Compound for treating microwave radiation induced brain injury by regulating GABA-alpha5 and composition thereof

Abstract

The invention discloses that S44819 can relieve the down-regulation of hippocampus GABA-alpha5 receptor gene expression caused by microwave radiation. Meanwhile, the invention discloses a compound and a composition thereof, the GABA-alpha5 specific antagonist S44819 and the nasal temperature-sensitive gel composition thereof can improve the negative influence of microwave radiation on neural behaviors by acting on GABA receptor subtype GABA-alpha5, and the GABA-alpha5 specific antagonist S44819 and the nasal temperature-sensitive gel composition are potential therapeutic drugs for improving the influence of microwave radiation on the neural behaviors.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and more specifically, to a compound S44819 having a significant therapeutic effect on cognitive decline induced by microwave radiation and a composition thereof.

[0002] Background

[0003] Microwaves refer to electromagnetic waves with frequencies between 300MHz and 300GHz and wavelengths between 1m and 1mm. As a higher frequency band among radio waves, they are easy to gather into beams, highly directional, and propagate in a straight line. They can be used to transmit high-frequency signals in free space with unobstructed line of sight.

[0004] With the development of the information age, microwave radiation has been widely used in military, medical, communication, industrial, aerospace and other fields. Humans are more and more exposed to microwave radiation. The use of mobile communication stations, microwave ovens, mobile phones, computers, military radars and civilian radars will generate microwave radiation. Microwave radiation is a physical source of pollution that is not easily detected by people. The harm of microwave radiation to the human body has become a basic research content in the fields of occupational hygiene and environmental medicine. The nervous system is highly sensitive to microwave radiation, and the hippocampus is a key brain area responsible for cognitive functions such as learning and memory. Many studies have shown that microwave radiation can cause hippocampal-dependent learning and memory dysfunction.

[0005] In the early 1950s, physiological psychologists had already noticed the important role of the hippocampus in learning and memory. Subsequent studies have found that patients with diseases such as AD, epilepsy, and depression are accompanied by various physiological structural changes in the hippocampus, and the hippocampus is easily damaged by factors such as hypoxia, inflammation, and anemia. Therefore, the study of emotional cognition and its functions related to the hippocampus has always been a hot research topic. As the neural basis for learning and memory in mammals, the hippocampus is involved in the encoding, storage, and recall of memory. In recent years, the development of cutting-edge brain science technologies such as optogenetics, chemical genetics, viral tracing, fluorescence microscopy optical sectioning tomography, brain mesoscopic network functional imaging, single-cell transcriptomics, and genetically encoded neurotransmitter fluorescent probes has provided a powerful means for in-depth research on the hippocampus, and will also open up new ideas for the prevention and treatment of hippocampal-related neurological diseases.

[0006] Most of the existing drugs that have therapeutic effects on microwave radiation damage are traditional Chinese medicines, which lack specific therapeutic targets. In addition, the research on the preventive and therapeutic effects of these drugs on microwave radiation damage has not been in-depth, and the protective effects on the nervous system have not been clarified. There are few reports on their protection against microwave radiation brain damage, and they are not comprehensive. Therefore, the research on the prevention and treatment of microwave radiation damage drugs is crucial for medical protection against electromagnetic radiation damage.

[0007] Increased or decreased GABA release plays an important regulatory role in the progression of neurodegenerative diseases. Damage to GABAergic transmission by gene mutation or application of GABA receptor antagonists can induce epileptic seizures, while drugs that enhance GABAergic transmission are used for anti-epileptic treatment. In animal epilepsy models and tissues from patients with temporal lobe epilepsy, it was found that a subpopulation of hippocampal GABA neurons was partially lost and GABAergic transmission at certain synapses was compensatory. In addition, at the GABAAR level, neurodegeneration-induced receptor loss was accompanied by significant changes in the expression of DG GABA receptor subunits. Pregnenolone inhibits GABAAR function, leading to increased synaptic GABA release, which is due to the activation of TRPM3 receptors near presynaptic GABA release sites; at the same time, pharmacological blockade of TRPM3 receptors indicates that pregnenolone leads to a decrease in the frequency of inhibitory postsynaptic currents. Insufficient astrocyte metabolism can lead to reduced glutamine synthesis, which directly impairs GABA synthesis in the brains of AD mice. Astrocyte metabolic dysfunction may be the basis for the imbalance of synaptic excitation and inhibition in AD brains. However, there are few reports on the effects of microwave radiation on hippocampal GABA neurotransmitter metabolism. Therefore, reduced synthesis or release of hippocampal GABA neurotransmitters may lead to epilepsy, and reduced release of hippocampal GABA neurotransmitters may mediate cognitive dysfunction caused by microwave exposure.

[0008] GABA receptor subtypes include GABA A R and GABA B R, combined with the literature research in recent years, this paper only focuses on GABA A R. GABA A GABA R is a chloride ion channel protein. There are 19 types of GABA A R subunits, a total of 8 categories, including α1-6, β1-3, γ1-3, δ, ε, θ, π and ρ1-3, these subunits usually function in the form of pentamers and exist in 20% to 50% of synapses in the brain. By comparing the mRNA expression of 19 subunits in the hippocampus, combined with cognitive function and anxiety, 5 subunits, α1, α2, α5, β3 and γ2, were selected to observe the effect of microwave radiation on their mRNA expression.

[0009] For selective antagonists of GABA-α5, S44819 is a first-in-class compound that acts as a potent, competitive, and selective antagonist of recombinant and natural GABA-α5 ( Figure 1). S44819 can enhance the spatial recognition memory of SD rats in the eight-arm radial maze and improve the severe working memory deficit caused by acute injection of scopolamine. At the same time, S44819 can enhance the exploration time of mice for new objects and enhance the LTP in the CA1 area of ​​the hippocampus of mice. Permanent occlusion of the right common carotid artery caused chronic cerebral hypoperfusion in mice, resulting in decreased cognitive ability of mice. Oral administration of S44819 for 14 consecutive days after surgery can significantly improve the reference memory and working memory of mice with permanent occlusion of the right common carotid artery. Mice exposed to transient intraluminal occlusion of the middle cerebral artery can cause stroke. Oral administration of S44819 was started 72 hours after stroke. The results showed that a dose of 10 mg / kg could continuously improve motor coordination and spatial memory. 42 days after treatment, 10 mg / kg S44819 could reduce striatal atrophy and increase the long-term survival rate of striatal neurons around the infarct focus. At the same time, delayed neuroprotection was associated with a decrease in astrocytes around the infarct area, an increase in the density of brain capillaries around the infarct area, and an increase in the proliferation and differentiation of neural progenitor cells near the subventricular zone on the affected side.

[0010] Based on the successful establishment of a microwave radiation mouse model in the early stage, the present invention further explored the effect of microwave radiation on the gene expression of hippocampal GABA receptor subtypes. By screening microwave radiation-sensitive hippocampal GABA receptor subtypes and using receptor drug intervention, the regulatory mechanism of microwave radiation affecting neurobehavior and its drug prevention and treatment targets were analyzed from the perspective of neurotransmitter receptor activity regulation. Summary of the invention

[0011] The present invention discloses that microwave radiation causes down-regulation of hippocampal GABA-α5 receptor gene expression, but has no significant effect on GABA-α1, GABA-α2, GABA-β3, and GABA-γ2 receptor gene expression. Compound S44819 is administered at a dose of 1.5 mg / kg, which can significantly improve the cognitive decline of mice induced by microwave radiation and restore the cognitive level of mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 S44819 chemical structure

[0013] Figure 2 Effects of microwave radiation on the expression of hippocampal GABA receptor subtype genes A. Melting peaks, melting curves and q-PCR amplification curves of GABA receptor subtype genes; B. Expression of hippocampal GABA receptor subtype genes in each experimental group of mice 6h and 7d after microwave radiation (n=6). Independent sample t test, *P<0.05

[0014] Figure 3Effects of S44819 on cognitive function changes in mice induced by microwave radiationA. Flow chart of microwave radiation and novel object recognition experiment; B. Example of mouse movement trajectory and heat map; C. Changes in novel object recognition index and discrimination index of mice in each experimental group 6 hours after microwave radiation (n=10). Two-way ANOVA, *P<0.05, **P<0.01, ***P<0.001 DETAILED DESCRIPTION

[0015] The technical solution of the present invention will be described below in conjunction with specific embodiments, but the protection of the present invention is not limited thereto.

[0016] Example 1: The regulatory effect of microwave radiation on hippocampal CA3 GABA neurons

[0017] The whole body of mice was irradiated using the S-band microwave radiation source built by the Military Medical Research Institute. The mice were placed in a round transparent glass box with holes in a free position. The radiation center frequency was 2.856 GHz, and the average power density was 8 mW / cm 2 SAR value was 6.12W / kg, the radiation source was 85cm away from the animal's body surface, and the single radiation lasted 15min. The mice in the control group (Con) were sham-irradiated under the same conditions.

[0018] 8-9 week old SPF grade C57BL / 6N mice, half male and half female, weighing 20g-24g, were used and purchased from Spef (Beijing) Biotechnology Co., Ltd., license number: SCXK (Beijing) 2019-0010. The mice were kept in the Experimental Animal Center of the Academy of Military Medical Sciences at a temperature of 18-22°C with sufficient water and food. The ethics of animal experiments were reviewed and approved by the Medical Ethics Committee of the Experimental Animal Center of the Academy of Military Medical Sciences (ethics number: IACUC-DWZX-2021-685).

[0019] For the qPCR experiment, the mice were randomly divided into three groups: control group (Con), 6h post-radiation group (MW-6h), and 7d post-radiation group (MW-7d), with 6 mice in each group.

[0020] GABA receptor administration combined with behavioral experiments The mice were randomly divided into 4 groups: control group (Con-NS), control antagonist group (Con-S), radiation group (MW-NS) and radiation antagonist group (MW-S), with 10 mice in each group.

[0021] 1. Primer Design

[0022] Six pairs of primers, including GABA-α1, GABA-α2, GABA-α5, GABA-β3, GABA-γ2 and β-Actin, were designed using the Primer Bank database and verified by comparison with the NCBI BLAST database. The primers were synthesized by TaKaRa Company of Japan. The primer sequences are shown in Table 1.

[0023] Table 1 Primer sequences

[0024]

[0025] 2. Total mRNA Extraction from Hippocampal Tissue

[0026] ① After the mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg), the brains were removed by decapitation, the hippocampal tissue was separated and added with 0.5 ml Trizol, and then placed on ice for 2 min. The tissue was homogenized at 60 Hz for 30 s for 3 times (the crushing can be stopped after two times, and the tissue was taken out and placed in a -20°C refrigerator for 3 min), and then placed at 4°C for 10 min after shaking.

[0027] ② Add 200 μl of chloroform, gently turn upside down for 15-20 seconds, and let stand on ice for 3 minutes;

[0028] ③ Centrifuge at 4°C, 12000rpm for 20min;

[0029] ④ Take the upper aqueous phase (be careful not to suck the middle layer), let it stand on ice for 2 minutes, add isopropanol with the same volume as the aqueous phase, shake gently 5-8 times, and let it stand on ice for 15 minutes;

[0030] ⑤ Centrifuge at 4°C, 12000rpm for 10min (fix the orientation of the EP tube to facilitate observation of the RNA precipitation position after centrifugation);

[0031] ⑥ Aspirate the supernatant (absorb from the inside of the placement angle, avoiding the white precipitate), add 1 ml of 75% ethanol (prepared with RNase-free ddH2O), gently turn upside down to wash the precipitate; centrifuge at 4°C, 12000rpm for 5 minutes;

[0032] ⑦ Aspirate and discard the supernatant, add 1 ml of 75% ethanol (prepared with RNase-free ddH2O), gently turn upside down to wash the precipitate; centrifuge at 4°C, 12000 rpm for 5 minutes;

[0033] ⑧Discard the supernatant without adding any reagents; centrifuge at 4°C, 12000rpm for 5min;

[0034] ⑨Open the EP tube and dry it naturally in a fume hood for 4 minutes (to allow residual ethanol to evaporate), then add 20 μl of RNase-free ddH2O to resuspend the precipitate.

[0035] ⑩ RNA content and concentration determination:

[0036] Take 2 μl of the above solution for determination. Adjust the instrument to RNA determination mode, add ddH2O to zero and clean three times, measure and record the concentration and A260 / 280, A260 / 230 ratios, and select samples with A260 / 280 ratios in the range of 2.0-2.2 and A260 / 230 ratios in the range of 2.0-2.4 for the next experiment.

[0037] 3. Reverse Transcription

[0038] ① To remove genomic DNA, prepare a mixed solution in an RNase-free centrifuge tube (see Table 2), mix it by gently pipetting, and incubate at 42°C for 2 min;

[0039] Table 2 Preparation method of genomic DNA removal solution

[0040]

[0041] ② Prepare the reverse transcription reaction system (see Table 3), add 5×HiScript IIIqRT SuperMix directly to the reaction tube in step ①, and mix gently by pipetting;

[0042] Table 3 Reverse transcription reaction system

[0043]

[0044] ③ Carry out reverse transcription reaction. The reaction conditions are shown in Table 4. The product cDNA can be directly used in qPCR reaction to avoid repeated freezing and thawing of cDNA.

[0045] Table 4 Reverse transcription reaction conditions

[0046]

[0047] 4. Real-time fluorescence quantitative PCR detection of DA / GABA receptor subtype gene expression

[0048] ① Prepare the mixed solution in a qPCR tube (see Table 5), then place the sample into a real-time fluorescence quantitative PCR instrument for amplification reaction;

[0049] Table 5 qPCR reaction system formula

[0050]

[0051] ②qPCR reaction conditions are shown in Table 6.

[0052] Table 6 qPCR reaction conditions

[0053]

[0054] Microwave radiation has a significant effect on the gene expression of hippocampal GABA receptor subtypes. The results of qPCR experiments showed that 6 hours after microwave radiation, the expression of GABA-α5 mRNA in the hippocampus of mice in the radiation group was significantly reduced compared with the control group (P < 0.05, see Figure 2 A, B), but no significant differences were found in the expression of GABA-α1 mRNA, GABA-α2 mRNA, GABA-β3 mRNA and GABA-γ2 mRNA. 7 days after microwave radiation, there were no statistically significant changes in the expression of GABA-α1 mRNA, GABA-α2 mRNA, GABA-α5 mRNA, GABA-β3 mRNA and GABA-γ2 mRNA. The above results suggest that microwave radiation can cause the downregulation of hippocampal GABA-α5 receptor gene expression, but has no significant effect on the expression of GABA-α1, GABA-α2, GABA-β3, GABA-γ2 receptor genes. Therefore, microwave radiation can cause the downregulation of hippocampal GABA-α5 receptor gene expression, but has no significant effect on the expression of GABA-α1, GABA-α2, GABA-β3, GABA-γ2 receptor genes.

[0055] Example 2: Preparation of S44819 nasal in situ gel

[0056] Accurately weigh 2g of poloxamer 188 and 12g of poloxamer 407, add appropriate amount of water and fully swell at 4°C to obtain a temperature-sensitive gel; weigh 1g of S44819, add 6mL of 1,2-propylene glycol and 8ml of ethanol, and mix well; add the above S44819 solution to the above temperature-sensitive gel, continue to add water to 50mL, and mix well to obtain the S44819 nasal in situ gel.

[0057] Example 3: The therapeutic effect of S44819 on brain damage caused by microwave radiation

[0058] 1. Drug preparation and administration method

[0059] Accurately weigh 1 mg of the GABA-α5 specific antagonist S44819 standard, add 1 ml of DMSO and 9 mL of normal saline to dissolve, and prepare a 0.1 mg / mL working solution. The control group (Con-S) and the radiation group (MW-S) were intraperitoneally injected 4 hours after sham radiation and radiation, respectively, with a dose of 1.5 mg / kg. The control group (Con-NS) and the radiation group (MW-NS) were given an equal volume of normal saline.

[0060] 2. New object recognition experiment

[0061] S44819 was tested 30 minutes after intraperitoneal injection.

[0062] A 40cm×40cm×40cm open field box was used. The mice were placed in the laboratory environment for 30 minutes for 3 consecutive days before the experiment. The formal experiment was divided into two stages. In the first stage, the mice were placed in an open field box with two identical old objects (4cm×4cm×4cm, purple) with their backs to the experimenter to explore freely for 5 minutes. The second stage of the experiment was carried out after an interval of 1 hour. In the second stage, one of the old objects was replaced with a new object (4cm×4cm×4cm, green) of the same shape but different color, and it was moved to the diagonal position of the old object. The mice were placed in the open field box with their backs to the experimenter to explore freely for 5 minutes. The ANY-maze video analysis software was used to record the time and number of times the mice touched the new and old objects in each stage, and their movement trajectory diagrams and heat maps were recorded. The novel object discrimination index (DI) and cognitive index (RI) of mice were calculated and analyzed: discrimination index = (time touching the new object - time touching the old object) / (time touching the new object + time touching the old object); cognitive index = time touching the new object / (time touching the new object + time touching the old object).

[0063] GABA-α5 antagonist S44819 has a significant effect on the cognitive function changes of mice induced by microwave radiation. The experimental protocol of intraperitoneal injection of S44819 after microwave radiation combined with novel object recognition is shown in Figure 3 A. The results showed that 6 hours after microwave radiation, compared with the control group, the novel object discrimination index and novel object cognition index of the mice in the radiation group were both reduced, and the difference was statistically significant (P < 0.001). Compared with the radiation group, the novel object discrimination index and novel object cognition index of the mice in the radiation administration group were significantly increased (P < 0.05), and the cognitive level of normal mice was restored. In addition, compared with the control group, the novel object discrimination index and novel object cognition index of the mice in the control administration group were reduced, and the difference was statistically significant (P < 0.01) (see Figure 3 B, C), and no statistical differences were found in the rest.

[0064] Therefore, microwave radiation can lead to a decline in the novel object exploration behavior and cognitive ability of mice, while S44819 intervention after microwave radiation significantly improves the cognitive ability of irradiated mice and restores them to the cognitive level of normal mice. This shows that the administration of GABA-α5 specific antagonist S44819 can significantly improve the cognitive decline of mice induced by microwave radiation.

Claims

1. A temperature-sensitive gel composition for treating brain damage caused by microwave radiation, characterized in that The composition containing S44819 and temperature-sensitive gel can alleviate the down-regulation of hippocampal GABA receptor gene caused by microwave radiation.

2. The temperature sensitive gel composition according to claim 1, characterized in that The active ingredient in the drug solution is S44819.

3. The temperature sensitive gel according to claim 1 or 2, characterized in that The raw material contains 0.1 to 10 parts of poloxamer 188, 1 to 10 parts of poloxamer 407 and 15 to 35 parts of water by weight.

4. The method for preparing the nasal preparation composition for treating microwave radiation brain damage according to any one of claim 3, comprising the step of mixing the drug solution and the temperature-sensitive gel.

5. A method for preparing a nasal preparation composition for treating microwave radiation brain damage according to claim 4, comprising the following steps: 1) adding poloxamer 407 and poloxamer 188 contained in the nasal preparation composition to ultrapure water at 4°C to fully swell, continuing to add water to the full amount, and mixing to obtain a temperature-sensitive gel; 2) adding the drug solution contained in the nasal preparation composition to the temperature-sensitive gel and mixing to obtain a nasal gel preparation.

6. Use of the nasal preparation composition according to any one of claims 2 to 5 in the preparation of a medicament for treating microwave radiation brain damage.

7. According to claim 1, microwave radiation can significantly down-regulate the expression of hippocampal GABA-α5 receptor gene.

8. Use of compound S44819 in the preparation of a drug for improving the cognitive decline of mice induced by microwave radiation and restoring the cognitive level of mice, characterized in that The dose was 1.5 mg / kg.

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