Reagent for detecting aging neutrophils in blood and use

Abstract

A reagent for detecting aging neutrophils in blood and the use thereof. The reagent can be used for assisting in diagnosis of mental disorders, and for predicting and / or judging the therapeutic effects of a drug for mental disorders. By using the number of aging neutrophils in blood as a blood diagnosis marker for mental disorders, the reagent for detecting aging neutrophils in blood can rapidly diagnose mental disorders, and also provides therapeutic targets and important bases for clinical applications such as gene therapy and drug therapy.

Description

Reagents for detecting senescent neutrophils in blood and their applications Technical Field

[0001] The present invention belongs to the field of biomedical detection, and more specifically, relates to a reagent for detecting senescent neutrophils in blood and its application. Background Art

[0002] With the rapid socioeconomic development of my country, people are experiencing widespread pressure from all sides, leading to an upward trend in the prevalence of mental disorders. According to the National Epidemiological Survey on Mental Disorders, the lifetime prevalence of various mental disorders among people aged 18 and over in my country is 16.57%, posing a significant threat and burden to society and families. Mental disorders are defined as disorders in which normal brain function is disrupted under the influence of various biological, psychological, and social environmental factors, leading to impairments in perception, thinking, emotion, behavior, and other mental activities. Common mental disorders include schizophrenia, bipolar disorder, depression, and anxiety. Mental disorders are highly heterogeneous, presenting clinical challenges such as difficulty in diagnosis and low cure rates.

[0003] To date, the pathogenesis, early diagnosis, and treatment of mental disorders remain key and challenging clinical issues. Current clinical diagnosis of mental disorders is based on the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders (DSM-5), fifth edition, or the International Statistical Classification of Diseases and Related Health Problems, 11th edition (ICD-11). Due to the lack of clarity regarding the etiology and pathomechanisms of mental disorders, the significant heterogeneity among patients, and the current reliance on subjective evidence such as patient self-report, accurate diagnosis and treatment of patients with mental disorders is difficult. Therefore, the development of objective and specific biomarkers is crucial for the early and accurate diagnosis, treatment, and prognosis and rehabilitation assessment of mental disorders. Although genetic and neurodevelopmental factors play a dominant role in the development and progression of mental disorders, the results of such research have been difficult to translate into easily detectable biomarkers in the clinic. However, increasing evidence indicates that the peripheral immune system plays a key role in regulating central nervous system homeostasis and disease. Cytokines released by the peripheral immune system can cross the blood-brain barrier, assist in activating microglia and astrocytes, and directly induce neuroinflammation. On the other hand, peripheral immune cells can also infiltrate the brain and participate in neuroinflammation and the progression of psychiatric disorders. Therefore, researchers have been searching for blood biomarkers for diagnosing mental disorders as an effective alternative. Applying objective biomarkers to the diagnosis of mental disorders can effectively supplement the uncertain subjective factors in current diagnoses, reduce the rate of misdiagnosis, and provide promising targets for the treatment of mental disorders.

[0004] Therefore, there is an urgent need in the art for blood biomarkers that can be used to diagnose mental disorders.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a reagent for detecting senescent neutrophils in blood and its application, which is not only simple to operate but also can quickly diagnose mental disorders with reliable results.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] In a first aspect, the present invention provides a reagent for detecting senescent neutrophils for use as an auxiliary diagnostic preparation for mental disorders.

[0009] Preferably, the mental disorder includes schizophrenia, bipolar disorder, major depression, and anxiety disorder.

[0010] Preferably, compared with normal people, the senescent neutrophils are significantly increased in patients with mental disorders; the test sample includes blood, preferably plasma or whole blood.

[0011] Preferably, the reagent matching method for detecting senescent neutrophils is based on the principle of cell counting technology, including electrical resistance method, light scattering method, special cell staining method, microscopic imaging method, volume conductance and light scattering combined detection method, electrical impedance and radiofrequency technology combined detection method, light scattering or cytochemical technology; the cytochemical technology includes at least one method suitable for the following: flow cytometry, immunofluorescence, immunohistochemistry, real-time fluorescence quantitative PCR, digital PCR or sequencing.

[0012] Preferably, the reagent for detecting senescent neutrophils consists of reagent A, reagent B, reagent C, and reagent D, wherein reagent A is MIgG2a-FITC and MIgG1-PE, reagent B is CD45-APC-Cy7, CD3-FITC, CD66b-APC, CXCR4-PE, and CD62L-BV421, and reagent C is Zombie Aqua TM Fixable Viability Kit-BV510, reagent D is 10× RBC Lysing Buffer.

[0013] In a second aspect, the present invention provides a preparation for auxiliary diagnosis of mental disorders, which is a reagent for detecting senescent neutrophils in the blood.

[0014] Preferably, the mental disorder includes schizophrenia, bipolar disorder, major depression, and anxiety disorder.

[0015] Preferably, compared with normal people, the senescent neutrophils are significantly increased in patients with mental disorders; the test sample includes blood, preferably plasma or whole blood.

[0016] Preferably, the reagent matching method for detecting senescent neutrophils is based on the principle of cell counting technology, including electrical resistance method, light scattering method, special cell staining method, microscopic imaging method, volume conductance and light scattering combined detection method, electrical impedance and radiofrequency technology combined detection method, light scattering or cytochemical technology; the cytochemical technology includes at least one method suitable for the following: flow cytometry, immunofluorescence, immunohistochemistry, real-time fluorescence quantitative PCR, digital PCR or sequencing.

[0017] Preferably, the reagent for detecting senescent neutrophils consists of reagent A, reagent B, reagent C, and reagent D, wherein reagent A is MIgG2a-FITC and MIgG1-PE, reagent B is CD45-APC-Cy7, CD3-FITC, CD66b-APC, CXCR4-PE, and CD62L-BV421, and reagent C is Zombie Aqua TM Fixable Viability Kit-BV510, reagent D is 10× RBC Lysing Buffer.

[0018] In a third aspect, the present invention provides a reagent for detecting senescent neutrophils as a reagent or product for predicting and / or judging the effect of a drug for treating mental disorders.

[0019] In a fourth aspect, the present invention provides the use of a substance capable of reducing the content of senescent neutrophils in the blood for use in the preparation of a drug for treating mental disorders, or the use of an agent that blocks the secretion of glucocorticoids in the preparation of a preparation for blocking neutrophil aging and / or improving the peripheral inflammatory response associated with schizophrenia.

[0020] In a fifth aspect, the present invention provides a reagent for detecting senescent neutrophils, which is composed of reagent A, reagent B, reagent C, and reagent D, wherein reagent A is MIgG2a-FITC and MIgG1-PE, reagent B is CD45-APC-Cy7, CD3-FITC, CD66b-APC, CXCR4-PE, and CD62L-BV421, and reagent C is Zombie Aqua TM Fixable Viability Kit-BV510, reagent D is 10× RBC Lysing Buffer.

[0021] In a sixth aspect, the present invention provides a method for treating peripheral inflammatory reactions associated with schizophrenia, comprising: blocking the secretion of glucocorticoids and / or reducing the content of senescent neutrophils in the blood.

[0022] Preferably, the treatment method is to block the secretion of glucocorticoids and / or reduce the content of senescent neutrophils in the blood by metyrapone.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] 1. The senescent neutrophils in the present invention have high specificity and sensitivity for the diagnosis of mental disorders, and the indicator is easy to obtain and easy to promote and use.

[0025] 2. The present invention assists in the diagnosis of mental disorders by detecting the number of senescent neutrophil populations. It is not only simple to operate, but also has reliable test results and good differential diagnostic significance.

[0026] 3. The present invention discovered for the first time that senescent neutrophils have high specificity and sensitivity for the diagnosis of mental disorders, and are a peripheral blood indicator that can be easily obtained.

[0027] 4. The present invention adopts a single-index detection method, which only requires measuring one indicator, namely the number of senescent neutrophil populations, which is convenient for future promotion and use, and can also be conveniently combined with other detection methods to obtain more reliable results.

[0028] 5. The present invention also found that substances that reduce the content of senescent neutrophils in the blood can be used as drugs to treat mental disorders, providing a new approach for the treatment of mental disorders.

[0029] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings, wherein like reference numerals generally represent like components throughout the exemplary embodiments of the present invention.

[0031] FIG1 shows the behavioral phenotype characteristics of Lrrc4 gene knockout mice.

[0032] A. Open field assay: WT and Lrrc4 - / - Representative activity maps of mice; B. Open field test: WT mice and Lrrc4 - / - The distance that mice traveled in the central area of ​​the open field; C. Open field experiment: WT mice and Lrrc4 - / - The percentage of time mice spent active in the central area of ​​the open field; D. Three-box social test: WT and Lrrc4 - / - The contact time between the Strange1 mouse (S1) and the empty metal cage (Empty) and the calculated ratio of the difference to the sum; E. Three-box social experiment: WT mice and Lrrc4 - / - Representative movement trajectories of mice; F. Three-box social experiment: WT and Lrrc4 - / -The contact time of mice with Strange1 mice (S1) and Strange2 mice (S2) and the calculated difference to sum ratio; G. Prepulse inhibition: all sexes of WT mice and Lrrc4 - / - PPI values ​​of mice; H. Prepulse inhibition: WT mice and Lrrc4 - / - PPI values ​​of male mice; I. Water maze test: WT and Lrrc4 - / - Representative movement trajectory of mice; J. Water maze test: WT mice and Lrrc4 - / - The distance of mice in the target area; K. Water maze experiment: WT mice and Lrrc4 - / - Percentage of time mice spent in the target area; Eight-arm maze test, WT and Lrrc4 - / - The number of times the mouse entered the correct arm (food arm) each day (reference memory).

[0033] FIG2 shows a statistical graph showing changes in the number of mature neutrophils in the peripheral blood of schizophrenia mouse models and control mice.

[0034] FIG3 shows a statistical graph showing changes in the number of senescent neutrophils in the peripheral blood of schizophrenia mouse models and control mice.

[0035] FIG4 shows the results of the diagnostic efficacy evaluation of mature and senescent neutrophils in diagnosing the schizophrenia mouse model.

[0036] FIG5 shows a statistical graph showing changes in the number of senescent neutrophils in the blood of patients with mental disorders and healthy controls.

[0037] FIG6 shows the immunofluorescence results of senescent neutrophils in the blood of patients with mental disorders and healthy controls.

[0038] FIG7 shows the effect of drug treatment on inhibiting neutrophil senescence in a mouse model.

[0039] FIG8 shows the effect of drug treatment on blocking neutrophil senescence in a mouse model to assist in the treatment of peripheral inflammation associated with schizophrenia. DETAILED DESCRIPTION

[0040] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0041] Senescent neutrophils are a subpopulation of cells that exist during the development and differentiation of neutrophils. Neutrophils differentiate and mature in the bone marrow and are released into the peripheral circulation. Once in the blood, neutrophils undergo circadian aging and then return to the bone marrow to be eliminated by macrophages. During this process, the function of neutrophils also changes, that is, from a defensive role to a role in maintaining blood homeostasis and tissue infiltration. In 2013, Andrés Hidalgo et al. low CXCR4 high Neutrophil population is defined as senescent neutrophils, which are characterized by obvious excessive nuclear division, changes in cell size and granules, and strong ROS production and NET generation capabilities. Currently, the detection of senescent neutrophils is based on their surface-specific molecular markers, namely CD62L in neutrophil populations. low CXCR4 high Studies have shown that during the aging process of neutrophils, accompanied by changes in biological processes such as cell activation, adhesion and migration, protein degradation and cell death, the pro-inflammatory activity of neutrophils is positively correlated with their aging process in the circulation, that is, aging neutrophils represent a group of cells with strong pro-inflammatory activity.

[0042] Example 1 Animal model screening

[0043] LRRC4 is a relatively specific and highly expressed gene in the central nervous system. It participates in the development and differentiation of brain tissue, promotes the formation of excitatory synapses, regulates information input to the hippocampus, and mediates long-term potentiation of synapses in the hippocampal CA1 region. Loss of LRRC4 expression is closely associated with central nervous system diseases such as glioma, autism, and encephalomyelitis. In addition, there are two strong linkage disequilibrium schizophrenia susceptibility SNP sites on the LRRC4 gene, and Lrrc4 gene knockout (Lrrc4 - / -) mice display behavioral phenotypes characteristic of schizophrenia. The main symptoms of schizophrenia can be categorized into three categories: positive symptoms (including auditory and visual hallucinations, delusions, conceptual disorganization, and thought disorder); negative symptoms (emotional blunting, social withdrawal, anhedonia, depression, impoverished thought, and poorly expressed speech); and cognitive impairment (including impaired executive function, working memory, and attention). First, we used an open field experiment to investigate the effects of Lrrc4 knockout on anxiety-related behaviors and motor skills in mice. Due to their tactile tendency, animals tend to explore the edges of the open field. The open field is an open, rectangular plastic box 30 cm long, 30 cm wide, and 20 cm high, with a bottom divided into nine identical squares. Experimental mice were placed from the center of the box floor and allowed to freely explore the box while video and route maps were recorded using Smart software for 3 minutes. Smart software was used to analyze the time the mice spent in the peripheral and central areas, comparing differences between WT and Lrrc4- / - mice. Figure 1A shows the relationship between WT mice and Lrrc4 - / - Representative activity maps of mice, Figure 1B for WT mice and Lrrc4 - / - The distance that mice traveled in the central area of ​​the open field. Figure 1C shows the distance that mice traveled in the central area of ​​the open field. - / - The percentage of time mice spend active in the central area of ​​the open field, Lrrc4 - / - The distance and time of mice's activities in the center of the open field were significantly higher than those of the WT group, indicating that Lrrc4 - / - The mice showed increased spontaneous activity. Secondly, we used a three-chamber social experiment to investigate the effects of Lrrc4 knockout on their social behavior. The three-chamber social experiment leverages the innate social preferences of mice and their tendency to explore new objects to assess their sociability and social novelty. The sociability test is based on the principle that animals with normal social behavior prefer to interact with individuals of their own species compared to inanimate objects; the social novelty test is based on the principle that animals spend more time exploring unfamiliar companions than familiar companions, which also relies on their intact memory of familiar companions. Decreased sociability and social novelty are often considered negative symptoms of schizophrenia. In the three-chamber experiment, the time mice spent interacting with different objects in each of the three chambers was recorded. Mice typically exhibited normal social behavior. In the first phase of the experiment, mice interacted significantly more frequently and for significantly longer with Stranger1 than with an empty cage. In the second phase, due to their improved memory and preference for novelty, mice preferred interacting with Stranger2, a previously unfamiliar companion. Figure 1E shows the representative movement trajectories of WT mice and Lrrc4- / - mice, and Figure 1D shows the representative movement trajectories of WT mice and Lrrc4 - / -The contact time of mice in the Strange1 mouse (S1) and empty metal cage (E) and the calculated ratio of difference to sum. Figure 1F shows the contact time of WT mice and Lrrc4 - / - The contact time of mice in Strange1 mice (S1) and Strange2 mice (S2) and the calculated difference to sum ratio. - / - In the first phase of the experiment, mice spent more time in contact with the Strange1 mouse than with the empty metal cage, showing normal social ability; Lrrc4 - / -In the second phase of the experiment, mice spent more time in contact with Strange1 than with Strange2, showing lower social novelty. Next, we used the prepulse inhibition experiment to evaluate the processing ability and attention of mice to external stimuli after knocking out Lrrc4. The central nervous system has the ability to filter and screen information. After processing and classifying the input information, animals will not respond to all startle reflexes, but will respond selectively according to the size to ensure the coherence of thinking. When multiple information appears at the same time, the central nervous system processes the information that appears first, and the information that appears later will be inhibited even if the intensity is greater. This mechanism is called "prepulse inhibition (PPI)". The experiment includes the following steps: (1) The mice are placed in a startle conditioning box and quietly adapt to 65dB white noise in dim conditions for a total of 4 days, with a time of 5 minutes per day. To ensure the accuracy of sound stimulation and experimental data, the equipment is calibrated with a standard instrument (decibel meter) before each test experiment. (2) Next, the startle reaction test experiment begins. At the beginning of each mouse experiment, there was a 5-minute white noise (65dB) adaptation period, followed by 10 consecutive 120dB bursts of white noise to induce a startle response (30ms), and the amplitude of the platform reflected the degree of startle of the mouse. (3) Then the input / output-function (IO function) test was carried out, with an adaptation time of 2 minutes. During this stage, there were 12 trails, each with different decibel test types: 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125 or 130dB, which appeared randomly. The stimulation duration was 20ms, and the time interval between each stimulation varied randomly between 10 and 30s to prevent the impact on the next round of testing. There was no prepulse stimulation, and the background noise remained at 65dB. (4) Next is the PPI phase. The PPI test is divided into three stages: adaptation period, Block I and Block II, namely no stimulation (no stimulation test, 20 trails); single startle test (120dB, 30 trails); prepulse combination (prepulse-pulse test, 30 trails). Mice are randomly tested in these three types of tests. The prepulse intensity of the prepulse combination is 68, 71, 74, 77 and 80dB, respectively, and the duration is 10ms. Each PPI phase includes 10 random trials of different types. The PPI of each animal is evaluated as a percentage (%PPI) value calculated as: 100×(startle average value - single pulse startle average value) / startle average value. After each round of experiments, the experimental device should be wiped with 75% alcohol and washed with water to remove the residual smell of the previous mouse. It should be wiped dry with cotton cloth before the next round of operations.We set the prepulse intensity to 68, 71, 74, 77, and 80 dB, and the startle signal intensity to 120 dB for WT and Lrrc4. - / - The PPI test was performed on mice, and the results showed that compared with WT mice, Lrrc4 - / - The PPI of mice was reduced, and the PPI reduction was statistically significant when the prepulse was 71dB (Figure 1G). - / - We further compared WT mice with Lrrc4 - / - Mice were separated by sex and found that Lrrc4 - / - Male mice showed a greater decrease in PPI (Figure 1H), consistent with the more severe symptoms seen in male patients with schizophrenia. Finally, we used water maze and eight-arm maze tests to investigate the effects of Lrrc4 knockout on learning and memory in mice. The water maze test is used to assess spatial learning and memory in mice. The experimental process consists of a training phase and a testing phase. A pool of water is filled to a point 0.5 cm above the platform. An appropriate amount of food whitening agent is added and stirred until it turns milky white. The water temperature is maintained at approximately 20°C. The pool is divided into four quadrants, with the platform placed in the third quadrant. On the first day of training, mice are gently lowered headfirst into the pool wall from the first quadrant. The time it takes for the mice to find the submerged platform is recorded. If the mice fail to find the platform within 60 seconds, they are guided to the platform, and the latency is recorded as 61 seconds. During each training session, the mice remain on the platform for 30 seconds. Mice are then lowered from the second, third, and fourth quadrants, and their latency is recorded. On the second day of training, mice were placed in the second quadrant, then in the third, fourth, and first quadrants, and the training continued for 7 days. Testing began on the eighth day. Before the test, the platform was removed and the mice were placed in water from the opposite side of the quadrant where the original platform was located. Smart software was used to automatically record the number of times the mice crossed the target quadrant where the original platform was located, the total time spent in the quadrant, and the number of times they passed the platform within 60 seconds. Figure 1I shows the results of WT mice and Lrrc4 mice. - / - Representative movement trajectories of mice. Figure 1J, K shows WT mice and Lrrc4 - / - The distance and time percentage of mice in the target area. - / -Mice spent less time and distance exploring the quadrant containing the platform, as well as crossed the platform less frequently, than WT mice, indicating that Lrrc4 knockout mice exhibit impaired learning and memory abilities. Furthermore, the eight-arm maze, consisting of eight identical arms radiating evenly from a central platform, is used to assess spatial learning and memory in mice and can also be used to distinguish between long-term reference memory and short-term working memory. Unlike the water maze, the radial arm maze is suitable for repeated testing or long-term memory testing in mice. Prior to testing, mice were trained once daily by placing sweet food in baited pockets at the end of fixed arms of the eight-arm maze to encourage memory formation. On the first day, food pellets (approximately 4-5 per arm) were evenly distributed throughout the maze arms and the central platform area, allowing mice to consume clean food or explore freely for ten minutes. This was repeated twice daily. On the second day, a food pellet was randomly placed at the end of four arms, and the mice were placed in the center of the maze and allowed to explore freely until they consumed clean food or had spent ten minutes. Training sessions were conducted in the morning and afternoon, with at least one hour between each session. The test was carried out from the third to the ninth day, and the mice were fasted for 12 hours before the experiment. During training, the mice were placed in the center of the maze under the camera, and were allowed to randomly choose to enter the arms of the eight directions to explore and find food. The training was completed until the 5-minute time limit was up or the mice completed all the foraging in the eight arms ahead of time. Later, the main test indicators were recorded based on the video: the number of reference memory errors, that is, the total number of errors made by the mice in the eight-arm maze within 5 minutes. Each repeated entry into the arm that had already eaten bait was counted as an error; working memory errors, the number of correct errors before the first error, that is, the number of correct arms completed in succession when the error first occurred. These two indicators are used to evaluate working memory and reference memory, respectively. Figure 1L shows WT and Lrrc4 - / - The number of times mice entered the correct arm (food arm) every day. During the test days, the number of correct mistakes before the first error of WT mice was higher overall, while Lrrc4 - / - However, the mice showed a low number of correct answers before the first error, and there was a significant difference on the third day, indicating that Lrrc4 - / - The reference memory ability of mice was impaired. - / - The mice exhibited behavioral characteristics such as increased locomotor activity, decreased social novelty, loss of prepulse inhibition, decreased spatial memory, and reference memory deficits.

[0044] Targeting Lrrc4 - / - mice and WT mice, in which WT mice had Lrrc4 - / -8 mice each. Cut off the whiskers of the mice, collect blood from the eyeballs with pointed tweezers, press the mouse heart gently, and place the blood in an anticoagulant tube. Aspirate the blood into a 15mL centrifuge tube, add red blood cell lysis buffer according to the ratio of blood: red blood cell lysis buffer = 1:10, turn over and mix, and let it stand at room temperature for 8-10 minutes. Immediately after lysis, add 1×PBS solution to stop red blood cell lysis, and centrifuge at 3800rpm / min for 5 minutes. Pour out the supernatant, add 1×PBS solution to resuspend the cell pellet, centrifuge at 3800rpm / min for 5 minutes, and resuspend with 1×PBS solution to obtain a peripheral blood single cell suspension. The single cell suspension was centrifuged at 3800rpm / min for 5 minutes, and an appropriate amount of 1×PBS solution was added according to the amount of cell pellet, and counted using a cell counter. Transfer 1×10 6 Transfer cells to a new 1.5 mL centrifuge tube and centrifuge at 3800 rpm for 5 minutes. Carefully aspirate the supernatant. Prepare an antibody mixture at a ratio of 1:100 flow cytometry antibody to 1× PBS, and add 100 μL of the antibody mixture to each tube. The antibody incubation sequence is as follows: incubate with the live / dead antibody mixture for 15 minutes, centrifuge at 3800 rpm for 5 minutes, and carefully aspirate the supernatant; incubate with the blocking antibody mixture for 15 minutes, centrifuge at 3800 rpm for 5 minutes, and carefully aspirate the supernatant; incubate with the marker antibody mixture (CD45, CD11b, Ly6G, CD62L, CXCR4) for 30 minutes, and centrifuge at 3800 rpm for 5 minutes. All incubations were performed at 4°C in the dark. After adding the flow cytometry antibodies, all steps were kept away from light until analysis. Resuspend the cell pellet in 200 μL of 1× PBS, centrifuge at 3800 rpm for 5 minutes, and carefully aspirate the supernatant. Repeat this cycle twice to remove nonspecific staining. Finally, 200 μL of 1×PBS solution was added to resuspend the cells. The number of immune cells in the sample was detected using a Cytek Athena flow cytometer. The readout result was the percentage of neutrophil subpopulations. The experimental results are shown in Figures 2 and 3.

[0045] As shown in Figures 2 and 3, compared with the WT group, Lrrc4 - / - The levels of mature neutrophils (t-test p=0.0156) and senescent neutrophils (t-test p=0.0007) in the peripheral blood of mice were significantly increased.

[0046] Receiver operating characteristic (ROC) curves (AUCs), along with sensitivity and specificity, were constructed using Graphpad Prism 8 software to assess the diagnostic efficacy of senescent neutrophils for mental disorders. An AUC closer to 1 is considered more reliable, and an AUC > 0.5 is generally considered diagnostically reliable. Specificity and sensitivity were determined by analyzing the percentage of senescent neutrophil subpopulations, with the optimal cutoff value, corresponding to the maximum Youden index (sensitivity + specificity - 1), being selected.

[0047] The results showed that the AUC for mature neutrophils in diagnosing schizophrenia animal models was 0.8594, p=0.0157, specificity was 0.875, and sensitivity was 0.875; the AUC for senescent neutrophils in diagnosing schizophrenia animal models was 1.0000, p=0.0008, specificity was 1.000, and sensitivity was 1.000 (Figure 4).

[0048] Based on the above experimental results, mature neutrophils and senescent neutrophils are planned to be used as auxiliary diagnostic indicators for mental disorders including schizophrenia, and the disease will be diagnosed in combination with clinical symptoms.

[0049] Example 2 Development of a Senescent Neutrophil Detection Kit

[0050] Based on Example 1, senescent neutrophils in peripheral blood can be used as an auxiliary diagnostic indicator for mental disorders including schizophrenia. The development of a detection kit for this indicator is of great significance for the immune status and prognosis guidance of mental disorders.

[0051] This example further develops a kit for detecting senescent neutrophil subsets. The kit is based on flow cytometry, and its specific components and application methods are as follows:

[0052] 1. Departments that use this test kit: Department of Psychiatry, Department of Hematology, and Department of Immunology.

[0053] 2. The components of the kit are shown in Table 1:

[0054] Table 1 Composition of the kit

[0055] 3. Reagent function:

[0056] Reagent A: MIgG2a-FITC and MIgG1-PE, isotype controls; used to set the negative range and eliminate interference from nonspecific fluorescence.

[0057] Reagent B: CD45-APC-Cy7, CD3-FITC, CD66b-APC, CXCR4-PE, and CD62L-BV421. CD45-APC-Cy7 and CD3-FITC are used to gate myeloid cells. CD45-FITC identifies all leukocytes; CD3-PE identifies all mature T lymphocytes. CD66b-APC, CXCR4-PE, and CD62L-BV421 are used to identify senescent neutrophils (CXCR4 high CD62L low ) as a percentage of total neutrophils. CD66b-APC identifies total human neutrophils; CXCR4-PE and CD62L-BV421 identify senescent neutrophils.

[0058] Reagent C: Identify live / dead cells.

[0059] Reagent D: used to lyse red blood cells.

[0060] 4. Instructions for using the kit:

[0061] (1) Specimen collection and preparation

[0062] For EDTA anticoagulated venous blood (at least 0.5 mL), it is recommended that the specimen be stained and analyzed within 6 hours after collection. The peripheral blood leukocyte concentration to be tested is 3.0×10 3 ~10.0×10 3 / μL. (If higher than 10.0×10 3 / μL, the blood sample needs to be diluted; if it is less than 10.0×10 3 / μL, it is necessary to increase the sample volume or separate the white blood cells)

[0063] (2) Dilution of red blood cell lysis buffer

[0064] Dilute solution D to 1× working solution with deionized water. The hemolytic efficiency of red blood cell lysis solution is affected by temperature. It should be pre-equilibrated to room temperature (20℃~25℃) before use.

[0065]

Note

[0066] (3) Staining and fixing cells

[0067] 1) Use three 12×75 mm flow cytometry tubes for each patient specimen, labeling them with the sample number (1, 2, 3) and tube number (A, B, C).

[0068] 2) Add 20 μL of Reagent A mixture to Tube A of each sample, add 20 μL of Reagent B+C mixture to Tube B of each sample, and add 20 μL of PBS to Tube C of each sample.

[0069] 3) Add 100 μL of anticoagulated whole blood to each specimen tube, mix thoroughly, and incubate in a 4°C refrigerator in the dark for 20-30 minutes.

[0070] 4) Add 2 mL of 1× Solution D (20°C–25°C) to each tube, mix thoroughly, and incubate at room temperature in the dark for 10–12 minutes until the liquid becomes transparent.

[0071] 5) Centrifuge at 1000 rpm for 5 min and discard the supernatant.

[0072] 6) Add 2 mL of PBS for washing, centrifuge at 1000 rpm for 5 min, and discard the supernatant.

[0073] 7) Add 0.5 mL of 2% formaldehyde solution to fix the cells, mix well, and analyze by flow cytometry within 24 hours.

[0074] [Note] If cells are to be analyzed by flow cytometry immediately after staining, formaldehyde fixation is not necessary. Cells can be washed and resuspended in 0.2 mL of PBS before analysis.

[0075] (4) Flow cytometry

[0076] When using flow cytometry for detection, the PE fluorescence excitation wavelength is Ex=488nm, and it is recommended to use the Blu FL2 channel for detection; the FITC fluorescence excitation wavelength is Ex=488nm, and it is recommended to use the Blu FL1 channel for detection; the APC fluorescence excitation wavelength is Ex=640nm, and it is recommended to use the Red FL1 channel for detection; the APC-Cy7 fluorescence excitation wavelength is Ex=640nm, and it is recommended to use the Red FL3 channel for detection; the BV-421 fluorescence excitation wavelength is Ex=405nm, and it is recommended to use the Vio Fl1 channel for detection; the BV510 fluorescence excitation wavelength is Ex=405nm, and it is recommended to use the Vio Fl2 channel for detection.

[0077] 5. Interpretation of the inspection report

[0078] 1) Inspection findings

[0079] Specific values ​​of senescent neutrophil subsets were determined.

[0080] 2) Common diagnoses

[0081] No abnormal results, schizophrenia, bipolar disorder, major depression, or anxiety disorder were found.

[0082] 2-1) indicates normal results (immunofluorescence, flow cytometry):

[0083] Senescent neutrophils: 3.178% ± 1.490%;

[0084] 2-2) Indicates abnormal results (immunofluorescence, flow cytometry):

[0085] Senescent neutrophils: 10.709% ± 5.276%;

[0086] Possible diseases with abnormal results: schizophrenia, bipolar disorder, major depressive disorder, anxiety disorder.

[0087] Analysis of abnormal results: An increase in senescent neutrophils may indicate excessive mental stress and elevated glucocorticoid levels, which are common in mental disorders such as schizophrenia, bipolar disorder, major depression, and anxiety.

[0088] Diagnostic and treatment recommendations: A comprehensive diagnosis and treatment plan should be made based on the clinical symptoms of different perceptions, thinking, emotions, volitional behaviors, and cognitive functions in combination with this result. The purpose of treatment is to control inflammation, alleviate some symptoms, and improve the clinical symptoms of mental disorders.

[0089] Example 3 Clinical population verification

[0090] To verify the technical effect of Example 2, 40 patients with schizophrenia and 9 patients with bipolar disorder who visited Hunan Brain Hospital from October 25, 2022 to November 25, 2022 were collected; 22 patients with severe depression and 5 patients with anxiety who visited Xiangya Second Hospital of Central South University in Hunan Province from May 22, 2024 to June 27, 2024 were collected; the controls were healthy people with normal indicators from the Physical Examination Department of Hunan Brain Hospital and Xiangya Second Hospital of Central South University in Hunan Province during the same period, and a total of 49 cases were collected.

[0091] Inclusion criteria for the schizophrenia group: ① Subjects confirmed by SC1D met the diagnostic criteria for schizophrenia in the American Psychiatric Association's 5th edition Diagnostic and Statistical Manual of Mental Disorders (DSM-5) and were diagnosed with schizophrenia by two or more attending psychiatrists; ② Aged between 18 and 65 years; ③ Han Chinese; ④ Junior high school education or above, and able to give informed consent and relevant testing; ⑤ Not taking medication for the first episode or not taking medication in the past 2 weeks; ⑥ Informed consent to participate in this study (and signed the informed consent form).

[0092] Inclusion criteria for the bipolar disorder group: ① Participants who met the DSM-5 diagnostic criteria for bipolar disorder through SC1D confirmation, had no history of manic or hypomanic episodes, and were diagnosed with bipolar disorder by two or more attending psychiatrists; ② Aged between 18 and 65 years; ③ Han ethnicity; ④ Junior high school education or above, and were able to give informed consent and relevant testing; ⑤ Not taking medication for the first episode or not taking medication in the past 2 weeks; ⑥ Informed consent to participate in this study (and signed the informed consent form).

[0093] Inclusion criteria for the major depressive disorder group: ① Those who passed the SC1D confirmation test met the DSM-5 diagnostic criteria for a major depressive episode, had no history of manic or hypomanic episodes, and were diagnosed with major depressive disorder by two or more attending psychiatrists; ② were between 18 and 65 years old; ③ were of Han ethnicity; ④ had a junior high school education or above and were able to give informed consent and relevant testing; ⑤ had not taken medication for the first episode or had not taken medication in the past 2 weeks; ⑥ had given informed consent to participate in this study (and signed the informed consent form).

[0094] Inclusion criteria for the anxiety disorder group: ① The current episode met the diagnostic criteria for anxiety disorders in ICD-11 and DSM-5 and was diagnosed as an anxiety disorder by two or more attending psychiatrists; ② The total score of the 17 HAMD items was ≥17 points; ③ Age between 18 and 65 years old; ④ Han nationality, right-handed; junior high school education or above; ⑤ No medication was taken for the first episode or in the past 2 weeks; ⑥ Informed consent to participate in this study (and signed the informed consent form).

[0095] The above mental disorder exclusion criteria are as follows: ① history of neurological disease or major somatic disease or endocrine disease; ② current or past history of other mental disorders that meet the diagnostic criteria of DSM-5, mental disorders caused by alcohol or drug dependence; ③ pregnancy and lactation period; ④ history of coma due to craniocerebral trauma or other diseases that may interfere with this study; ⑤ severe suicidal ideation or suicide attempts; ⑥ current acute infection or autoimmune or inflammatory disease; ⑦ ECT treatment within the past 6 months; ⑧ hearing impairment (rough measurement: finger twirling sound); color blindness; ⑨ other conditions that the researcher believes are unsuitable for participation in this study.

[0096] Inclusion criteria for the healthy control group: ① Age between 18 and 65 years; ② Junior high school education or above; ③ Signed informed consent. Exclusion criteria: ① Current or past diagnosis of any mental disorder (MINI 6.0.0 diagnosis) as defined in the Diagnostic and Statistical Manual of Mental Disorders, Vth Edition (DSM-V); ② History of organic brain disease, epilepsy, or head trauma; ③ History of drug or substance abuse in the past six months; ④ Current severe physical illness; ⑤ Pregnant or lactating women; ⑥ Hearing impairment (gross hearing; finger-twirling sounds); Color blindness; ⑦ Other conditions deemed by the researchers to be unsuitable for participation in this study.

[0097] (1) Clinical verification and auxiliary differential diagnosis method for patients with mental disorders - flow cytometry

[0098] 2 ml of cubital venous blood was obtained from all the subjects mentioned above. The number of senescent neutrophils in the samples was detected using the kit and its method of use of Example 2 and a Cytek Athena flow cytometer. The results were read as the percentage of neutrophil subpopulations. The experimental results are shown in Figure 5.

[0099] As shown in Figure 5, compared with the healthy control group, the number of senescent neutrophils in the blood of patients with schizophrenia (P = 2e-11), bipolar disorder (P = 0.018), major depression (P = 5.6e-08), and anxiety (P = 0.0063) was significantly increased.

[0100] (2) Clinical verification and auxiliary differential diagnosis method for patients with mental disorders - immunofluorescence

[0101] 2 mL of blood was obtained from the mid-cubital vein of each subject and diluted to 3 mL with 1× PBS. A Cytiva Percoll density gradient was prepared using a 9:1 ratio of Percoll stock solution to 10× PBS to prepare a 100% Percoll working solution. The 100% Percoll working solution was diluted with 1× PBS to concentrations of 78% and 65%. Each 15 mL centrifuge tube was first layered with 3 mL of 78% Percoll solution, followed by 3 mL of 65% Percoll solution. Finally, the tube was tilted 45° and 3 mL of cell suspension was layered dropwise onto the Percoll gradient. The tubes were centrifuged at 3200 rpm for 35 minutes. Important: The blood layer must not mix with the Percoll solution. After centrifugation, the suspension above the 65% Percoll solution was carefully aspirated, and the 65%-78% cloud layer containing neutrophils was collected and centrifuged at 3800 rpm for 5 minutes. The cells were resuspended in an appropriate amount of 1× PBS and centrifuged at 3800 rpm for 5 minutes. Add an appropriate amount of red blood cell lysis buffer based on the cell pellet size, mix thoroughly by inverting, and incubate at room temperature for 8-10 minutes. Immediately after lysis, add 1× PBS solution to terminate red blood cell lysis. Centrifuge at 3800 rpm for 5 minutes to obtain mouse peripheral blood neutrophils. Neutrophil pellets were isolated from the sample, embedded in paraffin, and sectioned. The number of senescent neutrophils (positive cells) was determined using staining with CXCR4 (ab124824, Abcam) and CD62L (A25759, Abclonal). The results are shown in Figure 6.

[0102] As shown in Figure 6, compared with healthy controls, the number of senescent neutrophils in the blood of patients with schizophrenia, bipolar disorder, major depression, and anxiety disorders increased significantly.

[0103] In summary, it has been confirmed in clinical populations that senescent neutrophils in peripheral blood can be used as an auxiliary diagnostic indicator for mental disorders including schizophrenia, bipolar disorder, major depression, and anxiety.

[0104] Example 4 Evaluation of the Effect of Blockade System-Assisted Therapy in Animal Models

[0105] Based on Example 1, for Lrrc4 - / - Mice were injected with metyrapone to block glucocorticoid synthesis, thereby blocking neutrophil senescence and reducing the number of senescent neutrophils in peripheral blood. 30 minutes after intraperitoneal injection of metyrapone / PBS buffer, Lrrc4 - / - Blood was collected from the eye sockets of the control group (Con) and the drug-treated group (Met) of WT mice. The number of neutrophils in the samples was detected using a Cytek Athena flow cytometer (the flow cytometry detection process was the same as in Example 1), and the result was read as the number of cells. The experimental results are shown in Figure 7. - / - The brain, lung, spleen and liver of mice in the Met group were stained with HE. The experimental results are shown in Figure 8.

[0106] As shown in Figure 7, compared with the Con group mice, - / - In the peripheral blood of mice in the -Met group, the number of senescent neutrophils was significantly reduced (t test, p = 0.0167). - / - The mice showed lymphocyte infiltration in the brain and lungs; the number of lymph nodes in the spleen decreased, and the immune system was impaired; and the liver showed a certain inflammatory response. However, Lrrc4, which reduces the number of senescent neutrophils in peripheral blood by blocking glucocorticoid synthesis, - / - Schizophrenia mice (Lrrc4 - / - -Met group) the systemic inflammatory response was restored.

[0107] The above experiments show that blocking the synthesis and secretion of glucocorticoids can effectively inhibit neutrophil aging and improve the peripheral inflammatory response associated with schizophrenia.

[0108] In summary, the present invention uses senescent neutrophils in the blood for aiding the diagnosis of mental disorders, demonstrating significant value in aiding differential diagnosis and can be used for the diagnosis of mental disorders including schizophrenia and bipolar disorder. Furthermore, reducing senescent neutrophils in the blood for aiding the treatment of mental disorders demonstrates significant therapeutic benefits, providing a therapeutic target and important basis for clinical applications such as gene therapy and drug therapy.

[0109] While various embodiments of the present invention have been described above, the above description is intended to be illustrative, not exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. Application of reagents for detecting senescent neutrophils as auxiliary diagnostic preparations for mental disorders.

2. The use according to claim 1, characterized in that: The mental disorders include schizophrenia, bipolar disorder, major depression, and anxiety disorders.

3. The use according to claim 1 or 2, characterized in that: Compared with normal people, the senescent neutrophils are significantly increased in patients with mental disorders; the detection sample includes blood, preferably plasma or whole blood.

4. The use according to claim 3, characterized in that: The method of detecting senescent neutrophils with reagents is based on the principle of cell counting technology. Including, resistance method, light scattering method, special cell staining method, microscopic imaging method, volume conductivity light scattering combined detection method, electrical impedance radiofrequency technology combined detection method, light scattering or cytochemical technology; the cytochemical technology includes at least one of the following methods: flow cytometry, immunofluorescence, immunohistochemistry, real-time fluorescence quantitative PCR, digital PCR or sequencing.

5. The use according to claim 3, characterized in that: The reagent for detecting senescent neutrophils is composed of reagent A, reagent B, reagent C, and reagent D, wherein reagent A is MIgG2a-FITC and MIgG1-PE, reagent B is CD45-APC-Cy7, CD3-FITC, CD66b-APC, CXCR4-PE, and CD62L-BV421, and reagent C is Zombie Aqua TM Fixable Viability Kit-BV510, reagent D is 10×RBC Lysing Buffer.

6. A preparation for assisting diagnosis of mental disorders, characterized in that: A reagent for detecting senescent neutrophils in the blood.

7. The preparation for auxiliary diagnosis of mental disorders according to claim 6, characterized in that: The mental disorders include schizophrenia, bipolar disorder, major depression, and anxiety disorders.

8. The preparation for auxiliary diagnosis of mental disorders according to claim 6 or 7, characterized in that: Compared with normal people, the senescent neutrophils are significantly increased in patients with mental disorders; the detection sample includes blood, preferably plasma or whole blood.

9. The preparation for auxiliary diagnosis of mental disorders according to claim 8, characterized in that: The method of detecting senescent neutrophils with reagents is based on the principle of cell counting technology. Including, resistance method, light scattering method, special cell staining method, microscopic imaging method, volume conductivity light scattering combined detection method, electrical impedance radiofrequency technology combined detection method, light scattering or cytochemical technology; the cytochemical technology includes at least one of the following methods: flow cytometry, immunofluorescence, immunohistochemistry, real-time fluorescence quantitative PCR, digital PCR or sequencing.

10. The preparation for auxiliary diagnosis of mental disorders according to claim 9, characterized in that: The reagent for detecting senescent neutrophils is composed of reagent A, reagent B, reagent C, and reagent D, wherein reagent A is MIgG2a-FITC and MIgG1-PE, reagent B is CD45-APC-Cy7, CD3-FITC, CD66b-APC, CXCR4-PE, and CD62L-BV421, and reagent C is Zombie Aqua TM Fixable Viability Kit-BV510, reagent D is 10×RBC Lysing Buffer.

11. Use of a reagent for detecting senescent neutrophils as a reagent or product for predicting and / or judging the effect of a drug for treating mental disorders.

12. Use of a substance capable of reducing the content of senescent neutrophils in the blood in the preparation of a drug for treating mental disorders, or use of an agent that blocks the secretion of glucocorticoids in the preparation of a preparation for blocking neutrophil senescence and / or improving the peripheral inflammatory response associated with schizophrenia.

13. A reagent for detecting senescent neutrophils, characterized in that: It is composed of reagent A, reagent B, reagent C, and reagent D, wherein reagent A is MIgG2a-FITC and MIgG1-PE, reagent B is CD45-APC-Cy7, CD3-FITC, CD66b-APC, CXCR4-PE, and CD62L-BV421, and reagent C is Zombie Aqua TM Fixable Viability Kit-BV510, reagent D is 10×RBC Lysing Buffer.

14. A method for treating peripheral inflammatory reactions associated with schizophrenia, characterized in that: include: Blocking the secretion of glucocorticoids and / or reducing the level of senescent neutrophils in the blood.

15. The method of claim 14, wherein: The treatment method is to block the synthesis of glucocorticoids and / or reduce the content of senescent neutrophils in the blood by metyrapone.

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