A method for constructing an animal model of depression-like behavior induced by nonylphenol and application thereof

Abstract

The application discloses a kind of n-nonyl phenol (NP) induced depressive behavior animal model construction method and its application.The method is divided into control group, NP low, medium and high dose group, knockout control group and NP high dose combined knockout group by wild type and Parkin gene knockout mice, continuous gavage 90 days;Through behavior, neurotransmitter, histopathology, mitochondrial electron microscopy, autophagy protein, intestinal barrier and flora 16S sequencing detection, comprehensive evaluation of the neurotoxicity and intestinal brain axis injury of NP.The results show that NP exposure dose-dependent induces depressive behavior, and causes hippocampal neuron damage, colon barrier damage, inflammation increases and flora disorder;Parkin knockout significantly aggravates the above changes, confirms that PINK1 / Parkin pathway function loss is the key susceptible factor of NP induced depression.The application first establishes mitochondria autophagy-intestinal brain axis combined evaluation system, provides experimental basis for environmental pollutant induced depression risk assessment and drug screening with Parkin as target.

Description

Technical Field

[0001] This invention belongs to the fields of environmental toxicology and neurobiology, specifically relating to a method for constructing an animal model of nonylphenol-induced depressive-like behavior and its application. Background Technology

[0002] Nonylphenol (NP) is a typical alkylphenol endocrine disruptor, widely used in the industrial production of surfactants, emulsifiers, pesticide adjuvants, and plastic additives. Due to its strong lipophilicity and recalcitrant nature, NP can persist in the environment and accumulate through the food chain. Humans primarily ingest NP through contaminated food and drinking water, and it has been widely detected in various environmental media, including surface water, groundwater, soil, and air. NP exhibits significant estrogen-like activity, interfering with the endocrine system and producing toxic effects on multiple systems, including the reproductive, immune, nervous, and cardiovascular systems. In recent years, the neurotoxicity of NP has received increasing attention. Existing studies have shown that NP can cause neurotoxicity through pathways such as affecting membrane permeability, autophagy, oxidative damage, mitochondrial function, and DNA breaks in neurons in brain regions such as the cerebral cortex, hippocampus, and pituitary gland. Low-dose exposure can lead to behavioral abnormalities, memory impairment, and central nervous system cell apoptosis in rats. However, current research on the mechanisms of NP neurotoxicity remains relatively limited, lacking in-depth and systematic explanation.

[0003] Depression is a prevalent mental disorder worldwide, with a complex pathogenesis. Besides the classic neurotransmitter imbalance and neuroinflammation hypotheses, the regulation of depression by the gut microbiota through the "microbe-gut-brain" axis has become a research hotspot in recent years. The "microbe-gut-brain" axis achieves bidirectional regulation through three pathways: neural, endocrine, and immune. Gut microbiota can secrete neuroactive substances such as serotonin and dopamine; their metabolites, short-chain fatty acids, can maintain intestinal barrier integrity and reduce inflammatory responses; increased intestinal permeability can trigger "leaky gut syndrome," leading to lipopolysaccharide translocation into the bloodstream, excessive activation of the immune system, and a large release of pro-inflammatory factors, thereby activating the kynurenine pathway, reducing 5-HT synthesis, and ultimately inducing depression. Previous studies have confirmed that gut microbiota dysbiosis is common in patients with depression, manifested as an increased relative abundance of Bacteroidetes and a significant decrease in Firmicutes. Studies by Pan et al. have shown that NP exposure can dose-dependently alter the structure of the rat gut microbiota, reduce microbial diversity related to short-chain fatty acid synthesis, and impair intestinal barrier function. Not only does NP have adverse effects on the structure of the gut microbiota, it can also exacerbate central nervous system damage through the gut-brain axis. However, whether NP induces depression by interfering with the homeostasis of the gut-microbiota axis, and the underlying molecular regulatory mechanisms, remain unclear.

[0004] The PINK1 / Parkin signaling pathway is one of the core pathways regulating mitophagy. Together, they recognize and clear damaged mitochondria, playing a crucial role in maintaining mitochondrial quality control and cellular homeostasis. In recent years, the role of this pathway in the development of depression has received increasing attention. Studies have shown that PINK1 deficiency can lead to abnormal hippocampal neuronal differentiation and reduce the tolerance of mice to stress-induced depression; mitochondrial dysfunction is considered a key factor in the development of depression. In the gut, PINK1 / Parkin-mediated mitophagy can maintain intestinal mucosal barrier function by clearing damaged mitochondria and mitigating oxidative stress damage. However, existing research largely focuses on the role of this pathway in neurodegenerative diseases such as Parkinson's disease, and its regulatory function in environmentally pollutant-induced depression lacks systematic exploration. In particular, whether the PINK1 / Parkin pathway participates in the process of NP interference with the gut-microbe axis homeostasis and inducing depression currently lacks direct evidence.

[0005] In summary, the existing technology has at least the following shortcomings: (1) Although NP is known to be neurotoxic and can affect the gut microbiota, there is a lack of systematic verification on whether NP induces depressive-like behavior through the "microbe-gut-brain" axis; (2) The specific role of PINK1 / Parkin-mediated mitophagy in NP-induced intestinal barrier damage, neuroinflammation, and microbiota dysbiosis is still unclear; (3) Currently, there is a lack of a joint evaluation system that integrates mitophagy, intestinal barrier function, gut microbiota, and neurobehavioral endpoints, making it difficult to fully reveal the complete mechanism by which environmental pollutants induce depression through the gut-brain axis. Therefore, it is urgent to establish an animal model and evaluation method that can systematically evaluate NP-induced depression through the "microbe-gut-brain" axis, and clarify the key regulatory role of the PINK1 / Parkin pathway in this process, so as to provide an experimental basis for risk assessment and targeted intervention of environmental pollutant-induced depression. Summary of the Invention

[0006] This invention discloses a method for constructing an animal model of nonylphenol-induced depressive-like behavior and its application. By administering nonylphenol via gavage to wild-type and Parkin gene knockout mice, combined with behavioral, histopathological, mitochondrial autophagy protein, intestinal barrier, and microbiome sequencing analyses, it was found that nonylphenol induces depression by blocking mitophagy and disrupting gut-brain axis homeostasis through the PINK1 / Parkin pathway, with Parkin deficiency exacerbating the damage. This model provides an experimental basis for risk assessment of environmental pollutant-induced depression and screening for antidepressant targets.

[0007] On the one hand, the present invention provides a method for constructing an animal model of nonylphenol-induced depressive-like behavior, which adopts the following technical solution: A method for constructing an animal model of nonylphenol-induced depressive-like behavior, characterized by comprising the following steps: S1. Animal grouping: Wild-type C57BL / 6 mice and Parkin gene knockout C57BL / 6 mice were randomly divided into the following six groups: control group, low-dose NP group, medium-dose NP group, high-dose NP group, Parkin knockout control group, and high-dose NP combined with Parkin knockout group. S2. Treatment with poison: Mice in each group were given corn oil as a solvent and administered the poison by gavage at a volume of 10 mL / kg for 90 consecutive days. S3. Behavioral evaluation: After the drug exposure ended, the mice were assessed for depressive-like behavior through the forced swimming test and the tail suspension test. S4. Histological examination: Mouse hippocampal and colon tissues were taken, and histopathological morphological changes were observed by HE staining. The ultrastructure of mitochondria was observed by transmission electron microscopy. S5. Molecular biological detection: Serum dopamine and serotonin levels, as well as IL-6 and TNF-α levels in hippocampal and colon tissues, were detected by ELISA; the expression levels of PINK1, Parkin, p62, and LC3-I / II proteins in hippocampal and colon tissues were detected by Western blotting. S6. Intestinal barrier function detection: The levels of diamine oxidase and secretory immunoglobulin A in colonic tissue were detected by ELISA; the expression levels of tight junction proteins Claudin-1 and Occludin in colonic tissue were detected by Western blotting. S7. Intestinal flora analysis: Mouse feces were collected, and the abundance and diversity of intestinal flora were analyzed by 16S rDNA sequencing. The content of short-chain fatty acids in feces was detected by gas chromatography.

[0008] Preferably, in step S1, the dose of the low-dose NP group is 0.25 mg / kg, the dose of the medium-dose NP group is 2.5 mg / kg, and the dose of the high-dose NP group is 25 mg / kg; the dose of the high-dose NP combined with the Parkin knockout group is 25 mg / kg; and the control group and the Parkin knockout control group are given an equal volume of corn oil.

[0009] Preferably, the conditions for the forced swimming experiment in step S3 are: water temperature 25℃, and the time the mouse remains still within 5 minutes is observed and recorded; the conditions for the tail suspension experiment are: the mouse is suspended with its tail fixed at 1 / 3 of its length, and the time the mouse remains still is recorded.

[0010] Preferably, the specific conditions for transmission electron microscopy observation in step S4 are as follows: take a 1 mm... 3 Tissue samples were fixed, dehydrated, embedded, and polymerized to prepare 70-80 nm ultrathin sections. The cross sections of the CA1 region of the hippocampus and the distal colon were observed under a transmission electron microscope. One sample was used in each group, and three fields of view were randomly selected at 8000x magnification.

[0011] Preferably, the dilution ratio of the primary antibody in step S5 using Western blotting is: Parkin 1:1000, PINK1 1:1000, p62 1:1000, LC3-I / II 1:1000, and the internal control is GAPDH 1:1000.

[0012] Preferably, the detection indicators for short-chain fatty acids in step S6 include acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid, with 2-ethylbutyric acid as an internal standard, and quantitative detection is performed by gas chromatography.

[0013] Preferably, the 16S rDNA sequencing in S7 uses the V3-V4 hypervariable region, and PCR amplification is performed using primers 341F / 806R. Sequencing is completed on the Illumina NovaSeq 6000 platform in PE250 paired-end mode.

[0014] Preferably, the genotype identification of Parkin gene knockout C57BL / 6 mice is performed by PCR, and the primer sequences used for identification include wild-type upstream primer, mutant upstream primer and common downstream primer.

[0015] On the other hand, this invention also provides an application of a method for constructing an animal model of nonylphenol-induced depressive-like behavior, employing the following technical solution: The application of a method for constructing an animal model of nonylphenol-induced depressive-like behavior in assessing the neurotoxicity of nonylphenol via the "microbe-gut-brain" axis.

[0016] Application of a method for constructing an animal model of nonylphenol-induced depressive-like behavior in the screening of antidepressant drugs targeting the PINK1 / Parkin pathway.

[0017] In summary, the beneficial effects of the present invention are as follows: This invention establishes for the first time a joint evaluation system integrating mitophagy, intestinal barrier function, gut microbiota, and neurobehavioral endpoints, enabling a systematic assessment of the complete mechanism by which nonylphenol (NP) induces depressive-like behavior via the "microbe-gut-brain" axis. Through comparative experiments with wild-type and Parkin knockout mice, this invention clearly demonstrates for the first time that NP exposure can block mitophagy by inhibiting the PINK1 / Parkin pathway, leading to hippocampal neuronal damage, colonic mucosal barrier disruption, increased systemic and central inflammatory responses, and gut microbiota dysbiosis (such as decreased abundance of Lactobacillus and Bacteroides, and reduced propionic acid production), ultimately inducing depressive-like behavior. Parkin deficiency significantly exacerbates these pathological changes, thus confirming the PINK1 / Parkin pathway as a key regulatory node in NP-induced depression. This evaluation system has the significant advantages of multi-indicator linkage and a clear mechanism, overcoming the shortcomings of single-indicator evaluation in existing technologies and providing a reliable technical means for assessing the neurotoxicity risk of endocrine disruptors.

[0018] Meanwhile, this invention confirms the crucial protective role of Parkin in maintaining gut-brain axis homeostasis, providing a novel animal model and intervention validation platform for screening antidepressants targeting the PINK1 / Parkin pathway. Based on the key bacterial metabolic abnormalities revealed in this invention, such as reduced propionic acid and decreased abundance of Lactobacillus, further intervention studies can be conducted through exogenous supplementation of short-chain fatty acids or specific probiotics, providing new potential strategies for the clinical prevention and treatment of depression caused by environmental pollutants. This invention not only provides a theoretical basis for elucidating the toxicological mechanisms of depression induced by environmental pollutants, but also provides a practical tool for environmental health risk assessment and early warning of neuropsychiatric diseases in the public health field, possessing significant scientific research value and translational application prospects. Attached Figure Description

[0019] Figure 1 Behavioral experiments and serum neurotransmitter levels after NP exposure and Parkin knockout.

[0020] in, Figure 1 A in the diagram represents the trajectory of the forced swimming experiment. Figure 1 B is a bar chart showing the forced swimming stillness time; Figure 1 C is a bar chart of the resting time in the tail suspension experiment; Figure 1 The bar chart in section D represents the serum dopamine (DA) content; Figure 1 The bar chart in section E shows the serum 5-hydroxytryptamine (5-HT) content. Figure 2 The levels of inflammatory factors in the hippocampus and colon of mice after NP exposure and Parkin knockout; in, Figure 2 The bar chart in section A shows the colonic IL-6 content. Figure 2 The bar chart in section B shows the colonic TNF-α content. Figure 2 The middle bar chart (C) shows the hippocampal IL-6 content. Figure 2 The middle D is a bar chart of hippocampal TNF-α content; Figure 3 The levels of indicators related to colonic mucosal barrier function in mice after NP exposure and Parkin knockout; in, Figure 3 In the middle, A is a bar chart showing the content of diamine oxidase (DAO) in colon tissue; Figure 3 Bar chart B shows the content of secretory immunoglobulin A (sIgA) in colonic tissue; Figure 4 HE staining results of the colon of mice after NP exposure and Parkin knockout; in, Figure 4 In section A, colon tissue stained with hematoxylin and eosin (HE) under a 200× microscope; Figure 4 Medium B is colon tissue stained with HE under a 400× microscope; Figure 5 HE staining results of the hippocampus in mice after NP exposure and Parkin knockout; in, Figure 5 Middle A is hippocampal tissue stained with hematoxylin and eosin (HE) under a 200× microscope; Figure 5 Middle B is hippocampal tissue stained with HE under a 400× microscope; Figure 6 Electron transmission microscopy results of hippocampal and colonic mitochondria after NP exposure and Parkin knockout, and the number of mitochondria and autophagosomes. in, Figure 6 A in the middle represents the results of transmission electron microscopy of the hippocampus; Figure 6 B represents the results of a colonic transmission electron microscope. Figure 6 In the middle, C represents the number of mitochondria in the hippocampus; Figure 6 In the middle, D represents the number of mitochondria in the colon; Figure 6 In this context, E represents the number of autophagosomes; Figure 7 The expression levels of autophagy proteins in the hippocampus after NP exposure and Parkin knockout; in, Figure 7Image A shows the Western blotting bands of PINK1, Parkin, p62, and LC3-I / II proteins in the hippocampus. Figure 7 The bar chart in section B shows the relative expression level of PINK1 protein in the hippocampus. Figure 7 The bar chart in section C represents the relative expression level of Parkin protein in the hippocampus. Figure 7 The middle section (D) is a bar chart showing the LC3-II / LC3-I ratio in the hippocampus. Figure 7 The bar chart in section E shows the relative expression level of p62 protein in the hippocampus. Figure 8 The expression levels of colonic autophagy proteins after NP exposure and Parkin knockout; in, Figure 8 Image A shows the Western blotting bands of PINK1, Parkin, p62, and LC3-I / II proteins in colon tissue. Figure 8 Bar chart B shows the relative expression levels of Parkin protein in colon tissue; Figure 8 The bar chart in section C represents the relative expression level of PINK1 protein in colon tissue; Figure 8 The bar chart in section D shows the relative expression level of p62 protein in colon tissue; Figure 8 E is a bar chart showing the LC3-II / LC3-I ratio in colon tissue; Figure 9 The expression levels of Claudin-1 and Occludin in the colon; in, Figure 9 Image A shows the Western blotting of Claudin-1 and Occludin proteins in colon tissue. Figure 9 B in the chart represents the horizontal histogram of Claudin-1 expression. Figure 9 In the bar chart, C represents the Occludin expression. Figure 10 Bar chart showing the content of short-chain fatty acids in the feces of mice after NP exposure and Parkin knockout; in, Figure 10 In the middle, A represents a bar chart showing the acetic acid content; Figure 10 B is a bar chart showing the propionic acid content; Figure 10 The bar chart in section C represents the butyric acid content. Figure 10The bar chart in section D shows the total short-chain fatty acid content. Figure 11 Bar chart showing the α-diversity index of gut microbiota in mice after NP exposure and Parkin knockout; in, Figure 11 A in the middle is a bar chart of the Chao1 index; Figure 11 B in the chart represents the ACE index histogram; Figure 11 C in the middle is the Shannon index histogram; Figure 11 D in the middle is a bar chart of the Simpson index; Figure 12 This represents the β-diversity of the mouse gut microbiota. Figure 13 Stacked plot of relative abundance of gut microbiota at the phylum level in mice after NP exposure and Parkin knockout; Figure 14 Stacked plot of relative abundance of gut microbiota genera in mice after NP exposure and Parkin knockout; Figure 15 Bar chart showing the relative abundance of specific species in the gut microbiota of mice after NP exposure and Parkin knockout; in, Figure 15 A in the middle represents Lactobacillus ( ) Lactobacillus A relative abundance histogram of ). Figure 15 B in the middle is Bacteroides ( Bacteroides A relative abundance histogram of ). Figure 15 C is Alloprevotella Relative abundance histogram Figure 15 D is Lachnospiraceae NK4A136 group Relative abundance histogram; Figure 16 A taxonomic branching diagram for the effect size analysis (LEfSe) in linear discriminant analysis; Figure 17 A bar chart showing the distribution of LDA values ​​for species with significant differences in effect size analysis in linear discriminant analysis; Figure 18 A diagram illustrating the mechanism by which PINK1 / Parkin-mediated mitophagy plays a role in NP-mediated depression induced by interference with gut-microbe axis homeostasis. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to the embodiments.

[0022] Example 1 The specific steps for feeding and grouping mice and preparing reagents are as follows: Thirty-two 28-day-old SPF-grade wild-type C57BL / 6 mice (purchased from the Laboratory Animal Center of Zunyi Medical University, with the experimental animal production license number: SYXK (Qian) 2021-0004) and 16 28-day-old Parkin knockout C57BL / 6 mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd., SCXK (Su) 2018-0008) with a body weight of 18-25 g were selected. After one week of adaptive feeding, the mice were randomly divided into six groups: control group (corn oil, C), NP low-dose group (0.25 mg / kg, L), NP medium-dose group (2.5 mg / kg, M), NP high-dose group (25 mg / kg, H), Parkin knockout group (commercial corn oil, KC), and NP high-dose + Parkin knockout group (25 mg / kg, KH), with 8 mice in each group. The mice were housed in an SPF-grade animal room. After weighing every morning, the mice were exposed by gavage at a volume of 10 mL / kg for 90 consecutive days. Dose basis: NP is widely present in food, drinking water and water environment, with a concentration of 0.1-100 μg / kg in food and 0.0195-8.54 μg / L in surface water, and the EU freshwater limit is 0.3 μg / L. Combined with previous studies showing that 400 μg / kg NP can induce brain damage and anxiety-like behaviors in rats, the doses in this study were set by comprehensively considering the environmental concentration, bioaccumulation effect and species difference: the L group was set at 0.25 mg / kg. This dose is mainly based on the environmental exposure level, and with reference to the dose (400 μg / kg) at which toxic effects were observed in rats in the previous study of the research group, it was determined after species conversion and adjustment: Dmouse = Drat×Kmouse / Krat×F = 0.4 mg / kg×3 / 6×1.25 = 0.25 mg / kg, where Drat = 0.4 mg / kg (effective dose for rats), Kmouse / Krat = 0.5 (species conversion factor), F = 1.25 (correction factor for environmental concentration and accumulation). Basis for setting the M group: the M group was set at 2.5 mg / kg. This dose was converted based on the Tolerable Daily Intake (TDI) of NP for Chinese people: equivalent dose for mice = human TDI × species factor × uncertainty factor = 0.025 mg / kg × 100 = 2.5 mg / kg. Among them, the species factor (10) is used to correct the metabolic difference between humans and mice, the uncertainty factor (10) is used to compensate for individual differences and the uncertainty of species extrapolation, and the total conversion factor is 100. Basis for setting the high-dose group (H group): the high-dose group was set at 25 mg / kg. This dose was determined based on the median lethal dose (LD50) of NP via oral exposure in mice: high dose = LD50 / 50 = 1231 mg / kg ÷ 50 ≈ 25 mg / kg.Solvent selection and control group setup: Due to the strong lipid solubility of nonylphenol, corn oil was selected as the gavage solvent in this study. Mice in the blank control group were simultaneously administered an equal volume of corn oil by gavage to eliminate solvent interference. This study was approved by the Animal Experiment Ethics Committee of Zunyi Medical University (ZMU21-2302-269).

[0023] Example 2 Behavioral experiments were conducted on six groups of mice (n=8 per group): C, L, M, H, KC, and KH. The specific steps are as follows: S1, Tail Suspension Test In the experiment, the tail section of the mouse was fixed in place, leaving it suspended head down. The mouse struggled in this environment, attempting to escape the predicament. The duration of stillness after the mouse failed to escape was directly proportional to its degree of depression.

[0024] S2, Forced Swimming Experiment Before the experiment, fill the cylinder with water to 2 / 3 of its height and adjust the water temperature to about 25℃. Place the mice in the water and observe and record the duration of stillness for 5 minutes. Stillness is defined as the mouse's limbs remaining still and its head floating on the surface of the water. The duration of stillness is directly proportional to the degree of depression.

[0025] Figure 1 The forced swimming experiment in China and Figure 1 The results of the BC tail suspension test showed that, compared with the control group, the resting time of mice in the high-dose NP group (H group), the Parkin knockout control group (KC group), and the high-dose NP combined with Parkin knockout group (KH group) were significantly prolonged. Among them, the forced swimming resting time of the KH group was longer than that of the H group and the KC group, indicating that both NP exposure and Parkin knockout can induce depressive-like behavior and that the two have a synergistic aggravating effect.

[0026] Example 3 The ELISA method was used to detect the levels of relevant indicators in serum, colon, and hippocampus tissues. The specific steps are as follows: S1. Sample pretreatment: Serum sample: Take out the mouse serum stored at -80℃, centrifuge at 2000 r / min for 10 min, and collect the supernatant for later use.

[0027] Tissue homogenate samples: Colon and hippocampal tissues were collected and pre-cooled PBS (pH 7.4) was added at a mass-to-volume ratio of 1:9. The samples were homogenized in an ice-water bath and centrifuged at 4°C and 3000 r / min for 15 min. The supernatant was collected for later use.

[0028] S2, ELISA detection steps: Add 50 μL of standard or sample (serum or tissue homogenate supernatant) to each well, with 10 μL of sample followed by 40 μL of sample diluent in the sample wells; except for the blank wells, add 100 μL of enzyme-labeled reagent to each well and incubate at 37°C for 60 min; discard the liquid in the wells, wash the plate 5 times with 20-fold diluted washing buffer, soaking for 20 s each time, and pat dry; add 50 μL each of chromogenic reagent A and B to each well sequentially, mix well, and develop color at 37°C in the dark for 15 min (the color intensity is directly proportional to the content of the target indicator); add 50 μL of stop solution to each well to terminate the reaction, and immediately measure the absorbance (OD value) of each well at 450 nm using an enzyme-linked immunosorbent assay reader; plot a standard curve using the standard concentrations and corresponding OD values, and calculate the content of each indicator based on the sample OD values.

[0029] Serum levels of dopamine (DA) and serotonin (5-HT) were detected using a DA ELISA kit and a 5-HT ELISA kit, respectively. The contents of inflammatory factors IL-6 and TNF-α in colon and hippocampal tissue homogenates were detected using IL-6 ELISA kit and TNF-α ELISA kit, respectively. The contents of secretory immunoglobulin A (sIgA) and diamine oxidase (DAO) in colon tissue were detected using sIgA ELISA kit and DAO ELISA kit, respectively.

[0030] Figure 1 Serum DA levels in the D group showed that DA levels were lower in all NP-exposed groups and Parkin knockout groups than in the control group, and DA levels in the KH group were lower than those in the H and KC groups. Figure 1 Serum 5-HT levels in patients with acute extremity leukemia (ALE) were measured, and all groups except group H showed elevated 5-HT levels compared to the control group. These results indicate that NP exposure and Parkin knockout can lead to disordered serum neurotransmitter secretion, manifested as a significant decrease in dopamine (DA) and an abnormal increase in 5-HT. Furthermore, Parkin deficiency can exacerbate NP-induced damage to the dopaminergic system.

[0031] Figure 2 The effects of NP exposure and Parkin knockout on the levels of inflammatory factors in the hippocampus and colon tissues of mice were demonstrated. Figure 2 The level of IL-6 in the middle A colon and Figure 2 The colonic TNF-α content in the middle B colon showed that, compared with the control group, the IL-6 and TNF-α content in all NP exposure groups except KC group was significantly increased; the colonic IL-6 and TNF-α content in KH group was higher than that in H group and KC group. Figure 2 Hippocampal IL-6 content and Figure 2The analysis of hippocampal TNF-α levels in the KH group showed that, except for the KC group, the levels of IL-6 and TNF-α in the hippocampus were higher than those in the control group. The TNF-α level in the hippocampus of the KH group was higher than that of the H group. These results indicate that NP exposure can induce inflammatory responses in the colon and hippocampus, and Parkin knockout can significantly exacerbate NP-induced systemic and central inflammatory damage, as evidenced by the fact that the levels of inflammatory factors in the KH group were generally higher than those in the NP exposure or gene knockout groups alone.

[0032] Figure 3 The effects of NP exposure and Parkin knockout on indices related to colonic mucosal barrier function in mice were demonstrated. Figure 3 The DAO content in the colon tissue of the middle A group showed that the DAO levels in the M group, H group, KC group and KH group were higher than those in the control group, and the DAO content in the KH group was higher than that in the H group and KC group. Figure 3 The sIgA content in the mid-B colon tissue showed that the sIgA levels in groups M, H, and KH were higher than those in the control group, with the sIgA content in group KH being higher than that in groups H and KC. DAO and sIgA are core serological indicators for evaluating intestinal barrier function, and elevated levels suggest impaired intestinal mucosal barrier function. These results indicate that NP exposure can damage intestinal mucosal barrier function, and Parkin knockout can further exacerbate NP-induced intestinal barrier damage, making it easier for pro-inflammatory substances such as lipopolysaccharide to enter the bloodstream, thereby aggravating systemic and central inflammatory responses.

[0033] Example 4 Colon and hippocampal tissues from each group of mice (n=3 per group) were collected for HE staining. The specific steps are as follows: Fresh hippocampal and distal colon tissues were collected and fixed in 50 mL centrifuge tubes containing formaldehyde fixative. The tissues were then transferred to a 15% sucrose solution A for dehydration, followed by a transfer to a 30% sucrose solution B. This experiment employed a cryo-embedding method, where the fully sucrose-embedded tissues were rapidly frozen in liquid nitrogen and stored at -80°C. After sectioning, the tissues were either directly fixed or stored at -20°C for later use. Hematoxylin-eosin (HE) staining was used to observe the cross-sections of the hippocampal CA1 region and the distal colon.

[0034] Figure 4 The pathological changes in the colon tissue of mice after NP exposure and Parkin knockout are shown. Figure 4 In Figure A, the colon was stained with HE under a 200x microscope. It can be seen that the colon tissue structure of the control group was intact, while the colon tissue of the H and KH groups showed local mucosal degeneration and necrosis. Figure 4In Figure B, observed under a 400x microscope, necrotic cells showed shrunken and fragmented nuclei, cytoplasmic dissolution, and blurred structure. The degree of necrosis was more pronounced in the KH group than in the H group. These results indicate that subchronic NP exposure can lead to colonic mucosal damage and induce intestinal epithelial cell necrosis, and that Parkin knockout can exacerbate NP-induced pathological damage to colonic tissue. This is consistent with... Figure 3 The elevated levels of DAO and sIgA corroborated each other, indicating that NP exposure and Parkin deficiency can disrupt the integrity of the intestinal mucosal barrier.

[0035] Figure 5 The pathological changes in the hippocampus of mice after NP exposure and Parkin knockout are shown. Figure 5 In the middle section, the hippocampus was stained with HE under a 200x microscope, and neuronal degeneration was observed in all groups except the control group. Figure 5 The image in section B, viewed under a 400x microscope, shows shrunken neuronal cell bodies, deeply basophilic staining of the cytoplasm and nucleus, nuclear pyknosis, and indistinct nucleoli. Neuronal degeneration in the KH group was more severe than in the KC group. The hippocampus is a key brain region regulating emotion and cognition, and neurons in its CA1 region are particularly sensitive to stress and inflammation. These results indicate that NP exposure can induce hippocampal neuronal degeneration and necrosis, and Parkin knockout significantly exacerbates NP-induced neuronal damage, providing direct neuropathological evidence for NP exposure and Parkin deficiency inducing depressive-like behavior.

[0036] Example 5 The ultrastructure of mitochondria in hippocampal and colonic tissues was observed using transmission electron microscopy. The specific steps are as follows: For each group, tissue from the same location in the hippocampus and colon was rapidly cut into 1 mm pieces within 1-3 minutes. 3 Samples were fixed in electron microscopy fixative. They were pre-cooled at 4°C and rinsed three times on ice with PBS (pH 7.4) for 10 min each time. After stepwise dehydration with 30%–100% graded ethanol, samples were infiltrated at 4°C for 1 h, 3 h, and 17–20 h at ethanol:pure resin ratios of 2:1, 1:1, and 1:2, respectively, followed by two infiltrations at 4°C with pure resin (17–20 h and 1 h). Samples were then encapsulated in pure resin, vacuum-sealed for 1 h, and then polymerized under UV light at -20°C for at least 48 h. After polymerization, 70–80 nm sections were prepared using an ultramicrotome, retrieved using a nickel-coated mesh, and stored at 4°C. Sections were stained with 2% uranium acetate-saturated alcohol in the dark for 8 min, washed three times each with 70% alcohol and ultrapure water, blotted dry with filter paper, and then dried overnight at 37°C. Transmission electron microscopy was used to observe and photograph the CA1 region of the hippocampus and the transverse section of the distal colon. One sample was used in each group, and three fields of view were randomly selected at 8000x magnification.

[0037] Figure 6 The changes in the ultrastructure of mitochondria in hippocampal and colon tissues after NP exposure and Parkin knockout were shown. Figure 6 Transmission electron microscopy results of the hippocampus showed that the mitochondrial structure of the hippocampal neurons in the control group was normal. The NP exposure group and the Parkin knockout group showed varying degrees of mitochondrial swelling, cristae breakage and matrix flocculentization. The KH group was the most severely damaged, showing highly swollen mitochondria, matrix vacuolation, and a large number of autophagosomes and lysosomes. Figure 6 The number of mitochondria in the hippocampus of group C showed that, except for group M, the number of mitochondria in all groups was lower than that in the control group. Figure 6 The number of autophagosomes in the H, KC and KH groups was higher than that in the control group. Figure 6 Transmission electron microscopy of the colon in the middle B group showed that the number of mitochondria in the colonic epithelial cells of the H, KC and KH groups was increased compared with the control group, but no obvious mitophagy was observed. Figure 6 The statistical analysis of colonic mitochondrial numbers further validated the above changes. These results indicate that NP exposure can induce mitochondrial damage in hippocampal neurons and compensatory activation of mitophagy, while Parkin knockout obstructs autophagic flux and leads to the accumulation of damaged mitochondria. Although the number of mitochondria in colonic tissue increased compensatorily, typical autophagosomes were not observed, suggesting that the colon may respond to mitochondrial stress through other mechanisms.

[0038] Example 6 The expression of Parkin, PINK1, p62, LC3I / II, Occludin, and Claudin-1 proteins in hippocampal and colon tissues was detected by Western blotting. The specific steps are as follows: The tissue was homogenized at low temperature, centrifuged after standing, and the supernatant was collected for later use. Protein quantification was performed according to the BCA quantitative kit. Gel preparation and loading were performed; electrophoresis was performed first, followed by electroporation. After blocking with milk, the membrane was incubated overnight with primary antibody. Primary antibody concentrations were prepared as follows: Parkin (1:1000), PINK1 (1:1000), p62 (1:1000), LC3I / II (1:1000), Occludin (1:1000), Claudin-1 (1:1000), and GAPDH (1:1000). The membrane was incubated overnight at 4°C. The membrane was washed three times the next day, followed by incubation with secondary antibody for 1 hour.

[0039] Figure 7 The expression levels of mitochondrial autophagy-related proteins in hippocampal tissue were demonstrated after NP exposure and Parkin knockout. Figure 7 A in the image is a Western blotting plot. Figure 7 China B to Figure 7E represents the quantitative analysis of the relative expression levels of each protein. The results showed that compared with the control group, the expression of PINK1, p62, and LC3-II proteins was significantly increased in the KH group; compared with the H group, PINK1 expression was decreased and p62 protein expression was increased in the KH group, while LC3-II protein expression was decreased in the KC group; compared with the KC group, both LC3-II and p62 protein expression were increased in the KH group. PINK1 is a key sensor for initiating mitophagy, LC3-II reflects the level of autophagosome formation, and p62 is a biomarker for autophagy substrates. These results indicate that NP exposure can activate hippocampal mitophagy, but Parkin knockout leads to impaired autophagic flux (manifested as p62 accumulation and abnormally increased LC3-II), preventing the effective clearance of damaged mitochondria and thus inducing neuronal damage.

[0040] Figure 8 The expression levels of mitochondrial autophagy-related proteins in colon tissue were shown after NP exposure and Parkin knockout. Figure 8 A in the image is a Western blotting plot. Figure 8 China B to Figure 8 E represents the quantitative analysis of the relative expression levels of each protein. The results showed that compared with the control group, the expression of PINK1 and p62 proteins was increased in all groups, while the expression of LC3-II protein was decreased; compared with the H group, the LC3-II protein expression in the KH group was further decreased; compared with the KC group, the LC3-II protein expression in the KH group was also significantly decreased. These results indicate that NP exposure and Parkin knockout can induce the initiation of mitophagy in colonic tissue (increased PINK1), but autophagic flux is blocked in the later stages (decreased LC3-II with increased p62), and mature autophagosomes cannot be cleared normally. Figure 6 Electron microscopy of the midcolon did not reveal typical autophagosomes, further suggesting that the colonic tissue was in a state of impaired autophagic flux, which may be an important mechanism by which Parkin deficiency leads to abnormal mitochondrial DNA dynamics and inflammatory activation.

[0041] Figure 9 The expression levels of the tight junction proteins Claudin-1 and Occludin in colonic tissue were shown after NP exposure and Parkin knockout. Figure 9 A in the image is a Western blotting plot. Figure 9 The bar chart in section B shows the relative expression levels of Claudin-1 protein, indicating that Claudin-1 expression in group H was significantly higher than that in the control group, while no significant changes were observed in groups KC and KH. Figure 9The bar chart in Figure C represents the relative expression levels of Occludin protein, showing that while Occludin expression increased across groups, there were no statistically significant differences. Claudin-1 and Occludin are core proteins for maintaining the integrity of tight junctions in the intestinal epithelium. This study found that high concentrations of NP exposure led to an abnormal increase in Claudin-1, which is not a sign of enhanced barrier function, but rather a compensatory response of the intestinal mucosa under inflammatory stress. This compensatory mechanism was impaired after Parkin knockout (Claudin-1 was not significantly upregulated in the KH group), combined with... Figure 3 The elevated levels of DAO and sIgA in the middle jiao (a traditional Chinese medicine) indicate that Parkin deficiency can exacerbate NP-induced intestinal barrier dysfunction.

[0042] Example 7 The specific steps for detecting short-chain fatty acid content in mouse feces using GC are as follows: This study established a quantitative detection method for short-chain fatty acids (SCFAs) such as acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid in mouse feces using an Agilent 7890B gas chromatograph (FID detector). 2-Ethylbutyric acid was used as an internal standard, extracted with ultrapure water and acidified with 10% metaphosphoric acid. Chromatographically pure standards were prepared into a mixed stock solution, serially diluted, and an equal volume of internal standard was added to plot a standard curve. Fecal samples were kept on ice throughout the process. 50-100 mg of frozen feces were taken, mixed with ultrapure water containing the internal standard and grinding beads, homogenized at low temperature, and centrifuged at 12000 rpm for 15 min at 4°C. The supernatant was acidified with metaphosphoric acid, incubated on ice for 30 min, centrifuged again, filtered through an aqueous filter membrane, and then analyzed. An HP-INNOWax column was used, with both the injection port and detector temperature set at 250℃. High-purity helium was supplied at a constant flow rate of 1.0–1.5 mL / min, a split ratio of 10:1, and an injection volume of 1 μL. The column temperature program was as follows: 90℃ for 1 min, ramping up to 150℃ at 10℃ / min and holding for 1 min, then ramping up to 220℃ at 20℃ / min and holding for 5 min. Qualitative analysis was performed using retention time, and quantification was performed using an internal standard curve. The SCFA content (μg / g) in feces was calculated based on the sample weight.

[0043] Figure 10 The study showed changes in the content of short-chain fatty acids (SCFAs) in mouse feces after NP exposure and Parkin knockout. Figure 10 China A to Figure 10The bar charts for acetic acid, propionic acid, butyric acid, and total SCFAs are shown in Figures D and D, respectively. The results showed that compared with the control group, the total SCFAs content in each group did not change significantly. However, the propionic acid content in group H was significantly lower than that in the control group, showing a certain dose-response relationship. While the contents of acetic acid and butyric acid showed a decreasing trend among the groups, these differences were not statistically significant. Short-chain fatty acids are important metabolites of the gut microbiota, playing a role in maintaining the intestinal barrier and regulating immune and neuroendocrine functions. As an important component of SCFAs, the reduction in propionic acid reflects the inhibitory effect of NP exposure on the metabolic function of the gut microbiota, which may indirectly participate in the occurrence of depressive-like behaviors through the gut-brain axis.

[0044] Example 8 The specific steps for detecting the gut microbiota in mice are as follows: This study used 16S rDNA high-throughput sequencing technology to analyze the abundance and diversity of mouse gut microbiota. Fecal genomic DNA was extracted using the ZymoBIOMICS DNA Microprep Kit (Cat# D4301), and its integrity was checked by 0.8% agarose gel electrophoresis. Nucleic acid concentration was measured using a Tecan F200 microplate reader for quality control. The hypervariable region of 16S rDNA V3-V4 was selected and amplified by PCR using tagged primers 341F / 806R. Amplification was performed using KOD-Plus-Neo enzyme in a 50 μL system on an ABI 9700 microplate reader. The reaction program was: 94℃ pre-denaturation for 1 min, 30 cycles (94℃ 20 s, 54℃ 30 s, 72℃ 30 s), and final extension for 5 min. Each sample was amplified three times. The products from the linear amplification phase were mixed in equal volumes, verified by 2% agarose gel electrophoresis, and then recovered by gel excision using the Zymoclean Gel Recovery Kit. Quantification was performed using Qubit® 2.0, and the samples were mixed in equimolar proportions. Libraries were constructed using the NEBNext Ultra II library preparation kit, and sequencing was finally performed on the Illumina NovaSeq 6000 platform in PE250 paired-end mode.

[0045] Figure 11 This study demonstrates the changes in α-diversity of the gut microbiota in mice after NP exposure and Parkin knockout. Figure 11 China A to Figure 11The bar charts in the middle and D represent the Chao1, ACE, Shannon, and Simpson indices, respectively. The results showed that compared to the control group, the Simpson indices of groups M, KC, and KH were significantly increased. While there were no significant differences in the Chao1, ACE, and Shannon indices, they all showed an upward trend with increasing exposure concentration. α-diversity reflects the species richness and evenness of the gut microbiota; an increased Simpson index indicates decreased community diversity and increased concentration of dominant species. These results indicate that NP exposure can lead to a dose-dependent increase in α-diversity of the mouse gut microbiota (i.e., decreased diversity), and Parkin knockout itself can also cause similar changes, suggesting that both NP exposure and Parkin deficiency can disrupt the balance of the gut microbiota.

[0046] Figure 12 The changes in β-diversity of the mouse gut microbiota after NP exposure and Parkin knockout are presented as Bray-Curtis dissimilarity scatter plots. The results show that, compared with the control group, the dispersion of sample points on the principal axis increased with increasing NP concentration in each NP exposure group, but the differences between groups did not reach statistical significance. β-diversity reflects the degree of difference in species composition among different samples. These results indicate that NP exposure and Parkin knockout did not produce a significant deviation effect on the overall community structure of the mouse gut microbiota, suggesting that the impact of NP on the gut microbiota may mainly manifest as changes in the abundance of specific genera rather than a directional shift in the overall community structure.

[0047] Figure 13 This study demonstrates the changes in the relative abundance of gut microbiota at the phylum level in mice after NP exposure and Parkin knockout. Results showed that the gut microbiota in the control group was mainly composed of Bacteroidetes (approximately 55%), Firmicutes, Epsilonbacteraeota, and Proteobacteria. After NP exposure, the ratio of Bacteroidetes to Firmicutes became imbalanced, with the relative abundance of Firmicutes decreasing and the relative abundance of Proteobacteria increasing with increasing NP concentration. Alterations in the phylum-level microbiota structure are an important marker of gut microbiota imbalance, and a decreased Firmicutes / Bacteroidetes ratio is closely associated with inflammatory bowel disease and neuropsychiatric disorders. These results indicate that NP exposure can induce structural remodeling of the mouse gut microbiota at the phylum level, which is an important microbiological basis for NP-induced depression via the gut-brain axis.

[0048] Figure 14 This study demonstrates the changes in the relative abundance of Lactobacillus genus at the genus level in the gut microbiota of mice after NP exposure and Parkin knockout. Results showed that the abundance of Lactobacillus genus (…) was significantly higher in the control group. Lactobacillus ) and Bacteroides ( BacteroidesThe abundance of bacteria was relatively high, and the relative abundance of several genera changed after NP exposure. The abundance of *Lactobacillus* was significantly reduced in groups M, KC, and KH, while the abundance of *Bacteroides* was significantly reduced in all groups except group H. The abundance of *Lachnospiraceae* NK4A136 group and... Alloprevotella The average relative abundance of *Lactobacillus* showed an increasing trend, but the individual differences within the group were large and did not reach statistical significance. Changes at the genus level are more likely to reflect functional changes in specific bacterial communities. *Lactobacillus* and *Bacteroides* are important beneficial bacteria in the gut, and a decrease in their abundance can affect the production of short-chain fatty acids and the maintenance of intestinal barrier function, and is closely related to NP-induced depressive-like behavior.

[0049] Figure 15 The study demonstrates the changes in the relative abundance of specific species in the gut microbiota of mice after NP exposure and Parkin knockout. Figure 15 A in the middle represents Lactobacillus ( ) Lactobacillus The relative abundance of M, KC and KH groups was significantly lower than that of the control group. Figure 15 B in the middle is Bacteroides ( Bacteroides The relative abundance showed that all groups except group H had a significant decrease; Figure 15 C is Alloprevotella Relative abundance showed an increasing trend among groups, but there was no statistically significant difference. Figure 15 In the middle, D represents the relative abundance of the Lachnospiraceae NK4A136 group, with no significant changes among groups. Lactobacillus is a key probiotic for maintaining the intestinal barrier and immune homeostasis; a decrease in its abundance can lead to increased intestinal permeability. Bacteroides participates in polysaccharide metabolism and short-chain fatty acid production; its reduction is closely related to inflammation and metabolic disorders. These results indicate that Parkin knockout itself can lead to a decrease in Lactobacillus abundance and exacerbate NP exposure-induced gut microbiota dysbiosis, suggesting that Parkin plays an important role in maintaining gut microbiota homeostasis.

[0050] Figure 16 This is a taxonomic clade diagram for the effect size analysis in linear discriminant analysis. In the diagram, circles from the inside out represent taxonomic levels from phylum to genus; yellow nodes represent species with no significant differences; and colored nodes represent marker species significantly enriched in their corresponding groups. The results show that group KH is dominated by Tannerellaceae family and... Parabacteroides It belongs to the characteristic enrichment group, group H is characterized by Prevotella_9 Characterized by the genera and family Veillonellaceae, group M is characterized by Prevotellaceae_UCG_001, etc. This analysis indicates that Parkin knockout combined with NP exposure can induce the presence of Bacteroidetes core flora (such as...) in the mouse gut. ParabacteroidesAbnormal abundance of specific microbiota (DMP) may be an important mechanism mediating intestinal barrier damage and thus inducing central nervous system inflammation.

[0051] Figure 17 A bar chart showing the distribution of LDA values ​​for species with significant differences in the linear discriminant analysis effect size analysis. A higher LDA score (threshold typically set >2) indicates a greater contribution of the species to distinguishing different groups. Results showed that several marker bacterial groups with LDA scores greater than 2 were identified in each group: the KH group was characterized by Tannerellaceae and... Parabacteroides The genus is the most important identifying group; group H is based on Prevotella_9 The M group was represented by Veillonellaceae; the KC group also had independent marker bacteria. LEfSe analysis statistically confirmed that NP exposure and Parkin knockout could induce abnormal enrichment of specific gut microbiota, further validating... Figures 13 to 15 The discovery that NP and Parkin knockout can synergistically induce targeted dysbiosis of the gut microbiota provides key microbiological evidence for elucidating the cascade mechanism of "microbiota dysbiosis-barrier damage-neuroinflammation-depressive behavior".

[0052] Figure 18 This diagram illustrates the mechanism by which PINK1 / Parkin-mediated mitophagy plays a role in NP-induced depression by disrupting gut-microbe-brain axis homeostasis, synthesizing the core findings of this study. The diagram demonstrates that NP exposure induces depressive-like behavior through two interconnected pathways: First, NP inhibits the PINK1 / Parkin pathway, blocking mitophagy in hippocampal neurons, leading to the accumulation of damaged mitochondria, impaired autophagic flux, and triggering neuroinflammation and neuronal degeneration. Second, NP induces disordered mitophagy in colonic tissue, damaging the intestinal mucosal barrier (elevated DAO and sIgA, abnormal tight junction proteins), inducing intestinal inflammation, and altering the composition of the gut microbiota (decreased abundance of Lactobacillus and Bacteroides, reduced propionic acid production). Following intestinal barrier damage, harmful substances such as LPS translocate into the bloodstream, further exacerbating systemic and central nervous system inflammation, and exacerbating neurotransmitter imbalances (decreased DAO, abnormal 5-HT) and depressive-like behavior through the gut-microbe-brain axis. Parkin gene knockout leads to loss of PINK1 / Parkin pathway function, significantly exacerbating the aforementioned pathological changes at both the brain and gut levels, confirming that Parkin plays a key protective role in maintaining mitochondrial quality control and gut-brain axis homeostasis.

[0053] Example 9 The statistical analysis, specifically the steps, is as follows: All experimental data are expressed as mean ± standard error (Mean ± SEM). Statistical analysis was performed using SPSS 26.0 software. One-way ANOVA was used for comparisons among multiple groups; the LSD method was used for post-hoc pairwise comparisons when variances were homogeneous, and the Games-Howell method was used when variances were unequal. Independent samples t-tests were used for comparisons between two groups. p <0.05 was considered statistically significant. All Western blotting bands were standardized using GAPDH as an internal reference before calculating relative expression levels. The Kruskal-Wallis rank-sum test was used to compare gut microbiota α-diversity among groups, and the PERMANOVA test (999 permutations) was used to compare β-diversity among groups.

[0054] The above are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for constructing an animal model of nonylphenol-induced depressive-like behavior, characterized in that, Includes the following steps: S1. Animal grouping: Wild-type C57BL / 6 mice and Parkin gene knockout C57BL / 6 mice were randomly divided into the following six groups: control group, low-dose NP group, medium-dose NP group, high-dose NP group, Parkin knockout control group, and high-dose NP combined with Parkin knockout group. S2. Treatment with poison: Mice in each group were given corn oil as a solvent and administered the poison by gavage at a volume of 10 mL / kg for 90 consecutive days. S3. Behavioral evaluation: After the drug exposure ended, the mice were assessed for depressive-like behavior through the forced swimming test and the tail suspension test. S4. Histological examination: Mouse hippocampal and colon tissues were taken, and histopathological morphological changes were observed by HE staining. The ultrastructure of mitochondria was observed by transmission electron microscopy. S5. Molecular biological detection: Serum dopamine and serotonin levels, as well as IL-6 and TNF-α levels in hippocampal and colon tissues, were detected by ELISA; the expression levels of PINK1, Parkin, p62, and LC3-I / II proteins in hippocampal and colon tissues were detected by Western blotting. S6. Intestinal barrier function detection: The levels of diamine oxidase and secretory immunoglobulin A in colonic tissue were detected by ELISA; the expression levels of tight junction proteins Claudin-1 and Occludin in colonic tissue were detected by Western blotting. S7. Intestinal flora analysis: Mouse feces were collected, and the abundance and diversity of intestinal flora were analyzed by 16S rDNA sequencing. The content of short-chain fatty acids in feces was detected by gas chromatography.

2. The method according to claim 1, characterized in that, In step S1, the dose of the low-dose NP group was 0.25 mg / kg, the dose of the medium-dose NP group was 2.5 mg / kg, and the dose of the high-dose NP group was 25 mg / kg; the dose of the high-dose NP combined with the Parkin knockout group was 25 mg / kg; the control group and the Parkin knockout control group were given an equal volume of corn oil.

3. The method according to claim 1, characterized in that, The conditions for the forced swimming experiment in step S3 are: water temperature 25℃, and the time the mouse remains still within 5 minutes; the conditions for the tail suspension experiment are: the mouse is suspended with its tail fixed at 1 / 3 of its length, and the time the mouse remains still is recorded.

4. The method according to claim 1, characterized in that, The specific conditions for transmission electron microscopy observation in step S4 are as follows: take a 1 mm... 3 Tissue samples were fixed, dehydrated, embedded, and polymerized to prepare 70-80 nm ultrathin sections. The cross sections of the CA1 region of the hippocampus and the distal colon were observed under a transmission electron microscope. One sample was used in each group, and three fields of view were randomly selected at 8000x magnification.

5. The method according to claim 1, characterized in that, The dilution ratios of the primary antibodies in the Western Blot method described in step S5 are: Parkin 1:1000, PINK1 1:1000, p62 1:1000, LC3-I / II 1:1000, and the internal control is GAPDH 1:1000.

6. The method according to claim 1, characterized in that, The detection indicators for short-chain fatty acids mentioned in step S6 include acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid. 2-Ethylbutyric acid is used as an internal standard, and gas chromatography is used for quantitative detection.

7. The method according to claim 1, characterized in that, In step S7, the 16S rDNA sequencing used the V3-V4 hypervariable region, and PCR amplification was performed using primers 341F / 806R. Sequencing was completed on the Illumina NovaSeq 6000 platform in PE250 paired-end mode.

8. The method according to claim 1, characterized in that, The genotype of the Parkin gene knockout C57BL / 6 mice was determined by PCR. The primer sequences used for identification included wild-type upstream primer, mutant upstream primer, and common downstream primer.

9. The use of the method according to any one of claims 1-7 in evaluating the neurotoxicity of nonylphenol via the "microbe-gut-brain" axis.

10. The application of the method according to any one of claims 1-7 in the screening of antidepressants targeting the PINK1 / Parkin pathway.

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