Use of nadph in preparation of drug for treating major depression, bipolar disorder, post-stroke depression or neuroinflammation

Abstract

Use of NADPH in the preparation of a drug for treating major depression, bipolar disorder, post-stroke depression or neuroinflammation, and use of NADPH or a pharmaceutically acceptable salt thereof in the preparation of a P2X7 receptor antagonist. Research finds that NADPH inhibits the rise of an inward current in microglia caused by ATP-induced calcium influx and P2X7 receptor activation. It is indicated that NADPH can inhibit ATP-induced P2X7R activation and inflammasome activation and it is shown that NADPH can be used as a P2X7 receptor inhibitor, and can be further used for preventing or treating diseases caused by P2X7 receptor activation and inflammasome activation, wherein these diseases comprise major depression, bipolar disorder, post-stroke depression or neuroinflammation-related diseases.

Description

Use of NADPH in the preparation of drugs for treating major depression, bipolar depression, post-stroke depression or neuroinflammation

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application filed with the China Patent Office on October 12, 2023, with application number 202311321959.0, and invention name “USE OF NADPH IN THE PREPARATION OF DRUGS FOR TREATING MAJOR DEPRESSION, BIDIRECTIONAL DEPRESSION, POST-STROKE DEPRESSION OR NEUROINFLAMMATORY”, the entire contents of which are incorporated herein by reference; this application also claims priority to the Chinese patent application filed with the China Patent Office on April 26, 2024, with application number 202410514820.6, and invention name “USE OF NADPH IN THE PREPARATION OF DRUGS FOR TREATING MAJOR DEPRESSION, BIDIRECTIONAL DEPRESSION, POST-STROKE DEPRESSION OR NEUROINFLAMMATORY”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application relates to the field of medical technology, and specifically to the use of NADPH in the preparation of drugs for treating major depression, bipolar depression, post-stroke depression or neuroinflammation-related Alzheimer's disease, experimental allergic encephalomyelitis or multiple sclerosis. Background Art

[0004] Depression is the most common mental illness today, characterized by a persistent and long-term low mood. It is the most important type of mental illness in modern people. There are many types of depression, clinically classified as major depression, bipolar depression, psychotic depression, reactive depression, menopausal depression, secondary depression, postpartum depression, seasonal depression, and post-stroke depression.

[0005] Major depressive disorder (MDD) is a common chronic illness affecting approximately 4.5% of the global population. It is typically characterized by extreme sadness, depressed mood, and loss of interest. In addition to these nonspecific symptoms, anhedonia makes it a leading cause of disability worldwide. Evidence from recent decades suggests that the neurobiology of depression extends beyond the catecholamine hypothesis. Current hypotheses suggest that MDD is caused by an interplay between genetic and environmental factors, leading to neuroendocrine dysregulation, neurochemical alterations (including impaired monoaminergic neurotransmission and enhanced glutamate release), neuroimmune responses, and decreased neuroplasticity (e.g., synaptogenesis and neurogenesis). Clinically available antidepressants primarily target monoaminergic signaling, particularly by inhibiting the reuptake of serotonin (5-HT) and norepinephrine (NA). However, these drugs are associated with significant potential side effects, with patients often experiencing weight gain, nausea, headaches, and sexual dysfunction. Therefore, the development of novel molecular targets to guide the development of more effective therapeutics is urgently needed.

[0006] Bipolar disorder, also known as bipolar affective disorder, is one of the most challenging psychiatric disorders. Bipolar disorder is characterized by recurrent episodes of elevated and depressed mood, accompanied by changes in activity or energy, and is associated with characteristic cognitive, physical, and behavioral symptoms. BD is a highly heritable disorder. Genomic data on BD-associated genetic factors have identified a series of molecules involved in calcium signaling. Current research focuses on VGCC genes, particularly L-type VGCC and its accessory subunits (encoded by CACNx). Pathway analysis has shown that CACNA1D and CACNB3, along with other VGCC genes, are highly associated with BD. Furthermore, altered calcium signaling has been found in neurons derived from induced pluripotent stem (iPSC) cells (iPSCs) with BD-associated phenotypes, suggesting that BD may be a channelopathy. The L-type voltage-gated calcium channel (VGCC) antagonist pregabalin is being evaluated for the treatment of BD. Lamotrigine, another antiepileptic drug that may block calcium channels, is also an effective treatment for BD. In summary, the development of effective calcium channel blockers may be a promising approach to combating BD. Post-stroke depression (PSD), also known as post-stroke depression, is a common mental health problem affecting approximately 33% of stroke survivors. Patients present with a range of affective disorders beyond stroke symptoms, characterized by low mood and loss of interest, often accompanied by physical symptoms. Severe cases may develop suicidal thoughts, and if not promptly addressed, some patients may end up committing suicide. The etiology of PSD remains unclear, but it is believed to be multifactorial, encompassing both biological and psychosocial factors. Numerous other biological factors are also thought to be associated with PSD-like inflammation, responses to ischemia, genetic susceptibility, neurogenesis, and activation of the hypothalamic-pituitary-adrenal (HPA) axis. Evidence suggests that cytokines play a crucial role in neuroinflammation and neurodegeneration, and are also involved in the pathogenesis of PSD. Inhibiting cytokine production may help improve PSD-related symptoms.

[0007] The P2X7 receptor (P2X7R) is an ATP-gated ion channel receptor expressed primarily in blood and brain immune cells. Substantial evidence suggests that the P2X7R plays a key role in the pathogenesis of depression. P2X7R knockout mice exhibit increased hippocampal 5-HT levels, increased 5-HT transporter binding sites and 5-HT uptake, and behavioral adaptations to stress. Stimulation of P2X7R in hippocampal nerve terminals induces glutamate release and leads to kainate receptor-mediated GABA efflux from interneurons. However, hippocampal slices from P2X7R knockout mice lack glutamate and GABA release induced by P2X7R activation. P2X7R activation reduces expression of the glutamate / sodium aspartate-dependent transporter (GLAST), a mechanism directly involved in glial clearance of glutamate. These findings consistently suggest that the P2X7R may regulate depression by modulating the uptake and release of various neurotransmitters. Furthermore, P2X7R has long been linked to inflammation and immunity, and the discovery of the NLRP3 inflammasome ultimately placed P2X7R at the heart of many neuropsychiatric diseases. To date, P2X7R is one of the most potent activation pathways of the NLRP3 inflammasome. NLRP3 inflammasome assembly activates Caspase-1, which cleaves the IL-1β protein precursor into a mature form, which is released extracellularly and triggers neuroinflammation. This signaling pathway is now believed to play a crucial role in disorders such as depression, including MDD (Figure 5), BD, PSD, Alzheimer's disease, Parkinson's disease, experimental allergic encephalomyelitis, and multiple sclerosis. More importantly, studies have found that the P2X7 gene is located in a region on chromosome 12q24.31, which has been identified as a susceptibility locus for affective disorders through linkage and association studies. A single nucleotide polymorphism (SNP) substitution at codon 460 of P2X7, from glutamine (Gln, Q) to arginine (Arg, R), has been found to be associated with BD and significantly associated with MDD (P = 0.0019). Mice carrying this variant exhibit altered sleep quality, resembling signs of the prodromal stage of MDD. Furthermore, healthy heterozygous human subjects also exhibit mild changes in sleep parameters. These results suggest that heterozygosity for wild-type P2X7 and its mood disorder-associated variant, P2X7Gln460Arg, represents a genetic risk factor that reflects susceptibility to mood disorders. Therefore, targeted inhibition of P2X7R may represent an approach to finding more effective antidepressants in clinical practice. P2X7R antagonists have been under development for many years, and several candidate compounds have been identified. However, due to the large number of P2XR isoforms and the problem of blood-brain barrier permeability, effective and specific P2X7R antagonists are currently lacking.

[0008] Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is a key component of the cellular antioxidant system and an essential electron donor for many antioxidant enzymes and biosynthetic reactions, such as nucleotide synthesis, lipid synthesis, and fatty acid chain elongation. However, there are currently no reports on NADPH antagonism of P2X7R and its use as an antidepressant drug. Summary of the Invention

[0009] Therefore, the technical problem to be solved by the present application is to provide a use of NADPH in the preparation of drugs for treating major depression, bipolar depression, post-stroke depression or neuroinflammation, Alzheimer's disease, experimental allergic encephalomyelitis or multiple sclerosis.

[0010] To this end, this application provides the following technical solutions:

[0011] Use of NADPH or a pharmaceutically acceptable salt thereof in the preparation of a P2X7 receptor antagonist.

[0012] Optionally, the P2X7 receptor antagonist is used to prevent or treat diseases associated with P2X7 receptor activation and inflammasome activation.

[0013] Optionally, the disease includes major depression, bipolar disorder, post-stroke depression, neuroinflammation, Alzheimer's disease, experimental allergic encephalomyelitis or multiple sclerosis.

[0014] Use of NADPH or a pharmaceutically acceptable salt thereof in preparing a product for alleviating or treating major depression, bipolar depression, post-stroke depression or neuroinflammation.

[0015] Optionally, the antagonist or the product comprises an effective amount of NADPH or a derivative thereof or a pharmaceutically acceptable salt thereof, with or without the addition of a pharmaceutically acceptable excipient;

[0016] And / or, the product includes medicine, food or health care product.

[0017] Optionally, the antagonist or the product uses NADPH or its derivatives or pharmaceutically acceptable salts thereof as an active ingredient, and conventional excipients are added to prepare a clinically acceptable solid preparation, semisolid preparation or liquid preparation.

[0018] Optionally, the dosage form of the antagonist or the product includes tablets, capsules, powders, mixtures, pills, granules, syrups, suppositories, aerosols, dry powder inhalers, ointments or injections;

[0019] Optionally, the dosage form includes a nanoformulation or a liposome preparation.

[0020] Optionally, the antagonist or the drug is administered by at least one of oral administration, injection, sublingual administration, nasal administration, rectal administration, transdermal administration, and pulmonary inhalation.

[0021] A pharmaceutical composition for preventing, alleviating or treating major depression, bipolar depression, post-stroke depression or neuroinflammation, comprising NADPH or its derivatives or pharmaceutically acceptable salts as the active ingredient, and conventional excipients to prepare the composition into a clinically acceptable solid preparation, semisolid preparation or liquid preparation.

[0022] Optionally, the dosage form of the pharmaceutical composition includes tablets, capsules, powders, mixtures, pills, granules, syrups, suppositories, aerosols, drops, dry powders, ointments or injections.

[0023] A method for preventing or treating diseases caused by P2X7 receptor activation and inflammasome activation, characterized by administering NADPH or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0024] Optionally, the disease includes major depression, bipolar disorder, post-stroke depression, neuroinflammation-related Alzheimer's disease, experimental allergic encephalomyelitis or multiple sclerosis.

[0025] Optionally, the administration subjects include humans and animals other than humans.

[0026] Optionally, the administration dosage of NADPH is 4 mg / kg-8 mg / kg.

[0027] Optionally, when the subject of administration is a human, the dosage of NADPH is 0.4 mg / kg-0.9 mg / kg.

[0028] Optionally, when the subject of administration is a human, the dosage of NADPH is 0.44 mg / kg-0.88 mg / kg.

[0029] The technical solution of this application has the following advantages:

[0030] 1. Use of NADPH or its derivatives or pharmaceutically acceptable salts provided herein in the preparation of P2X7 receptor antagonists. Studies in this application have found that NADPH inhibits ATP-induced calcium influx and the increase in inward current in microglia caused by P2X7 receptor activation, indicating that NADPH can inhibit ATP-induced P2X7R activation. NADPH can be used as a P2X7 receptor antagonist to inhibit inflammasome activation, and further, NADPH can be used to prepare drugs for the prevention or treatment of diseases caused by P2X7 receptor activation.

[0031] 2. The use of NADPH or its derivatives or pharmaceutically acceptable salts provided in the present application in the preparation of products for preventing, alleviating or treating major depression, bipolar depression, post-stroke depression, neuroinflammation, Alzheimer's disease, experimental allergic encephalomyelitis, and multiple sclerosis; the research of the present application found that NADPH can significantly improve major depression, bipolar depression, post-stroke depression or neuroinflammation, and thus NADPH can be used to prepare products for preventing, alleviating or treating major depression, bipolar depression, post-stroke depression or neuroinflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific implementation methods or the description of the prior art. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0033] FIG1 is the result of the effect of exogenous NADPH on ATP-induced calcium influx in Experimental Example 1 of the present application;

[0034] Figure 2 shows the effect of reduced endogenous NADPH content on ATP-induced P2X7R activity in Experimental Example 2 of the present application; Figure A shows the knockdown of G6PD in the si-G6PD group; Figure B shows the expression of G6PD in the si-G6PD group and the si-Con group; Figure C shows the relative fluorescence intensity of Inap1; Figure D shows the relative intracellular calcium ion fluorescence intensity; Figure E shows the bar graphs of the si-Con and si-G6PD groups, from left to right, corresponding to Vehicle, ATP, and NADPH;

[0035] FIG3 shows the effect of NADPH on ATP-induced inflammasome activation in Experimental Example 3 of the present application;

[0036] FIG4 is an investigation result of the therapeutic effect of NADPH on major depression in Experimental Example 4 of the present application;

[0037] FIG5 is a schematic diagram of the present invention showing that activation of the P2X7 receptor leads to major depression, bipolar depression, post-stroke depression, and neuroinflammation;

[0038] FIG6 is the result of the tail suspension experiment in investigating the therapeutic effect of NADPH on post-stroke depression in Experimental Example 6 of the present application;

[0039] FIG7 is the result of the sugar water preference experiment in investigating the therapeutic effect of NADPH on post-stroke depression in Experimental Example 6 of the present application;

[0040] Figure 8 shows the results of the forced swimming test in Experimental Example 6 of the present application to investigate the therapeutic effect of NADPH on post-stroke depression; Figure A shows the results of the total swimming distance; Figure B shows the results of the average swimming speed; and Figure C shows the results of the stationary time. DETAILED DESCRIPTION

[0041] The following examples are provided to further better understand the present application, but are not limited to the best implementation mode described herein, and do not limit the content and protection scope of the present application. Any product identical or similar to the present application obtained by anyone under the inspiration of the present application or by combining the features of the present application with other prior arts shall fall within the scope of protection of the present application.

[0042] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0043] Example 1 P2X7R Antagonists

[0044] This embodiment provides a P2X7R antagonist, which uses NADPH as the active ingredient, is added with conventional excipients, and is prepared according to conventional processes into clinically acceptable tablets, capsules, powders, mixtures, pills, granules, syrups, suppositories, aerosols, ointments or injections.

[0045] Example 2 Pharmaceutical Composition

[0046] This embodiment provides a pharmaceutical composition for preventing, alleviating or treating major depression, bipolar disorder, post-stroke depression or neuroinflammation, which uses NADPH as the active ingredient, is added with conventional excipients, and is prepared according to conventional processes into clinically acceptable tablets, capsules, powders, mixtures, pills, granules, syrups, suppositories, aerosols, ointments or injections.

[0047] Experimental Example 1 Antagonistic effect of NADPH on P2X7R

[0048] Calcium influx is one of the responses to ATP-activated microglia. This experiment explores the effects of exogenous NADPH on ATP-induced calcium influx. Fluo-3AM, a fluorescent calcium probe, will be used to monitor changes in calcium within microglia. The differential calcium response to ATP stimulation in the control and NADPH groups will be examined, preliminarily revealing whether NADPH is a P2X7R modulator / antagonist. The steps are as follows:

[0049] (1) Primary microglial cell culture: A routine method for microglial cell isolation and culture was established. Take C57BL / 6J mice that are 1-3 days old, mince the midbrain tissue, and digest it with 0.125% trypsin at 37°C for 10 minutes. After using complete culture medium to terminate the digestion, filter it through a 40μm cell mesh. Centrifuge at 1000rpm for 10 minutes at room temperature to collect the cells and inoculate them into cell culture flasks. After 3 days, replace the culture medium with fresh one and continue culturing. After 7 days, place the cells on a shaker at 220rpm and shake for 2 hours to obtain microglial cells.

[0050] (2) Experimental groups and methods

[0051] The microglial cells in step (1) were divided into four groups, namely, CON group, ATP group, ATP+NADPH group and NADPH group, with n=12. Primary microglial cells were evenly seeded in 96-well plates and placed in an incubator for more than 8 hours before subsequent experiments. The original culture medium in the 96-well plate was discarded, and Fluo-3AM (commercially available) was diluted to a working concentration of 5μM using phenol red-free DMEM. 100μL of the working solution was added to each well of the 96-well plate and placed in a cell incubator for incubation for 30 minutes for fluorescent probe loading, followed by washing three times with PBS. 100μL of phenol red-free DMEM was added to the control group, 100μL of 1mM ATP was added to the ATP group, and 100μL of a mixture of 1mM NADPH and 1mM ATP was added to the NADPH+ATP group. Finally, the intracellular calcium ion concentration was immediately measured using a microplate reader.

[0052] Intracellular calcium concentration measurement: Cells were incubated with a DMEM solution containing 5 μM Fluo-3 AM (a commercially available calcium fluorescent probe) in a 37°C cell culture incubator for 30 minutes to load the fluorescent probe. The cells were then washed three times with PBS and incubated for an additional 20-30 minutes to ensure complete conversion of Fluo-3 AM to Fluo-3 within the cells. Fluorescence was measured using a multifunctional microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm to reflect changes in intracellular calcium concentration.

[0053] Intracellular NADPH Content Detection: This study introduced the recently garnered genetically encoded fluorescent probe iNap (a gift from Professor Zhao Yuzheng of East China University of Science and Technology) to detect intracellular NADPH. iNap specifically binds to NADPH and is unaffected by other nucleotides and coenzymes. The probe was transfected into cells. After 24 hours, cells stably expressing iNap bound to NADPH. Fluorescence intensity was measured using a multifunctional microplate reader at excitation wavelengths of 420 nm / 485 nm and emission wavelengths of 528 nm, or using a fluorescence microscope at excitation wavelengths of 488 nm to reflect intracellular NADPH content.

[0054] Electrophysiological measurements: 24 h before the assay, cells were plated onto 13 mm glass coverslips. Membrane currents were recorded by whole-cell patch clamp at room temperature using an Axopatch 200B amplifier (Molecular Devices) and borosilicate glass electrodes (TF-150 World Precision Instruments). Pipettes (resistance range 7 to 10 MΩ) were filled with an intracellular solution containing 120 mM KCl, 30 mM NaCl, 0.5 mM CaCl2, 2 mM Mg-ATP, 10 mM Hepes, and 5 mM EGTA (pH 7.2). Cells were voltage-clamped at −60 mV. The extracellular recording solution contained 150 mM NaCl, 5 mM KCl, 10 mM glucose, 10 mM Hepes, 2 mM CaCl2, and 1 mM MgCl2 (pH 7.4). All drugs to be tested were dissolved in the extracellular recording solution and the cells were continuously perfused. The perfusion capillary was placed close to the cells to be studied.

[0055] Data statistics: One-way ANOVA, post hoc analysis by Tukey, *P<0.05, ***P<0.001.

[0056] (3) Test results

[0057] The results are shown in Figure 1A and B. Whole-cell patch clamp recordings showed that ATP-induced an increase in the inward current of microglial cells, while after NADPH treatment, the inward current of microglial cells decreased by about 20% compared with the ATP group, which is similar to the effect produced by the P2X7R antagonist A438079, preliminarily suggesting that NADPH inhibits ATP-induced P2X7R receptor activation.

[0058] The results are shown in Figure 1C. Using cell Ca 2+ Fluorescent probe Fluo-3AM was used to detect the effect of NADPH on microglial Ca2+ 2+ The results showed that ATP stimulation led to an increase in intracellular Ca 2+ The flux increased and the fluorescence intensity increased, while NADPH inhibited the Ca2+ induction induced by P2X7R activation. 2+ Increased inflow.

[0059] The results are shown in Figure 1D. Different concentrations of NADPH (0, 0.01, 0.1, 0.2, 0.5, 1, and 5 mM) were used to treat microglia after ATP stimulation to observe their Ca 2+ The results showed that compared with the ATP-only treatment group, the intracellular Ca2+ The flow rate did not change, but after the administration of 0.2, 0.5, 1 and 5 mM NADPH, the intracellular Ca 2+ The traffic volume shows a gradually decreasing trend.

[0060] These results consistently indicate that exogenous NADPH inhibits ATP-induced P2X7R activation.

[0061] Experimental Example 2: Decreased endogenous NADPH content leads to increased ATP-induced P2X7R activity

[0062] In Experimental Example 1, it was found that exogenous NADPH inhibited P2X7R function. In this experimental example, we further evaluated whether endogenous NADPH could have a similar effect on P2X7R function. G6PD (glucose-6-phosphate dehydrogenase) is the main synthase of NADPH in microglia, accounting for approximately 50%-60% of the total NADPH synthesis. In order to reduce the intracellular NADPH content, this experiment used small interfering RNA to silence the intracellular G6PD gene and then detected changes in ATP-induced microglial calcium influx. The steps are as follows:

[0063] (1) Primary microglial cell culture: Same as Experimental Example 1.

[0064] (2) Experimental groups and methods

[0065] For A, B, and C in Figure 2, the microglia in step (1) were evenly divided into two groups, namely the control knockdown group (si-Con) and the G6PD knockdown group (si-G6PD). One day before the knockdown, BV2 cells were evenly seeded in a 12-well plate to an initial density of 30%-40%, and the knockdown experiment was performed after culturing in the well plate for more than 8 hours. Prepare two centrifuge tubes, mix si-Con and si-G6PD with the transfection reagent according to the instructions of the transfection reagent (jetPRIME, Polyplus), shake and mix, and incubate at room temperature for 10 minutes. Wash the cells that were plated in advance with preheated PBS, and add 1 mL of complete culture medium to each well. Add the system after incubation to the well plate, mix gently, and place in a 37°C incubator for culture. After culturing for 4-6 hours, the complete culture medium can be replaced according to the cell state, and a series of subsequent experiments can be performed after 48 hours of knockdown. Figure 2 D divides BV2 cells into four groups, namely si-Con + Vehicle group, si-Con + ATP group, si-G6PD + Vehicle group and si-G6PD + ATP group, n = 6, and a knockdown experiment is performed with reference to Figure 2 A, and Fluo-3AM is added for calcium ion probe loading 48 hours after the knockdown, and then ATP (the amount added is the same as in Experimental Example 1) is added for intracellular calcium ion determination. Figure 2 E divides cells into 6 groups, namely si-Con + Vehicle group, si-Con + ATP group, si-Con + ATP + NADPH group, si-G6PD + Vehicle group, si-G6PD + ATP group and si-G6PD + ATP + NADPH group, and the amount of ATP and / or NADPH added to each group is the same as in Experimental Example 1, n = 5, and a knockdown experiment is performed with reference to Figure 2 A, and Fluo-3AM is added for calcium ion probe loading 48 hours after the knockdown, and then ATP (NADPH) is added for intracellular calcium ion determination.

[0066] Determination of intracellular calcium ion concentration: Same as Experimental Example 1.

[0067] Intracellular NADPH Content Detection: This study introduced the recently garnered genetically encoded fluorescent probe iNap (a gift from Professor Zhao Yuzheng of East China University of Science and Technology) to detect intracellular NADPH. iNap specifically binds to NADPH and is unaffected by other nucleotides and coenzymes. The probe was transfected into cells. After 24 hours, cells stably expressing iNap bound to NADPH. Fluorescence intensity was measured using a multifunctional microplate reader at excitation wavelengths of 420 nm / 485 nm and emission wavelengths of 528 nm, or using a fluorescence microscope at excitation wavelengths of 488 nm to reflect intracellular NADPH content.

[0068] Data statistics: Two-way ANOVA, post hoc analysis by Tukey, ***P<0.001.

[0069] (3) Test results

[0070] The results are shown in Figure 2A and B. Compared with si-Con, the G6PD protein level in the si-G6PD group was reduced by about 77%, indicating that G6PD knockdown was successful.

[0071] The results are shown in Figure 2C. Using the intracellular NADPH probe iNap, it was detected that the intracellular NADPH content in the G6PD knockdown group was significantly reduced compared with the control knockdown group.

[0072] The results are shown in Figure 2D. It was found that ATP treatment led to the 2+ The influx of Ca increased, but compared with the si-Con+ATP group, the Ca influx in the si-G6PD+ATP group was 2+ The influx was further enhanced, and this phenomenon was improved after exogenous NADPH was given (Figure 2E). The above results indicate that the reduction of endogenous NADPH content will lead to the enhancement of ATP-induced P2X7R activation, and conversely, NADPH can inhibit P2X7R activation.

[0073] Experimental Example 3 Effect of NADPH on ATP-induced inflammasome activation

[0074] The connection between P2X7R and inflammation and immunity has a long history. The discovery of the NLRP3 inflammasome finally placed P2X7R in the appropriate environment. The activation of the NLRP3 inflammasome requires two signaling processes. First, pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) promote the transcription of genes encoding IL-1β and inflammasome components such as NLRP3, thereby initiating inflammatory signals. The second signal is triggered by ATP, viral RNA, and particulate matter to activate the NLRP3 inflammasome. Among them, the downstream signal triggered by ATP activation of P2X7R is considered to be the key mechanism of NLRP3 inflammasome activation. This experiment explored whether NADPH affects P2X7R-mediated inflammatory signal transduction by studying the effect of NADPH on ATP-induced LPS-pretreated microglial inflammasome activation. The steps are as follows:

[0075] (1) Primary microglial cell culture: Same as Experimental Example 1.

[0076] (2) Construction of in vitro neuroinflammation model: The microglial cells in step (1) were pretreated with LPS (500 ng / ml) for 3 hours, the culture medium was replaced overnight, and 2.5 mM ATP was added for 30 minutes. After the cell treatment, the old culture medium was discarded, the cells were washed twice with PBS, and trypsin was added for digestion for 1-2 minutes, and then complete culture medium was added to terminate the reaction. All cells were blown up from the bottom of the culture dish and placed in a centrifuge tube. Centrifuged at 3000 rpm for 5 minutes, and the liquid was discarded to keep the cells. According to the amount of cell pellet, an appropriate amount of cell lysis buffer RIPA was added, and then the cell pellet was blown away with a pipette. The actual protein concentration of each sample was measured by the BCA method, and the cell lysis buffer was added to level each sample according to the sample volume and concentration. The sample loading buffer was added, mixed evenly, and placed in a 95°C metal bath and heated for 10 minutes to prepare the cell lysate loading buffer. To prepare the cell supernatant sample, first collect the cell supernatant into a centrifuge tube. Add an equal volume of methanol and 1 / 4 the volume of chloroform. Vortex rapidly and vigorously, and centrifuge at 15,000 rpm at 4°C for 5 minutes. Discard the supernatant, taking care not to aspirate the intermediate protein layer. Add 500 μL of methanol to the centrifuge tube, vortex rapidly to mix, and centrifuge at 15,000 rpm at 4°C for 5 minutes. Discard the supernatant, taking care not to aspirate any proteins adhered to the tube walls. Place the tube in a 37°C water bath to evaporate the methanol, which takes approximately 5-10 minutes. Finally, add a volume of cell lysate, pipette to disperse the proteins, add 5x loading buffer, vortex rapidly, and centrifuge. Heat the tube in a 95°C metal bath for 10 minutes to prepare the cell supernatant loading buffer. Preparation steps of cell supernatant and cell lysate: Collect cell supernatant and cell lysate and use Western blot and ELISA technology to detect inflammation-related indicators ((pro)IL-1β, (pro)Caspase-1, ASC). Combine immunofluorescence staining with stereology technology to evaluate microglial activation and determine whether the model is successful.

[0077] (3) Experimental groups and methods

[0078] The model was constructed according to step (2), and the primary microglial cells in step (1) were evenly seeded into 12-well plates and divided into four groups, namely, CON group, ATP group, ATP+NADPH group, and NADPH group. To construct a classic cellular NLRP3 inflammasome activation model, primary microglia were first pretreated with LPS (500 ng / mL) for 3 h, and the culture medium was replaced with fresh medium overnight. The CON group did not add any LPS, the ATP group was treated with ATP (2.5 mM) for 30 min, and the ATP+NADPH group was pretreated with NADPH (1 mM) for 30 min. Subsequently, the cells were treated with ATP (2.5 mM) for 30 min, and the NADPH group was pretreated with NADPH (1 mM) for 30 min. Western blotting was used to detect changes in the expression of NLRP3 inflammasome-related proteins (pro)IL-1β, (pro)Caspase-1, ASC, and NLRP3. ELISA was used to detect the content of mature IL-1β in the cell supernatant, and a Caspase-1 activity detection kit was used to detect Caspase-1 activity in cell lysates. (n=4-6).

[0079] Caspase-1 activity assay: Pro-Caspase-1 and Caspase-1 protein expression was detected by conventional immunoblotting. Caspase-1 activity was directly detected using a Caspase-1 activity assay kit (Biovision) to corroborate the immunoblotting results. The general procedure included: After the experimental treatment, cells were digested and centrifuged at 1000 g for 5 minutes, and the supernatant was aspirated. The cells were lysed with vortexing lysis buffer, incubated on ice for 10 minutes, and then vortexed again. The cells were centrifuged at 4°C (12,000 g for 10 minutes), and the supernatant was collected and absorbance was measured at 405 nm to determine caspase-1 activity.

[0080] Data statistics: One-way ANOVA, post hoc analysis by Tukey, *P<0.05, **P<0.01, ***P<0.001.

[0081] (4) Test results

[0082] The results are shown in Figure 3A to E. Primary microglia were pretreated with LPS (500 ng / ml) for 3 hours, then LPS was discarded and replaced with fresh culture medium overnight. Finally, 2.5 mM ATP was added for 30 minutes to induce NLRP3 inflammasome activation. Significant expression of mature IL-1β was detected in the cell supernatant, and IL-1β, Caspase-1, ASC, and NLRP3 protein expression was increased in the cell lysate. Before ATP treatment, cells were pretreated with NADPH (1 mM) for 30 minutes. The results showed that compared with the ATP group, the expression of NLRP3, IL-1β, Caspase-1, and ASC proteins in the NADPH+ATP group was significantly reduced.

[0083] The results are shown in Figure 3F. The IL-1β content in the supernatant was detected by ELISA, and the results also showed that NADPH significantly inhibited the maturation and secretion of IL-1β induced by ATP stimulation.

[0084] The results are shown in Figure 3G. The activity of Caspase-1 in cell lysates was detected. The results showed that compared with the untreated group, the activity of Caspase-1 was significantly increased after ATP stimulation, but the addition of NADPH inhibited the activity of Caspase-1, further indicating that exogenous NADPH inhibits the activation of NLRP3 inflammasome.

[0085] Experimental Example 4: Investigation of the therapeutic effect of NADPH on major depression

[0086] This study explores the effects of exogenous NADPH on the behavioral responses of rats in a major depressive disorder (MDD) model induced by chronic unpredictable stress (CUS). This model will be used to induce MDD in rats. Behavioral changes in sugar water preference and the forced swim test will be examined to preliminarily reveal the therapeutic effects of NADPH on this model. The steps are as follows:

[0087] CUS is one of the most well-documented and widely used animal models of depression. The model was constructed based on references and modified to suit actual experimental conditions. The final model was constructed using the following table. To create an unpredictable stressor, the same stressor was not repeated within two days. The details are as follows:

[0088] Table 1

[0089] (2) Experimental groups and methods

[0090] Three-month-old healthy male Sprague-Dawley rats were randomly divided into five groups based on body weight after one week of adaptive feeding: control (CON); chronic unpredictable stress model group (CUS); chronic unpredictable stress + fluoxetine group (CUS+F); chronic unpredictable stress + NADPH (4 mg / kg) group (CUS+N4); and chronic unpredictable stress + NADPH (8 mg / kg) group (CUS+N8). Rats in the CUS+F group were orally administered 5 mg / kg fluoxetine at 9:00 AM daily for 8 consecutive weeks. Rats in the CUS+N4 and CUS+N8 groups were intraperitoneally injected with 4 mg / kg and 8 mg / kg NADPH, respectively, at 9:00 AM daily. After 8 weeks of administration, the rats in each group underwent sucrose preference and forced swim tests to examine the behavioral changes of NADPH in the rat depression model.

[0091] Sucrose preference test (SPT): The SPT was conducted on the third day after 8 weeks of drug administration. Prior to testing, animals were allowed to acclimate for 24 hours to two bottles of 1% sucrose water (bottles of identical appearance, symmetrically placed relative to the cage). One of the bottles of sucrose water was then replaced with regular drinking water for another 24 hours, with the two bottles swapped once (to minimize the influence of positional preference). Following a 21-hour food and water deprivation period, sucrose water and pure water consumption were measured within one hour in each cage. Consumption was determined by weighing the sucrose and pure water bottles before and after each. The formula for calculation was: Percent sucrose preference = 1% sucrose water consumption / (1% sucrose water consumption + pure water consumption) × 100%.

[0092] Forced swimming test (FST): The FST was conducted on the sixth day after eight weeks of drug administration. After acclimating to the experimental room for at least one hour, the rats were placed in a transparent glass beaker with water at 25°C (50 cm high, 22 cm inner diameter, and 35 cm deep) for a swim. The test consisted of two parts: a 15-minute acclimation swim on the first day; and 24 hours later, each animal was placed back in the beaker for a 5-minute swim. The time to the first episode of immobility and the total time of immobility within the 5-minute period were recorded.

[0093] Data statistics: One-way ANOVA, post hoc analysis by Tukey, *P<0.05, **P<0.01, ***P<0.001.

[0094] Test results

[0095] The results are shown in Figure 4A. Anhedonia is one of the main symptoms of depression, and the SPT is a commonly used paradigm for detecting anhedonia in rat models of depression. The results showed that the CUS group had a significantly lower percentage preference for sucrose than the CON group. Compared with the CUS group, a 4 mg / kg dose of NADPH significantly increased the percentage preference for sucrose. Fluoxetine and an 8 mg / kg dose of NADPH also showed a nonsignificant trend toward an increase in the percentage preference for sucrose.

[0096] The results are shown in Figure 4B. The FST results showed that while CUS did not significantly increase the immobility time of rats, it significantly decreased their immobility latency. Compared with the CUS group, fluoxetine significantly decreased the immobility time and increased the immobility latency of rats. NADPH (4 mg / kg) only reversed the CUS-induced increase in immobility time, but NADPH (8 mg / kg) reversed both the CUS-induced increase in immobility time and decrease in immobility latency.

[0097] In summary, NADPH has an improving effect on CUS-induced anhedonia and decreased desire to survive in desperate situations, and can achieve similar anti-major depression effects as the positive control antidepressant drug fluoxetine.

[0098] Experimental Example 5: Investigation of the therapeutic effect of NADPH on bipolar depression

[0099] This example explores the behavioral effects of exogenous NADPH on a mouse model of bipolar depression. A bipolar depression model will be constructed using lipopolysaccharide (LPS). Behavioral changes in the mice's sugar preference, tail suspension test, and forced swim test will be assessed to reveal the therapeutic effects of NADPH on the bipolar depression model. The steps are as follows:

[0100] (1) Construction of bipolar depression model: A bipolar depression model was established by intraperitoneally injecting LPS (2 mg / kg) into C57BL / 6J male mice aged about 6-8 weeks for 5 consecutive days.

[0101] (2) Experimental Grouping and Methods: Mice were divided into three groups: saline group, LPS group, and NADPH+LPS group, with 6 mice in each group. The saline group received a conventional injection of saline, the LPS group received an intraperitoneal injection of 2 mg / kg LPS daily for 5 consecutive days, and the NADPH+LPS group received an intraperitoneal injection of NADPH (5 mg / kg) for 14 consecutive days. The mice in each group were then tested for sugar preference, tail suspension test, and forced swim test to determine the behavioral differences between the groups, thereby preliminarily determining the therapeutic effect of NADPH on the bipolar depression model.

[0102] Experimental Example 6: Investigation of the therapeutic effect of NADPH on post-stroke depression

[0103] This study investigates the effects of exogenous NADPH on the behavioral responses of mice with post-stroke depression. A transient middle cerebral artery occlusion (tMCAO) / reperfusion model will be established to induce depressive-like behaviors in mice. Behavioral changes in sucrose preference, tail suspension, and forced swim tests will be assessed to initially reveal the therapeutic effects of NADPH on this model of post-stroke depression. The steps are as follows:

[0104] (1) Construction of t-MCAO / R model: The mouse cerebral ischemia-reperfusion model was prepared by internal carotid artery occlusion method. First, a 20-25 g mouse was placed in an induction box with gas anesthetic to completely anesthetize the mouse. The anesthetized mouse was taken out and its limbs were fixed on the operating board, and the mouse head was fixed through the breathing mask of the anesthesia machine. The hair on the mouse neck was removed, the skin on the mouse neck was disinfected with 75% ethanol, and a long opening of about 1.5 cm was cut on the right side of the mouse neck with ophthalmic scissors. At this time, the mouse was placed under a stereomicroscope. Two 14 cm long microtweezers with a head width of 0.3 and 0.15 respectively were used to separate the soft tissue of the mouse neck, exposing the common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA). These three arteries were distributed in a Y shape. The common carotid artery was freed and tied with a slipknot using No. 6 medical braided wire to block the blood supply of the common carotid artery; the external carotid artery and internal carotid artery were separated upwards, taking care not to touch the vagus nerve. A slipknot was tied at the proximal end of the external carotid artery and the internal carotid artery. A knot was tied at the distal end of the external carotid artery, and the external carotid artery was transected distally using an electrocoagulator. A small incision was made between the knot and the slipknot using vascular scissors. A suture was inserted into the common carotid artery. The slipknot near the trifurcation at the proximal end of the external carotid artery was slightly tightened. The slipknot on the internal carotid artery was untied. The tail end of the suture was clamped and inserted into the internal carotid artery, starting at the trifurcation of the common carotid artery. If the suture felt difficult to advance, the insertion was stopped and the suture was loosened outward. The slipknot on the external carotid artery was tightened, and the skin was sutured. After 90 minutes of ischemia, the incision was opened, the slipknot on the external carotid artery was untied, the suture was immediately removed, the external carotid artery was ligated, and the slipknot on the common carotid artery was removed to allow cerebral blood reperfusion. No suture was inserted in the sham-operated mice, and all other procedures were identical. A constant-temperature electric blanket was used after surgery to ensure that the mice did not become hyperthermic or hypothermic. After the mice regained consciousness, they were placed in a clean cage to avoid trampling between awake and non-awakened mice, and food and water were provided.

[0105] (2) Experimental animals, groups, and methods

[0106] Experimental animals: SPF-grade adult ICR male mice weighing 25-28 g were purchased from Zhaoyan (Suzhou) New Drug Research Center Co., Ltd., license number: SCXK(Su)2018-0006. Household conditions included a temperature of 22°C, humidity of 50%-60%, good ventilation, an artificial day / night cycle (12 h / 12 ​​h), and free access to food and water.

[0107] Experimental Grouping: SPF-grade adult ICR male mice weighing 25-28 g were randomly divided into three groups: a sham-operated group (Sham group), a model group (Model group), and an NADPH group (n=6). Dosing regimen: The t-MCAO / R model was established according to the above-described method. Mice in the NADPH group received NADPH (7.5 mg / kg) via the tail vein at the moment of reperfusion, followed by daily administration of the same dose via the tail vein for 7 consecutive days. The sham-operated and model control groups received the corresponding vehicle once daily for 7 consecutive days.

[0108] Tail suspension test:

[0109] The tail suspension test is used to reflect the changes in the physical strength of mice after modeling and their desire to adapt to the stressful environment. The immobility time can reflect the degree of despair produced by mice in the stressful environment, which is similar to the manifestation of depression.

[0110] Seven days after modeling, the tail of the mouse (about 1 cm from the tip of the tail) was fixed to the top of the tail suspension box with medical tape, so that the mouse was in an upside-down state. Each mouse was suspended for 6 minutes, and the mouse's immobility time within the last 4 minutes was recorded.

[0111] Sugar water preference experiment

[0112] The sugar water preference test is used to assess the emotional state of mice, including their happiness and depression symptoms. Healthy mice prefer sweet food and water, while depressed mice experience a loss of pleasure and a reduced preference for sugar water.

[0113] The sugar water experiment was divided into two parts: adaptation period and test period.

[0114] During the acclimation period, mice were housed individually and given one bottle of 2% sucrose water and one bottle of pure water for 48 hours. Every 24 hours, the water bottles were swapped to allow the animals to adapt to the sugary water. After the acclimation period, they were deprived of water but not food for 24 hours. During the testing period, a sugar preference test was conducted after model establishment. The sugar and pure water bottles were weighed before the test. After 12 hours, the water bottles were swapped. After 24 hours, the bottles were weighed again at the end of the test. The sugar preference index (%) was calculated as: sugar water consumption (g) / total fluid consumption (g) x 100%.

[0115] Forced swim test

[0116] The forced swim test, also known as the behavioral despair test, is used to evaluate the effects of antidepressant drugs on mice. Depressed mice show reduced swimming motivation, swimming less distance and speed, and spending more time immobilized than healthy mice, reflecting a weakened response to stressful environmental stimuli and despair-like behavior. Mice are placed in a 30cm-high bucket (transparent cylinder) of water, where their hind legs cannot reach the bottom and the water environment prevents them from escaping. A camera is used to record swimming performance for 6 minutes, and software is used for analysis. The first 2 minutes are the adaptation period, and the total distance, speed, and immobility time of the mice in the last 4 minutes are recorded.

[0117] (3) Experimental results

[0118] Behavioral tests were performed 7 days after modeling, including tail suspension test, sugar water preference test and forced swimming test.

[0119] The results of the tail suspension test to detect the depressive-like behavior of mice are shown in Figure 6. In the last 4 minutes of the experiment, the immobility time of mice in the model group was significantly longer than that in the sham operation group, and depressive-like behavior appeared. After 7 days of continuous administration, the immobility time of mice was significantly reduced, indicating that the struggling time of mice in the suspension environment increased, suggesting that NADPH has an antidepressant effect.

[0120] The results of the sucrose preference experiment for detecting the anhedonia symptoms and depression levels in mice are shown in Figure 7. The healthy mice (sham operation group) showed a higher sucrose preference rate, while the sucrose preference rate of the mice in the model group was significantly reduced, indicating that the anhedonia of the mice in the model group was accompanied by depressive-like behavior. After 7 days of continuous administration of NADPH to the mice, the sucrose preference rate of the mice in the NADPH group increased significantly, and the anhedonia symptoms were alleviated to a certain extent, indicating that NADPH exerts its antidepressant effect by reducing anhedonia.

[0121] The results of the forced swim test are shown in Figure 8. The forced swim test was used to examine the depressive-like behavior and drug efficacy of mice. In the final 4 minutes of the experiment, the total swimming distance (A) and swimming speed (B) of the model group mice were significantly lower than those of the sham-operated group. After 7 days of continuous drug administration, the total swimming distance and swimming speed of the NADPH group mice were significantly higher than those of the model group mice. In addition, the swimming immobility time (C) of the model group mice was significantly higher than that of the sham-operated group, while the immobility time in the NADPH group was significantly lower than that of the model group. The total swimming distance and swimming speed of the NADPH group mice were significantly improved, and the swimming immobility time was reduced, reflecting that the NADPH group mice had an enhanced desire to survive under stressful conditions and weakened depressive-like behavior, suggesting that NADPH has an antidepressant effect.

[0122] The above reveals that NADPH can treat post-stroke depression.

[0123] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. Use of NADPH or a pharmaceutically acceptable salt thereof in the preparation of an antagonist of a P2X7 receptor.

2. The use according to claim 1, characterized in that The P2X7 receptor antagonist is used to prevent or treat diseases caused by P2X7 receptor activation and inflammasome activation.

3. The use according to claim 2, characterized in that: The diseases include major depression, bipolar depression, post-stroke depression, neuroinflammation-related Alzheimer's disease, experimental allergic encephalomyelitis or multiple sclerosis.

4. Use of NADPH or a pharmaceutically acceptable salt thereof in the preparation of a product for relieving or treating major depression, bipolar depression, post-stroke depression or neuroinflammation.

5. The use according to any one of claims 1 to 4, characterized in that: The antagonist or the product comprises an effective amount of NADPH or a derivative thereof or a pharmaceutically acceptable salt thereof, with or without the addition of a pharmaceutically acceptable excipient; And / or, the product includes medicine, food or health care product.

6. The use according to claim 5, characterized in that The antagonist or the product uses NADPH or its derivatives or pharmaceutically acceptable salts thereof as active ingredients, and conventional excipients are added to prepare clinically acceptable solid preparations, semisolid preparations or liquid preparations.

7. The use according to claim 6, characterized in that The dosage form of the antagonist or the product includes tablets, capsules, powders, mixtures, pills, granules, syrups, suppositories, aerosols, dry powder inhalers, ointments, nasal drops (nasal sprays), or injections; Optionally, the dosage form includes a nanoformulation or a liposome preparation.

8. The use according to claim 7, characterized in that The administration method of the antagonist or the drug is selected from at least one of oral administration, injection, sublingual administration, nasal administration, rectal administration, transdermal administration, and pulmonary inhalation.

9. A pharmaceutical composition for preventing, alleviating or treating major depression, bipolar depression, post-stroke depression or neuroinflammation-related diseases, characterized in that: NADPH or its derivatives or pharmaceutically acceptable salts thereof are used as active ingredients, and conventional excipients are added to prepare clinically acceptable solid preparations, semisolid preparations or liquid preparations.

10. The pharmaceutical composition according to claim 9, characterized in that The dosage form of the pharmaceutical composition includes tablets, capsules, powders, mixtures, pills, granules, syrups, suppositories, aerosols, drops, dry powders, ointments or injections.

11. A method for preventing or treating diseases caused by P2X7 receptor activation and inflammasome activation, characterized in that: NADPH or a pharmaceutically acceptable salt thereof is administered to a subject in need thereof.

12. The method according to claim 11, characterized in that The diseases include major depression, bipolar depression, post-stroke depression, neuroinflammation-related Alzheimer's disease, experimental allergic encephalomyelitis or multiple sclerosis.

13. The method according to claim 11, characterized in that The subjects of administration include humans and animals other than humans.

14. The method according to claim 11, characterized in that The administration dosage of NADPH is 4 mg / kg-8 mg / kg.

15. The method according to claim 11, characterized in that When the subject of administration is a human, the administration dosage of the NADPH is 0.4 mg / kg-0.9 mg / kg.

16. The method according to claim 15, characterized in that When the subject of administration is a human, the administration dosage of the NADPH is 0.44 mg / kg-0.88 mg / kg.

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