Application of NADPH (Nicotinamide Adenine Dinucleotide Phosphate) in preparation of medicine for treating major depression, bidirectional depression, post-stroke depression or neuroinflammation

Abstract

The invention relates to the technical field of medicines, in particular to application of NADPH (nicotinamide adenine dinucleotide phosphate) to preparation of medicines for treating major depression, bidirectional depression, post-stroke depression or neuroinflammation. The invention also provides an application of the NADPH or the pharmaceutically acceptable salt thereof in preparation of the P2X7 receptor antagonist. The research finds that the NADPH inhibits ATP-induced calcium ion internal flow and P2X7 receptor activation-induced microglial cell internal current rise, which indicates that the NADPH can inhibit ATP-induced P2X7R activation and inflammasome activation, so that the 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, and the NADPH can be used for preventing or treating diseases caused by P2X7 receptor activation and inflammasome activation. Wherein the depressive disorders comprise major depressive disorders, bidirectional depressive disorders, post-stroke depressive disorders or neuroinflammation related diseases.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to use of NADPH in preparing medicines for treating major depression, bipolar depression, post-stroke depression or neuroinflammation-related Alzheimer's disease, experimental allergic encephalomyelitis or multiple sclerosis. Background Art

[0002] Depression is the most common mental illness nowadays. It is characterized by continuous and long-term low mood and is the most important type of mental illness in modern people. There are many types of depression. Clinically, depression is divided into major (degree) depression, bipolar depression, psychotic depression, reactive depression, menopausal depression, secondary depression, postpartum depression, seasonal depression, post-stroke depression and other types.

[0003] Major depressive disorder (MDD) is a common chronic disease that affects approximately 4.5% of the world's population. It is usually characterized by extreme sadness, low mood, and loss of interest. In addition to these non-specific symptoms, lack of pleasure makes it the leading cause of disability in the global population. Evidence in recent decades has shown that the neurobiological occurrence of depression is no longer limited to the catecholamine hypothesis. The current hypothesis is that MDD is caused by the interaction between genetic and environmental factors, which leads to neuroendocrine disorders, neurochemical changes (including impaired monoamine neurotransmission and enhanced glutamate release), neuroimmune responses, and reduced neuroplasticity (such as synaptogenesis and neurogenesis). To date, clinically available antidepressants mainly target monoaminergic signaling, especially by inhibiting the reuptake of serotonin (5-HT) and norepinephrine (NA), but it is worrying that these drugs have considerable potential side effects. Patients usually show symptoms such as weight gain, nausea, headache, and sexual dysfunction. Therefore, there is an urgent need to develop new molecular targets to guide the production of drugs with better therapeutic effects.

[0004] Bipolar disorder, also known as bipolar disorder, is one of the most challenging mental disorders. Bipolar disorder is characterized by recurrent episodes of elevated and depressed moods, accompanied by changes in activity or energy, and is associated with characteristic cognitive, physical, and behavioral symptoms. BD is a highly heritable disease. A series of molecules involved in calcium signaling have been identified in genomic data of BD-related genetic factors. Current research focuses on VGCC genes, especially L-type VGCC and its accessory subunits (encoded by CACNx). Pathway analysis shows that CACNA1D and CACNB3 and other VGCC genes are highly associated with BD. In addition, changes in calcium signaling were also found in neurons derived from induced pluripotent stem cells (IPSCs) with BD-related phenotypes. These findings suggest that BD may be an ion channel disease. The L-type voltage-gated calcium channel (VGCC) antagonist pregabalin is being evaluated for the treatment of BD. Lamotrigine, another anti-epileptic drug that may block calcium channels, is also an effective drug for the treatment of BD. In summary, the development of effective calcium channel blockers may be an effective drug to combat BD. Post-stroke depression (PSD), also known as post-stroke depression, is a common mental health problem, affecting about 33% of stroke survivors. Patients show a series of affective disorder syndromes characterized by low mood and loss of interest in addition to stroke symptoms, often accompanied by physical symptoms. Severe patients may have suicidal thoughts. If not prevented in time, some patients may lead to suicide. The cause of PSD is still unclear, and it is considered to be multifactorial, including biological factors and psychosocial factors. Many other biological factors are also considered to be related to PSD-like inflammation, response to ischemia, genetic susceptibility, neurogenesis, and activation of the hypothalamus-pituitary-adrenal (HPA) axis. There is evidence that cytokines play an important role in neuroinflammation and neurodegeneration, and are also involved in the occurrence of PSD. Inhibiting the production of cytokines will help improve PSD-related symptoms.

[0005] The P2X7 receptor (P2X7R) is an ATP-gated ion channel receptor that is mainly expressed in blood cells and immune cells in the brain. A large body of evidence suggests that P2X7R plays a key role in the pathogenesis of depression. P2X7R knockout mice show increased hippocampal 5-HT levels, increased 5-HT transporter binding sites and 5-HT uptake, and behavioral adaptation to stress. Stimulation of P2X7R in hippocampal nerve terminals induces glutamate release and leads to kainate receptor-mediated GABA outflow from interneurons, while glutamate and GABA release induced by P2X7R activation is absent in hippocampal slices of P2X7R knockout mice. P2X7R activation reduces the expression of glutamate / sodium aspartate-dependent transporter (GLAST), a mechanism directly involved in the clearance of glutamate by glial cells. The above evidence consistently indicates that P2X7R can regulate depression by regulating the uptake and release of different neurotransmitters. In addition, P2X7R has long been associated with inflammation and immunity, and the discovery of the NLRP3 inflammasome ultimately puts P2X7R at the heart of many neuropsychiatric diseases. To date, P2X7R is one of the most effective activation pathways for the NLRP3 inflammasome, which assembles and activates Caspase-1, which cleaves the IL-1β protein precursor into a mature form, which is released outside the cell and triggers neuroinflammation. It is now believed that this signaling pathway plays a role in depression such as MDD (eg Figure 5 ), BD, PSD, Alzheimer's disease, Parkinson's disease, experimental allergic encephalomyelitis, multiple sclerosis and other diseases. More importantly, the study 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. The single nucleotide polymorphism substitution of P2X7 codon 460 from glutamine (Gln, Q) to arginine (Arg, R) was found to be associated with BD and significantly associated with MDD (P = 0.0019). Mice carrying this variant showed changes in sleep quality, similar to signs of the prodromal stage of MDD disease. In addition, sleep parameters of healthy heterozygous human subjects also changed slightly. These results indicate 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 be a way to find more effective antidepressants in clinical practice. P2X7R antagonists have been developed for many years, and some candidate compounds have been discovered. However, due to the existence of a large number of P2XR subtypes and the problem of blood-brain barrier permeability, there is currently a lack of effective and specific P2X7R antagonists.

[0006] Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is a key component of the cellular antioxidant system and an indispensable electron donor in many antioxidant enzymes and biosynthetic reactions, such as nucleotide synthesis, lipid synthesis, and fatty acid chain elongation. However, there are no reports on NADPH antagonism of P2X7R and its use in antidepressant drugs. Summary of the invention

[0007] Therefore, the technical problem to be solved by the present invention 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.

[0008] To this end, the present invention provides the following technical solutions:

[0009] Use of NADPH or a pharmaceutically acceptable salt thereof in preparing an antagonist of a P2X7 receptor.

[0010] Optionally, the method is used to prevent or treat a disease associated with P2X7 receptor activation and inflammasome activation.

[0011] Optionally, the disease includes major depression, bipolar depression, post-stroke depression, neuroinflammation, Alzheimer's disease, experimental allergic encephalomyelitis or multiple sclerosis.

[0012] Use of NADPH or a pharmaceutically acceptable salt thereof in preparing a product for relieving or treating major depression, bipolar depression, post-stroke depression or neuroinflammation.

[0013] 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;

[0014] And / or, the product includes medicine, food or health care product.

[0015] 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.

[0016] 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;

[0017] Optionally, the dosage form includes a nanoformulation or a liposome preparation.

[0018] 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.

[0019] A pharmaceutical composition for preventing, alleviating or treating major depression, bipolar depression, post-stroke depression or neuroinflammation, which uses NADPH or its derivatives or pharmaceutically acceptable salts thereof as an active ingredient and is added with conventional excipients to be prepared into a clinically acceptable solid preparation, semisolid preparation or liquid preparation.

[0020] Optionally, the dosage form of the pharmaceutical composition includes tablets, capsules, powders, mixtures, pills, granules, syrups, suppositories, aerosols, drops, dry powders, ointments or injections.

[0021] The technical solution of the present invention has the following advantages:

[0022] 1. The use of NADPH or its derivatives or pharmaceutically acceptable salts provided by the present invention in the preparation of antagonists of P2X7 receptors. The present invention has found that NADPH inhibits ATP-induced calcium ion influx and the increase in inward current of microglia caused by P2X7 receptor activation, indicating that NADPH can inhibit ATP-induced P2X7R activation, NADPH can be used as an antagonist of P2X7 receptors to inhibit inflammasome activation, and NADPH can be used to prepare drugs for preventing or treating diseases caused by P2X7 receptor activation.

[0023] 2. The use of NADPH or its derivatives or pharmaceutically acceptable salts provided by the present invention 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 drugs; the present invention has 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

[0024] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 This is the result of the effect of exogenous NADPH on ATP-induced calcium influx in Experimental Example 1 of the present invention;

[0026] Figure 2 It is the result of the effect of the reduced endogenous NADPH content on the ATP-induced P2X7R activity in Experimental Example 2 of the present invention; 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 fluorescence intensity of intracellular calcium ions; Figure E shows the bar graphs of the si-Con and si-G6PD groups corresponding to Vehicle, ATP and NADPH from left to right;

[0027] Figure 3 This is the result of the effect of NADPH on ATP-induced inflammasome activation in Experimental Example 3 of the present invention;

[0028] Figure 4 This is the result of investigating the therapeutic effect of NADPH on major depression in Experimental Example 4 of the present invention;

[0029] Figure 5 It is a schematic diagram of the present invention that P2X7 receptor activation leads to major depression, bipolar depression, post-stroke depression, and neuroinflammation;

[0030] Figure 6 This is the result of the tail suspension experiment in the investigation of the therapeutic effect of NADPH on post-stroke depression in Experimental Example 6 of the present invention;

[0031] Figure 7 It is the result of the sugar water preference experiment in the investigation of the therapeutic effect of NADPH on post-stroke depression in Experimental Example 6 of the present invention;

[0032] Figure 8 These are the results of the forced swimming test in Experimental Example 6 of the present invention in investigating the therapeutic effect of NADPH on post-stroke depression; Figure A shows the result of the total swimming distance; Figure B shows the result of the average swimming speed; and Figure C shows the result of the stationary time. DETAILED DESCRIPTION

[0033] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0034] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0035] Example 1 P2X7R Antagonists

[0036] This embodiment provides a P2X7R antagonist, which uses NADPH as an active ingredient, adds conventional excipients, and is prepared according to conventional processes into clinically acceptable tablets, capsules, powders, mixtures, pills, granules, syrups, suppositories, aerosols, ointments or injections.

[0037] Example 2 Pharmaceutical Composition

[0038] This embodiment provides a pharmaceutical composition for preventing, alleviating or treating major depression, bipolar depression, post-stroke depression or neuroinflammation, which uses NADPH as an 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.

[0039] Experimental Example 1 Antagonistic effect of NADPH on P2X7R

[0040] Calcium influx is one of the responses of ATP to microglia. This experiment explores the effect of exogenous NADPH on ATP-induced calcium influx. Fluo-3AM, a calcium ion fluorescent probe, will be used to monitor changes in calcium ions in microglia and detect the different responses of calcium levels in the control group and NADPH group to ATP stimulation, thereby preliminarily revealing whether NADPH is a P2X7R regulator / antagonist. The steps are as follows:

[0041] (1) Primary microglial cell culture: A routine method for isolating and culturing microglial cells 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 min. After terminating the digestion with complete culture medium, filter through a 40μm cell mesh. Centrifuge at 1000rpm for 10min at room temperature to collect the cells and inoculate them into a cell culture flask. After 3 days, replace the culture medium with fresh one and continue culturing. After 7 days, place the microglia on a shaker at 220rpm and shake for 2h to obtain microglia.

[0042] (2) Experimental groups and methods

[0043] The microglia in step (1) were divided into four groups, namely, CON group, ATP group, ATP+NADPH group and NADPH group, n=12. Primary microglia 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 5 μM using phenol red-free DMEM. 100 μL of the working solution was added to each well of the 96-well plate and placed in a cell culture incubator for 30 minutes for fluorescent probe loading, followed by washing three times with PBS. 100 μL of phenol red-free DMEM was added to the control group, 100 μL of 1 mM ATP was added to the ATP group, and 100 μL of a mixture of 1 mM NADPH and 1 mM ATP was added to the NADPH+ATP group. Finally, the intracellular calcium ion concentration was immediately measured using an ELISA reader.

[0044] Determination of intracellular calcium ion concentration: Use DMEM solution containing 5μM Fluo-3 AM (calcium ion fluorescent probe, commercially available) and cells to incubate in a 37°C cell culture incubator for 30 minutes to load the fluorescent probe, then wash with PBS 3 times, and then incubate for another 20-30 minutes after washing to ensure that Fluo-3AM is completely converted into Fluo-3 in the cells. Use a multifunctional microplate reader to detect the fluorescence value at an excitation wavelength of 488nm and an emission wavelength of 525nm to reflect the changes in intracellular calcium ion concentration.

[0045] Detection of intracellular NADPH content: This study introduced the gene-encoded fluorescent probe iNap, which has attracted much attention in recent years, to detect intracellular NADPH (donated by Professor Zhao Yuzheng of East China University of Science and Technology). iNap can specifically bind to NADPH and is not affected by other nucleotides and coenzymes. The probe was transfected into the cells. After 24 hours, the cells that stably expressed iNap bound to NADPH. The fluorescence value was detected at an excitation wavelength of 420nm / 485nm and an emission wavelength of 528nm using a multifunctional microplate reader, or the fluorescence intensity was detected at an excitation wavelength of 488nm using a fluorescence microscope to reflect the intracellular NADPH content.

[0046] Electrophysiological testing: 24 h before the measurement, the cells to be tested were seeded on 13 mm glass coverslips. Whole-cell patch clamps were used to record membrane currents at room temperature using an Axopatch 200B amplifier (Molecular Devices) and borosilicate glass electrodes (TF-150 World Precision Instruments). The pipette (resistance range of 7 to 10 MΩ) was 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). The cell voltage was 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 perfused continuously. The perfusion capillary was placed close to the cells to be studied.

[0047] Data statistics: One-way ANOVA, post hoc analysis by Tukey, *P<0.05, ***P<0.001.

[0048] (3) Test results

[0049] The results are as follows Figure 1 As shown in A and B, whole-cell patch clamp recordings showed that ATP-induced enhanced inward current in microglia, while after NADPH treatment, the inward current in microglia decreased by about 20% compared with the ATP group, which was similar to the effect produced by the P2X7R antagonist A438079, preliminarily suggesting that NADPH inhibits ATP-induced P2X7R receptor activation.

[0050] The results are as follows Figure 1 As shown in C, using cell Ca 2+ Fluorescent probe Fluo-3AM detects the effect of NADPH on microglial Ca 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+-induced Ca2+-induced P2X7R activation. 2+ Increased inflow.

[0051] The results are as follows Figure 1 As shown in D, different concentrations of NADPH (0, 0.01, 0.1, 0.2, 0.5, 1, 5 mM) were used to treat ATP-stimulated microglia to observe their response to Ca 2+The results showed that compared with the group treated with ATP alone, the intracellular Ca 2+ 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.

[0052] The above results consistently indicate that exogenous NADPH inhibits ATP-induced P2X7R activation.

[0053] Experimental Example 2 Decreased endogenous NADPH content leads to increased ATP-induced P2X7R activity

[0054] In Experimental Example 1, it was found that exogenous NADPH inhibited P2X7R function. In this experimental example, it was further evaluated whether endogenous NADPH could have a similar effect on P2X7R function. G6PD (6-phosphoglucose dehydrogenase) is the main synthase of NADPH in microglia, accounting for about 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 the changes in ATP-induced microglial calcium influx. The steps are as follows:

[0055] (1) Primary microglial cell culture: Same as Experimental Example 1.

[0056] (2) Experimental groups and methods

[0057] for Figure 2 In A, B, and C, 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 inoculated 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), oscillate and mix, and incubate at room temperature for 10 minutes. Wash the cells that have been 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 BV2 cells were divided into four groups, namely, si-Con+Vehicle group, si-Con+ATP group, si-G6PD+Vehicle group and si-G6PD+ATP group, n=6, refer to Figure 2A knockdown experiment was performed in A, and Fluo-3AM was added for calcium ion probe loading 48 hours after the knockdown, and then ATP (the amount added was the same as in Experimental Example 1) was added to measure intracellular calcium ions. Figure 2 In Figure E, the cells were divided 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. The amount of ATP and / or NADPH added to each group was the same as that in Experimental Example 1, n = 5, refer to Figure 2 A knockdown experiment was performed in Figure A, and Fluo-3AM was added for calcium ion probe loading 48 hours after knockdown, followed by the addition of ATP (NADPH) for intracellular calcium ion measurement.

[0058] Determination of intracellular calcium ion concentration: Same as Experimental Example 1.

[0059] Detection of intracellular NADPH content: This study introduced the gene-encoded fluorescent probe iNap, which has attracted much attention in recent years, to detect intracellular NADPH (donated by Professor Zhao Yuzheng of East China University of Science and Technology). iNap can specifically bind to NADPH and is not affected by other nucleotides and coenzymes. The probe was transfected into the cells. After 24 hours, the cells that stably expressed iNap bound to NADPH. The fluorescence value was detected at an excitation wavelength of 420nm / 485nm and an emission wavelength of 528nm using a multifunctional microplate reader, or the fluorescence intensity was detected at an excitation wavelength of 488nm using a fluorescence microscope to reflect the intracellular NADPH content.

[0060] Data statistics: Two-way ANOVA, post hoc analysis by Tukey, ***P<0.001.

[0061] (3) Test results

[0062] The results are as follows Figure 2 As shown in A and B, the G6PD protein level in the si-G6PD group was reduced by approximately 77% compared with that in si-Con, indicating that G6PD knockdown was successful.

[0063] The results are as follows Figure 2 As shown in C, the intracellular NADPH probe iNap detected that the intracellular NADPH content in the G6PD knockdown group was significantly reduced compared with the control knockdown group.

[0064] The results are as follows Figure 2 As shown in D, the results showed that ATP treatment led to Ca 2+ The influx of Ca in si-G6PD+ATP group was increased compared with that in si-Con+ATP group. 2+The influx was further enhanced, and this phenomenon was improved after exogenous administration of NADPH ( Figure 2 The above results indicate that the decrease of endogenous NADPH content will lead to the enhancement of ATP-induced P2X7R activation, and conversely, NADPH can inhibit P2X7R activation.

[0065] Experimental Example 3 Effect of NADPH on ATP-induced inflammasome activation

[0066] The connection between P2X7R and inflammation and immunity has a long history. The discovery of NLRP3 inflammasomes finally placed P2X7R in the appropriate environment. The activation of NLRP3 inflammasomes requires two signal 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 NLRP3 inflammasomes. 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:

[0067] (1) Primary microglial cell culture: Same as Experimental Example 1.

[0068] (2) Construction of in vitro neuroinflammation model: Add LPS (500 ng / ml) to the microglial cells in step (1) for pretreatment for 3 h, replace the culture medium overnight, and then add 2.5 mM ATP for 30 min. After the cell treatment, discard the old culture medium, wash twice with PBS, add trypsin for digestion for 1-2 min, and then add complete culture medium to terminate the reaction. Blow up all the cells from the bottom of the culture dish and place them in a centrifuge tube. Centrifuge at 3000 rpm for 5 min, discard the liquid and leave the cells. Add an appropriate amount of cell lysis buffer RIPA according to the amount of cell precipitation, and then use a pipette to blow away the cell precipitation. Calculate the actual protein concentration of each sample by the BCA method, and add cell lysis buffer to level each sample according to the sample volume and concentration. Add loading buffer, mix well, and heat in a 95°C metal bath for 10 min to prepare the cell lysate loading buffer. For the preparation of cell supernatant samples, first collect the cell supernatant into a centrifuge tube, add the same volume of methanol as the cell supernatant and 1 / 4 of the volume of chloroform as the cell supernatant, shake rapidly and vigorously, and centrifuge at 4°C 15000rpm for 5min. Then discard the supernatant, be careful not to absorb the middle protein layer, add 500μL methanol to the centrifuge tube, vortex and oscillate quickly, centrifuge at 4°C 15000rpm for 5min, aspirate and discard the supernatant, be careful not to absorb the protein attached to the tube wall, and then place it in a 37°C water bath to evaporate the methanol, which takes about 5-10min. Finally, add a certain volume of cell lysis solution, blow off the protein with a pipette, add 5x loading buffer, shake and centrifuge quickly, and heat it in a 95°C metal bath for 10min to make 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), and combine immunofluorescence staining with stereology technology to evaluate microglial activation and determine whether the model is successful.

[0069] (3) Experimental groups and methods

[0070] The model was constructed according to step (2), and the primary microglia in step (1) were evenly seeded into a 12-well plate and divided into four groups, namely, CON group, ATP group, ATP+NADPH group and NADPH group. In order to construct the 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 anything, 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, followed by treatment with ATP (2.5 mM) for 30 min, and the NADPH group was pretreated with NADPH (1 mM) for 30 min. WB was used to detect the expression changes 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 the Caspase-1 activity detection kit was used to detect the Caspase-1 activity of the cell lysate, n=4-6.

[0071] Caspase-1 activity assay: conventional immunoblotting was used to detect the expression of pro-Caspase-1 and Caspase-1 proteins; Caspase-1 activity was directly detected using the Caspase-1 activity assay kit (Biovision) to confirm the results of immunoblotting. The general process includes: digestion after the experimental treatment is completed and the cells are collected by centrifugation at 1000g for 5min, and the supernatant is aspirated. The lysis buffer is shaken and lysed, ice bathed for 10min, and shaken again. Centrifuged at 4℃ (12,000g, 10min), the supernatant is taken and the absorbance is measured at 405nm to determine the Caspase-1 enzyme activity.

[0072] Data statistics: One-way ANOVA, post hoc analysis by Tukey, *P<0.05, **P<0.01, ***P<0.001.

[0073] (4) Test results

[0074] The results are as follows Figure 3As shown in A to E, primary microglia were pretreated with LPS (500 ng / ml) for 3 h, then LPS was discarded and replaced with fresh culture medium overnight, and finally 2.5 mM ATP was added for 30 min to induce NLRP3 inflammasome activation. A large amount of IL-1β mature body expression in the cell supernatant was detected, and the expression of IL-1β, Caspase-1, ASC and NLRP3 proteins in the cell lysate was increased. Before ATP treatment, NADPH (1 mM) was given to pretreat cells for 30 min. 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.

[0075] The results are as follows Figure 3 As shown in F, 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.

[0076] The results are as follows Figure 3 As shown in G, 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.

[0077] Experimental Example 4 Investigation of the therapeutic effect of NADPH on major depression

[0078] This example explores the effect of exogenous NADPH on the behavior of rats with major depression (MDD) model induced by chronic unpredictable stimulation (CUS). The rat MDD model will be induced by chronic unpredictable stress, and the behavioral changes in rat sugar water preference and forced swimming test will be detected, thereby preliminarily revealing the therapeutic effect of NADPH on the major depression model. The steps are as follows:

[0079] CUS is one of the most well-documented and widely used animal models of depression. The model was constructed based on the references and partially modified according to the actual experimental conditions. The following table was used to construct the model. In order to generate unpredictable stressors, the same stressor will not appear repeatedly within two days. The details are as follows:

[0080] Table 1

[0081]

[0082] (2) Experimental groups and methods

[0083] Three-month-old healthy male SD rats were selected. After one week of adaptive feeding, the rats were randomly divided into 5 groups according to body weight, namely: control group (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). The rats in the CUS+F group were gavaged with 5 mg / kg fluoxetine at 9 am every day for 8 consecutive weeks. The rats in the CUS+N4 and CUS+N8 groups were intraperitoneally injected with 4 mg / kg and 8 mg / kg NADPH at 9 am every day. After 8 weeks of administration, the rats in each group were subjected to sugar water preference and forced swimming tests to detect the behavioral changes of NADPH on the rat depression model.

[0084] Sucrose preference test (SPT): The SPT experiment was conducted on the third day after 8 weeks of drug administration. Before the formal test, the animals were allowed to adapt to two bottles of 1% sucrose water for 24 hours (the bottles had the same appearance and were placed symmetrically relative to the mouse cage). Then one of the bottles of sucrose water was replaced with ordinary drinking water for the animals to adapt for 24 hours. The two bottles of water in the middle were exchanged once (to reduce the influence of position preference on the animals). After 21 hours of fasting and water deprivation, the sucrose water consumption and pure water consumption of the animals in each cage within 1 hour were tested. The consumption was obtained by measuring the weight of the sucrose water bottle and the pure water bottle before and after. The calculation formula is: Sugar water preference percentage = 1% sucrose water consumption / (1% sucrose water consumption + pure water consumption) × 100%.

[0085] Forced swimming test (FST): The FST experiment was conducted on the sixth day after 8 weeks of drug administration. After the rats adapted to the experimental room for at least 1 hour, they were placed in a transparent glass beaker with a water temperature of 25°C for swimming. The beaker was 50 cm high, 22 cm in inner diameter, and 35 cm deep. The experiment consisted of two parts: 15 minutes of adaptive swimming on the first day; 24 hours later, each animal was put back into the beaker to swim for 5 minutes, and the first immobility time and the total cumulative immobility time of the rats within 5 minutes were recorded.

[0086] Data statistics: One-way ANOVA, post hoc analysis by Tukey, *P<0.05, **P<0.01, ***P<0.001.

[0087] Test results

[0088] The results are as follows Figure 4As shown in A, "anhedonia" is one of the main symptoms of depression. SPT is a commonly used paradigm for detecting "anhedonia" in depression model rats. The results showed that the preference percentage of rats in the CUS group for sucrose was significantly lower than that in the CON group. Compared with the CUS group, the 4 mg / kg dose of NADPH significantly increased the sucrose preference percentage. Fluoxetine and 8 mg / kg doses of NADPH had an increasing but not significant trend in the sucrose preference percentage.

[0089] The results are as follows Figure 4 As shown in B, the FST results showed that although CUS did not significantly increase the immobility time of rats, it significantly reduced their immobility latency. Compared with the CUS group, fluoxetine significantly reduced the immobility time of rats and increased their immobility latency. 4 mg / kg NADPH only reversed the increase in immobility time induced by CUS, but 8 mg / kg NADPH reversed both the increase in immobility time and the decrease in immobility latency induced by CUS.

[0090] 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.

[0091] Experimental Example 5 Investigation of the therapeutic effect of NADPH on bipolar depression

[0092] This example explores the effect of exogenous NADPH on the behavior of a bipolar depression mouse model. A bipolar depression model will be constructed using lipopolysaccharide (LPS). The behavioral changes in the sugar water preference, tail suspension test, and forced swimming test of mice will be tested to reveal the therapeutic effect of NADPH on the bipolar depression model.

[0093] Here are the steps:

[0094] (1) Construction of bipolar depression model: A bipolar depression model was established by intraperitoneal injection of LPS (2 mg / kg) into C57BL / 6J male mice aged about 6-8 weeks for 5 consecutive days.

[0095] (2) Experimental groups and methods: The mice were divided into three groups, namely the saline group, LPS group and NADPH+LPS group, with 6 mice in each group. The mice in the saline group were routinely injected with saline, the LPS group was intraperitoneally injected with 2 mg / kg LPS every day for 5 consecutive days, and the NADPH+LPS group was treated by intraperitoneal injection of NADPH (5 mg / kg) into the bipolar depression model mice constructed for 14 consecutive days. Afterwards, the sugar water preference, tail suspension test and forced swimming test were performed on the mice in each group to detect the behavioral differences of the mice in each group, so as to preliminarily determine the therapeutic effect of NADPH on the bipolar depression model.

[0096] Experimental Example 6 Investigation of the therapeutic effect of NADPH on post-stroke depression

[0097] This example explores the effect of exogenous NADPH on the behavior of mice with post-stroke depression. A transient middle cerebral artery occlusion (tMCAO) / reperfusion model will be established to induce depressive-like behavior in mice. The behavioral changes in the sugar water preference, tail suspension test and forced swimming test of mice will be tested to preliminarily reveal the therapeutic effect of NADPH on the post-stroke depression model. The steps are as follows:

[0098] (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 of 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 by 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 stereo microscope. Two 14 cm long microtweezers with head widths 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 the common carotid artery was ligated with a slipknot using a 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 upward, and care was taken not to touch the vagus nerve. A slipknot was tied at the proximal end of the external carotid artery and the internal carotid artery. Tie a knot at the distal end of the external carotid artery, and cut the external carotid artery with an electric coagulator at the distal end of the knot. Use vascular scissors to cut a small opening between the knot and the slipknot, insert the thread plug into the common carotid artery, slightly tighten the slipknot near the trifurcation at the proximal end of the external carotid artery, untie the slipknot on the internal carotid artery, clamp the tail end of the thread plug and insert the thread plug into the internal carotid artery, and use the trifurcation of the common carotid artery as the starting point. When the thread plug feels difficult to push, stop pushing and loosen the thread plug outward, tighten the slipknot of the external carotid artery, and suture the skin. After 90 minutes of ischemia, open the incision, untie the slipknot of the external carotid artery, immediately remove the thread plug, ligate the external carotid artery, and remove the slipknot on the common carotid artery to achieve brain blood reperfusion. No thread plug was inserted into the mice in the sham operation group, and the other steps were consistent. A constant temperature electric heating blanket was used after surgery to ensure that the body temperature of the mice would not be too high or too low. 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.

[0099] (2) Experimental animals, groups, and methods

[0100] Experimental animals: SPF grade ICR male adult mice weighing 25-28 g were selected. They were purchased from Zhaoyan (Suzhou) New Drug Research Center Co., Ltd., license number: SCXK (Suzhou) 2018-0006. Conditions: temperature 22 ° C, humidity 50%-60%, good ventilation, artificial day and night (12h / 12h), free access to food and water.

[0101] Experimental grouping: SPF-grade ICR male adult mice weighing 25-28g were randomly divided into 3 groups: sham group, model group and NADPH group, n = 6. Dosage regimen: According to the above-mentioned method for constructing t-MCAO / R model, the first administration time of NADPH group mice was at the moment of reperfusion, and NADPH (7.5mg / Kg) was administered through the tail vein, and then the administration was administered through the tail vein every day, with the same dose as before, for 7 consecutive days. The sham group and the model control group were given the corresponding solvent once a day for 7 consecutive days.

[0102] Tail suspension test:

[0103] The tail suspension test is used to reflect the physical changes of mice after modeling and their desire to adapt to the stressful environment. The immobility time can reflect the degree of despair of mice in the stressful environment, which is similar to the manifestation of depression.

[0104] 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 to make the mouse hang upside down. Each mouse was suspended for 6 minutes, and the immobility time of the mouse within the last 4 minutes was recorded.

[0105] Sugar water preference experiment

[0106] The sugar water preference experiment is used to assess the emotional state of mice, and can evaluate the happiness level and depression symptoms of mice. Healthy mice prefer sweet food and water, while mice in a depressed state lose pleasure and have a reduced preference for sugar water.

[0107] The sugar water experiment was divided into two parts: adaptation period and test period.

[0108] During the adaptation period, mice were housed in a single cage and given 1 bottle of 2% sucrose water and 1 bottle of pure water for 48 hours. The positions of the water bottles were exchanged every 24 hours to allow the animals to adapt to the sugary drinking water. After the adaptation period, water was deprived but food was not fasted for 24 hours. During the test period, the sugar water preference test period began after modeling. The sugar water and pure water bottles were weighed before the test. The positions of the water bottles were exchanged after 12 hours. The bottles were weighed again after the test ended 24 hours later. The sugar water preference index (%) of mice was calculated as sugar water consumption (g) / total liquid consumption (g) x 100%.

[0109] Forced swimming test

[0110] The forced swimming test, also known as the behavioral despair test, is used to evaluate the effects of antidepressant drugs on mice. Depressed mice have reduced swimming enthusiasm and show less total swimming distance and swimming speed, as well as longer immobility time than healthy mice, reflecting that the mice are less sensitive to stressful environmental stimuli and have despair-like behaviors. The mice were placed in a 30-cm-high bucket (transparent cylinder). The hind limbs of the mice could not touch the bottom, and the water environment made it impossible for them to escape. The swimming performance was recorded for 6 minutes using a camera and analyzed using software. The first 2 minutes were the adaptation period, and the total swimming distance, swimming speed, and immobility time of the mice in the last 4 minutes were counted.

[0111] (3) Experimental results

[0112] Behavioral tests were performed 7 days after modeling, including tail suspension test, sugar water preference test and forced swimming test.

[0113] The results of the tail suspension test to detect the depressive-like behavior of mice are as follows Figure 6 As shown in the figure, 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 occurred. After 7 days of continuous administration, the immobility time of mice was significantly reduced, indicating that the struggling time of mice in the hanging environment increased, suggesting that NADPH has an anti-depressant effect.

[0114] The results of the sugar water preference experiment to detect the anhedonia symptoms and depression levels of mice are as follows Figure 7 As shown, the healthy mice (sham operation group) showed a higher preference rate for sugar water, while the preference rate for sugar water in the model group mice was significantly reduced, indicating that the anhedonia of the model group mice was accompanied by depressive-like behavior. After continuous administration of NADPH group mice for 7 days, the preference rate for sugar water in the NADPH group mice was significantly increased, and the symptoms of anhedonia were alleviated to a certain extent, indicating that NADPH exerts an antidepressant effect by reducing anhedonia.

[0115] The results of the forced swimming test are as follows Figure 8 As shown in the figure, the depressive-like behavior and drug efficacy of mice were detected by forced swimming test. In the last 4 minutes of the experiment, the total swimming distance (A) and swimming speed (B) of the mice in the model group were significantly lower than those in the sham operation group. After 7 days of continuous administration, the total swimming distance and swimming speed of the mice in the NADPH group were significantly higher than those in the model group. In addition, the swimming immobility time (C) of the mice in the model group was significantly higher than that in the sham operation group, while the immobility time in the NADPH group was significantly lower than that in the model group. The total swimming distance and swimming speed of the mice in the NADPH group were significantly improved, and the swimming immobility time was reduced, reflecting that the mice in the NADPH group had an enhanced desire to survive under the oppressive environment and weakened the depressive-like behavior, indicating that NADPH has an antidepressant effect.

[0116] The above reveals that NADPH can treat post-stroke depression.

[0117] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the 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 In the preparation for preventing or treating 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.

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