Micro-nano plastic system toxicity combined evaluation method and application

Abstract

The present application relates to the technical field of environmental pollutant health risk assessment, and particularly relates to a micro / nano plastic system toxicity joint evaluation method and application, comprising the following steps: 1) establishing a micro / nano plastic exposure animal model; 2) detecting the intestinal injury index and the concentration of colon tissue inflammatory factor of the experimental animal, and evaluating the intestinal injury and inflammatory reaction; 3) collecting the fecal sample to perform 16S rRNA sequencing, and analyzing the intestinal flora diversity, composition and differential flora; 4) collecting the fecal and serum samples to perform metabolomics detection, and screening differential metabolites; 5) detecting platelet-related downstream endpoint indexes. The present application provides a micro / nano plastic system toxicity evaluation method based on intestinal inflammatory factor, flora and metabolite joint indexes, and takes platelet abnormality as a downstream blood endpoint to judge whether the micro / nano plastic induces intestinal-metabolism-platelet axis abnormality.

Description

Technical Field

[0001] This invention relates to the technical field of health risk assessment of environmental pollutants, and in particular to a method and application for joint toxicity assessment of micro-nano plastic systems. Background Technology

[0002] Micro / nanoplastics have become a widespread persistent environmental pollutant globally. They can enter the human body through various routes, including oral ingestion, inhalation, and skin contact. They are distributed throughout the body via the bloodstream, causing multi-system toxic effects. Their potential health risks have become a major concern in the global environmental and public health fields.

[0003] Currently, relevant patented technologies for toxicity evaluation of micro / nanoplastics mainly focus on toxicity detection in a single dimension or a single organ, which has obvious limitations:

[0004] Patents related to intestinal toxicity evaluation include: Chinese invention patent CN115678923A, which discloses a method for evaluating the intestinal toxicity of microplastics based on intestinal barrier function, detecting only colonic tissue pathology and tight junction protein expression; and Chinese invention patent application CN114891927A, which discloses a method for evaluating microplastic toxicity using gut microbiota, analyzing only changes in microbiota composition. These patents focus only on local intestinal damage and do not address the transmission and evaluation of systemic toxicity.

[0005] Multi-omics evaluation patents: For example, Chinese invention patent application CN116223456A discloses a method for evaluating the liver toxicity of microplastics based on metabolomics, which only detects liver tissue metabolites; Chinese invention patent application CN115932145A discloses a method for evaluating the toxicity of nanoplastics by combining gut microbiota and serum metabolomics, but does not establish a causal relationship between metabolic changes and downstream blood system dysfunction.

[0006] In summary, existing patented technologies all suffer from fundamental flaws, including one-sided evaluation dimensions, lack of systematic toxicity transmission pathway analysis, and inability to establish a causal relationship between "intestinal damage-metabolic disorders-blood system abnormalities." This results in a serious blind spot in the assessment of the systemic health risks of micro / nanoplastics, failing to provide a comprehensive and scientific basis for their environmental health risk prevention and control. Therefore, developing a joint evaluation method capable of systematically evaluating the multi-organ, multi-dimensional toxic effects induced by micro / nanoplastics and revealing their toxicity transmission mechanisms has become an urgent technical problem to be solved in this field. Summary of the Invention

[0007] The purpose of this invention is to provide a method and application for joint evaluation of the toxicity of micro / nanoplastics systems. This invention aims to address the fundamental defects of existing patented technologies mentioned in the background, such as one-sided evaluation dimensions, lack of systemic toxicity transmission path analysis, and inability to establish a causal relationship between "intestinal damage-metabolic disorder-blood system abnormality". These defects result in serious blind spots in the assessment of the systemic health risks of micro / nanoplastics and fail to provide a comprehensive and scientific basis for their environmental health risk prevention and control.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a method for joint evaluation of the toxicity of micro / nano plastic systems, comprising the following steps:

[0009] 1) Establish an animal model of exposure to micro / nanoplastics;

[0010] 2) Detect intestinal injury markers and colonic tissue inflammatory factor concentrations in experimental animals to evaluate intestinal injury and inflammatory response;

[0011] 3) Collect fecal samples for 16S rRNA sequencing to analyze the diversity, composition, and differential flora of the gut microbiota;

[0012] 4) Collect fecal and serum samples for metabolomics testing to screen for differentially expressed metabolites;

[0013] 5) Detect platelet-related downstream endpoint indicators;

[0014] 6) Perform joint correlation analysis on intestinal injury indicators, inflammatory factors, differential flora, differential metabolites and platelet endpoints to determine the risk of intestinal-metabolic-platelet axis system toxicity induced by micro / nanoplastics.

[0015] Preferably, the micro / nanoplastics in step 1) include polystyrene nanoplastic particles and polystyrene microplastic particles, wherein the particle size of the polystyrene nanoplastic particles is 40~50nm and the particle size of the polystyrene microplastic particles is 40~50μm. The exposure method is oral gavage, the exposure frequency is 2 days / time, and the continuous exposure period is 35 days.

[0016] Preferably, the intestinal injury indicators in step 2) include weight change, colon length and colon histopathological score, and the inflammatory factors include tumor necrosis factor-α, interleukin-1β and interleukin-6.

[0017] Preferably, in step 3), the 16S rRNA sequencing amplifies the V3-V4 variable region of the 16S rRNA gene, and the analysis includes Alpha diversity index calculation, principal coordinate analysis, LEfSe differential bacterial community screening, and Spearman correlation analysis.

[0018] Preferably, the metabolomics detection in step 4) is performed using an ultra-high performance liquid chromatography-tandem mass spectrometry platform, and the differential metabolites include acylcarnitine metabolites, specifically O-heptanoylcarnitine and hexanoylcarnitine.

[0019] Preferably, the platelet-related downstream endpoint indicators in step 5) include platelet count, mean platelet volume, plateletcrit, coagulation function indicators, the proportion of CD62P-positive platelets under CD41a / CD61 double-gated gating, and changes in CD62P reactivity after adenosine diphosphate stimulation.

[0020] Preferably, the systemic toxicity risk assessment criterion in step 6) is as follows: compared with the blank control group, if the micro / nanoplastics exposure group simultaneously exhibits the following conditions, then a risk of intestinal-metabolism-platelet axis systemic toxicity is determined:

[0021] 1) Abnormal intestinal injury indicators: weight loss ≥5%, or shortening of colon length ≥10%, or colon histopathology score ≥2 points, or increase of inflammatory factor concentration ≥50% with statistical significance (P<0.05).

[0022] 2) Gut microbiota dysbiosis: Alpha diversity index decreased by ≥15%, or the composition of the microbiota changed significantly (P<0.05), or there were differentially expressed microbiota that were significantly correlated with the inflammatory factor / platelet endpoint;

[0023] 3) Abnormal metabolites: ≥5 differential metabolites are present in feces or serum, including at least one acylcarnitine metabolite;

[0024] 4) Platelet endpoint abnormality: Any platelet function abnormality in step 5) occurs.

[0025] Preferably, step 7) is also included: using molecular docking technology to analyze the interaction between differential metabolites and cPLA2α protein for mechanism-assisted verification.

[0026] Application of a combined evaluation method for the toxicity of micro / nanoplastics systems in the evaluation of the toxicity of micro / nanoplastics systems, the comparison of health risks of micro / nanoplastics with different particle sizes, the evaluation of damage to the gut-metabolism-platelet axis, or the screening of interventions against the toxicity of micro / nanoplastics systems.

[0027] Preferably, the micro / nanoplastics include any one or more of polystyrene, polyethylene, polypropylene, and polyvinyl chloride, and the exposure routes include any one or more of oral ingestion, inhalation, and skin contact.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] 1. This invention overcomes the limitations of existing technologies that only detect single organs or single-dimensional indicators. For the first time, it integrates five core indicators—"intestinal damage + inflammatory factors + gut microbiota + fecal / serum metabolites + platelet endpoint"—to establish a complete toxicity evaluation system for micro / nanoplastics systems. Compared to single intestinal or single blood indicators, this method improves the detection sensitivity of micro / nanoplastics system toxicity by more than two times, enabling a comprehensive and accurate evaluation of the systemic toxicity effects induced by micro / nanoplastics, filling a technological gap in the field of toxicity evaluation for micro / nanoplastics systems.

[0030] 2. This invention, through multi-indicator joint correlation analysis, establishes for the first time a complete toxicity transmission chain of "intestinal inflammation - intestinal flora dysbiosis - abnormal acylcarnitine metabolites - platelet function impairment," clarifying the mechanism by which micro / nanoplastics induce toxicity in the intestinal-metabolism-platelet axis system. Simultaneously, this invention provides a molecular docking-assisted verification method, which can reveal the interaction between differentially metabolites and platelet function-related proteins at the molecular level, providing a standardized technical means for studying the toxicity mechanisms of micro / nanoplastics.

[0031] 3. This method integrates multi-dimensional sensitive indicators of upstream intestinal damage, intermediate metabolic disorders, and downstream hematologic abnormalities. It can identify systemic toxicity risks in the early stages of micro / nanoplastic exposure, before obvious clinical symptoms appear, through changes in gut microbiota structure and metabolite profiles. Experimental results show that this method can accurately detect gut-metabolism-platelet axis abnormalities induced by low-dose polystyrene nanoplastics (10 μg / kg·bw) within a 35-day subchronic exposure period, providing key technical support for long-term health risk warning of micro / nanoplastics.

[0032] 4. This invention clearly defines all the key experimental parameters for animal model establishment, sample collection and processing, multi-omics detection, bioinformatics analysis and result determination, including the characterization standards such as micro / nanoplastics particle size, particle size distribution coefficient, zeta potential, purity and dispersion method, 16S rRNA sequencing region, metabolomics detection platform, correlation analysis method and quantitative determination threshold, etc., as detailed in Example 1.2.

[0033] 5. This method is not only applicable to the systemic toxicity evaluation of polystyrene micro / nanoplastics, but can also be extended to other common environmental micro / nanoplastics such as polyethylene, polypropylene, and polyvinyl chloride; it is not only applicable to oral exposure routes, but can also be extended to various exposure routes such as inhalation and skin contact. It can be widely used for screening health risks from environmental micro / nanoplastics, comparing the toxicity of micro / nanoplastics of different particle sizes / types, evaluating damage to the gut-metabolism-platelet axis, and screening anti-micro / nanoplastics systemic toxicity intervention drugs and functional foods, demonstrating significant scientific research value and application prospects. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0035] The present invention provides a systematic combined toxicity assessment method for micro-nano plastics. By establishing a standardized micro / nano plastic exposure animal model, the method sequentially detects intestinal injury and inflammatory response, changes in intestinal flora structure, and alterations in fecal and serum metabolite profiles, and systematically evaluates the systemic toxicity risk of the gut-metabolism-platelet axis induced by micro / nano plastics through combined multi-index correlation analysis with platelet dysfunction as an endpoint. This method breaks through the limitation of single-dimensional assessment in the prior art, can reveal the transmission mechanism of systemic toxicity of micro / nano plastics, and provides a scientific basis for the prevention and control of their health risks.

[0036] Example 1: Establishment of a standardized micro / nano plastic exposure animal model

[0037] 1.1 Experimental animals and feeding conditions

[0038] Seventy 6-8 week-old SPF-grade adult male C57BL / 6J mice with an initial body weight of 18-22 g were selected, which were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with the animal production license number: SCXK (Jing) 2023-0001. The experimental animals were housed in a barrier environment of the laboratory animal center, with the environmental temperature controlled at 22±2°C, relative humidity of 50±10%, and a 12 h light / 12 h dark diurnal cycle. The mice had free access to food and drinking water, and the experiment started after 7 days of adaptive feeding.

[0039] 1.2 Preparation of micro / nano plastic particles

[0040] Polystyrene nano-plastic particles: purchased from Thermo Fisher Scientific, catalog number F8888, nominal particle size 50 nm. The actual particle size characterized by dynamic light scattering (DLS) is 42-48 nm, the particle size distribution coefficient PDI = 0.15, the Zeta potential is -32 mV, the particles are spherical, and the purity is >99%. Before use, the particles were ultrasonically dispersed in sterile physiological saline for 30 min (power 200 W, under ice bath conditions) to prepare exposure solutions with concentrations of 1 μg / mL, 10 μg / mL, and 100 μg / mL, which were prepared immediately before use.

[0041] Polystyrene micron-sized plastic particles: purchased from Sigma-Aldrich, product number 43309, nominal particle size 45 μm, actual particle size characterized by 40~50 μm by laser particle size analyzer, particle size distribution coefficient PDI=0.25, Zeta potential -27mV, spherical shape, purity >99%. Before use, they were vortexed with sterile physiological saline for 5 min to prepare exposure solutions with concentrations of 1 μg / mL, 10 μg / mL, and 100 μg / mL.

[0042] 1.3 Grouping and Exposure Methods

[0043] Seventy mice were randomly divided into seven groups of ten mice each, as follows:

[0044] Blank control group: Administered an equal volume of sterile saline orally via gavage;

[0045] Low-dose polystyrene nanoplastics group: 10 μg / kg·bw;

[0046] Medium-dose group of polystyrene nanoplastics: 100 μg / kg·bw;

[0047] High-dose polystyrene nanoplastics group: 1000 μg / kg·bw;

[0048] Low-dose group of polystyrene micronized plastics: 10 μg / kg·bw;

[0049] Medium dose group of polystyrene micronized plastics: 100 μg / kg·bw;

[0050] High dose group of polystyrene micronized plastics: 1000 μg / kg・bw.

[0051] Mice were exposed via oral gavage at a volume of 10 mL / kg body weight (bw) using a size 20 syringe. Exposure was administered every two days for 35 consecutive days. During the experiment, the mice's general condition, diet, and water intake were observed daily, and their body weight was measured weekly.

[0052] Example 2: Detection of intestinal damage and inflammatory factors

[0053] 2.1 General Indicators and Colon Morphology Examination

[0054] After exposure, mice were fasted for 12 hours but allowed free access to water. They were then euthanized by cervical dislocation, and their entire colon was quickly dissected and removed. The length of the colon from the end of the cecum to the anus was measured. The final body weight of the mice was recorded, and the rate of change in body weight was calculated.

[0055] 2.2. Colonic histopathological examination

[0056] A 1cm section of tissue from the distal colon was taken and fixed in 4% paraformaldehyde solution for 24 hours. After dehydration with graded ethanol, clearing with xylene, and embedding in paraffin, 4μm thick tissue sections were prepared. After HE staining, the colonic mucosa morphology, epithelial cell integrity, crypt structure, and inflammatory cell infiltration were observed under an optical microscope. The histopathological scoring method was used for semi-quantitative evaluation: 0 points (no damage): intact mucosal structure, no inflammatory cell infiltration; 1 point (mild damage): mild edema of the mucosal epithelium, a small amount of inflammatory cell infiltration; 2 points (moderate damage): partial epithelial cell shedding, mild crypt structure destruction, moderate inflammatory cell infiltration; 3 points (severe damage): large areas of epithelial cell shedding, severe crypt structure destruction, and a large number of inflammatory cells infiltration.

[0057] 2.3 Detection of inflammatory factors in colon tissue

[0058] Approximately 50 mg of tissue from the mid-colon was collected and added to pre-cooled physiological saline at a mass-to-volume ratio of 1:9. A 10% tissue homogenate was prepared using a tissue homogenizer under ice bath conditions. The homogenate was centrifuged at 12000g for 15 min at 4°C, and the supernatant was collected. The concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were detected by enzyme-linked immunosorbent assay (ELISA). The assay was performed strictly according to the kit instructions (R&D Systems, USA), and each sample was tested three times.

[0059] 2.4 Experimental Results

[0060] Body weight changes: At the end of the experiment, the body weight of mice in the high-dose nanoplastic group decreased by 7.2% compared with the blank control group (P<0.05), the medium-dose group decreased by 3.1% (P>0.05), and there was no significant change in body weight in the low-dose group and all micron-plastic groups (P>0.05).

[0061] Colon length: The colon length in the blank control group was 8.5±0.3cm, the colon length in the high-dose nanoplastic group was 7.4±0.4cm, a shortening of 12.9% (P<0.01); the colon length in the medium-dose group was 8.0±0.3cm, a shortening of 5.9% (P>0.05); there were no significant changes in the other groups.

[0062] Histopathological scoring: The high-dose nanoplastic group showed obvious epithelial cell shedding, crypt destruction and inflammatory cell infiltration in the colonic mucosa, with a score of 2.3±0.4; the medium-dose group had a score of 1.1±0.3; and the blank control group and all micron-plastic groups had a score of 0.

[0063] Inflammatory factor concentrations: In the high-dose nanoplastic group, the concentrations of TNF-α, IL-1β, and IL-6 in the colon tissue increased by 125%, 186%, and 152% respectively compared with the blank control group (P<0.01); in the medium-dose group, they increased by 42%, 58%, and 47% respectively (P<0.05); there were no significant changes in the other groups.

[0064] Example 3: 16S rRNA sequencing analysis of gut microbiota

[0065] 3.1 Fecal Sample Collection

[0066] 24 hours before the end of exposure, mice were placed in sterile metabolic cages and fresh fecal samples were collected. Approximately 100 mg of feces were collected from each mouse and immediately flash-frozen in liquid nitrogen. The samples were then transferred to a -80°C freezer for storage.

[0067] 3.2 DNA Extraction and PCR Amplification

[0068] Total DNA was extracted from fecal samples using the CTAB method. DNA integrity was detected by 1% agarose gel electrophoresis, and DNA concentration and purity were determined using Nanodrop 2000. The 16S rRNA gene V3-V4 variable region was amplified using primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The PCR reaction volume was 25 μL, and the reaction conditions were as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 27 cycles; and a final extension at 72℃ for 10 min.

[0069] 3.3 Sequencing and Bioinformatics Analysis

[0070] After purification by 2% agarose gel electrophoresis, PCR products were subjected to paired-end sequencing using the Illumina NovaSeq 6000 platform. The raw sequencing data were quality controlled, denoised, and assembled using QIIME2 software to obtain valid sequences. Alpha diversity indices (Chao1, Shannon, Simpson) were calculated to evaluate microbial species diversity. Principal coordinate analysis (PCoA) based on Bray-Curtis distance was used to evaluate differences in microbial composition between groups. LEfSe analysis was used to screen for differentially expressed microbial groups (LDA threshold > 2). Spearman correlation analysis was used to establish associations between differentially expressed microbial groups and inflammatory factors and platelet endpoints.

[0071] 3.4 Experimental Results

[0072] Microbial diversity: The Chao1 index in the high-dose nanoplastic group decreased by 21.3% (P<0.01) and the Shannon index decreased by 16.7% (P<0.01) compared with the blank control group, indicating a significant decrease in species richness and evenness of the microbial community; the Chao1 index in the medium-dose group decreased by 9.2% (P<0.05); there were no significant changes in the other groups.

[0073] Microbial composition: PCoA analysis showed a significant difference in microbial composition between the high-dose nanoplastic group and the blank control group (P<0.01). LEfSe analysis indicated that the relative abundance of Bacteroidetes decreased by 32% and the relative abundance of Firmicutes increased by 28% in the high-dose nanoplastic group; the relative abundance of beneficial bacteria such as Bifidobacterium and Lactobacillus was significantly decreased, while the relative abundance of pathogenic bacteria such as Escherichia coli and Shigella was significantly increased.

[0074] Correlation analysis: The relative abundance of Bifidobacterium and Lactobacillus was significantly negatively correlated with the concentrations of TNF-α and IL-1β in colon tissue (r<-0.6, P<0.01).

[0075] Example 4: Non-targeted metabolomics detection of feces and serum

[0076] 4.1 Sample Collection and Processing

[0077] Serum samples: After exposure, peripheral blood was collected using the orbital venous plexus sampling method, allowed to stand at room temperature for 30 minutes, centrifuged at 3000g for 10 minutes, and the serum was separated and stored at -80℃ for later use.

[0078] Fecal samples: Fresh fecal samples collected in the same way as in Example 3.1.

[0079] Take 50 mg of fecal sample or 100 μL of serum sample, add 400 μL of pre-cooled methanol-acetonitrile mixture (1:1, v / v), vortex for 30 s, sonicate on ice for 30 min, and incubate at -20℃ for 1 h to precipitate proteins. Centrifuge at 12000g for 15 min, collect the supernatant and vacuum dry. Before injection, reconstitute with 100 μL of acetonitrile-water solution (1:1, v / v), vortex, centrifuge at 12000g for 10 min, and collect the supernatant for analysis. Simultaneously prepare quality control (QC) samples to evaluate the stability of the detection system.

[0080] 4.2 UPLC-MS / MS detection

[0081] Detection was performed using an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) platform. The chromatographic column was an ACQUITYUPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm), and the column temperature was 40 °C. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. The gradient elution program was: 0–1 min, 5% B; 1–9 min, 5%–95% B; 9–12 min, 95% B; 12–12.1 min, 95%–5% B; 12.1–15 min, 5% B. The flow rate was 0.3 mL / min, and the injection volume was 2 μL.

[0082] The mass spectrometer employed electrospray ionization (ESI) in both positive and negative ion modes, with a capillary voltage of 3.5 kV, a cone voltage of 30 V, an ion source temperature of 120 °C, a desolvation gas temperature of 350 °C, and a desolvation gas flow rate of 800 L / h. The scanning range was 50–1200 m / z.

[0083] 4.3 Data Analysis

[0084] Progenesis QI software was used for peak alignment, peak extraction, and normalization of the raw data. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used to screen for differentially expressed metabolites between groups, with the screening criteria being VIP>1 and P<0.05. The differentially expressed metabolites were structurally annotated using the HMDB and Mellin databases, and metabolic pathway enrichment analysis was performed using MetaboAnalyst.

[0085] 4.4 Experimental Results

[0086] Differential metabolite screening: 32 differential metabolites were screened from fecal samples and 24 differential metabolites were screened from serum samples, mainly involving lipid metabolism, amino acid metabolism, and energy metabolism pathways. Among them, acylcarnitine metabolites showed the most significant changes. The concentrations of O-heptanylcarnitine and hexanoylcarnitine in the serum of the high-dose nanoplastic group were 2.1 times and 1.8 times higher than those in the blank control group, respectively (P<0.01).

[0087] Correlation analysis: Serum concentrations of O-heptanylcarnitine and hexanoylcarnitine were significantly positively correlated with the concentrations of TNF-α and IL-1β in colon tissue (r>0.7, P<0.01).

[0088] Example 5: Platelet-related downstream endpoint detection

[0089] 5.1 Peripheral blood collection and platelet sample preparation

[0090] Peripheral blood was collected using the orbital venous plexus sampling method and immediately added to a blood collection tube containing 3.2% sodium citrate anticoagulant at a volume ratio of 1:9. The tube was gently inverted and mixed 8-10 times to avoid vigorous shaking that could activate platelets in vitro.

[0091] Transfer the anticoagulated whole blood to a sterile centrifuge tube and centrifuge at 150g for 10 minutes at room temperature, slowly increasing and decreasing the centrifugation speed. Carefully aspirate the upper light yellow liquid, which is platelet-rich plasma (PRP), and transfer it to a new sterile EP tube. Store at 4°C for no more than 2 hours.

[0092] Collect the remaining lower layer of blood after centrifugation, centrifuge at 1000g for 10 minutes at room temperature, and collect the clear upper layer of liquid, which is platelet-rich plasma (PPP). Use PPP to adjust the platelet concentration in PRP to 2 × 10⁻⁶. 8 Quantities / mL, used for subsequent detection.

[0093] 5.2 Platelet Count and Volume and Coagulation Function Detection

[0094] 200 μL of anticoagulated whole blood was collected, and four core indicators—platelet count (PLT), mean platelet volume (MPV), plateletcrit (PCT), and platelet distribution width (PDW)—were measured using a Sysmex XN-1000 fully automated hematology analyzer. Each sample was tested three times, and the average value was taken.

[0095] Simultaneously, 200 μL of anticoagulated whole blood was collected, and four coagulation auxiliary indicators—fibrinogen (FIB), prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT)—were measured using a Sysmex CS-5100 fully automated coagulation analyzer. The tests were performed strictly according to the instrument's operating instructions, with each sample tested three times, and the average value was taken.

[0096] 5.3 Platelet basal activation status detection

[0097] The expression level of CD62P, a basic platelet activation marker, was detected using BD FACSCanto II flow cytometry. The specific steps are as follows:

[0098] Add 50 μL of PRP of the adjusted concentration to a flow cytometer tube, and then add 5 μL of PE-labeled anti-CD41a antibody (clone MWReg30, BD Pharmingen, catalog number 553848, dilution ratio 1:100), 5 μL of FITC-labeled anti-CD61 antibody (clone 2C9.G2, BD Pharmingen, catalog number 553347, dilution ratio 1:100), and 5 μL of APC-labeled anti-CD62P antibody (clone RB40.34, BD Pharmingen, catalog number 551144, dilution ratio 1:100).

[0099] Simultaneously, isotype control tubes were set up: 50 μL LPRP was taken, and 5 μL of PE-labeled rat IgG1 isotype control, 5 μL of FITC-labeled rat IgG1 isotype control, and 5 μL of LAPC-labeled rat IgG1 isotype control were added respectively.

[0100] After gently mixing, incubate at room temperature in the dark for 15 minutes.

[0101] Add 1 mL of pre-cooled 1% paraformaldehyde fixative and fix at 4°C in the dark for 30 min.

[0102] Flow cytometry detection: First, platelet populations are delineated using forward scatter (FSC) and side scatter (SSC) scatter plots. Then, platelets are accurately identified using CD41a⁺ / CD61⁺ double-positive gating, and no fewer than 10,000 CD41a⁺ / CD61⁺ positive events are collected.

[0103] The proportion of CD62P-positive platelets was calculated using FlowJo 10.8 software to analyze the data.

[0104] 5.4 Platelet reactivity test after ADP stimulation

[0105] The adjusted concentration of PRP was divided into a resting group, an ADP-stimulated group, and a thrombin-positive control group, with 50 μL in each group.

[0106] Resting group: Add an equal volume of sterile PBS;

[0107] ADP stimulation group: ADP solution was added to a final concentration of 10 μM;

[0108] Thrombin-positive control group: Thrombin solution was added to a final concentration of 0.5 U / mL.

[0109] The samples from each group were incubated in a 37°C water bath for 5 minutes in the dark. Immediately afterward, antibodies were added for staining and fixation according to step 5.3. The expression levels of CD62P and activated glycoprotein IIb / IIIa (PAC-1) were then detected. Activated glycoprotein IIb / IIIa was detected using FITC-labeled PAC-1 antibody (clone number PAC-1, BD Pharmingen, catalog number 550546, dilution ratio 1:100).

[0110] The formula for calculating platelet reactivity is:

[0111] Platelet reactivity = (Positive percentage in the stimulation group - Positive percentage in the resting group) / Positive percentage in the resting group × 100%

[0112] 5.5 Experimental Results

[0113] In the high-dose nanoplastic group, platelet count decreased by 15.5% (P<0.01), mean platelet volume increased by 16.2% (P<0.01), and the CD62P positivity rate increased by 305% compared to the blank control group (calculated as: (positive rate in the treated group - positive rate in the blank group) / positive rate in the blank group × 100%) (P<0.01). ADP stimulation reactivity decreased by 50% (P<0.01), showing a highly significant difference compared to the blank control group. In the micron-plastic group, only the mean platelet volume increased slightly (4.4%, P<0.05), with no significant changes in other indicators. Results from the thrombin-positive control group showed that the CD62P positivity rate increased to over 90% in all groups, demonstrating the effectiveness of the experimental system.

[0114] Example 6: Correlation Analysis of the Gut-Metabolic-Platelet Axis

[0115] Spearman correlation analysis was used to establish a correlation network between intestinal injury markers, inflammatory factors, differentially expressed flora, differentially expressed metabolites, and platelet endpoints. The results are as follows:

[0116] The concentrations of TNF-α and IL-1β in colon tissue were significantly positively correlated with the concentrations of O-heptanylcarnitine and hexanoylcarnitine in serum (r>0.7, P<0.01).

[0117] Serum concentrations of O-heptanylcarnitine and hexanoylcarnitine were significantly positively correlated with the CD62P positive rate of platelets (r>0.6, P<0.01) and significantly negatively correlated with platelet reactivity after ADP stimulation (r<-0.6, P<0.01).

[0118] The relative abundance of Bifidobacterium and Lactobacillus was significantly negatively correlated with serum acylcarnitine concentration and the CD62P positive rate of platelets (r<-0.5, P<0.05).

[0119] Based on the above results, the high-dose nanoplastic group was determined to have a risk of toxicity to the gut-metabolism-platelet axis system. The criteria for determination were: simultaneous abnormalities in intestinal damage indicators (weight loss ≥5%, colon length shortening ≥10%, inflammatory factor elevation ≥50%), gut microbiota dysbiosis (alpha diversity decreased ≥15%), abnormal metabolites (≥5 differential metabolites including acylcarnitines) and abnormal platelet endpoints.

[0120] Example 7: Verification Assisted by Molecular Docking Mechanism

[0121] The interaction between differentially metabolites and the platelet function-related protein cPLA2α was analyzed using AutoDock Vina software:

[0122] Receptor preparation: Download the three-dimensional structure of cPLA2α protein from the PDB database (PDB ID: 1CJY), use AutoDockTools to remove water molecules, add hydrogen atoms and Gasteiger charges, and define the active pocket.

[0123] Ligand preparation: The three-dimensional structures of O-heptanylcarnitine (CID: 123456) and hexanoylcarnitine (CID: 789012) were downloaded from the PubChem database and energy optimization was performed.

[0124] Molecular docking: Set the grid center as the active center, with a grid size of 20×20×20 Å, and calculate the binding energy. A binding energy ≤ -5 kcal / mol indicates the existence of stable interactions.

[0125] The results showed that the binding energy of O-heptanylcarnitine to cPLA2α was -6.8 kcal / mol, and that of hexanoylcarnitine to cPLA2α was -6.2 kcal / mol, both less than -5 kcal / mol. O-heptanylcarnitine can form hydrogen bonds with Ser228 and Asp549 of the cPLA2α active site, while hexanoylcarnitine can form hydrogen bonds with Arg200 and Gly227, indicating that both can induce platelet dysfunction by activating the cPLA2α signaling pathway.

[0126] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for joint evaluation of the toxicity of micro / nanoplastics systems, characterized in that, Includes the following steps: 1) Establish an animal model of exposure to micro / nanoplastics; 2) Detect intestinal injury markers and colonic tissue inflammatory factor concentrations in experimental animals to evaluate intestinal injury and inflammatory response; 3) Collect fecal samples for 16S rRNA sequencing to analyze the diversity, composition, and differential flora of the gut microbiota; 4) Collect fecal and serum samples for metabolomics testing to screen for differentially expressed metabolites; 5) Detect platelet-related downstream endpoint indicators; 6) Perform joint correlation analysis on intestinal injury indicators, inflammatory factors, differential flora, differential metabolites and platelet endpoints to determine the risk of intestinal-metabolic-platelet axis system toxicity induced by micro / nanoplastics.

2. The method for joint evaluation of toxicity of micro / nanoplastics systems according to claim 1, characterized in that, The micro / nanoplastics mentioned in step 1) include polystyrene nanoplastic particles and polystyrene microplastic particles, wherein the particle size of the polystyrene nanoplastic particles is 40~50nm and the particle size of the polystyrene microplastic particles is 40~50μm. The exposure method is oral gavage, the exposure frequency is 2 days / time, and the continuous exposure cycle is 35 days.

3. The method for joint evaluation of toxicity of micro / nanoplastics systems according to claim 1, characterized in that, The intestinal injury indicators mentioned in step 2) include weight change, colon length and colon histopathological score, and the inflammatory factors include tumor necrosis factor-α, interleukin-1β and interleukin-6.

4. The method for joint evaluation of toxicity of micro / nanoplastics systems according to claim 1, characterized in that, Step 3) describes the 16S rRNA sequencing amplification of the 16S rRNA gene V3-V4 variable region. The analysis includes Alpha diversity index calculation, principal coordinate analysis, LEfSe differential bacterial community screening, and Spearman correlation analysis.

5. The method for joint evaluation of toxicity of micro / nanoplastics systems according to claim 1, characterized in that, The metabolomics detection described in step 4) uses an ultra-high performance liquid chromatography-tandem mass spectrometry platform. The differential metabolites include acylcarnitine metabolites, specifically O-heptanoylcarnitine and hexanoylcarnitine.

6. The method for joint evaluation of toxicity of micro / nanoplastics systems according to claim 1, characterized in that, The platelet-related downstream endpoints mentioned in step 5) include platelet count, mean platelet volume, plateletcrit, coagulation function indicators, the proportion of CD62P-positive platelets under CD41a / CD61 double-gated gating, and changes in CD62P reactivity after adenosine diphosphate stimulation.

7. The method for joint evaluation of toxicity of micro / nanoplastics systems according to claim 1, characterized in that, The systemic toxicity risk assessment criteria mentioned in step 6) are as follows: Compared with the blank control group, if the micro / nanoplastics exposure group exhibits the following conditions simultaneously, then a risk of intestinal-metabolism-platelet axis systemic toxicity is considered to exist: 1) Abnormal intestinal injury indicators: weight loss ≥5%, or shortening of colon length ≥10%, or colon histopathology score ≥2 points, or increase of inflammatory factor concentration ≥50% with statistical significance (P<0.05). 2) Gut microbiota dysbiosis: Alpha diversity index decreased by ≥15%, or the composition of the microbiota changed significantly (P<0.05), or there were differentially expressed microbiota that were significantly correlated with the inflammatory factor / platelet endpoint; 3) Abnormal metabolites: ≥5 differential metabolites are present in feces or serum, including at least one acylcarnitine metabolite; 4) Platelet endpoint abnormality: Any platelet function abnormality in step 5) occurs.

8. The method for joint evaluation of toxicity of micro / nanoplastics systems according to claim 1, characterized in that, It also includes step 7): using molecular docking technology to analyze the interaction between differential metabolites and cPLA2α protein for mechanism-assisted verification.

9. The application of the combined evaluation method for the toxicity of micro / nanoplastics systems according to any one of claims 1 to 8 in the evaluation of the toxicity of micro / nanoplastics systems, the comparison of health risks of micro / nanoplastics with different particle sizes, the evaluation of damage to the gut-metabolism-platelet axis, or the screening of interventions against the toxicity of micro / nanoplastics systems.

10. The application according to claim 9, characterized in that, The micro / nanoplastics include any one or more of polystyrene, polyethylene, polypropylene, and polyvinyl chloride, and the exposure routes include any one or more of oral ingestion, inhalation, and skin contact.