Use of alistipes in preparation of early warning marker of depressive-like behavior in offspring female mice

Abstract

This invention belongs to the field of biomedical technology and discloses the application of *Alistaplasma aristata* in the preparation of early warning biomarkers for depressive-like behavior in offspring female mice. Depressive-like behavior in offspring female mice is caused by exposure to endocrine disruptors during pregnancy and lactation. The *Alistaplasma aristata* of this invention, as an early warning biomarker for depressive-like behavior in offspring female mice, has advantages such as high specificity, early warning time, non-invasiveness, and strong sex specificity. It verifies the correlation between decreased *Alistaplasma aristata* abundance and damage to the colonic barrier, hippocampal neurons, and synaptic structures, constructing a complete logical chain of "microbiota dysbiosis - intestinal barrier disruption - hippocampal neurotoxicity - depressive-like behavior," providing solid theoretical support for the reliability of *Alistaplasma aristata* as an early warning biomarker for depressive-like behavior in offspring female mice.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of *Alistaplasma gondii* in the preparation of an early warning biomarker for depressive-like behavior in offspring female mice induced by exposure to environmental endocrine disruptors during pregnancy and lactation. Background Technology

[0002] Triclosan (TCS) is a synthetic broad-spectrum antibacterial agent that has been widely added to personal care products such as toothpaste, facial cleanser, and soap, as well as medical disinfectants, since the 1960s. Due to its strong lipophilicity (octanol-water partition coefficient logKow=4.76), TCS can enter the human body through the skin, oral mucosa, and gastrointestinal tract. It has been detected in human fat, liver, blood, breast milk, and amniotic fluid, and is classified by the World Health Organization as an environmental endocrine disrupting chemical (EDC).

[0003] In recent years, the developmental neurotoxicity of TCS has gradually become a research hotspot. Existing studies have shown that early-life TCS exposure can induce behavioral abnormalities and neurodevelopmental disorders in offspring mice, with male offspring often exhibiting attention deficit hyperactivity disorder-like phenotypes such as hyperactivity and impulsivity. The gut microbiota, as an important micro-ecological system, bidirectionally regulates the function of the central nervous system through the "gut-brain axis," and its dysregulation is closely related to mental illnesses such as depression and anxiety. Previous studies have confirmed that TCS exposure can cause changes in gut microbiota composition and disruption of gut homeostasis; however, current research mainly focuses on diseases such as intestinal inflammation and tumors, and the mechanism by which it affects offspring neurodevelopment through the gut microbiota remains unclear.

[0004] In studies on gut microbiota associated with depression, existing reports show that Bacteroidetes (Bacteroidetes) are prevalent in the gut of patients with major depressive disorder (MDD). Bacteroidota Proteobacteria ( Proteobacteria Increased abundance of Firmicutes ( Firmicutes The abundance of *Alistaplasma* decreased; at the genus level, *Alistaplasma* ( Alistipes Enterobacteriaceae ( Enterobacteriaceae Increased abundance of *Faecalibacterium* ( Faecalibacterium The abundance of anti-inflammatory bacteria such as [list of bacteria] decreased. However, the above studies all focused on adult patients with depression and have not yet addressed specific microbial biomarkers for early life environmental exposures leading to depressive-like behaviors in offspring, nor have they considered the impact of gender differences.

[0005] Currently, health risk assessment for TCS exposure mainly relies on the testing of maternal blood and urine samples, which cannot directly reflect neurodevelopmental damage in offspring. Furthermore, the diagnosis of existing neurodevelopmental abnormalities largely depends on behavioral observation, often only confirming the diagnosis after symptoms become apparent, thus missing the early intervention window. Simultaneously, existing gut microbiota biomarkers are mostly broad-spectrum indicators, easily affected by factors such as diet, age, and sex, lacking specific biomarkers for specific exposure factors, specific sexes, or specific disease phenotypes. The specific problems and limitations of existing technologies are as follows: Lack of sex-specific warning biomarkers: Existing TCS neurodevelopmental toxicity studies mainly focus on male offspring, while females are a high-risk group for depression (the prevalence rate is about 1.5 times that of males), and there are no specific gut microbiota biomarkers for female offspring depressive-like behaviors caused by TCS exposure during pregnancy and lactation.

[0006] Early warning timing is severely delayed: Existing assessment methods rely on behavioral testing of offspring more than 4 weeks after birth, which cannot provide early warning of risks in early life and makes it difficult to carry out early intervention.

[0007] Mechanisms are unclear: Existing studies have not elucidated the specific mechanisms by which TCS exposure induces depressive-like behavior in offspring through vertical transmission from mother to offspring microbiota, thus failing to provide a theoretical basis for targeted intervention. Summary of the Invention

[0008] To address the aforementioned issues, this invention provides the application of *Alistaplasma gondii* in the preparation of early warning biomarkers for depressive-like behavior in offspring female mice.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: Application of Alistairips bacterium in the preparation of an early warning biomarker for depressive-like behavior in offspring female mice, where the depressive-like behavior is caused by exposure to environmental endocrine disruptors during pregnancy and lactation.

[0010] Furthermore, the endocrine disruptor is triclosan; Depression-like behaviors include anxiety-like behaviors, cognitive memory impairment, and decreased social novelty preference; The application determines the relative abundance of Alistair 16S rRNA by detecting the V3-V4 region sequence. The application uses *Alistaplasma gondii* as an early warning marker to detect fresh feces from female offspring mice on day 28 after birth.

[0011] A fecal microbiota detection kit, 16S rRNA gene sequencing detection panel, or gene chip targeting Alistera bacteria to screen for and improve depressive-like behavior in offspring female mice induced by triclosan exposure during pregnancy and lactation.

[0012] Furthermore, the fecal microbiota detection kit, 16S rRNA gene sequencing detection panel or gene chip includes primers 343F / 798R, Adapter I5 and Adapter I7; The sequence of primer 343F is shown in SEQ ID NO: 1; The sequence of primer 798R is shown in SEQ ID NO: 2; The sequence of Adapter I5 is shown in SEQ ID NO: 3; The sequence of Adapter I7 is shown in SEQ ID NO:4.

[0013] Application of Alistair et al. as a target in screening probiotics that improve depressive-like behavior in offspring female mice induced by triclosan exposure during pregnancy and lactation.

[0014] A non-invasive early warning method is disclosed, wherein the method involves extracting microbial DNA from fecal samples of female offspring mice to be tested, and measuring the V3-V4 region of the 16S rRNA gene of the microbial DNA to quantify the relative abundance of *Alistaplasma*. When the relative abundance of *Alistaplasma* in the feces of female offspring mice is less than 0.6 times that in the feces of healthy female offspring mice, it is considered a high risk of depressive-like behavior; when the relative abundance of *Alistaplasma* in the feces of female offspring mice is greater than or equal to 0.6 times that in the feces of healthy female offspring mice, it is considered a low risk of depressive-like behavior.

[0015] Furthermore, the early warning method involves collecting fresh fecal samples from female offspring mice on day 28 after birth, extracting total genomic DNA from the feces, and performing quality control on concentration, purity, and integrity. Using the qualified total genomic DNA as template DNA, the hypervariable region of the bacterial 16S rRNA gene V3-V4 is amplified, purified with magnetic beads, and a sequencing library is constructed. Paired-end sequencing is performed on the Illumina NovaSeq 6000 platform. After removing primer and adapter sequences from the raw sequencing data, and then performing quality control, noise reduction, paired-end sequence splicing, and chimera removal, species annotation is performed based on the Silva 138 database, and the relative abundance of the genus *Alistera* is calculated.

[0016] Furthermore, the hypervariable region of the bacterial 16S rRNA gene V3-V4 was amplified using qualified total genomic DNA as template DNA. After the first round of PCR amplification with primers 343F / 798R, the amplified product was purified by magnetic beads. The purified product was then amplified in the second round using Adapter I5 and Adapter I7 to obtain the second round of PCR product.

[0017] Furthermore, the first round of PCR amplification system included 2×Gflex PCR Buffer, primers 343F / 798R, template DNA, and Tks Gflex DNA Polymerase; The system for the second round of PCR amplification included 2×Gflex PCR Buffer, Adapter I5, Adapter I7, template DNA, and Tks Gflex DNA Polymerase.

[0018] Furthermore, the magnetic bead purification process involves mixing magnetic beads at a volume ratio of 0.8:1 with the PCR product, limiting the DNA fragment cutoff range to 300-500bp, allowing it to stand at room temperature, adsorbing it with a magnetic rack and discarding the supernatant, washing with 80% ethanol, air-drying at room temperature, and then eluting with enzyme-free water.

[0019] The beneficial effects of the application of *Alistaplasma aristata* in the preparation of early warning biomarkers for depressive-like behavior in offspring female mice are as follows: High specificity: This invention found that the abundance of *Alistaplasma gondii* was highly significantly correlated with core indicators of depressive-like behavior in offspring female mice, negatively correlated with the resting time in the tail suspension test (r=-0.62), and extremely significantly positively correlated with the dwell time in the central region of the open field test (r=0.93). P <0.001), compared to the exploration time of the new object recognition experiment (r=0.73, P <0.05) and the three-box social experiment social novelty preference index (r=0.74, P <0.05) showed a significant positive correlation, which was significantly higher than that of other differentially expressed bacterial genera (such as Muribauculaceae and Parasutterella), and its specificity was superior to existing broad-spectrum bacterial biomarkers; Early warning time: The detection of this invention can be completed 28 days after the offspring are born, earlier than the obvious manifestation of behavioral abnormalities (usually 6-8 weeks after birth), which provides a critical time window for early intervention; Non-invasive: This invention only requires the collection of fecal samples to complete the test, avoiding the damage to experimental animals caused by invasive sampling such as blood and brain tissue. It is simple to operate and highly safe. Highly gender-specific: This invention provides a new early warning biomarker and method for the specific phenotype of female offspring depression-like behavior caused by TCS exposure during pregnancy and lactation, and provides a dedicated technical means for assessing the neurodevelopmental risks of female offspring. The mechanism is clear: This invention further verifies the correlation between the decrease in Alistair abundance and colonic barrier damage, hippocampal neuronal and synaptic structure damage, and constructs a complete logical chain of "flora dysbiosis-intestinal barrier disruption-hippocampal neurotoxicity-depressive-like behavior", providing solid theoretical support for the reliability of Alistair as an early warning biomarker of depressive-like behavior in progeny female mice. Attached Figure Description

[0020] Figure 1 This is a flowchart of the TCS exposure time during pregnancy and lactation and a statistical chart of the body weight of female offspring mice at PND 28 in Embodiment 2 of the present invention; wherein, Figure A is a flowchart of the TCS exposure time during pregnancy and lactation, and Figure B is a statistical chart of the body weight of female offspring mice at PND 28. Figure 2 These are the experimental results of female offspring behavior in each group in Embodiment 2 of the present invention; wherein, Figure A shows the results of the tail suspension experiment, Figure B shows the results of the open field experiment, and Figure C shows the results of the new object recognition experiment. Figure 3 This is a comparison chart of the gut microbiota α diversity index (Shannon, Simpson, Chao1) of female offspring mice in each group in Example 3 of the present invention; wherein, Figure A is a comparison chart of Shannon results, Figure B is a comparison chart of Simpson results, and Figure C is a comparison chart of Chao1 results. Figure 4 This is a PCoA analysis diagram of the β-diversity of gut microbiota in female offspring mice from each group in Example 3 of this invention; Figure 5 This is a box plot showing the relative abundance of *Alistela alisteris* in each group of female offspring mice in Example 3 of this invention. The results of this study show that there was no statistically significant difference in the relative abundance of *Alistela alisteris* in the intestines of offspring mice between the blank control group and the TCS-exposed groups; however, the relative abundance of *Alistela alisteris* in the high-dose TCS-exposed group was significantly lower than that in the low-dose TCS-exposed group, indicating that the change in *Alistela alisteris* abundance only showed a significant difference between different TCS exposure doses. This bacterium can be used as a characteristic microbiota marker of TCS-induced damage to offspring organisms. Figure 6 This is a Spearman correlation heatmap of Alistepikelius abundance and offspring behavioral indicators in Example 4 of the present invention; Figure 7 These are H&E staining images of colon tissue from female offspring mice in each group of Example 5 of this invention (scale bar: 100μm / 50μm). Figure 8 These are transmission electron micrographs (TEM) of the CA1 region of the hippocampus of female offspring mice in each group of Example 5 of this invention (scale bar: 2μm / 500nm); where Figure A is used to observe the morphology of presynaptic mitochondria and synaptic vesicles, and Figure B is used to observe the structure of the postsynaptic dense region and synaptic cleft. Figure 9 These are Western blot diagrams and quantitative analysis diagrams of SYN1 protein expression in the hippocampus of female offspring mice in each group in Example 5 of the present invention; wherein, Figure A is a Western blot diagram of hippocampal protein expression in each group of female offspring mice, Figure B is a statistical analysis of the relative expression level of BDNF protein in the hippocampus, Figure C is a statistical analysis of the relative expression level of SYN1 protein in the hippocampus, and Figure D is a statistical analysis of the relative expression level of PSD95 protein in the hippocampus. Detailed Implementation

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can also be implemented in other ways different from those described herein. Conventional adjustments or equivalent substitutions made by those skilled in the art to the types of drugs, dosages, detection methods, and experimental conditions without departing from the core ideas of the present invention should be included within the scope of protection of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The present invention will be further described in detail below with reference to specific embodiments to enable those skilled in the art to understand it.

[0022] Example 1: A specific gut microbiota early warning biomarker and a non-invasive early warning method for depressive-like behavior in offspring female mice induced by TCS exposure during pregnancy and lactation. I. Warning Signs The warning marker of this invention is the relative abundance of *Alistaplasma gondii* (belonging to the phylum Bacteroidetes, class Bacteroidetes, order Bacteroidetes, family Rikenbacteria). In female offspring mice exhibiting depressive-like behavior due to TCS exposure during pregnancy and lactation, the abundance of *Alistaplasma gondii* was significantly lower than that in the healthy control group, and its abundance was significantly negatively correlated with the severity of depressive-like behavior.

[0023] II. Early Warning Methods The early warning method based on the above-mentioned warning markers includes the following steps: S1. Fecal sample collection: Collect 3-5 fresh fecal samples, immediately place them in liquid nitrogen for flash freezing, and then transfer them to a -80℃ freezer for storage; among them, fresh fecal samples were collected from female offspring mice on postnatal day 28 (PND 28). S2. Genomic DNA Extraction: Total genomic DNA (i.e., microbial DNA) was extracted from fecal samples using the MagPure Soil DNA LQ Kit. The DNA concentration and purity were determined using a NanoDrop 2000 micro-spectrophotometer, and DNA integrity was detected by 1.2% agarose gel electrophoresis. S3 and 16S rRNA gene amplification and sequencing: Using qualified total genomic DNA as template DNA, universal primers 343F (as shown in SEQ ID NO: 1, specifically 5'-TACGGRAGGCAGCAG-3') and 798R (as shown in SEQ ID NO: 2, specifically 5'-AGGGTATCTAATCCT-3') with barcode labels were used as upstream and downstream primers to amplify the V3-V4 hypervariable region of the bacterial 16S rRNA gene by PCR; PCR amplification was performed in two rounds: The first round of PCR amplification consisted of a total volume of 30 μL, including: 15 μL of 2×Gflex PCR Buffer, 1 μL each of forward and reverse primers (10 μmol / L), ≥50 ng of template DNA, 0.6 μL of Tks Gflex DNA Polymerase, and enzyme-free water to a final volume of 30 μL. The program for the first round of PCR amplification was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 20 s, for a total of 26 cycles; final extension at 72℃ for 5 min; and storage at 4℃.

[0024] The purified product from the first round of PCR, after being purified by AMPure XP magnetic beads, was used as a template for the second round of PCR amplification.

[0025] The total volume of the second round of PCR amplification was 30 μL, including: 15 μL of 2×Gflex PCR Buffer, 1 μL of Adapter I5, 1 μL of Adapter I7, 50 ng of the first round purified product, 0.6 μL of Tks Gflex DNA Polymerase, and enzyme-free water to a final volume of 30 μL. The second round PCR program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 20 s, for a total of 7 cycles; 72℃ final extension for 5 min; and storage at 4℃. The sequence of Adapter I5 is shown in SEQ ID NO: 3, specifically 5'-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTC-3'; the sequence of Adapter I7 is shown in SEQ ID NO: 4, specifically 5'-CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTCGG-3'; NNNNNNNN in the sequence is an 8bp sample-specific index sequence, which distinguishes different sample index sequences and is used to split the sequencing data.

[0026] The second-round PCR products were purified again using AMPure XP magnetic beads. After quantification using Qubit and verification of single bands by agarose gel electrophoresis, the concentration was adjusted to construct a sequencing library. Paired-end sequencing (250 bp reads) was performed on the Illumina NovaSeq 6000 platform. During the AMPure XP magnetic bead purification process, the PCR products were mixed with AMPure XP magnetic beads at a volume ratio of 0.8:1. This parameter limited the DNA fragment cutoff range to 300-500 bp. After standing at room temperature for 5 min, the supernatant was discarded after magnetic adsorption, and the product was washed twice with 80% ethanol. After air drying at room temperature, the product was eluted with 25 μL of enzyme-free water. S4. Bioinformatics Analysis: The raw sequencing data is in FASTQ format. The primer and adapter sequences are removed using Cutadapt software. The primer matching error tolerance adopts the software's default matching rules. The minimum quality control threshold for sequencing bases is Q20. The remaining Cutadapt parameters adopt the software's default parameters.

[0027] The DADA2 plugin was used on the QIIME2 (version 2020.11) platform to complete quality control, noise reduction, paired-end sequence assembly, and chimeric removal. DADA2 used the software's default operating parameters: the maximum expected error value (maxEE) for both forward and reverse reads was 2.0. The paired-end read truncation length was dynamically set based on the sequencing quality distribution of a single batch of samples. Chimeric sequences were removed using the consensus algorithm to obtain amplicon sequence variants (ASVs). Sequencing depth flattening was performed on the ASV abundance table to achieve sample homogenization. The q2-feature-classifier classifier was used, with the classification confidence threshold set to 0.7. Species annotation was performed based on the Silva 138 database, and the relative abundance of the genus Alistipes was calculated.

[0028] S5. Risk assessment: The measured mean relative abundance of Alistair spp. in the healthy control group was 0.025027. When the relative abundance of *Alistaplasma* in the feces of female offspring mice is less than 0.6 times the mean relative abundance of *Alistaplasma* in the feces of healthy female offspring mice, it is considered a high risk of depressive-like behavior; that is, when the relative abundance of *Alistaplasma* in the sample is less than 0.0150162, it is considered a high risk of depressive-like behavior. In this example, the absolute abundance threshold of the female offspring mouse depression model is 0.015016, which is less than 0.0150162, and is therefore considered a high risk of depressive-like behavior. When the relative abundance of Alistera bacteria in the feces of female offspring mice is greater than or equal to 0.6 times the mean relative abundance of Alistera bacteria in the feces of healthy female offspring mice, it is considered to be at low risk of depressive-like behavior; that is, when the relative abundance of Alistera bacteria in the test sample is greater than or equal to 0.0150162, it is considered to be at low risk of depressive-like behavior.

[0029] III. Application of Early Warning Products Using *Alistaplasma aristata* as an early warning biomarker, an early warning product for depressive-like behavior in offspring female mice induced by TCS exposure during pregnancy and lactation was developed. This product includes a fecal microbiota detection kit, a 16S rRNA gene sequencing panel, and a gene chip. The fecal microbiota detection kit, the 16S rRNA gene sequencing panel, and the gene chip all contain: DNA extraction reagents, primers 343F / 798R, Adapter I5, Adapter I7, 2×Gflex PCR Buffer, Tks Gflex DNA Polymerase, and AMPure XP magnetic beads, as well as other necessary standard reagents.

[0030] This invention can also use Alistair as a target to screen for probiotics that improve depressive-like behavior in offspring female mice induced by triclosan exposure during pregnancy and lactation.

[0031] Example 2: Establishment of an animal model of TCS exposure during pregnancy and lactation and verification of depressive-like behavior in offspring. I. Laboratory Animals like Figure 1 As shown in Figure A, 40 8-week-old SPF-grade non-pregnant female C57BL / 6J mice (weighing 16-20g) and 10 male mice (weighing 20-25g) were purchased from the Experimental Animal Center of Ningxia Medical University. The experiment was approved by the Experimental Animal Welfare and Ethics Committee of Ningxia Medical University (Approval No.: IACUC-NYLAC-2024-104). All mice were acclimatized for one week under the following conditions: temperature 22±2℃, humidity 50±5%, 12-hour light-dark cycle, and free access to food and water.

[0032] II. Animal Grouping and Exposure The female mice were housed together at a female-to-male ratio of 4:1. Every morning at 7:00 AM, the vaginal opening and smears of the female mice were examined. The observation of vaginal plugs or a large number of sperm under a microscope was recorded as Gestational Day 0 (GD 0). Afterward, the males and females were separated into different cages. The pregnant mice were randomly divided into 4 groups of 10 mice each: Solvent control group (VG group): corn oil was administered by gavage at a volume of 0.1 mL / day; Low-dose TCS group (LT group): 8 mg / kg / d of TCS (Sigma-Aldrich, purity >97%) corn oil suspension was administered by gavage; Medium-dose TCS group (MT group): 50 mg / kg / d TCS corn oil suspension was administered by gavage; High-dose TCS group (HT group): 100 mg / kg / d TCS corn oil suspension was administered by gavage.

[0033] The mice were continuously exposed to gavage from GD 5 to GD 21 (gestation) and PND 3 to PND 21 (lactation), for a total exposure period of 36 days. After parturition, the birth and survival status of the offspring were recorded. At PND 21, the mice were weaned and housed separately by sex. Ten female offspring mice were randomly selected from each group for subsequent experiments. The body weight of the female offspring mice at PND 28 is shown below. Figure 1 As shown in Figure B, the results showed that the average body weight was 17.41±0.73g in the VG group, 16.38±0.52g in the LT group, 15.41±0.47g in the MT group, and 14.53±0.62g in the HT group. Body weight decreased with increasing TCS exposure dose, suggesting that high-dose TCS exposure during pregnancy and lactation has a certain impact on the growth and development of female offspring mice.

[0034] III. Offspring Behavioral Testing Behavioral tests were conducted from PND 28 to PND 36, and all experiments were performed in a quiet, softly lit environment. (1) Tail Suspension Test (TST): The distal 1 / 3 of the mouse tail is fixed and suspended, and the entire process is recorded for 6 minutes. The resting time is analyzed 4 minutes after the test. Figure 2 As shown in Figure A, compared with the VG group, the resting time of the LT group was significantly prolonged (P<0.05), and the resting time of the MT and HT groups was extremely significantly prolonged (P<0.001).

[0035] (2) Open field test (OFT): The mouse was placed in a 25×25×40cm space. 3 In the center of the plexiglass open space box, allow free exploration for 5 minutes, recording the total distance traveled and the time spent in the central area. For example... Figure 2 As shown in Figure B, compared with the VG group, the MT group had a significantly shorter central zone dwell time (P<0.01), while there was no significant difference in total activity among the groups (P>0.05).

[0036] (3) Novel Object Recognition (NOR) Experiment: Mice were allowed to acclimatize to an empty box 24 hours before the experiment. 24 hours later, two identical objects were placed in the box for 5 minutes of exploration. 24 hours later, a new object was placed in the box, and the exploration time for the new object was recorded within 5 minutes. Figure 2As shown in Figure C, compared with the VG group, the MT group had a significantly reduced new object exploration time (P<0.05).

[0037] (4) Three-box social experiment: Before the experiment, mice were acclimatized to the three-box setup for 10 minutes. During the social preference phase, a strange mouse (M1) was placed in the box, and the exploration time of M1 and the empty box was recorded within 10 minutes. During the social novelty phase, another strange mouse (M2) was placed in the box, and the exploration time of M1 and M2 was recorded. The social novelty preference index (NPI) was calculated. Figure 2 As shown in Figure D, the NPI in the LT group was significantly lower than that in the VG group (P<0.01).

[0038] Example 3: 16S rRNA sequencing analysis of maternal and offspring gut microbiota Following the early warning method in Example 1, 16S rRNA sequencing analysis of gut microbiota was performed on the maternal and progeny generations of the four experimental groups in Example 2, as detailed below: S1. Fecal sample collection Fecal samples from female mice were collected at GD 18 to GD 21, and fecal samples from female offspring mice were collected at PND 28.

[0039] S2, Genomic DNA Extraction Total genomic DNA was extracted using the MagPure Soil DNA LQ Kit, and DNA integrity was tested. S3 and 16S rRNA gene amplification and sequencing Using qualified total genomic DNA as template DNA, the V3-V4 region of the 16S rRNA gene was amplified, and the library was constructed and sequenced on the Illumina NovaSeq 6000 platform.

[0040] S4, Bioinformatics Analysis After quality control, the raw data yielded ASVs, and species were annotated based on the Silva 138 database. The results showed: Results of gut microbiota α diversity index detection in offspring female mice as follows Figure 3 As shown, compared with the VG group, the gut microbiota α diversity (Shannon, Simpson, Chao1 index) of female offspring mice in the TCS exposure group was significantly reduced (P<0.05). The results of the β-diversity PCoA analysis are as follows: Figure 4 As shown, the TCS-exposed group and the VG group samples were clearly separated in spatial distribution, indicating that the bacterial community structure had changed significantly. The results of the genus-level abundance difference analysis of Alistair ethopterinella are as follows: Figure 5 As shown, the abundance of Alistairypus increased in the LT group and decreased significantly in the MT and HT groups (P<0.05).

[0041] Example 4: Correlation analysis between Alistairypsy and depressive-like behavior Spearman correlation analysis was used to correlate the abundance of *Alistela alisteris* in each group of female offspring mice in Example 2 with behavioral indicators. The results are as follows: Figure 6 As shown. The results show: The abundance of Alistairops was negatively correlated with the quiescence time of the tail suspension experiment (r=-0.62). It showed a highly significant positive correlation with the dwell time in the central region of the open field experiment (r=0.93, P<0.001). It was significantly positively correlated with the time for exploring new objects (r=0.73, P<0.05). It showed a significant positive correlation with the social novelty preference index (r=0.74, P<0.05).

[0042] Other differentially expressed genera (e.g.) Muribaculaceae , Parasutterella The correlation between the bacteria and behavioral indicators was not statistically significant, confirming that *Alistaplasma gondii* is the most specific biomarker associated with depressive-like behavior in offspring.

[0043] Example 5: Validation of intestinal barrier and hippocampal neurotoxicity I. Colon tissue pathological examination Distal colon tissues from female offspring mice in each group of Example 2 were taken, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E and PAS. The results are as follows. Figure 7 As shown, the colonic mucosa structure of the VG group was intact and goblet cells were abundant; the TCS exposure group showed inflammatory cell infiltration, epithelial shedding, disordered crypt structure, and a significant reduction in the number of goblet cells.

[0044] II. Hippocampal Tissue Pathology and Ultrastructural Detection Hippocampal tissue was collected for H&E staining, Nissl staining, and transmission electron microscopy observation. The results are as follows: Figure 8 As shown, in the VG group, neurons in the CA1, CA3 and DG regions of the hippocampus were neatly arranged and Nissl bodies were clearly visible; in the MT group, the number of neurons decreased, cell bodies shrank, Nissl bodies dissolved, mitochondria showed vacuolar degeneration, synaptic clefts widened, and postsynaptic density decreased.

[0045] III. Hippocampal synapse-related protein detection Western blot analysis was used to detect the expression of BDNF, SYN1, and PSD95 proteins in the hippocampus. The results are as follows: Figure 9 As shown, compared with the VG group, the HT group showed a significant decrease in SYN1 protein expression (P<0.05), and a decreasing trend in BDNF and PSD95 protein expression.

[0046] The above results confirm that decreased abundance of *Alistaplasma alisteri* is accompanied by intestinal barrier damage and hippocampal neurotoxicity, further supporting the reliability of using *Alistaplasma alisteri* as an early warning biomarker for early warning of depressive-like behaviors in offspring female mice induced by triclosan exposure during pregnancy and lactation. These depressive-like behaviors include anxiety-like behaviors, cognitive memory impairment, and decreased social novelty preference.

[0047] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. Those skilled in the art can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An application of *Alistaplasma aristata* in the preparation of early warning biomarkers for depressive-like behavior in offspring female mice, characterized in that, The depressive-like behavior in offspring female mice was caused by exposure to environmental endocrine disruptors during pregnancy and lactation.

2. The application according to claim 1, characterized in that, The endocrine disruptor is triclosan; Depression-like behaviors include anxiety-like behaviors, cognitive memory impairment, and decreased social novelty preference; The application determines the relative abundance of Alistair 16S rRNA by detecting the V3-V4 region sequence. The application uses *Alistaplasma gondii* as an early warning marker to detect feces in female offspring mice on day 28 after birth.

3. A fecal microbiota detection kit, 16S rRNA gene sequencing detection panel, or gene chip targeting Alistera bacteria to screen for and improve depressive-like behavior in offspring female mice induced by triclosan exposure during pregnancy and lactation.

4. The fecal microbiota detection kit, 16S rRNA gene sequencing detection panel or gene chip according to claim 3, characterized in that, The fecal microbiota detection kit, 16S rRNA gene sequencing detection panel or gene chip includes primers 343F / 798R, Adapter I5 and Adapter I7; The sequence of primer 343F is shown in SEQ ID NO: 1; The sequence of primer 798R is shown in SEQ ID NO: 2; The sequence of Adapter I5 is shown in SEQ ID NO: 3; The sequence of Adapter I7 is shown in SEQ ID NO:

4.

5. Application of Alistair et al. as a target in screening probiotics that improve depressive-like behavior in offspring female mice induced by triclosan exposure during pregnancy and lactation.

6. A non-invasive early warning method, characterized in that, The early warning method involves extracting microbial DNA from fecal samples of female offspring mice to be tested, and measuring the V3-V4 region of the 16S rRNA gene of the microbial DNA to quantify the relative abundance of Alistairy. When the relative abundance of Alistairy in the feces of female offspring mice to be tested is less than 0.6 times that of Alistairy in the feces of healthy female offspring mice, it is considered a high risk of depressive-like behavior.

7. The non-invasive early warning method according to claim 6, characterized in that, The early warning method involves collecting fecal samples from female offspring mice on day 28 after birth, extracting total genomic DNA from the feces, and performing quality control on concentration, purity, and integrity. Using the qualified total genomic DNA as template DNA, the hypervariable region of the bacterial 16S rRNA gene V3-V4 is amplified, purified with magnetic beads, and a sequencing library is constructed. Paired-end sequencing is performed on the Illumina NovaSeq 6000 platform. Primers and adapter sequences are removed from the raw sequencing data, and after quality control, noise reduction, paired-end sequence splicing, and chimera removal, species annotation is performed based on the Silva 138 database, and the relative abundance of the genus *Alistera* is calculated.

8. The non-invasive early warning method according to claim 7, characterized in that, The amplification of the V3-V4 hypervariable region of the bacterial 16S rRNA gene was carried out using qualified total genomic DNA as template DNA. After the first round of PCR amplification with primers 343F / 798R, the product was purified by magnetic beads. The purified product was then amplified in the second round using Adapter I5 and Adapter I7 to obtain the second round of PCR product.

9. The non-invasive early warning method according to claim 8, characterized in that, The system for the first round of PCR amplification includes 2×Gflex PCR Buffer, primers 343F / 798R, template DNA, and Tks Gflex DNA Polymerase; The system for the second round of PCR amplification included 2×Gflex PCR Buffer, Adapter I5, Adapter I7, template DNA, and Tks Gflex DNA Polymerase.

10. The non-invasive early warning method according to any one of claims 7-9, characterized in that, The process of magnetic bead purification involves mixing the magnetic beads with the PCR product, limiting the DNA fragment cutoff range to 300-500 bp, allowing it to stand at room temperature, adsorbing the supernatant using a magnetic rack, washing, air-drying at room temperature, and then eluting again.

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