Application of inosine in treatment of anxiety disorder

Abstract

The invention relates to application of inosine in treatment of anxiety disorder, and relates to the technical field of medicines. The invention provides an application of inosine in preparation of a medicine for preventing and / or treating anxiety disorder, the inosine inhibits three inflammatory factors of NLRP3, CaspaseI and IL-18 by up-regulating 5-HT, GABA and serum dopamine of a hippocampus and a cortex, inhibits an NLRP3 / CaspaseI / IL-18 inflammatory pathway, increases the complexity of microglial cell morphology, activates neurons, achieves the effect of remarkably reversing anxiety behavior, and can be used for preparing a medicine for preventing and / or treating anxiety disorder. The inosine can be used for preventing and treating the anxiety disorder, and a new method and means are provided for clinically treating the anxiety disorder.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to application of inosine in treating anxiety. Background Art

[0002] Anxiety disorder is a common mental disorder, manifested as persistent tension, uneasiness, fear and other emotional reactions, which seriously affect the quality of life of patients. According to a survey by the World Health Organization, anxiety disorder is the most common mental disorder in the world. In 2019, 301 million people suffered from anxiety disorder, which usually appeared in childhood or adolescence, and only 1 in 4 patients with anxiety disorder received treatment. Currently, 4% of the global population suffers from anxiety disorder. In 2019, 301 million people suffered from anxiety disorder worldwide, making anxiety disorder the most common of all mental disorders. It is considered to be the most common type of mental disorder, and more than 60 million people are affected by anxiety disorder in Europe each year. However, according to epidemiological surveys, it is estimated that one-third of the population is affected by anxiety disorder in their lifetime. Anxiety disorders often interfere with daily activities such as work performance, academic studies and interpersonal relationships. Although there are very effective treatments for anxiety disorders, only about 1 / 4 of patients (27.6%) receive treatment.

[0003] Anxiety often arises from stress response mechanisms triggered by external environmental stimuli. When an organism encounters external stimuli, a complex and multi-level stress response system is activated inside it, which mainly operates through feedback regulation. In this system, some inflammatory pathways also play an important role in the development of anxiety disorders. Among them, proinflammatory cytokines, specifically interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), have been shown to be closely related to the pathogenesis of clinical depression and anxiety. The bioavailability and activity of IL-1β are regulated by caspase-1-dependent proteolysis relative to inflammasomes. Therefore, interventions that regulate caspase-1 activation or activity should reduce anxiety, especially in a state that promotes IL-1β maturation. Studies have reported that interleukin 18 (IL-18) is involved in mood changes caused by pyroptosis in rodent models of hemorrhagic shock and resuscitation. The role of IL-18 binding protein (IL-18BP), IL-18BP has excellent anti-inflammatory effects as a negative regulator of IL-18. IL-18BP significantly alleviated HSR-induced anxiety-like behavior, astrocyte activation, and the increase of pyroptosis-related factors, while the NLRP3 agonist nigericin partially reversed the IL-18BP-induced neuroprotection. IL-18BP exhibited anti-pyroptotic effects on astrocytes in an in vitro model of hypoxia-glucose deprivation. This suggests that inhibition of IL-18R-NLRP3 signaling by IL-18BP can attenuate astrocyte activation and pyroptosis.

[0004] Microglia are mononuclear macrophages present in the central nervous system and have important immune functions. Under stress, microglia undergo morphological changes and their complexity decreases. Danyang Chen et al. found that acute restraint stress leads to increased activity of GABAergic neurons (GABA CeA) in the CeA of male mice, leading to anxiety-like behavior within 12 hours; at the same time, GABA CeA neuronal CX3CL1 secretion is increased through MST4 (mammalian sterile 20-like kinase 4)-NF-κB-CX3CL1 signaling, thereby activating microglia in the CeA. Activated microglia, in turn, inhibit GABA CeA neuronal activity by phagocytosing dendritic spines, ultimately leading to the disappearance of anxiety-like behavior caused by restraint stress. These findings reveal a dynamic molecular and cellular mechanism in which microglia drive negative feedback to inhibit GABA CeA neuronal activity, thereby promoting the maintenance of brain homeostasis in response to acute stress.

[0005] At present, there are many kinds of drugs for treating anxiety disorders, but they have problems such as large side effects and strong dependence. Therefore, it is of great significance to develop a safe and effective treatment for anxiety disorders. Summary of the invention

[0006] In view of the above problems, the present invention provides an application of inosine in the preparation of a drug for preventing and / or treating anxiety. Inosine upregulates 5-HT, GABA and serum dopamine in the hippocampus and cortex, inhibits three inflammatory factors, NLRP3, CaspaseI and IL-18, inhibits the NLRP3 / CaspaseI / IL-18 inflammatory pathway, increases the complexity of microglia morphology, activates neurons, and achieves a significant reversal of anxiety behavior. Inosine can be used for the prevention and treatment of anxiety, providing new methods and means for the clinical treatment of anxiety.

[0007] In order to achieve the above object, the present invention provides a use of inosine in preparing a drug for preventing and / or treating anxiety.

[0008] Inosine is well known for its antioxidant properties. Its potential in disease treatment is mainly based on its antioxidant effect and ability to increase urate levels and increase energy resources by increasing ATP availability. Inosine has the potential to treat AD by regulating different brain mechanisms involved in neuroprotection, and can also prevent STZ-induced decrease in synaptophysin immunoreactivity in the CA3 hippocampus. The presence of endogenous ligands indicates that inosine and EBZ show the same activity in inhibiting [3H]FNZP binding, while FNZP binds to GABA receptors and increases GABA expression. Inosine can increase the expression of brain-derived neurotrophic factor (BDNF) in the brain through adenosine receptors, protect dopaminergic neurons from MPTP-induced neuronal damage, and improve dopaminergic neuron loss and MPTP-mediated astrocyte and microglia activation in the substantia nigra and striatum. Previous studies have found that the pathogenesis of anxiety disorders mainly focuses on neurotransmitter, neuroendocrine and immune dysfunction. Among them, the research content for neurotransmitters mainly includes 5-hydroxytryptamine, dopamine, norepinephrine, γ-aminobutyric acid, neuropeptide Y, etc. Especially 5-hydroxytryptamine (5-HT), after knocking out the 5-HT1AR gene in the mouse brain, the mouse showed anxiety-like behavior; atmospheric PM2. Exposure caused depression and anxiety-like behavior in mice, which was also related to the reduction of 5-HT levels. The inventors used a chronic restraint stress-induced anxiety model to explore the potential of inosine in treating anxiety. Through mouse behavioral experiments, pharmacodynamic studies and pathway studies, it was proved that inosine up-regulated 5-HT, GABA and serum dopamine in the hippocampus and cortex, inhibited NLRP3, CaspaseI and IL-18 three inflammatory factors, inhibited the NLRP3 / CaspaseI / IL-18 inflammatory pathway, increased the complexity of microglia morphology, activated neurons, and achieved a significant reversal of anxiety behavior. Inosine is a metabolite naturally present in the human body, has good biocompatibility and safety, can be used for the prevention and treatment of anxiety, and provides new methods and means for the clinical treatment of anxiety.

[0009] In one embodiment, the anxiety disorder is generalized anxiety disorder.

[0010] Generalized anxiety disorder is anxiety induced by chronic restraint stress, usually caused by long-term stressors in the external environment, such as work pressure, interpersonal relationship problems, or long-term negative life events. Unlike anxiety caused by other genetic and developmental factors, such as anxiety in complications of intellectually impaired color syndrome, anxiety is related to genetics, differences in brain structure and function, and limitations in cognitive and social abilities. It is also accompanied by other cognitive and behavioral challenges, such as learning disabilities, social disorders, and sleep problems. Therefore, the causes, principles, and symptoms of the two are essentially different. At the same time, although patients with depression are often accompanied by anxiety-like behaviors, patients with anxiety disorders rarely have depressive-like behaviors. The pathogenesis, treatment principles, behavioral manifestations and animal modeling methods of depression and anxiety are completely different. The main differences are as follows: 1. Depression may be related to the reduction of neurotrophic factors such as brain-derived neurotrophic factor (BDNF), which is related to hippocampal hypofunction, memory impairment and high risk of depression, while anxiety may be more related to the stress response system, especially the overactivation of the hypothalamic-pituitary-adrenal axis (HPA axis); 2. Although some drugs such as SSRIs and SNRIs can be used to treat depression and anxiety at the same time, not all antidepressants are suitable for anxiety. For example, some antidepressants may increase anxiety symptoms, such as fluvoxamine; 3. There are also significant differences in behavioral manifestations. Anxiety is mainly manifested as excessive worry, tension, fear, and anticipatory anxiety, which may be accompanied by physiological symptoms such as palpitations, sweating, and muscle tension, while depression is manifested as reduced vitality, changes in appetite and weight, and more immobility time in forced swimming tests or tail suspension tests. 4. The mainstream method for modeling depression is chronic unpredictable stress, while the commonly used modeling method for anxiety is chronic restraint stress.

[0011] In one embodiment, the drug further comprises a pharmaceutically acceptable excipient.

[0012] In one embodiment, the dosage form of the drug includes an injectable preparation or an oral preparation.

[0013] In one embodiment, the oral preparation includes tablets, capsules, powders, pills or oral liquid preparations.

[0014] In one embodiment, when the dosage form of the drug is an injection preparation, the dosage of the drug is 250-350 mg per kilogram of body weight.

[0015] The present invention also provides a medicine for preventing and / or treating anxiety disorders, wherein the medicine comprises a medicine active ingredient and excipients, and the medicine active ingredient is inosine.

[0016] In one embodiment, the auxiliary material includes at least one of an excipient, a lubricant, an antioxidant, a preservative, a binder, a filler, or a thickener.

[0017] In one embodiment, the anxiety disorder is generalized anxiety disorder.

[0018] In one of the embodiments, the drug upregulates 5-HT, GABA and serum dopamine in the hippocampus and cortex, inhibits three inflammatory factors, NLRP3, CaspaseI and IL-18, inhibits the NLRP3 / CaspaseI / IL-18 inflammatory pathway, increases the complexity of microglia morphology, activates neurons, and achieves the effect of reversing anxiety behavior.

[0019] The inventors administered the drug to individual mice by intraperitoneal injection, with each mouse being administered once a day, 1 hour before restraint stress every day, for 10 days. The experiment verified that inosine can increase the frequency and time of mice entering the open arm of the elevated plus maze, increase the number and time of central exploration of mice in the open field test; increase the levels of 5-HT and GABA in the hippocampus of the experimental mice, increase the levels of 5-HT and GABA in the cerebral cortex of mice, and increase the content of DA in serum; reduce the expression of three inflammatory indicators of Caspase-1, IL-18, and NLPR3 in the experimental mice; increase the complexity of microglia morphology and increase the density of C-FOS neurons, indicating that inosine can improve anxiety-like behavior, thereby preventing and treating anxiety, and provide a new and effective choice for the clinical prevention and treatment of anxiety.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The invention discloses an application of inosine in the preparation of a drug for preventing and / or treating anxiety disorder. Inosine inhibits three inflammatory factors, NLRP3, CaspaseI and IL-18, by upregulating 5-HT, GABA and serum dopamine in the hippocampus and cortex, inhibits the NLRP3 / CaspaseI / IL-18 inflammatory pathway, increases the complexity of microglial morphology, activates neurons, and achieves a significant reversal of anxiety behavior. Inosine can be used for the prevention and treatment of anxiety disorder, and provides a new method and means for the clinical treatment of anxiety disorder. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a scatter bar graph showing the frequency of each group of mice entering the center of the open field in the open field experiment in the embodiment;

[0023] Figure 2 This is a scatter bar graph showing the statistical residence time of each group of mice in the open field center detected in the open field experiment in the embodiment;

[0024] Figure 3 This is a scatter bar graph showing the frequency of each group of mice entering the open arm of the elevated plus maze in the elevated plus maze test in the embodiment;

[0025] Figure 4 This is a scatter plot of the statistical time each group of mice spend in the elevated plus maze open arm in the elevated plus maze test in the embodiment;

[0026] Figure 5 The elevated plus maze and open field test mouse behavior trajectory diagram in the embodiment;

[0027] Figure 6 It is a statistical scatter bar graph of the 5-TH content in the mouse hippocampus detected by high performance liquid chromatography-mass spectrometry in the embodiment;

[0028] Figure 7 It is a statistical scatter bar graph of GABA content in mouse hippocampus detected by HPLC-MS / MS in the embodiment;

[0029] Figure 8 It is a statistical scatter histogram of the 5-TH content in the mouse cortex detected by high performance liquid chromatography-mass spectrometry in the embodiment;

[0030] Fig. 9 This is a statistical scatter histogram of GABA content in the mouse prefrontal cortex detected by high performance liquid chromatography-mass spectrometry in the embodiment;

[0031] Fig.10 This is a statistical scatter bar graph of the DA content in mouse serum detected by HPLC-MS / MS in the embodiment;

[0032] Fig.11 This is a scatter plot of the statistical expression of Caspase1 mRNA in mouse hippocampus detected by RT-qPCR in the embodiment, wherein the internal reference is GAPDH;

[0033] Fig.12 This is a statistical scatter bar graph of IL-18 mRNA expression in mouse hippocampus detected by RT-qPCR in the embodiment, wherein the internal reference is GAPDH;

[0034] Fig.13 This is a statistical scatter bar graph of NLPR3 mRNA expression in mouse hippocampus detected by RT-qPCR in the embodiment, wherein the internal reference is GAPDH;

[0035] Fig.14 This is a graph showing the neuroprotective effect of inosine on mice in the examples. DETAILED DESCRIPTION

[0036] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0038] definition:

[0039] 1. The “individual”, “subject”, “mouse” and “experimental subject” in the embodiments of the present invention all have the same meaning, namely male C57BL / 6 mice.

[0040] 2. Open field test: also known as the open box test, it is an experimental method widely used to evaluate the autonomous movement, exploratory behavior and tension of rodents. This technology plays an important role in the study of neuropsychiatric diseases such as Parkinson's disease, depression, anxiety, and central nervous system drug screening. The principle of the open field test is based on the fact that anxiety levels affect the exploratory behavior of mice in open areas. Mice that have not experienced restraint stress usually have smooth hair and tight skin on their backs, and show strong activity. They will not stay in the corners for a long time, but tend to move to the center of the open field and explore. In contrast, mice that have repeatedly experienced restraint stress for a long time have dull and messy hair, loose skin on their backs, and show lower activity. They are more inclined to curl up in corners or walk along the edge of the open field, and their exploratory behavior is significantly reduced. These behavioral changes provide researchers with valuable information about the anxiety state and environmental adaptability of animals.

[0041] 3. Elevated plus maze: It is an experimental method used to evaluate the anxiety response of rodents. Its advantages are simple operation and intuitive reflection of the conditioned response of mice. The elevated plus maze consists of two open arms and two closed arms. The design principle is based on the conflict between the mouse's curiosity to explore the novel environment and its habit of avoiding light, which induces anxiety. By quantitatively analyzing the length and frequency of mice staying in the open and closed arms, their anxiety level can be assessed. Low-anxiety mice tend to explore the open arms, which is manifested by longer stay time and more entries; while high-anxiety mice prefer to curl up in the closed arms, showing obvious avoidance behavior. Therefore, the elevated plus maze experiment has become an effective means to assess the anxiety state of mice.

[0042] 4. The enterobacteria test process strictly follows the manufacturer's instructions, and uses the fecal DNA kit produced by Guangzhou Maibao Biotechnology Co., Ltd. (located in Guangzhou, China) to accurately extract microbial DNA from the colon contents. Subsequently, the concentration and DNA purity of the extracted 2μL sample were carefully analyzed using a microplate reader, and the sample was diluted to a standard concentration of 10ng / mL based on the analysis results. Next, after fully mixing cDNA, SYBR Green and primers, real-time PCR analysis was performed to ensure the accuracy of the results. The relative expression level of AKK was calculated by standardizing to the expression level of 16S rRNA mRNA. This step served as an internal control to enhance the reliability of the experimental results.

[0043] 5. Real-time fluorescence quantitative PCR (RT-qPCR): It is a molecular biology technique used to amplify and detect specific DNA or RNA sequences in real time. This method uses fluorescent dyes or probes that emit signals when bound to amplified DNA or RNA molecules, allowing the target sequence to be quantified. RT-qPCR is often used in applications such as gene expression analysis, viral load quantification, and gene mutation detection.

[0044] 6. Microglia: As an indispensable mononuclear macrophage in the central nervous system, it plays a vital immune role. When facing stress, these cells will undergo significant morphological changes, which are specifically manifested as a significant reduction in complexity. In order to deeply evaluate the degree of activation of microglia in the hippocampus, the present invention carefully designed the expression analysis of the IBA-1 marker, and with the help of the AnalyzeSkeleton tool, the two-dimensional morphology of IBA-1 positive cells was comprehensively and carefully evaluated. In the comparative analysis of the complexity of microglia morphology, the inventors covered a number of key indicators, including but not limited to the number of branches, maximum branch length, number of nodes, triple integrals, plate panels, endpoint imaginary numbers, and quadruple points, and also introduced the average branch length as an important reference. These multi-dimensional and multi-level considerations are intended to comprehensively and accurately characterize the morphological changes of microglia under stress. In addition, in view of the core position of PFC in memory and behavior regulation, the present invention also cleverly uses the expression of the C-FOS gene as an effective marker of the basal activity of neurons, and then compares and analyzes the density of C-FOS neurons. This innovative research method not only reveals the potential connection between microglia and neuronal activity in PFC, but also provides valuable reference and inspiration for future research in related fields.

[0045] Example

[0046] 1. Preparation of experimental animals.

[0047] 60 male C57BL / 6 mice, weighing 24g-27g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd. The mice were housed in an SPF-grade experimental room with an ambient temperature of 25±2°C, a humidity of 50±5%, a 12-hour light-dark cycle, and free access to drinking water.

[0048] After one week of adaptive feeding of mice, 30 adult male C57BL / 6 mice were randomly divided into three groups according to their body weight, namely, a blank group (Control), a model group (Anxiety), and an inosine group (Inosine).

[0049] Mouse modeling method:

[0050] Anxiety disorder was induced by restraining mice using chronic restraint stress. Male C57BL / 6 mice weighing 24-27 g were selected as the subjects. Successful modeling subjects should have signs or symptoms such as significantly reduced desire to explore, loose skin on the back, dark fur color, and a preference for curling up or walking in the edge area.

[0051] 2. Preparation of experimental reagents and materials.

[0052] 1. Experimental reagents.

[0053] Inosine was weighed and dissolved in physiological saline, and 300 mg / kg / day was administered by intraperitoneal injection.

[0054] 2. Experimental materials.

[0055] 50 ml centrifuge tube bundle, open field apparatus (Shanghai Xinruan Company, China), cross elevated maze (Shanghai Xinruan Company, China), fecal DNA kit (Guangzhou Maibao Biotechnology Co., Ltd., Guangzhou, China).

[0056] 3. Experimental methods.

[0057] 1. Open field experiment.

[0058] The open field apparatus (35cm×35cm×30cm) consists of five sides, with a white bottom and blue sides. The camera hanging above records the number of times each mouse enters the center within 5 minutes, the time spent exploring the center, and the distance moved in the center. After each mouse is tested, the instrument is wiped with 75% alcohol and the next mouse is tested after the alcohol evaporates completely. The entire experimental process is carried out in a quiet and unmanned environment. The open field experiment should be carried out after the stress ends on the 10th day.

[0059] 2. Elevated plus maze test.

[0060] The elevated plus maze consists of two opposite open arms (30cm×10cm) and two opposite closed arms (30cm×10cm×20cm), with an angle of 90° between the open arms and the closed arms, forming a central area in the middle, 70cm above the ground. Each mouse was placed in the central area facing the open arm one by one, and the activity of the mouse was recorded within 5 minutes, including the number of times it entered the open arm and the closed arm, the time spent in the open arm, and the distance walked in the open arm. After each mouse was tested, the instrument was wiped with 75% alcohol and waited for the alcohol to evaporate completely before testing the next mouse. The entire experimental process was carried out in a quiet and unmanned environment, and the elevated plus maze experiment should be carried out after the open field experiment.

[0061] 3. Hippocampal and cortical tissue analysis, microglial morphology analysis, intestinal flora analysis and related statistical measurements.

[0062] The mice were anesthetized with isoflurane, sacrificed, and subjected to hippocampal and cortical tissue analysis, microglial morphology analysis, intestinal flora analysis, and related statistical measurements.

[0063] In the mouse hippocampus and cortical tissue analysis experiment, after the mouse is fully anesthetized, the heart is immediately lavaged with PBS solution. The mouse hippocampus is quickly peeled off on ice, immediately put into liquid nitrogen for quick freezing, and stored at -80℃.

[0064] In the microglia morphological analysis experiment, the expression of the marker IBA-1 was mainly analyzed, and the AnalyzeSkeleton tool was used to evaluate the two-dimensional morphology of IBA-1 positive cells, and the expression of the C-FOS gene was used to identify the basic activity of neurons. Microglia, as an indispensable mononuclear macrophage in the central nervous system, plays a vital immune role. This embodiment is for the expression analysis of IBA-1 markers, and with the help of the AnalyzeSkeleton tool, the two-dimensional morphology of IBA-1 positive cells is comprehensively and carefully evaluated. In the comparative analysis of the complexity of microglia morphology, multiple key indicators are covered, including but not limited to the number of branches, maximum branch length, number of nodes, triple integrals, plate-like panels, endpoint imaginary numbers and quadruple points, etc., and the average branch length is also introduced as an important reference. These multi-dimensional and multi-level considerations are intended to comprehensively and accurately characterize the morphological changes of microglia under stress. In addition, given the central role of PFC in memory and behavior regulation, this study also used the expression of the C-FOS gene as an effective marker of basal neuronal activity, and then conducted a comparative analysis of the density of C-FOS neurons. This innovative research method not only reveals the potential connection between microglia and neuronal activity in PFC, but also provides valuable references and inspiration for future research in related fields.

[0065] For the enterobacteria detection experiment, a stool DNA kit was used. The stool examination process strictly followed the manufacturer's instructions. The stool DNA kit produced by Guangzhou Maibao Biotechnology Co., Ltd. (located in Guangzhou, China) was used to accurately extract microbial DNA from the colon contents. Subsequently, the concentration and DNA purity of the extracted 2 μL sample were carefully analyzed using a microplate reader, and the sample was diluted to a standard concentration of 10 ng / mL based on the analysis results. Next, after fully mixing cDNA, SYBR Green and primers, real-time PCR analysis was performed to ensure the accuracy of the results. The relative expression level of AKK was calculated by normalizing to the expression level of 16S rRNAmRNA. This step served as an internal control to enhance the reliability of the experimental results.

[0066] In this example, real-time fluorescence quantitative PCR (RT-qPCR) was used for PCR analysis.

[0067] Real-time fluorescence quantitative PCR is a molecular biology technique used to amplify and detect specific DNA or RNA sequences in real time. The method uses fluorescent dyes or probes that emit signals when bound to amplified DNA or RNA molecules, allowing the target sequence to be quantified. RT-qPCR is commonly used in applications such as gene expression analysis, viral load quantification, and gene mutation detection.

[0068] RT-qPCR main steps:

[0069] (1) Extraction of RNA:

[0070] 1.1 Take 30-50 mg of sample tissue, add 500 μL Trizol, mix and place in ice water mixture for thorough grinding.

[0071] 1.2 Add 100 μL of chloroform, cover the lid tightly, invert 25 times, mix thoroughly and let stand for 2 minutes.

[0072] 1.3 Centrifuge at 12000rcf, -4℃ for 15min and take 200μL supernatant.

[0073] 1.4 Add 200 μL of isopropanol solution to the supernatant, cover the lid tightly, invert 25 times, mix thoroughly and let stand for 10 minutes to precipitate RNA.

[0074] 1.5 Centrifuge at 12000rcf, -4℃ for 10 min and discard the supernatant.

[0075] 1.6 Add 1 mL of 80% ethanol (prepare 80% ethanol with DEPC water) to the precipitate.

[0076] 1.7 Centrifuge at 7500rcf, -4℃ for 5 min and discard the supernatant.

[0077] 1.8 Centrifuge again for 1 min and remove excess ethanol with a pipette.

[0078] 1.9 After the precipitate is dried, add an appropriate amount of DEPC water until the precipitate is just dissolved.

[0079] (2) RNA purity measurement and concentration adjustment:

[0080] 2.1 RNA purity test: After the RNA is completely dissolved, perform an RNA purity test immediately. If the OD260 / 280 value is between 1.8-2.2, it means that the RNA purity meets the standard and can be used for subsequent experiments.

[0081] 2.2 RNA concentration adjustment: After measuring the RNA concentration with an ELISA instrument, in order to ensure the same amount of sample in each well, the RNA concentration was adjusted with DEPC water.

[0082] (3) Reverse transcription (according to ReverTra qPCR RT kit instructions require the experiment):

[0083] 3.1 Take 6 μL of water and 1 μL of RNA, shake thoroughly to mix, and place in a PCR instrument to denature (65°C, 5 min).

[0084] 3.2 Add 2μL of Buffer, 0.5μL of Primer and 0.5μL of Enzyme, and shake thoroughly to mix. The primer sequences are shown in Table 1.

[0085] 3.3 Set the heating program of the PCR instrument (37℃15min; 98℃5min; 4℃40S), put the mixed solution into the PCR instrument again and heat it according to the pre-set program. Store at -20℃. The primer sequences are shown in the following table.

[0086] Table 1 Primer sequences

[0087]

[0088] In this example, statistical data were analyzed using GraphPad Prism software (version 8.0) and expressed as means ± SE. The differences between two groups were analyzed using t-test; the differences between more than two groups were analyzed using one-way analysis of variance followed by Tukey's multiple comparison test, and when P < 0.05, it was considered statistically significant.

[0089] 4. Experimental results.

[0090] 1. If Figure 1As shown in the open field test results, inosine effectively increased the frequency of mice exploring in the open field, and the data are expressed as mean ± standard deviation (n = 6 mice / group). Figure 2 As shown in the open field test, the results showed that inosine effectively increased the time mice spent exploring the open field, and the data were expressed as mean ± standard deviation (n = 6 mice / group). The individuals treated with inosine showed at least a significantly higher number of times entering the central area of ​​the open field, a significantly higher exploration time in the central area, and a significantly higher walking distance than the individuals not treated with inosine.

[0091] 2. If Figure 3 As shown in the elevated plus maze test results, inosine effectively increased the number of mice in the elevated plus maze, and the data are expressed as mean ± standard deviation (n = 6 / group). Figure 4 As shown in the figure, the results of the elevated plus maze test showed that inosine effectively increased the time mice spent in the elevated plus maze, and the data were expressed as mean ± standard deviation (n = 6 / group). Figure 5 As shown in the figure, individuals without inosine treatment at least preferred to stay in the closed arms or walk slowly at the ends of the closed arms, and did not dare to approach the open arms or immediately retreated to the ends of the closed arms once they reached the open arms. Their exploration was significantly reduced, while individuals with inosine treatment showed a significant increase in their exploration.

[0092] 3. If Figure 6 As shown in the figure, tissue extract detection showed that inosine effectively increased the 5-HT content in the mouse hippocampus, and the data were expressed as mean ± standard deviation (n = 6 / group). Figure 7 As shown in the figure, tissue extract detection showed that inosine effectively increased the GABA content in the mouse hippocampus, and the data were expressed as mean ± standard deviation (n = 6 / group). Figure 8 As shown in the figure, tissue extract detection showed that inosine effectively increased the 5-HT content in the mouse cerebral cortex, and the data were expressed as mean ± standard deviation (n = 6 / group). Fig. 9 As shown in the figure, tissue extract detection showed that inosine effectively increased the GABA content in the mouse cerebral cortex, and the data were expressed as mean ± standard deviation (n = 6 / group). Fig.10 As shown, tissue extract detection showed that inosine effectively increased the DA content in mouse serum, and the data are expressed as mean ± standard deviation (n = 6 mice / group).

[0093] 4. If Fig.11 As shown in the figure, RT-qPCR showed that inosine effectively reduced the Caspase-1 content in mice, and the data were expressed as mean ± standard deviation (n = 6 mice / group). Fig.12 As shown in the figure, RT-qPCR showed that inosine effectively reduced the IL-18 content in mice, and the data were expressed as mean ± standard deviation (n = 6 mice / group). Fig.13As shown, RT-qPCR showed that inosine effectively reduced the NLPR3 content in mice, and the data were expressed as mean ± standard deviation (n = 6 mice / group).

[0094] 5. If Fig.14 As shown, microglial morphological analysis showed that inosine could significantly increase the complexity of microglial morphology, and all indicators were significantly improved. Neurons were activated after inosine treatment. The data are expressed as mean ± standard deviation (n = 6 / group).

[0095] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0096] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of inosine in preparing a drug for preventing and / or treating anxiety disorders.

2. The use according to claim 1, characterized in that: The anxiety disorder is generalized anxiety disorder.

3. The use according to claim 1, characterized in that: The drug also includes pharmaceutically acceptable excipients.

4. The use according to claim 1, characterized in that: The dosage form of the drug includes an injection preparation or an oral preparation.

5. The use according to claim 4, characterized in that: The oral preparations include tablets, capsules, powders, drop pills or oral liquid preparations.

6. The use according to claim 4, characterized in that: When the dosage form of the drug is an injection preparation, the dosage of the drug is 250-350 mg per kilogram of body weight.

7. A drug for preventing and / or treating anxiety disorders, characterized in that: The medicine comprises active pharmaceutical ingredients and excipients, and the active pharmaceutical ingredient is inosine.

8. The drug according to claim 7, characterized in that The auxiliary materials include at least one of excipients, lubricants, antioxidants, preservatives, adhesives, fillers, or thickeners.

9. The drug according to claim 7, characterized in that The anxiety disorder is generalized anxiety disorder.

10. The drug according to claim 7, characterized in that The drug upregulates 5-HT, GABA and serum dopamine in the hippocampus and cortex, inhibits three inflammatory factors, NLRP3, CaspaseI and IL-18, inhibits the NLRP3 / CaspaseI / IL-18 inflammatory pathway, increases the complexity of microglial morphology, activates neurons, and achieves the effect of reversing anxiety behavior.

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