Drug screening and diagnostic kit based on the mechanism of microplastics aggravating depression

Abstract

The application discloses a drug screening and diagnostic kit based on a mechanism of microplastics aggravating depression, and relates to the technical field of biological medicine.At present, whether polystyrene nano plastic exposure increases the susceptibility of depression and further affects the occurrence and development of depression and the potential mechanism thereof are not clear.The application provides a drug screening method based on a mechanism of microplastics aggravating depression, and the method comprises the following steps: contacting a to-be-tested substance with microplastics-treated microglial cells or nerve cells in an autophagic flow blocked state; detecting changes in the autophagic flow level, the mitochondrial reactive oxygen species level, the NF-KB signal pathway activity or the TrkB / PI3K / Akt signal pathway activity of the microglial cells or the nerve cells; wherein a substance capable of restoring the autophagic flow level, reducing the mitochondrial reactive oxygen species level, inhibiting the NF-KB signal pathway activity or activating the TrkB / PI3K / Akt signal pathway activity is determined as a candidate drug.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to drug screening and diagnostic kits based on the mechanism by which microplastics exacerbate depression. Background Technology

[0002] Depression is a common severe mental disorder characterized by persistent low mood and a significant loss of interest or pleasure. It is a major risk factor for suicidal behavior worldwide, significantly shortening life expectancy and imposing a heavy burden on families and society. While significant progress has been made in research on depression, the focus has largely been on traditional areas such as genetic susceptibility, altered neurobiochemical mechanisms, and individual psychological factors. In recent years, the academic community has gained a deeper understanding of the role of environmental factors in the development and progression of depression.

[0003] Microplastics (MPs), as a new type of environmental pollutant, can be divided into micron-sized plastics and nano-sized plastics based on their particle size. Nanoplastics, due to their nanoscale size, have an exponentially increased specific surface area and possess extremely strong biomembrane permeability, allowing them to cross the blood-brain barrier and directly enter the bloodstream, accumulating in brain tissue. Polystyrene nanoplastics (PS-NPs) have become a research focus due to their high detection frequency, wide distribution, and more pronounced toxic effects compared to other polymers. Studies show that PS-NPs can be preferentially taken up by microglia, which, as immune effector cells in the central nervous system, play a crucial role in the immune defense against foreign substances.

[0004] Previous studies have shown that PS-NPs, once introduced into brain tissue, can induce a series of complex neurotoxic effects, including damage to neuronal structure and function, triggering excessive activation of central immune cells, and inducing persistent neuroinflammatory responses. These pathological processes bear a striking resemblance to those of depression. In the process of cellular response to endocytosis of foreign substances, the autophagy pathway plays a crucial role in homeostasis. The core biological function of autophagy ultimately manifests in the degradation and recycling processes, where the normal function of lysosomes is essential for the smooth flow of autophagic flux. However, PS-NPs are artificially synthesized high-molecular-weight foreign substances, possessing inert and difficult-to-biodegrade properties. Whether lysosomes can effectively degrade such substances, and whether degradation failure will induce lysosomal damage, remain to be investigated.

[0005] Furthermore, excessive activation of microglia is a core element in neuroinflammation-mediated neuronal damage. Activated microglia release large amounts of pro-inflammatory cytokines such as TNF-α and IL-1β, which can induce neuronal apoptosis and interfere with synaptic plasticity. These damage mechanisms are closely related to the inhibition of the PI3K / Akt signaling pathway mediated by brain-derived neurotrophic factor (BDNF) and its high-affinity receptor TrkB. This pathway is a key molecular pathway regulating neuronal survival, synaptic plasticity, and exerting antidepressant effects. Currently, whether exposure to polystyrene nanoplastics increases susceptibility to depression, thereby affecting the occurrence and development of depression, and its potential mechanisms of action, remain unclear. Summary of the Invention

[0006] To address the aforementioned problems, this invention aims to provide a drug screening and diagnostic kit based on the mechanism by which microplastics exacerbate depression. This kit can reveal the specific molecular mechanism by which PS-NPs exacerbate depression, and based on this mechanism, develop technical solutions for drug screening and diagnostic assessment, providing new strategies and tools for the prevention and treatment of depression related to environmental pollutants.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for screening candidate drugs for the prevention or treatment of depression exacerbated by microplastic exposure, the method comprising: The test substance is brought into contact with microglia or nerve cells that have been treated with microplastics and are in a state of inhibited autophagic flow. Detect changes in autophagic flux levels, mitochondrial reactive oxygen species levels, NF-κB signaling pathway activity, or TrkB / PI3K / Akt signaling pathway activity in microglia or nerve cells; Among them, substances that can restore autophagic flux levels, reduce mitochondrial reactive oxygen species levels, inhibit NF-κB signaling pathway activity, or activate TrkB / PI3K / Akt signaling pathway activity were identified as candidate drugs.

[0008] A kit for detecting the risk of microplastics exacerbating depression, characterized in that the kit comprises: Reagents for detecting the levels of autophagic flux inhibition markers in biological samples from test subjects; and / or Reagents for detecting the levels of markers of microplastic-induced microglial cell activation; and / or A reagent for detecting markers of neuronal damage or reduced synaptic plasticity.

[0009] The beneficial effects of this invention are: 1. This study is the first to fully elucidate the cascade signaling pathways by which PS-NPs exacerbate depression, providing a clear target for related interventions.

[0010] This invention, through in vivo animal experiments, network toxicology, GEO dataset analysis, and in vitro cell experiments, demonstrates for the first time that PS-NPs exacerbate depression through a complete pathway: "lysosomal damage → autophagic flux blockade → mitochondrial ROS burst → NF-κB inflammatory pathway activation → microglia activation → inhibition of neuronal TrkB / PI3K / Akt signaling → decreased synaptic plasticity and neuronal apoptosis." Based on this pathway, candidate drugs can be precisely screened and intervention strategies designed.

[0011] 2. This study provides a method for screening antidepressant drug candidates with a clear mechanism and well-defined targets.

[0012] The screening method of this invention, by detecting changes in autophagic flux levels (LC3-II / I ratio, p62 protein), mitochondrial reactive oxygen species levels, NF-κB pathway activity (p-P65), or TrkB / PI3K / Akt pathway activity (p-TrkB, p-PI3K, p-Akt), can rapidly and specifically screen small molecule compounds or biological agents that can restore autophagic flux, scavenge mitochondrial ROS, inhibit NF-κB, or activate the TrkB pathway. This method is based on a mechanism validated in vitro and in vivo, is simple to operate, and yields reliable results.

[0013] 3. An effective pharmaceutical composition was provided that can reverse PS-NPs-induced neuronal damage and depressive-like behavior.

[0014] The pharmaceutical composition proposed in this invention comprises a mitochondrial reactive oxygen species scavenger (e.g., MitoTEMPO), an autophagy flux restorer, an NF-κB inhibitor, or a TrkB / PI3K / Akt agonist. Experiments have demonstrated that MitoTEMPO can significantly reduce PS-NPs-induced intracellular ROS levels in BV2 cells and restore mitochondrial membrane potential.

[0015] 4. A diagnostic kit is provided for assessing the risk of microplastics exacerbating depression.

[0016] The diagnostic kit described in this invention can objectively assess an individual's risk of exacerbated depression due to microplastic exposure by detecting the expression levels of autophagic flux inhibition markers (LC3-II / I ratio, p62), microglial activation markers (IL-1β, TNF-α, iNOS), neuronal damage markers (Bax / Bcl-2 ratio), and synaptic plasticity markers (PSD95, BDNF) in biological samples (brain tissue, cerebrospinal fluid, or blood). All of these markers showed significant correlations with PS-NPs exposure doses in in vitro and in vivo experiments, demonstrating clinical diagnostic and epidemiological survey value.

[0017] 5. All technical solutions of this invention are supported by sufficient experimental data, are authentic and reliable, and have strong repeatability.

[0018] All conclusions of this invention are derived from standardized in vivo animal experiments (CUMS mouse model), network toxicology analysis, GEO dataset differential analysis, and in vitro cell experiments (CCK-8, Western blot, transmission electron microscopy, JC-1, DCFH-DA, autophagic flow intervention, etc.). The data are detailed and statistically processed, providing a solid foundation for the industrial applicability and feasibility of this invention.

[0019] In summary, this invention fills a gap in the field of molecular mechanisms by which microplastics exacerbate depression, and provides a complete application solution based on this mechanism, from mechanism research to drug screening, intervention and treatment, clinical diagnosis and environmental monitoring, which has significant scientific value and broad application prospects. Attached Figure Description

[0020] Figure 1 The diagram below illustrates the performance testing of microplastics according to the present invention. A. Representative electron microscope image of the microplastic; B. Fourier transform infrared spectrum of the microplastic at wavelengths of 500-3500 cm⁻¹; C. Particle size distribution of the microplastic; D. Zeta potential distribution of the microplastic. Figure 2 The effect of PS-NPs on body weight in depressed mice; A. Experimental design flowchart; B. Body weight change curves of mice in each group during the experiment. Compared with the control group, ***P<0.001, compared with the CUMS group, #P<0.05, ##P<0.01 (mean ± standard deviation, n=10); Figure 3 The baseline behavioral tests and experimental groupings for mice were as follows: A. Total water intake in the sucrose preference test; B. Sucrose preference rate in the sucrose preference test; C. Autonomous trajectory map and heat map of mice within 8 minutes; D. Total distance traveled by mice; E. Time spent immobile by mice; F. Number of times mice entered the central area; G. Distance traveled by mice in the central area; H. Time spent by mice in the central area; I. Time spent immobile in the tail suspension test. Figure 4 The effects of PS-NPs on the behavior of depressed mice in the early (week 2) phase: A. Total water intake in the saccharide preference test; B. Saccharide preference rate in the saccharide preference test; C. Autonomous trajectory map and heat map of mice within 5 minutes; D. Total distance of mouse movement; E. Total number of times mice entered the arm; F. Time spent in the open arm; G. Number of times mice entered the open arm; H. Percentage of time spent in the open arm; I. Percentage of times mice entered the open arm; J. Time spent in the closed arm. Figure 5The effects of PS-NPs on the behavior of depressed mice in the mid-term (week 5) study included: A. Total water intake in the saccharide preference test; B. Saccharide preference rate in the saccharide preference test; C. Autonomous trajectory map and heat map of mice within 8 minutes in the open field test; D. Total distance traveled by mice; E. Time spent immobile by mice; F. Number of times mice entered the central area; G. Distance traveled by mice in the central area; H. Time spent by mice in the central area; I. Autonomous trajectory map and heat map of mice within 5 minutes in the elevated cross maze test; J. Total distance traveled by mice; K. Total number of times mice entered the arms; L. Time spent in the open arms; M. Number of times mice entered the open arms; N. Percentage of mice spent in the open arms; O. Percentage of mice entered the open arms; P. Time spent in the closed arms. Compared with the control group, *P<0.05, **P<0.01, ***P<0.001; compared with the CUMS group, #P<0.05, ##P<0.01 (mean ± standard deviation, n=10). Figure 6 The effects of PS-NPs on the behavior of depressed mice were measured at the endpoint (week 8). The results included: A. Total water intake in the saccharide preference test; B. Saccharide preference rate in the saccharide preference test; C. Autonomous trajectory map and heatmap of mice within 8 minutes in the open field test; D. Total distance traveled by mice; E. Time immobile by mice; F. Number of times mice entered the central area; G. Distance traveled by mice in the central area; H. Time spent in the central area by mice; I. Time immobile in the tail suspension test. Compared with the Control group, *P<0.05, **P<0.01, ***P<0.001; compared with the CUMS group, #P<0.05, ##P<0.01 (mean ± standard deviation, n=10). Figure 7 Effects of PS-NPs on pathological changes in brain tissue of depressed mice (scale bar: 200 μm); Figure 8 Collection of core targets for PS-NPs that exacerbate depression; A. Sources of PS-NP-related targets; B. Venn diagram of PS-NPs and depression-related targets; Figure 9 A. Network analysis of the core target of PS-NPs in exacerbating depression; B. Construction of PPI network using the STRING database; C. Visualization and topology analysis of the PPI network using Cytoscape. Figure 10 GO and KEGG enrichment analysis for PS-NPs exacerbating depression; A. GO enrichment analysis of common targets; B. KEGG enrichment analysis of common targets; C. Schematic diagram of neurotrophic factor signaling pathways; Figure 11To predict PS-NPs activating inflammatory responses via the NF-κB signaling pathway in the GEO database; A. Volcano plot of differential gene expression between the Control group and polystyrene nanogroup in the GSE256038 dataset; B. Volcano plot of differential gene expression between the Control group and depression group in the GSE126512 dataset; C. Venn diagram of differentially expressed genes in the GSE256038 and GSE126512 datasets; D. KEGG pathway enrichment map of common targets of differentially expressed genes in the GSE256038 and GSE126512 datasets. Figure 12 Western blot analysis was used to detect the effect of PS-NPs on the inflammatory response in depressed mice. A. Western blot images of NLRP3, Arg-1, TNF-α, IL-18, and IL-1β proteins in mouse brain tissue; BF. Statistical graph of NLRP3, Arg-1, TNF-α, IL-18, and IL-1β protein expression. Compared with the control group, *P<0.05, **P<0.01, ***P<0.001; compared with the CUMS group, #P<0.05, ##P<0.01 (mean ± standard deviation, n=6). Figure 13 Western blot analysis was used to detect the effect of PS-NPs on mitophagy in depressed mice. A. Western blot images of P62, LC3, Parkin, and PINK1 proteins in mouse brain tissue; BF. Statistical graph of P62, LC3, Parkin, and PINK1 protein expression. Compared with the control group, *P<0.05, **P<0.01; compared with the CUMS group, #P<0.05, ##P<0.01 (mean ± standard deviation, n=6). Figure 14 Western blot analysis was used to detect the effect of PS-NPs on neuronal synaptic plasticity in depressed mice. A. Western blot images of PSD95 and BDNF proteins in mouse brain tissue; BC. Statistical graphs of PSD95 and BDNF protein expression. Compared with the control group, *P<0.05, **P<0.01; compared with the CUMS group, #P<0.05, ##P<0.01 (mean ± standard deviation, n=6). Figure 15 Western blot analysis was used to detect the effect of PS-NPs on neuronal apoptosis in depressed mice. A. Western blot image of Bcl2 and Bax proteins in mouse brain tissue; BC. Statistical graph of Bcl2 and Bax protein expression. Compared with the control group, *P<0.05, **P<0.01; compared with the CUMS group, #P<0.05, ##P<0.01 (mean ± standard deviation, n=6). Figure 16 Western blot analysis was used to detect the effects of PS-NPs on the TrkB / PI3K / Akt signaling pathway in depressed mice. A. Western blot images of p-TrkB, TrkB, p-PI3K, PI3K, p-Akt, and Akt proteins in mouse brain tissue; BF. Statistical graph of p-TrkB, TrkB, p-PI3K, PI3K, p-Akt, and Akt protein expression. Compared with the Control group, *P<0.05, **P<0.01, ***P<0.001. Compared with the CUMS group, #P<0.05, ##P<0.01 (mean ± standard deviation, n=6). Figure 17 The effect of PS-NPs on BV2 cell viability; compared with the 0 μg / mL group, *P<0.05, ***P<0.001 (mean ± standard deviation, n=3); Figure 18 To determine the optimal dose of PS-NPs to exacerbate the inflammatory response using Western blotting; Western blot analysis of iNOS, TGF-β, TNF-α, and IL-18 proteins in A.BV2 cells; statistical plot of iNOS, TGF-β, TNF-α, and IL-18 protein expression in BE. Compared with the control group, *P<0.05, **P<0.01; compared with the LPS group, #P<0.05 (mean ± standard deviation, n=3); Figure 19 Effects of PS-NPs on autophagy-related proteins in BV2 cells; A. Western blot images of P62 and LC3 proteins in BV2 cells; BC. Statistical graphs of P62 and LC3II / I protein expression. Compared with the Control group, *P<0.05, **P<0.01, ***P<0.001; compared with the CUMS group, #P<0.05, ##P<0.01, ###P<0.001 (mean ± standard deviation, n=6); Figure 20 To verify via transmission electron microscopy that PS-NPs cause autophagic flux blockage in BV2 cells. Red arrows indicate PS-NP accumulation in lysosomes; yellow arrows indicate damaged mitochondria. (Scale bar: 5 μm (top) and 2 μm (bottom)); Figure 21To verify that PS-NPs cause autophagic flux blockage in BV2 cells using chloroquine; A. Western blot images of P62 and LC3 proteins; B. Statistical graph of P62 and LC3 proteins. Compared with the control group, **P<0.01, ***P<0.001; compared with the LPS group, #P<0.05, ##P<0.01; compared with the LPS+PS-NPs group, nsP>0.05 (mean ± standard deviation, n=6). Figure 22 To verify that PS-NPs cause autophagic flux blockage in BV2 cells with rapamycin; A. Western blot of P62 and LC3 proteins; B. Statistical graph of P62 and LC3 proteins. Compared with the control group, **P<0.01, ***P<0.001; compared with the LPS group, #P<0.05, ##P<0.01; compared with the LPS+PS-NPs group, nsP>0.05 (mean ± standard deviation, n=6); Figure 23 Western blotting analysis of the effect of PS-NPs-induced autophagic flux obstruction on mitophagy; A. Immunoblot images of PINK1 and Parkin proteins; BC. Statistical plots of PINK1 and Parkin proteins. Compared with the control group, **P<0.01, ***P<0.001; compared with the LPS group, #P<0.05, ##P<0.01 (mean ± standard deviation, n=6); Figure 24 The effect of PS-NPs-induced autophagic flux blockage on mitochondrial membrane potential; A. JC-1 staining results (scale bar: 100 μm); B. Statistical graph of JC-1 results. Compared with the control group, *P<0.05; compared with the LPS group, #P<0.05, ##P<0.01 (mean ± standard deviation, n=6); Figure 25 To detect the effect of autophagic flux blockage on intracellular ROS levels using the DCFH-DA fluorescent probe; A. ROS staining results (scale bar: 1 mm); B. ROS results statistical graph. Compared with the Control group, **P<0.01, ***P<0.001; compared with the LPS group, #P<0.05, ##P<0.01 (mean ± standard deviation, n=6); Figure 26To detect the effect of autophagic flow obstruction on the NF-κB signaling pathway using Western blot experiments; to obtain immunoblot images of Ap-P65 and P65 proteins; and to generate statistical plots of B-p-P65 and P65 proteins. Compared with the Control group, ***P<0.001; compared with the LPS group, ##P<0.01 (mean ± standard deviation, n=6). Figure 27 To verify the effect of PS-NPs on ROS production induced by MitoTEMPO in BV2 microglia; A. ROS staining results (scale bar: 1 mm); B. ROS statistical graph. Compared with the control group, **P<0.01, ***P<0.001; compared with the LPS group, #P<0.05, ##P<0.01 (mean ± standard deviation, n=6); Figure 28 To verify the effect of PS-NPs on the decrease in mitochondrial membrane potential induced by MitoTEMPO in BV2 microglia; A. JC-1 staining results (scale bar: 2mm); B. Statistical graph of JC-1 results. Compared with the control group, *P<0.05; compared with the LPS group, #P<0.05, ##P<0.01 (mean ± standard deviation, n=6); Figure 29 Screening for the optimal concentration of conditioned medium for treating neuronal cells; Figure 30 Western blot analysis was performed to detect the effect of conditioned medium on neuronal apoptosis. A. Western blot image of Bcl2 and Bax proteins in neurons conditioned in medium; BC. Statistical graphs of Bcl2 and Bax protein expression. Compared with the Control group, *P<0.05, **P<0.01, **P<0.001; compared with the LPS group, #P<0.05, ##P<0.01 (mean ± standard deviation, n=6). Figure 31 Western blot analysis was used to detect the effect of conditioned medium on neurotrophic factor signaling pathways in neurons. A. Immunoblotting of p-TrkB, TrkB, p-PI3K, PI3K, p-Akt, and Akt proteins in neurons. B. Statistical graph of p-TrkB, TrkB, p-PI3K, PI3K, p-Akt, and Akt protein expression. Compared with the Control group, *P<0.05, **P<0.01, ***P<0.001. Compared with the CUMS group, #P<0.05, ##P<0.01 (mean ± standard deviation, n=6). Figure 32 This is a schematic diagram illustrating the mechanism of action of the present invention. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. The components of the embodiments of this application described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0022] Example 1: Microplastic Performance Testing To characterize the physical and chemical properties of the microplastics used in the experiment, the performance of the microplastics was analyzed using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), and nanoparticle zeta potential analysis. Figure 1 .

[0023] The microplastic solution was ultrasonically dispersed for 10 min at approximately 15°C. It was then dropped onto a copper mesh, allowed to dry naturally, sputtered with gold, and observed under a scanning electron microscope. The results are as follows: Figure 1 As shown in Figure A, the microplastic particles exhibit a good spherical appearance, with relatively regular morphology, clear edges, and a relatively uniform particle size distribution, showing high uniformity and dispersion overall.

[0024] Fourier transform infrared spectroscopy was performed using the potassium bromide pellet method, with a scanning range of 4000-4000. The result is as follows Figure 1 As shown in B, at 3023 1613 1483 755 and 697 The presence of characteristic absorption peaks of polystyrene confirms that the material is polystyrene. This identification result lays a materials science foundation for subsequent research on environmental behavior and biological effects.

[0025] The hydrated particle size was determined using a dynamic light scattering particle size analyzer, and the results are as follows: Figure 1 As shown in C, the average hydrated particle size is 66.49±0.86 nm, the polydispersity index (PDI) is <0.2, and it exhibits a unimodal distribution, indicating that the particles are uniformly distributed in the aqueous phase without obvious polydispersity or agglomeration.

[0026] Surface potential was measured using a nanoparticle size zeta potential analyzer, and the results are as follows: Figure 1 As shown in Figure D, the average Zeta potential is -71.64 ± 0.16 mV, indicating that the nanoplastic has good dispersion stability in aqueous solution and can meet the material stability standards for subsequent cytotoxicity experiments and environmental behavior studies.

[0027] The above results show that the microplastics used in this example are polystyrene (i.e., PS-NPs), with uniform particle size and good dispersion stability in aqueous solution.

[0028] Example 2: In vivo animal model verification that PS-NPs aggravate depression This example is used to establish a CUMS mouse model and verify that PS-NPs exposure aggravates depression-like behaviors, providing an in vivo efficacy evaluation model basis for pharmaceutical compositions.

[0029] 2.1 Laboratory animals In this study, a total of 50 8-week-old clean-grade male C57BL / 6J mice were selected as experimental subjects, with their body weight controlled in the range of 22±2 grams. All animals required for the experiment were obtained from Hunan Silaikejingda Laboratory Animal Co., Ltd., whose animal use license number is SCXK (Beijing) 2020-0010. Before the formal experiment started, all mice were housed in a clean-grade laboratory animal facility for 7 days of adaptive feeding. During this adaptation period, the experimental animals had free access to food and drinking water, and the feeding environment maintained a 12-hour light-dark cycle. All animal experimental operations involved in this study were carried out in strict accordance with the relevant provisions of the national "Regulations on the Administration of Laboratory Animals", and the entire experimental protocol was formally reviewed and approved by the Laboratory Animal Ethics Committee of Shihezi University (Approval No.: A2025-1230).

[0030] 2.2 Experimental design and grouping After 1 week of adaptive feeding, 50 C57BL / 6 mice were subjected to behavioral tests at baseline, and were divided into groups by randomized block according to the sucrose preference rate: Control group (n=10), CUMS group (n=10), low-dose group (CUMS+5mg / kg / day PS-NPs, n=10), medium-dose group (CUMS+10mg / kg / day PS-NPs, n=10), and high-dose group (CUMS+20mg / kg / day PS-NPs, n=10).

[0031] 2.3 Construction of chronic unpredictable mild stress model 1. Stress protocol design The stress stimuli include the following 9 types: Fasting: 24h (remove food, no water restriction) Water deprivation: 24h (remove drinking water, no food restriction) Diurnal inversion: 24h (dark from 10:00 a.m. to 10:00 p.m., light from 10:00 p.m. to 10:00 a.m. the next day) Inclined rearing: 24 hours (cage tilted at 45°) Wet bedding: 24 hours (add water to the cage just enough to soak the bedding, making the bedding damp). Tail clamp: 1 min (clamp the mouse's tail 1 cm from the heart using a long tail clamp) Restraint stress: 2 hours (the mouse was placed in a 50 mL centrifuge tube with holes punched in the tube wall to ensure ventilation). Noise stimulation: 3 hours (playing white noise, intensity approximately 80dB) Empty cage: 24 hours (remove bedding and place in an empty cage). 2. Stress Procedure One to two stressors were randomly administered daily for eight consecutive weeks. Figure 2 Figure A shows the entire experimental procedure. To prevent the animals from adapting, the same stimulus was not applied consecutively, and the order of the stimuli was randomly adjusted weekly. The stress stimulus was administered between 10:00 and 12:00 each day.

[0032] 2.4 Microplastic Exposure Scheme Control group: administered an equal volume of normal saline (0.2 mL / 20 g) by gavage. Model group (CUMS group): CUMS stimulation, followed by gavage administration of an equal volume of physiological saline (0.2 mL / 20 g). Low-dose group (LowPS-NPs group): CUMS stimulation, followed by gavage administration of PS-NPs solution (5 mg / kg, dissolved in physiological saline). Medium-dose group (Medium PS-NPs group): CUMS stimulation, followed by infusion of PS-NPs solution (10 mg / kg, dissolved in physiological saline). High-dose group (HighPS-NPs group): CUMS stimulation, followed by gavage administration of PS-NPs solution (20 mg / kg, dissolved in physiological saline). 2.5 Behavioral Testing 1. Sugar water preference experiment Adaptation training: Before conducting the formal saccharide water preference test, a two-day single-cage single-mouse saccharide water adaptation training should be carried out. On the first day, each mouse is given two bottles of 2% saccharide water solution for 24 hours. On the second day, one of the bottles of 2% saccharide water solution is replaced with one bottle of ordinary deionized water, and the positions of ordinary deionized water and saccharide water are switched every 12 hours.

[0033] Fasting and water restriction: After the acclimatization period with sucrose, the mice were fasted and deprived of water for 12 hours.

[0034] Sugar water preference test: In the sugar water preference test, each mouse was tested individually in one cage. Each mouse was given one bottle of weighed 2% sugar water solution and one bottle of weighed ordinary deionized water for 12 hours. The positions of the ordinary deionized water and 2% sugar water solution were switched every 6 hours. After 12 hours, the mice were weighed again. The sugar water preference rate for each mouse within 12 hours was calculated as: (Sugar water consumption / (Sugar water consumption + Deionized water consumption)) × 100%.

[0035] 2. Open field experiment The complete open field experimental system consists of an open field enclosure, a camera system, and video recording and analysis software. The open field enclosure measures 40×40×40cm. After the equipment is set up, the mice are removed from their cages and placed in the central area of ​​the open field, facing away from the experimenter. The experimenter quickly leaves, and simultaneously starts the video capture software or video recording system to record the animal's activity in the open field for 8 minutes. After the experimental monitoring period, the mice are removed and returned to their cages. The entire open field enclosure is cleaned with 75% alcohol, allowed to dry, and then replaced with another mouse to repeat the experiment. The total distance the mice traveled in the open field, the time they remained stationary, the number of times they entered the central area, the time they spent in the central area, and the distance they traveled in the central area are recorded.

[0036] 3. Elevated Cross Maze Experiment The entire elevated cross-maze experimental system consists of a cross-maze structure, a camera system, and video recording and analysis software. The elevated cross-maze structure comprises two open arms and two closed arms. Each arm is 70cm long and 5cm wide, while the closed arm walls are 20cm high. The central area is a 5cm x 5cm square open area. After the equipment is properly configured, each mouse is placed in a uniform area with its back to the experimenter. The camera records the mouse's movement as it freely explores the elevated cross-maze for 5 minutes. After the experiment, the mice are returned to their cages, and the elevated cross-maze is cleaned with 75% alcohol. Excess alcohol and mouse excrement are then wiped dry with tissue paper. The next mouse is tested after the odor has dissipated. The total distance the mouse travels in the elevated cross-maze experiment, the total number of arm entries, the number of times it enters an open arm, the time spent in an open arm, the percentage of time spent in an open arm, the percentage of times it enters an open arm, and the time spent in a closed arm are recorded.

[0037] 4. Tail Suspension Test An opaque box measuring 20cm x 20cm and 50cm high was constructed. A horizontal bar was placed 30cm from the bottom. Mice were hung upside down on the bar using white medical tape, 5mm from the tail tip. A camera recorded the mice's behavior for the first 6 minutes of the experiment. The "immobility time" for each mouse during the last 4 minutes of the experiment was calculated using double-blind data analysis. In this behavioral experiment, immobility time refers to the time during which all of the mouse's limbs remain still, except for respiration.

[0038] 2.6 Sample Collection After the final behavioral test, mice were fasted for 12 hours but allowed free access to water. They were then anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg), and their brains were rapidly harvested after perfusion of pre-cooled physiological saline to the heart. The brain tissue was fixed in 4% paraformaldehyde for HE and Nissl staining. Brain tissue used for Western blotting was directly cryopreserved at -80 degrees Celsius.

[0039] 2.7 Results Analysis (1) Effects of PS-NPs on body weight in depressed mice During the 8-week experiment, changes in mouse body weight were monitored and recorded weekly, and behavioral tests were performed on mice in each group every two weeks to assess the dynamic changes in their depressive-like behaviors. The results of the experiment are as follows: Figure 2 As shown in Figure B, throughout the experiment, the body weight of the healthy control group mice showed a continuous and stable upward trend. Compared with the healthy control group, the body weight of the CUMS group mice was significantly reduced (P < 0.001). More notably, compared with the CUMS group, the body weight of the PS-NPs exposed mice was further significantly reduced (P < 0.01). Based on the above experimental results, it can be concluded that PS-NPs exposure can significantly aggravate the body weight loss in depressed mice.

[0040] (2) Baseline behavioral testing To control for the interference of individual differences on the experimental results, baseline behavioral tests were performed on the mice used in the experiment before the experiment began, and they were randomly assigned to groups based on their sucrose preference rate. The results are as follows: Figure 3 As shown, there were no significant differences in the performance of the mice in the sucrose preference test, open field test, and tail suspension test (P>0.05), indicating that the mice in each group had the same level of anhedonia, anxiety, and despair before the experiment.

[0041] (3) Effects of early (week 2) PS-NPs on the behavior of depressed mice To assess the effects of combined intervention of CUMS and PS-NPs on mouse behavior, the sucrose preference test and elevated cross maze test were used to detect changes in the behavior of mice in each group two weeks into the experiment.

[0042] from Figure 4 As observed in the AB study, the sucrose preference experiment showed that, compared to the control group, the sucrose preference rate in the CUMS group mice increased, indicating that after short-term stress, the mice may exhibit compensatory activity by enhancing their response to reward stimuli, rather than a typical state of anhedonia. Compared to the CUMS group, the sucrose preference rate in the low- and medium-dose PS-NPs intervention groups did not show significant changes, while the sucrose preference rate in the high-dose PS-NPs intervention group decreased slightly, but the difference was not statistically significant (P>0.05), indicating a potential inhibitory effect on stress-induced activity. This result suggests that the intervention efficacy of PS-NPs may have a dose-related characteristic, with a more pronounced inhibition of compensatory reward responses under high-dose exposure.

[0043] Elevated cross maze experiment ( Figure 4 CJ further evaluated the anxiety-like behavior and exploratory activities of the mice. Compared with the control group, the CUMS group mice had increased dwell time and number of entries into the open arm, indicating that they exhibited higher exploratory activity and lower anxiety-like behavior under short-term stress. Compared with the CUMS group, mice in different doses of PS-NPs intervention groups showed varying degrees of decrease in dwell time and number of entries into the open arm, and this decrease showed a certain dose-related trend, that is, as the PS-NPs intervention dose increased, the open arm-related exploratory indicators continued to decrease, but the differences between groups did not reach a statistically significant level (P>0.05). No obvious regular trends were observed in the dwell time, total number of entries, and total distance traveled in the closed arm among the groups of mice, indicating that there was no significant difference in the overall motor ability of the mice in each group, ruling out the possibility that activity ability interfered with the experimental results.

[0044] The above results indicate that short-term exposure to CUMS can induce behavioral hyperactivity in mice, manifested as increased sensitivity to rewards and increased exploratory behavior. PS-NPs intervention has a potential inhibitory effect on this hyperactivity, especially in the high-dose group. Although the differences between the various indicator groups have not reached statistical significance, the behavioral changes show a consistent trend, suggesting that PS-NPs exposure may have a certain inhibitory effect on emotion-related behaviors in mice under short-term stress.

[0045] (4) Effects of PS-NPs on the behavior of depressed mice during the mid-term (week 5) Five weeks into the experiment, behavioral changes in mice in each experimental group were assessed using the sucrose preference test, open field test, and elevated cross maze test.

[0046] A classic indicator for evaluating anhedonia behavior is the sugar water preference rate, such as... Figure 5As shown in Figures AB, the saccharin preference rate in the CUMS group mice showed a significant decreasing trend compared to the healthy control group (P<0.05). This result indicates that after five weeks of CUMS stimulation, the mice's sensitivity to reward stimuli decreased significantly, successfully inducing typical anhedonia-like behavior, demonstrating that the depression-related behavioral phenotype had been stably constructed. Compared to the control group, the saccharin preference rate of mice exposed to low, medium, and high doses of PS-NPs further decreased (P<0.01), and the saccharin preference rate tended to gradually decrease as the PS-NPs dose increased. These results suggest that long-term exposure to PS-NPs can significantly aggravate anhedonia behavior in depressed mice, indicating that nanoplastics may act as an environmental risk factor, exacerbating the severity of the depression-like phenotype.

[0047] The open field test and the elevated cruciate maze test were used to comprehensively assess the anxiety-like behaviors and self-exploration abilities of each group of mice. In the open field test ( Figure 5 Compared with the control group, mice in the CUMS group showed a significant decrease in total movement distance, a reduction in the number of times and duration of exploration in the central area, and an increase in immobile time, indicating that they exhibited significant anxiety-like behavior and decreased exploration motivation under stress. Compared with the CUMS group, mice in each dose of PS-NPs intervention group showed a continued trend of decreasing total movement distance in the open field, a decrease in the time spent in the central area and the number of entries, and a slight increase in the time spent immobile, but these differences did not reach the statistically significant standard (P>0.05). 0.05).

[0048] In the elevated cross maze experiment ( Figure 5 Compared with the control group, mice in the CUMS group showed an increased trend in open arm exploration activity, with an increase in the number of times they entered the open arm and the duration of their stay. This phenomenon may be related to impulsive or maladaptive exploratory behaviors in animals under long-term stress. Compared with the CUMS group, mice in each PS-NPs intervention group showed varying degrees of decrease in the duration and number of times they entered the open arm, and the decrease slightly increased with increasing dosage, indicating that PS-NPs exposure may correct abnormal exploratory behavior caused by stress to some extent. However, the differences between the groups did not reach a statistically significant level (P>0.05). No significant differences were found among the experimental groups in the number of times they entered the closed arm and the total distance they moved (P>0.05), indicating that overall motor function was not significantly affected.

[0049] Based on the above behavioral experimental results, five weeks of PS-NPs exposure significantly aggravated CUMS-induced anhedonia in mice, indicating that nanoplastics may exacerbate depression-related phenotypes by disrupting reward pathway function. In terms of anxiety-like behaviors, there were no statistically significant differences among the PS-NPs intervention groups, but certain trends were observed in various indicators of the open field and elevated cross maze, suggesting that PS-NPs exposure may have a potential aggravating effect on anxiety.

[0050] (5) Effects of late-stage (week 8) PS-NPs on the behavior of depressed mice After eight weeks of exposure to CUMS and PS-NPs, this study systematically evaluated the behavioral changes in mice in each experimental group using sucrose preference test, open field test, and tail suspension test. The results of the sucrose preference test are as follows: Figure 6 As shown in AB, compared with the CUMS model group, the total water intake and sucrose preference rate of mice in the PS-NPs exposure group were significantly reduced (P<0.05). This result indicates that PS-NPs exposure can significantly aggravate anhedonia behavior in CUMS-induced depressed mice. The results of the open field experiment (…) Figure 6 The results of the CH test showed that, compared with the CUMS group, the PS-NPs exposed mice had a significantly reduced total distance traveled in the open field. Simultaneously, the number of times these mice entered the central region, the distance they traveled within the central region, and the duration of their stay were all significantly reduced (P<0.05). These results suggest that PS-NPs exposure can significantly exacerbate anxiety-like behavior in depressed mice. The results of the tail suspension test (…) Figure 6 I) showed that, compared with the CUMS model group, the PS-NPs exposed group had a significantly increased immobility time in the tail suspension test (P<0.05). This finding indicates that PS-NPs exposure can significantly exacerbate hopeless behavior in a mouse model of depression.

[0051] Based on the above behavioral experimental results, it can be concluded that eight weeks of PS-NP exposure significantly exacerbated anhedonia, anxiety-like behavior, and hopelessness behavior in CUMS-induced depressed mice. These results suggest that PS-NP exposure may be a potential risk factor for the development and progression of depression.

[0052] (6) Effect of HE staining on pathological changes in brain tissue of depressed mice After the 8-week experiment, brain tissue was collected from each group, and HE staining was used to examine the pathological damage of the mouse brain tissue. The relevant results are shown in the figure below. Figure 7In the control group, neurons in the brain tissue of mice were neatly arranged, with no damage to cell structure, and no obvious edema or inflammatory cell infiltration. Compared with the control group, the brain tissue of mice in the CUMS group showed obvious pathological damage, with disordered neuronal arrangement, enlarged intercellular spaces, and some neurons showing shrinkage and darkening in color. Nuclear shrinkage and karyolysis were very significant, and edema in the surrounding tissues was very significant. Compared with the CUMS group, the pathological damage in the CUMS + low-dose PS-NPs group was slightly aggravated, manifested by an increase in the number of shrunken neurons and more obvious inflammatory cell infiltration. The above changes were further aggravated in the CUMS + medium-dose PS-NPs group, with scattered necrotic neurons and vacuolar degeneration. The damage was most severe in the CUMS + high-dose PS-NPs group, with a large number of neurons shrinking and dying, obvious structural destruction, and extensive tissue edema, suggesting that PS-NPs exposure can dose-dependently aggravate CUMS-induced pathological damage to brain tissue.

[0053] The results showed that PS-NPs significantly aggravated CUMS-induced depressive-like behaviors.

[0054] Example 3: Key Prediction Mechanisms of Network Toxicology and GEO Dataset Using the CTD, GeneCards, SwissTargetPrediction, and ChEMBL databases, 2746 PS-NP-related targets, genes, and proteins were obtained. Using the GeneCards and OMIM databases, 545 depression-related targets were obtained. Taking the intersection, 195 common targets were found. Figure 8 B).

[0055] 195 common target points were imported into the STRING database (confidence > 0.7) to construct a PPI network. Figure 9 A), imported into Cytoscape for topological analysis, revealed that the top-ranked targets by degree value included inflammation-related (IL6, IL10, TNF, TGFB1), apoptosis-related (TP53, CASP3), BDNF associated with neurotrophic factor signaling pathways, and PINK1 closely related to mitophagy. Figure 9 B).

[0056] GO enrichment analysis showed that common targets were enriched in biological processes such as behavioral regulation, chemical synaptic transmission regulation, synaptic plasticity regulation, and neuronal death regulation. Figure 10 A) further confirms its crucial role in neuronal signal transduction and synaptic function regulation. KEGG enrichment analysis showed significant enrichment in the neurotrophic factor signaling pathway and the PI3K-Akt pathway (A). Figure 10B). Combining PPI network analysis with KEGG pathway enrichment results, it is preliminarily speculated that PS-NPs may participate in the pathological progression of depression by interfering with the transduction of BDNF / TrkB-mediated PI3K-Akt signaling, thereby reducing the role of neurotrophic support.

[0057] Two transcriptome datasets, GSE256038 and GSE126521, were obtained from the public gene expression database Gene Expression Omnibus (GEO). The GSE256038 dataset is a dataset on the inflammatory gene expression profiles induced by polystyrene nanoparticles and microplastic particles in astrocytes derived from neural stem cells. Figure 11 A), the GSE126512 dataset is about changes in serotonergic circuits in neurons of SSRI-resistant depression patients ( Figure 11 B). Further analysis of the differentially expressed genes in the GSE256038 and GSE126521 datasets yielded 115 common genes. Figure 11 C), KEGG enrichment showed significant enrichment of the NF-κB signaling pathway (C), Figure 11 D).

[0058] These results indicate that inflammation, autophagy (PINK1), the BDNF / TrkB / PI3K / Akt pathway, and the NF-κB pathway are key factors in the exacerbation of depression by PS-NPs.

[0059] Example 4: In vivo experimental verification of key mechanisms (Western blot) Brain tissue from mice in each experimental group in Example 2 was collected, proteins were extracted by RIPA lysis, quantified by BCA, subjected to SDS-PAGE electrophoresis, transferred to PVDF membranes, blocked, and incubated with the corresponding primary antibodies (NLRP3, ...). TNF-α, IL-18, IL-1β, P62, LC3, Parkin, PINK1, PSD95, BDNF, Bax, Bcl-2, p-TrkB, TrkB, p-PI3K, PI3K, p-Akt, Akt, p-P65, P65), incubated overnight at 4°C, then incubated with secondary antibody at room temperature for 1 h, followed by ECL imaging.

[0060] Results: Compared with the CUMS group, the expression levels of IL-1β, IL-18, TNF-α, Arg-1, and NLRP3 in the inflammatory proteins of the PS-NPs group were significantly increased (P < 0.05). Figure 12 The expression levels of the autophagy-related protein P62 and the LC3-II / I ratio were significantly increased, indicating that autophagy flux was blocked; the expression levels of the mitochondrial autophagy proteins PINK1 and Parkin were significantly decreased. Figure 13This indicates that mitophagy is inhibited; the expression levels of synaptic plasticity proteins PSD95 and BDNF were significantly decreased (P < 0.05), suggesting that PS-NP exposure inhibits neuronal synaptic plasticity. Figure 14 The anti-apoptotic protein Bcl-2 was decreased, while the pro-apoptotic protein Bax was significantly increased. Figure 15 In the group exposed to PS-NPs, the expression levels of TrkB / PI3K / Akt were significantly lower than those in the Control and CUMS groups (P < 0.05), indicating that PS-NPs can inhibit the TrkB / PI3K / Akt signaling pathway. Figure 16 ).

[0061] The above results indicate that PS-NPs induce autophagy flux blockade, mitophagy inhibition, NF-κB inflammatory activation, TrkB / PI3K / Akt pathway inhibition, decreased synaptic plasticity, and neuronal apoptosis in vivo.

[0062] Example 5: In vitro cell experiments to verify the mechanism and autophagy flow inhibition This embodiment was used to verify PS-NPs-induced autophagy arrest, mitochondrial damage, and ROS burst in BV2 microglia, and to verify the intervention effect of MitoTEMPO.

[0063] 5.1 Effect of PS-NPs on BV2 cell viability: BV2 cells were seeded in 96-well plates ( The samples were treated with 0, 5, 25, 50, 100, 200, 300, and 400 μg / mL PS-NPs for 24 h, respectively, followed by incubation with CCK-8 for 40 min and measurement of OD450. The results showed ( Figure 17 ): Control group: Cell viability ≥95% within the range of 5-200 μg / mL; subsequent control groups: 5, 25, and 50 μg / mL.

[0064] 5.2 Effects of PS-NPs on BV2 inflammatory factors: Western blot was used to detect iNOS, TGF-β, TNF-α, and IL-18. The results showed ( Figure 18 When PS-NPs are at 40 μg / mL or higher, iNOS and IL-18 are significantly elevated; TGF-β is compensatorily elevated at 10-20 μg / mL; and TNF-α is significantly elevated at 160 μg / mL.

[0065] 5.3 Effects of PS-NPs on autophagy-related proteins: Western blot analysis of P62 and LC3. Results showed ( Figure 19 Compared with the control group, the PS-NPs group showed significantly increased P62 expression and a significantly upregulated LC3-II / I ratio.

[0066] 5.4 Transmission electron microscopy (TEM) observation of ultrastructure: Cells were collected, fixed with glutaraldehyde, then fixed with osmium tetroxide, dehydrated, embedded, ultrathinly sectioned, stained with uranium acetate and lead citrate, and observed under a TEM. Results are as follows: Figure 20 As shown: the mitochondrial cristae in the control group were clear and intact; the mitochondrial cristae in the LPS group were disordered; the low-dose PS-NPs group had a small number of particles in lysosomes; the medium-dose group had an increase in the number and size of particles in lysosomes; and the high-dose group had lysosomal membrane rupture and mitochondrial cristae breakage and vacuolization.

[0067] 5.5 Validation of autophagic flux blockade by chloroquine and rapamycin intervention: Control group, PS-NPs group (50 μg / mL), chloroquine group (CQ, 10 μM), and PS-NPs+CQ group were set up. Results are as follows... Figure 21 As shown: The PS-NPs+CQ group showed no significant increase in P62 and LC3-II / I compared to the PS-NPs group (no additive effect). Control group, PS-NPs group, rapamycin group (Rapa, 100 nM), and PS-NPs+Rapa group were set up. Results are as follows... Figure 22 As shown, the PS-NPs+Rapa group had significantly higher P62 and LC3-II / I levels compared to the PS-NPs group (P<0.05).

[0068] 5.6 Mitochondrial autophagy, membrane potential, and ROS detection: Western blot analysis of PINK1 and Parkin showed that PS-NPs reduced the expression of both. Figure 23 JC-1 staining was used to detect mitochondrial membrane potential, and the results showed that PS-NPs decreased the red-green fluorescence ratio in a dose-dependent manner. Figure 24 DCFH-DA staining for ROS detection showed that PS-NPs exhibited dose-dependent enhancement of green fluorescence ( ). Figure 25 The p-P65 / P65 ratio increases ( Figure 26 ).

[0069] 5.7 MitoTEMPO intervention: Pretreatment with MitoTEMPO (5 μM) for 1 h, followed by treatment with PS-NPs (50 μg / mL) for 24 h. Results showed ( Figure 27 , 28 MitoTEMPO can significantly reverse PS-NPs-induced increases in ROS and decreases in mitochondrial membrane potential.

[0070] Example 6: Conditioned culture medium experiment to verify the effect of microglia on neurons This embodiment is used to verify that activated microglia inhibit the neuronal TrkB / PI3K / Akt pathway by releasing inflammatory factors.

[0071] Supernatant from BV2 cells treated with different concentrations of PS-NPs (0, 5, 25, 50 μg / mL) for 24 h was collected, centrifuged to remove debris, and conditioned medium was obtained. SH-SY5Y neurons were then treated with the conditioned medium at a 1:50 ratio for 24 h. Figure 29 Western blot was used to detect Bax, Bcl-2, p-TrkB, p-PI3K, and p-Akt in neurons.

[0072] The results are as follows Figure 30 , 31 As shown, compared with the control group and the LPS group, the PS-NPs conditioned medium group showed significantly decreased Bcl-2 and significantly increased Bax (P<0.05), and significantly decreased levels of p-TrkB, p-PI3K, and p-Akt (P<0.05). This indicates that inflammatory factors secreted by activated microglia inhibit the neuronal TrkB / PI3K / Akt pathway and induce apoptosis.

[0073] In summary, PS-NPs first impair lysosomal function, leading to impaired autophagic degradation and blocked autophagic flux. This, in turn, triggers mitochondrial damage and ROS bursts, activates the NF-κB signaling pathway, promotes microglia activation and the release of inflammatory factors. These released inflammatory factors further inhibit the neuronal TrkB / PI3K / Akt signaling pathway, ultimately resulting in reduced synaptic plasticity and neuronal apoptosis, thereby exacerbating the development and progression of depression. Figure 32 As shown.

[0074] Example 7 A method for screening candidate drugs for the prevention or treatment of depression exacerbated by microplastic exposure, comprising: The test substance is brought into contact with microglia or nerve cells that have been treated with microplastics and are in a state of inhibited autophagic flow. Detect changes in autophagic flux levels, mitochondrial reactive oxygen species levels, NF-κB signaling pathway activity, or TrkB / PI3K / Akt signaling pathway activity in microglia or nerve cells; Among them, substances that can restore autophagic flux levels, reduce mitochondrial reactive oxygen species levels, inhibit NF-κB signaling pathway activity, or activate TrkB / PI3K / Akt signaling pathway activity were identified as candidate drugs.

[0075] Among them, microplastics are polystyrene nanoplastics.

[0076] Impaired autophagic flux manifests as an increased LC3-II / I ratio and / or increased p62 protein levels in cells.

[0077] NF-κB signaling pathway activity was assessed by detecting p-P65 protein expression levels; The activity of the TrkB / PI3K / Akt signaling pathway was assessed by detecting the expression levels of p-TrkB, p-PI3K, and / or p-Akt proteins.

[0078] Example 7 A kit for detecting the risk of microplastics exacerbating depression, the kit comprising: Reagents for detecting the levels of autophagic flux inhibition markers in biological samples from test subjects; and / or Reagents for detecting the levels of markers of microplastic-induced microglial cell activation; and / or A reagent for detecting markers of neuronal damage or reduced synaptic plasticity.

[0079] The markers of autophagic flux obstruction include the LC3-II / I ratio and p62 protein level; The microglia activation markers include the protein expression levels of IL-1β, TNF-α, and / or iNOS; The neuronal damage markers include the Bax / Bcl-2 ratio. The markers of reduced synaptic plasticity include the protein expression levels of PSD95 and / or BDNF.

[0080] The biological sample is a brain tissue sample, a cerebrospinal fluid sample, or a blood sample.

[0081] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for screening candidate drugs for the prevention or treatment of depression exacerbated by microplastic exposure, characterized in that, The method includes: The test substance is brought into contact with microglia or nerve cells that have been treated with microplastics and are in a state of inhibited autophagic flow. Detect changes in autophagic flux levels, mitochondrial reactive oxygen species levels, NF-κB signaling pathway activity, or TrkB / PI3K / Akt signaling pathway activity in microglia or nerve cells; Among them, substances that can restore autophagic flux levels, reduce mitochondrial reactive oxygen species levels, inhibit NF-κB signaling pathway activity, or activate TrkB / PI3K / Akt signaling pathway activity were identified as candidate drugs.

2. The method according to claim 1, characterized in that, The microplastics are polystyrene nanoplastics.

3. The method according to claim 1 or 2, characterized in that, Impaired autophagic flux manifests as an increased LC3-II / I ratio and / or increased p62 protein levels in cells.

4. The method according to claim 1, characterized in that, NF-κB signaling pathway activity was assessed by detecting p-P65 protein expression levels; The activity of the TrkB / PI3K / Akt signaling pathway was assessed by detecting the expression levels of p-TrkB, p-PI3K, and / or p-Akt proteins.

5. A reagent kit for detecting the risk of microplastics exacerbating depression, characterized in that, The kit contains: Reagents for detecting the levels of autophagic flux inhibition markers in biological samples from test subjects; and / or Reagents for detecting the levels of markers of microplastic-induced microglial cell activation; and / or A reagent for detecting markers of neuronal damage or reduced synaptic plasticity.

6. The reagent kit according to claim 5, characterized in that, The markers of autophagic flux obstruction include the LC3-II / I ratio and p62 protein level; The microglia activation markers include the protein expression levels of IL-1β, TNF-α, and / or iNOS; The neuronal damage markers include the Bax / Bcl-2 ratio. The markers of reduced synaptic plasticity include the protein expression levels of PSD95 and / or BDNF.

7. The reagent kit according to claim 6, characterized in that, The biological sample is a brain tissue sample, a cerebrospinal fluid sample, or a blood sample.

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