with pre-cooled PBS. Tissue Homogenization Preparation: An appropriate amount of brain tissue (wet weight) was weighed and added to 4℃ PBS buffer at a ratio of 1:9 (w / v). The tissue homogenizer was used with the following parameters: single homogenization time: 30 s, running speed: 6.50 m / s, repeated for 4 cycles. Centrifugation and Aliquoting: Centrifugation was performed at 12000 rpm for 15 min at 4℃. The supernatant was carefully transferred to new EP tubes to prepare a 10% tissue homogenate suspension. Protein Quantification and Preservation: Total protein concentration was determined using a BCA assay kit (Beyotime P0012). After aliquoting, the tissue was stored at -80℃ for later use. Following the ELISA kit instructions, the levels of TNF-α (E-EL-H0109) and IL-6 (E-EL-H6156) in the brain tissue were detected using enzyme-linked immunosorbent assay (ELISA).

[0037] Experimental results are as follows Figure 4 China A Figure 4 As shown in Figure B, ELISA analysis revealed that LPS combined with hypoxia (HY) treatment significantly promoted the release of pro-inflammatory factors in the mouse cerebral cortex: TNF-α levels increased sharply from 38.583±0.278 pg / mL in the control group to 126.716±0.378 pg / mL in the model group. ### P<0.001; IL-6 content increased from 139.397±0.601 pg / mL to 236.024±0.294 pg / mL (P<0.001); ### P<0.001, confirming that hypobaric hypoxia can induce neuroinflammation. Drug intervention experiments showed that high and medium doses of CT-6 and the positive control drug dexamethasone (DEX) effectively inhibited the overexpression of the aforementioned inflammatory factors, with the CT-6-H group, CT-6-M group, and DEX group showing the most significant anti-inflammatory effects. P<0.01 to P<0.001). This indicates that CT-6 has a dose-dependent neuro-inflammatory effect.

[0038] Example 5: Pathological analysis of hippocampal region in brain tissue of HACE model mice with different concentrations of CT-6 Experimental Methods: One animal that successfully underwent modeling according to the method in Example 2 was randomly selected from each group and quickly euthanized. Intact brain tissue was immediately removed, rinsed with physiological saline under ice bath conditions, and fixed in 4% paraformaldehyde solution at 4℃ for 24 hours. Tissue Dehydration and Clearing: Gradient dehydration program: 60% ethanol (24 h) → 80% ethanol (24 h) → 90% ethanol (12 h), 95% ethanol I (12 h) → 95% ethanol II (12 h), anhydrous ethanol I (1 h) → anhydrous ethanol II (1.5 h), ethanol / xylene mixture (1:1, 15 min), xylene I (15 min) → xylene II (15 min). Embedding and Sectioning: Paraffin embedding: soft paraffin for 1 h, hard paraffin for 1 h, 4 μm thick sections were cut using a paraffin microtome and dried in a 60℃ oven for 4 h. Staining program. Staining procedure: Xylene dewaxing (I and II, 20 min each); graded ethanol rehydration: 100% ethanol (5 min × 2) → 95% → 90% → 80% → 75% (2 min each); hematoxylin staining (3 min) → differentiation (20 s) → blueing (30 s), eosin counterstaining (5 min); graded ethanol dehydration: 85% → 95% ethanol (5 min each), xylene clearing (5 min × 2). Mounting and observation. Mounting treatment: Neutral resin mounting; observation of pathological changes in the hippocampus under an optical microscope.

[0039] Experimental results are as follows Figure 5 As shown, in the normal control group, the neurons in the hippocampus of rats exhibited intact morphology and structure, regular cell layering, and clear and intact nuclear membranes, with no obvious pathological changes. After LPS combined with hypoxia (HY) treatment, the experimental group showed typical characteristics of neuronal injury: reduced neuronal nuclear volume, chromatin condensation (nuclear pyknosis), significantly widened pericellular and perivascular spaces, disordered cell arrangement in the hippocampus, and localized cell loss. Notably, both the CT-6 group and the positive control drug dexamethasone (DEX) group showed significant neuroprotective effects: neuronal nuclear morphology basically returned to normal, pericellular edema was significantly reduced, hippocampal cell arrangement became more orderly, and perivascular spaces narrowed. This pathological evidence fully confirms that CT-6 can effectively alleviate inflammation- and hypoxia-induced brain tissue damage through multiple mechanisms, including reducing neuroinflammatory responses, improving microcirculatory disturbances, and maintaining the integrity of the blood-brain barrier.

[0040] Example 6: Immunoblotting and immunofluorescence staining analysis of the effects of different concentrations of CT-6 on the expression of AQP4 and MMP-9 proteins in brain tissue of HACE model mice. Experimental methods: The expression of AQP4 and MMP9 in mouse brain tissue was analyzed using Western blotting and immunofluorescence staining techniques.

[0041] Immunoblotting method, (1) Protein extraction: Take brain tissue samples from each group into animal grinding tubes, add pre-prepared lysis buffer, set the grinding program as: 30s, 6.50m / s, 4 cycles, after grinding, centrifuge at 12000r, 4℃ for 15min, and aspirate the supernatant into a new EP tube for later use. (2) Protein concentration determination: The protein content of each group was detected using the BCA protein detection kit (Beyotime P0012). Protein standards were prepared to the required concentration using protein standard preparation solution, and BCA working solution was prepared at a ratio of A:B=50:1. An appropriate amount of the sample to be tested was added to a 96-well plate, and the protein was diluted with PBS and the volume was increased to 20µL. Three replicates were set up. 200µL of BCA working solution was added to each well of the 96-well plate, mixed well, and then transferred to 37℃ and incubated for 30min. After incubation, the absorbance was measured at 562nm wavelength using a microplate reader. A standard curve was plotted based on the protein standards, and the protein content in the sample was calculated. According to the protein content obtained, 4×Loading buffer was added to the sample, and the sample was boiled in a water bath for 10min to completely denature the protein sample. After cooling to room temperature, the sample was stored at -20℃ for later use. (3) Preparing the adhesive: Install the cleaned glass plate and glass tank, test for leaks, pour out the water, and dry the edges to prepare the adhesive. Prepare an 8% or 10% concentration of separating adhesive and a 4% concentration of concentrating adhesive, pour the adhesive into the center of the glass clamp, and insert the comb. After the adhesive has solidified, remove the comb; (4) Electrophoresis: After preparing the gel, add the sample and marker using a micro-syringe. Add the prepared electrophoresis solution to the inner and outer tanks. Initially, use a low voltage of 80V. After about 20 minutes, when the proteins enter the separating gel, change the voltage to 120V. When the desired proteins are separated, stop the electrophoresis when the bromophenol blue is about 1 cm from the bottom edge. (5) Transfer: Before electrophoresis, cut the PVDF membrane and soak the sponge pad and filter paper in TBST (Beyotime 9997S) in advance. Soak the PVDF membrane in pure methanol for 15 seconds beforehand, and then put it into the transfer solution. Arrange the membrane in the following order: clamp + sponge + filter paper + gel + membrane + filter paper + sponge + clamp. Carefully place the membrane on the gel, gently remove air bubbles, clamp it, and put it into the transfer tank with the membrane side facing the positive electrode. Transfer the membrane at a constant current of 300mA for 2 hours. After the transfer is completed, remove the PVDF membrane and wash it in TBST for 3 minutes. (6) Immunological reaction: The membrane was placed in a dish and blocked with 5% skim milk (prepared with TBST) for 2 hours. Rabbit anti-MMP9 (1:1000) (Aibotek A0289) and AQP4 (1:1000) (Aibotek A11210) were added and incubated overnight at 4°C on a shaker. The membrane was washed with TBST for 10 minutes each time, 3 times. CY3-labeled goat anti-rabbit IgG (1:300) (Beyotime A0516) prepared with TBST was added and incubated at room temperature for 2 hours. The membrane was washed with TBST for 10 minutes each time, 3 times to reduce background. (7) Exposure: Prepare the ECL luminescent reagent according to A:B=1:1 and expose it using a chemiluminescence analyzer. Quantify using ImageJ1.52a software.

[0042] Immunofluorescence method, (1) Dewaxing to water: Place the paraffin sections in xylene (15min×2), anhydrous ethanol (5min×2), 85% alcohol (5min), and 75% alcohol (5min) in sequence, and finally wash with distilled water; (2) Antigen retrieval: Add an appropriate amount of 0.01M citrate antigen retrieval buffer (pH=6.0) to the retrieval box, heat it in the microwave oven until boiling, then turn off the heat and soak the tissue sections for about 8 minutes. Then turn on the medium-low heat for 7 minutes and let it cool naturally to room temperature. Then place the sections in PBS and wash them three times on a decolorizing shaker for 5 minutes each time. (3) Serum blocking: Carefully aspirate excess liquid from the slide, draw a closed circle around the tissue using a histochemical pen, add autofluorescence quencher inside the circle for 5 min, rinse with running water for 10 min, and then add 3% BSA solution and incubate for 30 min. (4) Add primary antibody: Gently shake off the remaining blocking solution on the dry section, add rabbit anti-MMP9 (1:1000) (Aibotek A0289) and AQP4 (1:1000) (Aibotek A11210) diluted with PBS, and incubate overnight at 4°C in a humidified chamber; (5) Add secondary antibody: Wash 3 times with PBS, 5 min each time. After gently shaking dry the section, add CY3-labeled goat anti-rabbit IgG (1:300) (Beyotime A0516) to the circle and incubate at room temperature in the dark for 50 min; (6) DAPI counterstaining: Wash 3 times with PBS, 5 min each time. After gently shaking off the excess water, add DAPI staining solution to the circle and incubate at room temperature in the dark for 10 min; (7) Mounting: Wash 3 times with PBS, 5 min each time. Gently shake off excess water and then mount with anti-fluorescence quenching mounting medium; (8) Image acquisition: Images were observed and acquired using a fluorescence microscope. Dewaxing and rehydration of paraffin sections: soaking in xylene (15 min × 2 times), treatment with anhydrous ethanol (5 min × 2 times); gradient alcohol hydration: 85% ethanol (5 min), 75% ethanol (5 min), final rinsing with distilled water; antigen retrieval: retrieval solution preparation: 0.01 M sodium citrate buffer (pH 6.0); (9) Repair process: Microwave heating to boiling, then stand for 8 min, maintain on medium-low heat for 7 min, cool naturally to room temperature, wash with PBS (shake on a shaker, 5 min × 3 times). Blocking and antibody incubation; (10) Background treatment: The tissue area was circled and sealed, treated with autofluorescence quencher for 5 minutes, rinsed with running water for 10 minutes, and blocked with 3% BSA for 30 minutes; (11) Primary antibody reaction: antibody dilution ratio MMP9 (1:200), AQP4 (1:200), incubate overnight at 4°C; (12) Secondary antibody reaction and nuclear staining: Secondary antibody treatment, CY3-labeled goat anti-rabbit IgG (1:300), incubated at room temperature in the dark for 50 minutes; nuclear staining, DAPI solution staining in the dark for 10 minutes, washed with PBS (5 min × 3 times).

[0043] (13) Mounting and observation: The sample is sealed with anti-fluorescence quenching mounting medium; image acquisition, observation and recording under a fluorescence microscope, and multi-channel image acquisition.

[0044] The results of the immunoblotting experiment showed that ( Figure 6 Compared with the normal control group, the expression levels of AQP4 and MMP9 in the brain tissue of mice in the HACE model group were significantly upregulated (confirmed by gray value analysis after β-actin normalization). CT-6-H and the positive control drug dexamethasone (DEX) significantly downregulated the expression of AQP4 protein. P<0.05, while CT-6-M and CT-6-L did not show significant regulatory effects (P>0.05); in terms of MMP9 expression regulation, high and medium doses of CT-6 and DEX all showed extremely strong inhibitory effects (P>0.05). P<0.001. Immunofluorescence assay results showed that ( Figure 7 The significantly reduced green and red fluorescence in the CT-6-H and CT-6-M groups indicates decreased expression of AQP4 and MMP9 proteins. These results suggest that CT-6 may play a role in protecting the integrity of the blood-brain barrier by dose-dependently regulating the expression of AQP4 and MMP9, which may be one of the important molecular mechanisms by which it alleviates high-altitude cerebral edema.

[0045] The above embodiments are merely illustrative examples of the present invention, and those skilled in the art can make modifications or optimizations according to actual needs. It should be particularly noted that any such modifications or optimizations, as long as they do not deviate from the core ideas or basic principles of the present invention, should be considered to fall within the protection scope of the present invention.

Claims

1. Use of a xanthine amide compound or a salt thereof in the manufacture of a medicament for the prevention and / or treatment of a nervous system disease associated with hypoxic neuroinflammation, wherein, The compound has the following structure: 。 2. The use according to claim 1, wherein, The nervous system disease related to hypoxic neuroinflammation includes high altitude cerebral edema, ischemic stroke, hemorrhagic stroke, ischemia-hypoxia brain injury, traumatic brain injury, Alzheimer's disease, Parkinson's disease, Huntington's disease, epilepsy, post-traumatic stress disorder.

3. The use according to claim 2, wherein, The nervous system disease related to hypoxic neuroinflammation is high altitude cerebral edema.

4. The use according to any one of claims 1 to 3, wherein, The compound can inhibit the production of inflammatory factors of LPS+hypoxia-induced human microglial HMC3 cell hypoxic neuroinflammation model.

5. Use according to claim 4, wherein, The inflammatory factors include NO, TNF-alpha and IL-1beta.

6. The use according to any one of claims 1 to 3, wherein, The compound can promote the cell growth of LPS+hypoxia-induced human microglial HMC3 cell hypoxic neuroinflammation model.

7. The use according to any one of claims 1 to 3, wherein, The compound can inhibit the weight loss, brain tissue water content increase, inflammatory factor production, histopathological change and / or brain edema related protein expression of LPS+hypoxia-induced high altitude cerebral edema animal model.

8. Use according to claim 7, wherein, The inflammatory factors include TNF-alpha and IL-6, and the brain edema related proteins include AQP4 and MMP-9.

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