Application of Cristinia sp. As biomarker in preparation of product for diagnosing or monitoring neurodevelopment injury

Abstract

The invention discloses application of Cristinia sp. As a biomarker in preparation of a product for diagnosing or monitoring neurodevelopment injury, and belongs to the technical field of microbial marker detection. The invention discloses an application of a protection marker of a progeny neurodevelopment injury caused by the exposure of a beneficial bacterium, namely, Cristinia sp. To an endocrine disrupter (EDCs) in a maternal environment. The invention further discloses an application of the protection marker of the progeny neurodevelopment injury caused by the exposure of the endocrine disrupter (EDCs) in the maternal environment. According to the application disclosed by the invention, the maternal prenatal EDCs exposure can obviously reduce the abundance of Cristseneleae bacteria in the intestinal tract of a filial generation by more than or equal to 99%, so that the protective effect of the Cristseneleae bacteria on neurodevelopment is weakened. By detecting the abundance decreasing level of the bacterium in excrement, the risk of neurodevelopmental disorder can be pre-warned, and the pre-warning accuracy rate reaches 85% or above.

Description

Technical Field

[0001] This invention relates to the field of microbial biomarker detection technology, and in particular to the application of Christensenella as a biomarker in the preparation of products for the diagnosis or monitoring of neurodevelopmental damage. Background Technology

[0002] Neurodevelopmental disorders (NDDs), such as autism and attention deficit hyperactivity disorder (ADHD), are a group of complex diseases caused by abnormal neurodevelopment. They are influenced by a combination of factors, including genetic susceptibility, epigenetic modifications, and environmental exposures, such as triclosan, bisphenol A (BPA), and parabens. Triclosan (TCS), a widely used antibacterial agent, has received considerable attention in recent years and is considered an emerging endocrine disruptor. TCS can enter the intrauterine environment through maternal ingestion and affect fetal neurodevelopment. Previous studies have found that TCS exposure during pregnancy and lactation can lead to neurodevelopmental abnormalities in offspring, but the specific warning markers and mechanisms remain unclear.

[0003] Gut microbiota are closely related to neurodevelopment, and maternal environmental exposure may affect offspring neurodevelopment by altering the gut microbiota. 16S rRNA sequencing technology provides an effective means of screening differentially expressed microbiota, and combined with proteomics analysis, it can reveal the association between microbiota and neurodevelopmental disorders. Christensenellaceae, as a beneficial gut bacterium, has been shown to exert neuroprotective effects by regulating intestinal immunity, maintaining barrier function, and participating in energy metabolism (such as producing short-chain fatty acids). Currently, existing technologies mostly focus on blood biomarkers, but fecal testing has the advantage of being non-invasive, and no studies have yet linked Christensenellaceae to offspring neurodevelopmental disorders caused by maternal exposure to endocrine disruptors (EDCs). Therefore, screening for specific microbiota biomarkers has important clinical significance. Summary of the Invention

[0004] The purpose of this invention is to provide the application of Christensenella as a biomarker in the preparation of products for the diagnosis or monitoring of neurodevelopmental impairment, thereby addressing the problems existing in the prior art. Specifically, this invention aims to provide the application of Christensenella in the preparation of products for improving neurodevelopmental impairment caused by prenatal environmental EDC (Exposure to Drugs and Chemicals) exposure. Screening for the abundance of Christensenella in fecal samples of offspring can provide early warning of neurodevelopmental risks.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] One of the technical solutions of this invention is the application of Christensenella as a biomarker in the preparation of products for the diagnosis or monitoring of neurodevelopmental damage.

[0007] The second technical solution of the present invention is a product for diagnosing or monitoring neurodevelopmental damage, including a reagent for specific detection of Christensenella.

[0008] Based on the above technical solution, the present invention has the following technical effects:

[0009] This invention discloses the application of beneficial bacteria *Christensenellaceae* as a protective biomarker against neurodevelopmental damage in offspring induced by maternal exposure to environmental endocrine disruptors (EDCs). This invention reveals that prenatal maternal exposure to EDCs significantly reduces the abundance of *Christensenellaceae* in the offspring gut, leading to a weakened protective effect against neurodevelopmental disorders. By detecting the level of decreased abundance of this bacterium in feces, the risk of neurodevelopmental disorders can be predicted. Attached Figure Description

[0010] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0011] Figure 1 To establish a model of prenatal EDC exposure-induced neurodevelopmental damage in offspring by administering triclosan (TCS) orally via gavage.

[0012] Figure 2 This represents the social novelty index of male offspring. The bar chart shows the mean ± standard error. Group VG has n=6, and all other groups have n=8. * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001.

[0013] Figure 3 Comparison of recognition index differences in new object recognition experiments for male offspring.

[0014] Figure 4 A graph showing the difference in total open field activity among male offspring.

[0015] Figure 5 HE staining of the brain and hippocampus of male offspring.

[0016] Figure 6 Box plot showing the relative abundance of the top 10 differentially abundant species at the male offspring genus level.

[0017] Figure 7 Boxplot showing the differences in male progeny Christenellaceae.

[0018] Figure 8 This is a cumulative species curve of meconium 16S rRNA sequencing at birth for offspring in the Triclosan-exposed group (TE) and Triclosan-unexposed group (TU) of pregnant women in the population, obtained by liquid chromatography-mass spectrometry. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0025] This invention provides the application of Christensenella as a biomarker in the preparation of products for the diagnosis or monitoring of neurodevelopmental disorders.

[0026] In some specific implementations, the neurodevelopmental damage refers to neurodevelopmental damage in offspring caused by exposure to endocrine disruptors in the maternal prenatal environment.

[0027] In some specific implementation plans, the level of Christensenella abundance recovery was measured after a tryptophan-based dietary intervention to assess the intervention's effectiveness.

[0028] In some specific implementations, the endocrine disruptor includes triclosan.

[0029] This invention also provides a product for diagnosing or monitoring neurodevelopmental damage, including a reagent for the specific detection of Christensenella.

[0030] In some specific implementations, the product includes reagents or kits.

[0031] In some specific implementations, the product uses the feces of the patient to be tested as the test sample.

[0032] The abundance of Christensenellaceae was positively correlated with the offspring’s neurodevelopmental protective capacity. In offspring with neurodevelopmental disorders caused by maternal EDC exposure, the abundance of Christensenellaceae was significantly lower than that in the healthy control group.

[0033] This invention establishes an animal model of maternal exposure to endocrine disruptors (EDCs) (using triclosan as a biomarker): pregnant and lactating female mice were fed diets of 0 mg / kg / day (solvent control group: Vehicle Group, denoted as VG), 50 mg / kg / day (triclosan exposure group: Medium Triclosan, denoted as MT; based on the No Obvious Adverse Effect Level (NOAEL) of 50 mg / kg / day), and 50 mg / kg / day + 0.08% tryptophan (tryptophan intervention group: Tryptophan Group, denoted as TG). Fecal samples were collected from offspring male mice, DNA was extracted, and 16S rRNA was sequenced to detect the abundance of Christensenellaceae. Behavioral tests (three-box socialization, open field test, and novel object recognition) were used to determine the neurodevelopmental impairment phenotype of the offspring. Pathological staining was used to determine hippocampal damage.

[0034] This invention elucidates for the first time the neuroprotective effect of Christensenellaceae; provides a direct target for probiotic intervention in neurodevelopmental disorders; and establishes an assessment model of "beneficial bacteria abundance-neuroprotective capacity," promoting the development of non-invasive detection technology.

[0035] Example 1

[0036] I. Establishment of a mouse model of prenatal environmental endocrine exposure

[0037] This invention selected 30 SPF-grade C57 BL / 6J female mice (8 weeks old, weighing 16-20g) and 10 SPF-grade C57 BL / 6J male mice (8 weeks old, weighing 20-25g). Intervention began after one week of acclimatization at an experimental animal center. Female mice were randomly divided into three groups of 10 each: ① Solvent control group (Vehicle Group, denoted as VG); ② Neurodevelopmental impairment group (exposed to a dose of triclosan NOAEL = 50mg / kg / day, denoted as MT); ③ Tryptophan intervention group (50mg / kg / day TCS gavage + dietary intervention with 0.08% tryptophan supplementation, denoted as TG). Male mice were raised under normal conditions and used only for mating. Pregnant mice were housed individually around day 18 of gestation.

[0038] Figure 1 The modeling process was described. TCS (97%, Sigma-Aldrich, USA) was dissolved in corn oil (Aladdin, China) used as a medium and stored at room temperature. Adult pregnant mice were exposed to the drug via gavage at a dose of 0.1 mL / day. The day a vaginal plug was found was designated GD0 (gestational day). Gavage was continued from GD5 to GD21. The first day of delivery was designated PND0 (postnatal day). Gavage continued from PND3 to PND21, for a total exposure of 33 days.

[0039] Although not all mother mice gave birth on the same day, all births were completed within 5 days. The number of offspring and surviving pups was counted, and stillborn pups were discarded. At day 21 postpartum (PND), the mice were weaned, and the mothers and pups were separated into different cages, with male and female pups housed separately. Ten male and ten female pups were randomly selected from each group. Behavioral tests were conducted at day 28 postpartum, and the mice were culled for testing after the experiment.

[0040] II. Determination of Neurodevelopmental Damage in Offspring Mice

[0041] (I) Behavioral Experiments

[0042] The three-box social experiment, the new object recognition experiment, and the open field experiment were conducted on the 28th day after the mice were born (PND28). The PND28 mentioned below does not refer to the exact 28th day after the mice were born, but rather to the period from PND28 to PND36.

[0043] Behavioral data are expressed as mean ± standard deviation (SEM). One-way ANOVA was used to assess differences among the three groups. All charts were created using GraphPadPrism 10.0.

[0044] (1) Three-Chamber Social Test: This test examines the social behavior of mice and their preference for novelty. The three-chamber setup consists of three plexiglass chambers, each 20cm wide × 40cm long × 22cm high, with small square openings (10×5cm) in the partition walls, allowing mice to freely enter each chamber. Two cylindrical plastic cages (17cm high, 8cm in diameter at the base, with 1cm spacing between the bars) are placed in the corners of the two side chambers to confine unfamiliar mice.

[0045] Evaluation indicators:

[0046] Social Novety Index = Time spent exploring a strange mouse (T) NS - Exploration time for familiar mice (T) FS ) / Exploration time for unfamiliar mice (T) NS )+ exploration time for familiar mice (T) FS ).

[0047] like Figure 2 As shown, the social novelty index of offspring in the prenatal exposure group was significantly lower than that in the solvent control group, indicating that offspring exhibited social behavioral disorders after triclosan exposure.

[0048] (2) Novel Object Recognition Test (NORT): This test assesses the learning and memory abilities of mice. First, mice are placed in an open area with two identical objects (2cm wide × 5cm long × 9cm high) and allowed to explore freely for 5 minutes. After 24 hours, one of the objects is replaced with a new object (different in shape and color from the old one), and the mice are placed in that area to explore for another 5 minutes. The time the mice spend interacting with the new and old objects (sniffing or exploring within 2 cm of the object) is recorded.

[0049] The evaluation indicators are:

[0050] Recognition Index (RI) = Time to explore new objects (T)N ) / (New object exploration time (T) N ) + Old object exploration time (T) F ).

[0051] like Figure 3 As shown, the cognitive index of offspring in the prenatal exposure group was significantly lower than that in the solvent control group, indicating that the offspring had impaired learning and memory abilities.

[0052] (3) Open-field test (OFT): The open-field test chamber used in this experiment was 50 cm in length, width, and height. Timing began 3 seconds after the mice were placed in the central area of ​​the open field. The Smart 3.0 system captured video, recording and analyzing the time taken for the experimental animals to enter the central area, the distance they traveled through the central area, and their average speed throughout the open field. Each mouse was recorded for 5 minutes. Throughout the process, the environment had to be kept quiet, and the test chamber was wiped clean with 75% alcohol before placing the next mouse in.

[0053] like Figure 4 As shown, the total activity level of offspring in the prenatal exposure group was significantly lower than that in the solvent control group, indicating that the offspring exhibited depressive-like emotional problems.

[0054] Based on the results of the above behavioral experiments, prenatal exposure to endocrine disruptors can lead to neurodevelopmental damage in offspring.

[0055] (II) Collection of offspring mouse samples

[0056] (1) Collection of mouse feces

[0057] The feces of the rats were collected on PND 28. Between 9:00 and 11:00 AM, the experimenters stayed in the laboratory to collect 3-5 fresh feces at any time and stored them in 2mL sterilized centrifuge tubes. The tubes were then flash-frozen in liquid nitrogen and transferred to a -80℃ freezer for later use.

[0058] (2) Brain tissue sample collection

[0059] After blood collection, hippocampal tissue was extracted. The brain was dissected on ice, the hippocampus was separated, blood was absorbed with filter paper, and the left and right hippocampi were quickly removed, placed separately into cryovials, and labeled. The left hippocampus was used for hippocampal tissue structure observation. All right hippocampi were collected, placed into cryovials, labeled, and quickly placed in liquid nitrogen for preservation. After all hippocampi were removed, they were frozen at -80°C for later analysis.

[0060] (III) HE staining to observe hippocampal morphology

[0061] Mouse brain and colon tissues were subjected to gradient dehydration and clearing treatment, then embedded in paraffin, sectioned, dewaxed, and stained with hematoxylin and eosin (H&E). The stained sections were then dehydrated and cleared again, mounted, and finally observed under a microscope.

[0062] like Figure 5 As shown, the hippocampal structure in the solvent control group was clear, with relatively regular cell arrangement in each layer, revealing the typical layered outline of the hippocampal region, and showing no obvious abnormal morphological changes, indicating a normal tissue structure. In contrast, some cells in the neurodevelopmental injury group exhibited irregular morphology, varying nuclear staining depths, cell swelling and atrophy, and localized abnormal cell density, reflecting cellular damage caused by exposure. The cell morphology and nuclear staining in the tryptophan diet intervention group were better than those in the neurodevelopmental injury group, closely resembling the cell state of the solvent control group, and the cell density was more regular, indicating that cell damage was reduced and the histopathological state improved after intervention.

[0063] III. Christensenella screening

[0064] Total genomic DNA was extracted from fecal samples using the MagPure Soil DNA LQ kit (Magan) according to the manufacturer's instructions. The concentration and integrity of the extracted DNA were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) and agarose gel electrophoresis, respectively. After quantification, the DNA samples were stored at -20°C for later use.

[0065] Using extracted DNA as a template, the V3-V4 variable region of the 16S rRNA gene was amplified by PCR. This amplification process employed barcoded primers, Takara Ex Taq polymerase (Takara), and universal primers 343F (5'-TACGGRAGGCAGCAG-3') and 798R (5'-AGGGTATCTAATCCT-3'). After amplification, the quality of the PCR products was first checked by agarose gel electrophoresis. The amplicons were then purified using AMPure XP magnetic beads (Agencourt) and a second round of PCR amplification was performed. After a second purification using the same magnetic beads, the final concentration of the amplicons was precisely measured using the Qubit dsDNA detection kit (Thermo Fisher Scientific, USA). The concentration-adjusted amplicons were sequenced on an Illumina NovaSeq 6000 platform using 250 bp paired-end read mode (Illumina, San Diego, California, USA; Shanghai Ouyi Biotechnology Co., Ltd., China).

[0066] Microbial community diversity was analyzed using representative sequences and amplicon sequence variants (ASV) abundance tables. Statistical analysis was performed using the R software package. Christensenellaceae was screened from significantly differentially expressed microbial communities in different sample groups using the Kruskal-Wallis test.

[0067] like Figure 6 As shown, the Kruskal-Wallis test was used to screen the top 10 bacteria with significant differences in abundance at the genus level in the three groups. Christensenellaceae was then further identified, with the mean value for the VG group being 0.00016479, the mean value for the MT group being 0, and the mean value for the TG group being 0.00001225. (See...) Figure 7 result).

[0068] IV. Evidence from Population Verification

[0069] like Figure 8 As shown, we collected serum samples from pregnant women who underwent prenatal checkups at a tertiary hospital in Ningxia in 2023 after obtaining informed consent. We then used liquid chromatography-mass spectrometry (LC-MS) to detect triclosan exposure in the serum of these women. They were divided into two groups based on whether they had triclosan exposure: a triclosan-exposed group (TE) and a triclosan-unexposed group (TU), with 5 women in each group. Fetal feces were collected within 24 hours of delivery for 16S rRNA detection. The results were consistent with animal experiments, showing that Christenella ceae had a higher relative expression abundance in the non-EDCs exposure group.

[0070] The serum liquid chromatography-mass spectrometry method is as follows:

[0071] First, liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis.

[0072] Triple quadrupole mass spectrometer (TRIPLE QUAD) TMThe Sciex Instruments 5500 system (Foster City, California, USA) was used to analyze the target compounds in multiple reaction monitoring (MRM) mode. Scheduled MRM was used to improve the sensitivity and selectivity of the analytical method. Parameters for each ion pair were automatically obtained through a compound optimization program and confirmed by manually injecting a standard solution into the ion source. The ion source was optimized by flow injection analysis and set as follows: electrospray ionization voltage set to -5.5 kV, curtain gas and collision gas (nitrogen) flow rates set to 40 psi, ion source heater temperature set to 500 °C, nebulizing gas (ion source gas 1) flow rate set to 50 psi, and heating gas (ion source gas 2) flow rate set to 40 psi.

[0073] Liquid chromatography parameters were optimized for analyte analysis. Acetonitrile (ACN) and ultrapure water were selected as the mobile phases. In this section, an Agilent G6460 triple quadrupole mass spectrometer coupled with a 1290 high-performance liquid chromatography system was used. The chromatographic column was a BETASIL C18 (2.1 mm × 100 mm, 5 μm, Thermo Fisher Scientific, Massachusetts, USA). Chromatographic separation was performed using an ultra-high performance liquid chromatography system (Exion). TM The analysis was performed using a BETASIL C18 analytical column (LC). The mobile phase consisted of Milli-Q water (solvent A) and acetonitrile (solvent B). The gradient conditions were as follows: starting with 10% B, increasing to 30% B in 0.51 min and holding for 2.5 min, then increasing to 95% B in 4 min and holding for 3 min, and finally decreasing to 10% B in 0.1 min and holding for 2.89 min. The total analytical run time was 13 min. The injection volume was 5 μL, the flow rate was 0.3 mL / min, and the column oven temperature was set to 30 °C.

[0074] Secondly, liquid-liquid extraction combined with solid-phase extraction purification (LLE+SPE) is used.

[0075] For solid-phase extraction (SPE), we selected Oasis MAX (60 mg / 3 cc, Waters Corporation, Massachusetts, USA). In a glass tube, 25 ng of the analyte standard and 5 ng of the internal standard mixture were added to 0.5 mL of methanol (MeOH). For the MCX extraction column, it was diluted with water (H₂O, 4.5 mL) or formic acid solution (FA, 4.5 mL, 0.1% (v / v)) and vortexed before SPE purification. Other procedures were performed according to the product instructions, except for the washing step. Subsequently, the MCX extraction column was washed with water or formic acid solution. Therefore, there are four treatment methods for the extraction column (dilution-washing), namely H₂O-H₂O, H₂O-FA, FA-H₂O, and FA-FA for MCX. The elution solvents for the MCX extraction column were 2 mL methanol / acetonitrile (1:1, v / v) and 2 mL methanol / acetonitrile containing 1% ammonia (1:1, v / v) (interval three times). After drying under nitrogen, the combined extracts were concentrated to 0.5 mL of methanol / acetonitrile (1:1, v / v) and injected for analysis. Each treatment was repeated three times. Extraction columns with higher recoveries were selected for SPE extraction.

[0076] After three liquid-liquid extractions (LLE), the collected extract was evaporated to near dryness, and 0.5 mL of methanol / acetonitrile (1:1, volume ratio) was added. Then, the SPE step was performed according to the operating procedure determined during the MCX selection process.

[0077] In summary, this invention demonstrates that Christensenellaceae has a targeted detection function against EDCs-induced neurodevelopmental damage in mice, showing a reduced abundance of Christensenella in mice with EDCs-induced neurodevelopmental damage. Therefore, detecting Christensenella in offspring feces can be used to predict neurodevelopmental damage at an early stage, and further preventive and improvement measures can be taken through dietary interventions and other means.

[0078] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. Application of Christensenella as a biomarker in the preparation of products for the diagnosis or monitoring of neurodevelopmental damage.

2. The application according to claim 1, characterized in that, The neurodevelopmental impairment refers to the neurodevelopmental impairment in offspring caused by exposure to endocrine disruptors in the maternal prenatal environment.

3. The application according to claim 1, characterized in that, After combining tryptophan-based dietary intervention, the level of Kristensenella abundance recovery was measured to assess the intervention effect.

4. The application according to claim 2, characterized in that, The endocrine disruptors include triclosan.

5. A product for diagnosing or monitoring neurodevelopmental impairment, characterized in that, This includes reagents for the specific detection of Christensenella.

6. The product according to claim 5, characterized in that, The products include reagents or kits.

7. The product according to claim 5 or 6, characterized in that, The product uses the feces of the patient being tested as the test sample.

Images