An experimental method for evaluating the hepatotoxicity of nanoplastics in the laboratory

Abstract

This invention provides a laboratory method for evaluating the hepatotoxicity of nanoplastics. The method comprehensively evaluates the long-term toxic effects of nanoplastic ingestion on the liver using hepatotoxicity indicators, oxidative stress indicators, mouse liver biochemical indicators, and inflammatory indicators. This method is convenient, rapid, low-cost, and simple to operate. The invention employs long-term gavage administration to expose mice to toxic substances, simulating the daily intake of nanoplastics in humans. It confirms that oral ingestion of nanoplastics exceeding a certain amount will exhibit hepatotoxicity and induce liver damage. This invention provides a comprehensive and effective laboratory evaluation method for the hepatotoxicity caused by daily nanoplastic ingestion, offering a reference for scientifically assessing the potential impact of nanoplastics on human health.

Description

Technical Field

[0001] This invention belongs to the field of toxicological evaluation of nanoplastics, and specifically relates to an experimental method for evaluating the hepatotoxicity of nanoplastics in the laboratory. Background Technology

[0002] With the increasing production and consumption of plastic products globally, plastic pollution is becoming one of the world's most pressing environmental problems. While plastic products have brought numerous conveniences to people's daily lives, their difficulty in degradation and improper disposal have caused significant environmental damage. Larger plastic fragments in the environment gradually degrade into smaller fragments through weathering, such as microplastics (<1000 μm) and nanoplastics (<1000 nm), eventually accumulating in the deep sea, farmland, and organisms. Plastic fragments in the environment have already impacted global carbon cycles, nutrient cycles, biodiversity, and ecosystems. In the long term, they will pose a serious threat to human health. Current research indicates that nanoplastics, when accumulated in organisms, can cause cytotoxicity, oxidative stress, immunotoxicity, neurotoxicity, and reproductive toxicity. Therefore, the toxicity testing of nanoplastics is particularly important.

[0003] Polystyrene (PS) is a polymer of styrene monomers and is widely used in the production of foamed plastics and other products, such as toys, CDs, food packaging boxes, and cup lids. Therefore, it is a representative plastic pollutant widely present in the environment. Compared to microplastics, polystyrene nanoplastics have smaller sizes and larger specific surface areas, allowing them to adsorb more harmful substances. After entering the body, they form protein crowns with proteins in blood plasma, thus facilitating translocation within the organism. Simultaneously, the large specific surface area also promotes the excessive generation of reactive oxygen species and the release of more pollutants, leading to membrane damage and protein dysfunction. Therefore, studying the toxic effects and mechanisms of action of nanoplastics is crucial.

[0004] The liver is a vital organ responsible for various physiological functions, including metabolism, detoxification, blood clotting, and immunity. Ingested nanoplastics can cross the intestinal barrier and enter the liver via the portal vein, accumulating there. However, to date, research on hepatotoxicity has primarily focused on aquatic organisms, with relatively little research on rodents (such as mice). Therefore, this invention utilizes a mouse model to investigate liver damage induced by long-term nanoplastic intake and constructs an experimental method for comprehensively evaluating the hepatotoxicity of nanoplastics in the laboratory. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide an experimental method for evaluating the hepatotoxicity of nanoplastics in the laboratory. The method provided by the present invention is fast, efficient, easy to operate, and has good reproducibility.

[0006] This invention provides a laboratory method for evaluating the hepatotoxicity of nanoplastics, the method comprising the following steps:

[0007] (1) Mice that were acclimatized for one week were given drugs with nanoplastics of different sizes and fed continuously for 6-8 weeks.

[0008] (2) Detection of CD11b in mouse liver tissue + Ly6C + Cells and CD45 + Ly6G + Cell infiltration status;

[0009] (3) Determine the hepatotoxicity of the nanoplastics based on the test results.

[0010] Specifically, an experimental method for comprehensively evaluating the hepatotoxicity of nanoplastics in the laboratory includes the following steps:

[0011] (S1) Mouse normal hepatocytes were cultured with a culture medium containing a wide range of nanoplastic particles. The control group was a combination of mouse normal hepatocytes cultured with a culture medium without nanoplastic particles. Cell viability was detected and cell survival rate was calculated.

[0012] (S2) The culture medium containing a certain concentration of nanoplastic particles was co-cultured with normal mouse liver cells, and the cell apoptosis was detected.

[0013] (S3) Culture medium containing a certain concentration of nanoplastic particles was co-cultured with normal mouse hepatocytes to detect cellular oxidative stress and mitochondrial membrane potential damage.

[0014] (S4) Six-week-old C57BL / 6 mice were acclimatized for one week and then administered the same concentration of nanoplastics. The mice were observed for six consecutive weeks, and their weight and food intake were recorded at fixed times each day.

[0015] (S5) Detection of inflammatory response and histological lesions in mouse liver tissue, and detection of CD11b in mouse liver tissue. + Ly6C + Cells and CD45 + Ly6G + Cell infiltration status.

[0016] (S6) Detection of oxidative stress and lipid metabolism in mouse liver tissue.

[0017] (S7) Detection of liver function in mouse serum.

[0018] (S8) CD11b in mouse liver tissue + Ly6C + Cells and CD45+ Ly6G + The degree of liver damage in mice was comprehensively evaluated and the extent of hepatotoxicity was determined by combining the cell infiltration status with oxidative stress indicators, lipid metabolism indicators, and liver function indicators in mouse serum.

[0019] In step (S1), the final concentration of the nanoplastic particles is 0-800 μg / ml. Preferably, cell viability is detected by the MTT assay.

[0020] Nanoplastic particles include plastic particles such as polystyrene, polyethylene, and polyvinyl chloride. The nanoplastic particles selected in this invention are derived from polystyrene plastic particles, and their size ranges from 20 to 500 nm.

[0021] The normal mouse liver cell line was the AML-12 cell line.

[0022] The final concentration of the nanoplastic particles in step (S2) is 200 μg / ml.

[0023] In step (S3), the nanoplastics induce the generation of reactive oxygen species while inhibiting glutathione activity and promoting superoxide dismutase activity. The final concentration of the nanoplastic particles is 300 μg / ml.

[0024] In step (S4), 6-week-old C57BL / 6 mice, weighing 16-18 g and all female, were randomly grouped according to experimental needs, with no fewer than 5 animals in each group, and were given nanoplastic particles by gavage.

[0025] Furthermore, the administration of nanoplastic particles of different sizes at a concentration of 75-100 mg / kg via gavage was carried out for 6-8 weeks.

[0026] The liver tissue sample used in step (S6) for detecting oxidative stress and lipid metabolism is 0.2-0.3 g in size.

[0027] The liver function indicators mentioned in step (S7) are preferably alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

[0028] Compared with existing technologies, this invention provides an experimental method for evaluating the hepatotoxicity of nanoplastics in the laboratory. This invention comprehensively evaluates the toxic effects of ingested nanoplastics on cells and the liver by examining multiple toxicity-related indicators, and correlates liver tissue damage with CD11b levels in mouse liver tissue. + Ly6C + Cells and CD45 + Ly6G +This invention correlates cell infiltration patterns to evaluate the hepatotoxicity of nanoplastics by detecting the infiltration of two cell types, providing an effective method for laboratory analysis of nanoplastic hepatotoxicity. This method is convenient, rapid, low-cost, and simple to operate. This invention discovers and confirms that when ingested nanoplastics exceed a certain threshold, they exhibit hepatotoxicity and induce liver damage. Based on this invention, a comprehensive and effective laboratory evaluation method can be provided for the hepatotoxicity caused by daily nanoplastic intake, offering a reasonable scientific assessment of its potential impact on human health. Attached Figure Description

[0029] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings:

[0030] Figure 1 Transmission electron microscope images of polystyrene nanoparticles of different sizes.

[0031] Figure 2 The figure shows the effect of different sizes and concentrations of nano-polystyrene plastic particles on the viability of AML-12 cells.

[0032] Figure 3 The figure shows the effect of different sizes of polystyrene nanoparticles on apoptosis in AML-12 cells.

[0033] Figure 4 The figure shows the effect of different sizes of nano-polystyrene plastic particles on oxidative stress in AML-12 cells.

[0034] Figure 5 Figure 1 shows the results of co-localization of different sized polystyrene nanoparticles with AML-12 cell mitochondria and their effect on membrane potential.

[0035] Figure 6 The graph shows the changes in body weight, food intake, liver weight, and liver coefficient of mice in the experimental and control groups.

[0036] Figure 7 The figure shows the effects of oxidative stress on liver and lipid metabolism in mice in the experimental and control groups.

[0037] Figure 8 The figure shows the effect of the experimental group and the control group on liver function.

[0038] Figure 9 Images of liver tissue sections from the experimental and control groups of mice.

[0039] Figure 10 This diagram shows the changes in liver inflammation-related immune cells in mice of the experimental and control groups. Implementation

[0040] The present invention will be further described in detail below through specific embodiments.

[0041] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0042] Unless otherwise specified, the materials and reagents used in this embodiment are commercially available. Example

[0043] Effects of nano-polystyrene particles on the viability of normal mouse hepatocytes AML-12

[0044] Experimental materials

[0045] The normal mouse hepatocyte AML-12 cell line was obtained from Yubo Biotechnology Co., Ltd. (Shanghai, China), and nano-polystyrene plastic particles were purchased from Huizhi Biotechnology Co., Ltd. (Shanghai, China) in an aqueous solution with a concentration of 50 mg / ml for later use.

[0046] Cell culture and treatment of nano-polystyrene plastic particles

[0047] Mouse normal hepatocytes AML-12 were cultured in F-12 / DMEM medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 10 μg / ml insulin, 5.5 μg / ml transferrin, 5 ng / ml selenium, and 40 ng / ml dexamethasone, and then incubated at 37°C in a humidified 5% CO2 incubator. AML-12 cells were treated for 4 h with different concentrations (0, 10, 25, 50, 100, 200, and 500 nm) of polystyrene nanoparticles.

[0048] Cell viability assay

[0049] AML-12 cells were loaded at 1×10 4 Cells were seeded at a density of [number] cells / ml in 96-well plates and incubated overnight, then treated with different sizes and concentrations of polystyrene nanoparticles for 4 hours. AML-12 cells treated with culture medium containing no polystyrene nanoparticles served as a control group. Absorbance values ​​measured at 490 nm using an MTT assay were recorded. Cell viability results were analyzed to elucidate the toxic effects of polystyrene nanoparticles on AML-12 cells.

[0050] Experimental results

[0051] The results are as follows Figure 2The results showed that smaller particle sizes were associated with greater cytotoxicity to AML-12 cells. Specifically, 20 nm polystyrene particles significantly reduced cell viability at high concentrations, while other sizes of polystyrene nanoparticles did not affect AML-12 cell viability within the 0-800 μg / ml range. For 20 nm polystyrene particles, AML-12 cell viability decreased with increasing concentration, indicating that AML-12 cytotoxicity was concentration- and size-dependent. Example

[0052] Nanoparticles of polystyrene plastic induce apoptosis in normal mouse hepatocytes AML-12.

[0053] Experimental methods

[0054] AML-12 cells were grown at a rate of 1.5 × 10⁻⁶. 5 After seeding AML-12 cells at a density of 1 cell / well in 48-well plates and culturing for 12 h, apoptosis assays were performed. Different sizes of polystyrene nanoparticles with a final concentration of 200 μg / ml were co-incubated with the cells for 4 h. The cell culture medium was then transferred to 2 ml centrifuge tubes. Adherent cells were washed once with PBS, and cells were digested with an appropriate amount of trypsin. The cells were incubated at room temperature, and culture medium was added to terminate the digestion. The cells were gently pipetted and transferred to centrifuge tubes, and the cell pellet was collected. Following the Annexin V-PI apoptosis kit instructions, 195 μl of Annexin V-FITC binding buffer was added to resuspend the cells, followed by 5 μl of Annexin V-FITC dye and 10 μl of PI dye. The cells were incubated at room temperature in the dark for 10 min, and immediately analyzed by flow cytometry. Early and late apoptosis in AML-12 cells were analyzed by detecting Annexin V and PI signals. 1 × 10⁶ cells were collected each time. 5 The data were analyzed using FlowJo10 software.

[0055] Experimental results

[0056] To investigate the effects of exposure to different sizes of nano-polystyrene plastic particles at the same concentration on cell apoptosis, this experiment used Annexin V-PI flow cytometry to quantitatively analyze early and late apoptosis in AML-12 cells. The experimental results are as follows: Figure 3 As shown. Compared with the control group, the number of early apoptotic AML-12 cells (Annexin V) was significantly reduced after treatment with 20 nm polystyrene plastic particles. + PI - The number of late-stage apoptotic AML-12 cells (Annexin V) increased significantly. + PI +No significant changes were observed. However, after treatment with nano-polystyrene plastic particles of different sizes at the same concentration, the number of AML-12 cells undergoing early and late apoptosis remained largely unchanged compared to the control group. Furthermore, compared to the control group, the early apoptosis rate increased by 42.4% in the 20 nm polystyrene plastic particle treatment group, while the early apoptosis rate in other treatment groups showed no significant change. This indicates that the smaller the size of the nano-polystyrene plastic particles, the greater the damage to AML-12 cells, i.e., the greater the toxicity to AML-12 hepatocellular carcinoma. Example

[0057] Nanoparticles of polystyrene plastic induce oxidative stress in normal mouse hepatocytes AML-12.

[0058] Experimental methods

[0059] The oxidative stress in AML-12 cells exposed to polystyrene nanoparticles of different sizes was detected using a reactive oxygen species (ROS) assay kit, a superoxide dismutase (SOD) assay kit (Beyotime Biotechnology, Shanghai, China), and a glutathione (GSH) assay kit (Nanjing Jiancheng Biotechnology Institute, Nanjing, China). Intracellular ROS production was detected using the 2,7-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe. DCFH-DA itself is non-fluorescent but can cross the cell membrane. After entering the cell, DCFH-DA is hydrolyzed by intracellular esterases to generate DCFH. Intracellular ROS can oxidize the non-fluorescent DCFH into fluorescent DCF. The fluorescence intensity of DCF is directly proportional to the ROS content in the cell. After cell adhesion, DCFH-DA was co-incubated with AML-12 cells for 45 min for in situ probe loading. The DCFH-DA probe was washed away, and then different sizes of polystyrene nanoparticles at a final concentration of 300 μg / ml were co-incubated with the cells. Imaging analysis was performed using an inverted fluorescence microscope, and average fluorescence intensity was analyzed using ImageJ. Superoxide dismutase (SOD), an enzyme that catalyzes the detoxification of superoxide free radicals, was detected using the S0101M Superoxide Dismutase Detection Kit according to the manufacturer's instructions, and the absorbance at 450 nm was measured using a microplate reader. Glutathione (GSH), an antioxidant that maintains cellular redox homeostasis, was detected using a glutathione detection kit (microplate method), and the absorbance at 405 nm was measured using a microplate reader.

[0060] Experimental results

[0061] To assess the oxidative stress damage caused by nano-polystyrene plastic particles, this example examined various oxidative stress indicators in normal mouse hepatocytes AML-12 treated with nano-polystyrene particles. The experimental results are as follows: Figure 4As shown in the figure, compared with the control group, at the same concentration, treatment with 20 nm polystyrene plastic particles significantly increased the intracellular ROS level of AML-12 cells, treatment with 50 nm polystyrene plastic particles slightly increased the intracellular ROS level, while treatment with other sizes of polystyrene nanoparticles did not significantly change the intracellular ROS level of AML-12 cells. This indicates that 20 nm polystyrene plastic particles have a stronger stimulating effect on intracellular ROS in AML-12 cells. Moreover, compared with the control group, treatment with 20 nm polystyrene plastic particles significantly decreased the intracellular GSH content of AML-12 cells by ~41%, while the intracellular SOD antioxidant enzyme level significantly increased by ~27%. These results indicate that exposure of AML-12 cells to 20 nm polystyrene plastic particles significantly alters the activity of intracellular antioxidant enzymes (GSH and SOD). Due to the imbalance between intracellular oxidation and antioxidation, oxidative stress is ultimately induced in cells. This further suggests that the smaller the size of the polystyrene nanoparticles, the greater the damage to AML-12 cells, i.e., the greater the toxicity to AML-12 hepatocellular cells. Example

[0062] Nanoparticles of polystyrene plastic induce mitochondrial membrane potential damage in normal mouse hepatocytes AML-12.

[0063] Experimental methods

[0064] To determine the effects of nano-polystyrene plastic on mitochondria, a co-localization experiment was first performed. AML-12 cells were co-incubated with 20 nm and 100 nm red fluorescent polystyrene plastic particles, followed by incubation with a 100 nm MitoTracker. ® GreenFM (Yisheng Biotechnology Co., Ltd., Shanghai, China) staining was performed at 37 ℃ for 30 min. Imaging analysis was performed using an FV1000 confocal microscope. To investigate the effect of nano-polystyrene plastic particles on mitochondrial membrane damage, mitochondrial membrane potential (MMP) was detected using a 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole carbonylcyanine iodide (JC-1) assay kit (Beyotime, Shanghai, China) according to the manufacturer's instructions. Imaging analysis was performed using an inverted fluorescence microscope. Red fluorescence represented JC-1 polymers, indicating normal mitochondrial membrane potential; green fluorescence represented JC-1 monomers, indicating decreased mitochondrial membrane potential. The ratio of green to red fluorescence intensity was analyzed using ImageJ software.

[0065] Experimental results

[0066] Mitochondria are the primary site of cellular energy metabolism and have long been considered a major target organ for oxidative damage. The co-localization of nano-polystyrene plastic particles with AML-12 cell mitochondria and their impact on mitochondrial membrane potential damage are shown in the following results. Figure 5 As shown. The study found that both 20 nm and 100 nm red fluorescent polystyrene plastic particles were associated with MitoTracker. ® Green fluorescence overlaps well. Furthermore, 20 and 100 nm polystyrene plastic particles show good overlap with MitoTracker. ® The changes in the linear region of interest (ROI) intensity distribution of Green were almost synchronous. These results indicate that 20 and 100 nm polystyrene plastic particles readily enter the mitochondria of normal mouse hepatocytes. Mitochondrial dysfunction under oxidative stress leads to apoptosis. To determine the extent of damage to the mitochondrial membrane potential, JC-1 dye was used for detection. The results showed that, compared with the control group, JC-1 still emitted red fluorescence after treatment with 100 nm polystyrene plastic particles, while after treatment with 20 nm polystyrene plastic particles, JC-1 exhibited predominantly green fluorescence with a small amount of red fluorescence. The change in fluorescence from red to green indicates that 20 nm polystyrene plastic particles can cause a decrease in the mitochondrial membrane potential of AML-12 cells. Therefore, 20 nm polystyrene plastic particles can enter the mitochondria of cells, leading to a decrease in mitochondrial membrane potential, causing mitochondrial dysfunction, and promoting apoptosis, indicating that nano-polystyrene plastic particles have greater toxicity to normal mouse hepatocytes. Example

[0067] The toxic effects of nano-polystyrene plastic particles on the liver of C57BL / 6 mice

[0068] Effects of nano-polystyrene plastic particles on mouse body weight, food intake, and liver coefficient

[0069] Experimental methods

[0070] Six-week-old female C57BL / 6 mice were selected for the experiment and randomly divided into six groups: a control group and groups containing 20, 50, 100, 200, and 500 nm polystyrene plastic particles. Five different particle sizes of polystyrene plastic particles (20, 50, 100, 200, and 500 nm) were administered via gavage at a dose of 75 mg / kg body weight every two days to investigate the toxic effects of different sizes of polystyrene nanoparticles on mice. The control group received an equal volume of PBS via gavage. After initial exposure to the polystyrene nanoparticles, the body weight of each mouse in the control, 20, 50, 100, 200, and 500 nm polystyrene plastic particle groups was measured daily at the same time using an electronic balance. The weight of food consumed by each group was also measured. The average food intake was calculated using the obtained data, and the behavioral status of the mice was observed. After humane sacrifice of all mice, liver tissue was harvested and weighed using an electronic balance. The liver organ coefficient was then calculated using the following formula:

[0071] Liver organ coefficient = Liver organ weight (g) / Mouse body weight (g) × 100

[0072] Experimental results

[0073] To investigate whether oral administration of nano-polystyrene plastic particles of different sizes affects the body weight and feeding behavior of mice, we monitored changes in the mice's body weight and food intake. The experimental results are as follows: Figure 6 As shown in the figure. The results indicate that exposure to nano-polystyrene plastic particles of different sizes did not significantly affect the body weight or food intake of mice. To investigate the liver damage caused by oral administration of different nano-polystyrene plastic particles in mice, liver tissue was weighed, and the liver coefficient was obtained according to the ratio of liver weight to mouse body weight. The experimental results are shown in the figure. Figure 6 As shown, it was clearly observed that liver weight increased significantly after exposure to 200 and 500 nm polystyrene plastic particles, and the liver coefficient showed the same trend. These results indicate that 200 and 500 nm polystyrene plastic particles caused initial damage to the mouse liver.

[0074] Effects of nano-polystyrene plastic particles on oxidative stress and lipid metabolism in mouse liver

[0075] Experimental methods

[0076] Take 0.2-0.3g of liver tissue and add PBS buffer at a weight (g):volume (ml) ratio of 1:9. Place the centrifuge tube containing the liver tissue on ice and homogenize mechanically using a handheld tissue homogenizer. After the tissue block is completely dissolved, centrifuge at 4℃ and collect the supernatant. Determine the protein concentration using the BCA method. Protein concentration is directly proportional to absorbance. The protein concentration in the sample can be calculated using a standard curve and the measured absorbance. The determination of SOD, GSH, and triglycerides (TG) in the liver should be performed according to the manufacturer's instructions. Record the absorbance at 450, 405, and 510 nm using an ELISA reader, and finally calculate the values ​​of SOD, GSH, and TG in the liver using the formulas provided in the instructions.

[0077] Experimental results

[0078] To investigate whether polystyrene nanoparticles of different sizes induce oxidative stress in mouse liver, oxidative stress-related indicators in liver homogenate were measured using a detection kit. The experimental results are as follows: Figure 7 As shown in the figure. Experimental results indicated that ingestion of 500 nm polystyrene plastic particles significantly reduced GSH levels in mouse livers, while other sizes of polystyrene plastic particles showed no significant change compared to the control group. Ingestion of polystyrene plastic particles did not significantly affect liver SOD levels. The significant change in GSH indicates that exposure to large-sized polystyrene plastic particles causes oxidative stress damage to the mouse liver. Further testing of liver lipid metabolism-related indicators—triglycerides (TG)—revealed that exposure to 200 nm polystyrene plastic particles resulted in a slight increase in liver TG compared to the control group, while exposure to 500 nm polystyrene plastic particles resulted in a significantly increased liver TG compared to the control group. Other sizes of polystyrene plastic particles showed no significant change in liver TG, indicating that ingestion of 200 and 500 nm polystyrene plastic particles disrupts lipid metabolism in the mouse liver. Therefore, 200 and 500 nm polystyrene plastic particles can damage mouse liver tissue.

[0079] Effects of nano-polystyrene plastic particles on liver function in mice

[0080] Experimental methods

[0081] Collect 1-2 ml of blood and place it in a 2 ml centrifuge tube. Incubate the blood sample at 4°C until coagulation. Centrifuge at 3500 rpm for 20 min at 4°C. Collect the serum for testing. The determination of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels is performed according to the kit instructions (Nanjing Jiancheng Engineering Research Institute, Nanjing, China). First, prepare standard curves for AST and ALT. Then, use a microplate reader to detect the absorbance at 510 nm. Calculate the absolute absorbance based on the measured absorbance and the control absorbance. Refer to the standard curve to obtain the corresponding AST / ALT activity units.

[0082] Experimental results

[0083] To confirm that the accumulation of polystyrene nanoparticles in the liver can lead to liver function impairment, the effects of polystyrene nanoparticles of different sizes on liver function indicators were tested. The experimental results are as follows: Figure 8 As shown in the figure, compared with the control group, treatment with 20, 50, and 100 nm polystyrene plastic particles slightly increased the levels of serum AST and ALT, indicators related to liver function; while treatment with 200 and 500 nm polystyrene plastic particles significantly increased serum AST and ALT levels. Serum AST and ALT levels are important indicators of liver function; significantly elevated serum AST and ALT levels indicate impaired liver function in mice, further demonstrating that 200 and 500 nm polystyrene plastic particles cause hepatotoxicity and liver tissue damage.

[0084] Histopathological analysis of liver tissue after exposure to nano-polystyrene plastic particles

[0085] Experimental methods

[0086] After humane euthanasia of mice in each group, liver tissue was harvested and fixed in 4% paraformaldehyde. The fixed tissue specimens were then embedded in paraffin wax, cut into 3-5 μm thick sections, adhered to glass slides, dried, and preserved for further hematoxylin-eosin staining. Hematoxylin-eosin (HE) staining is a commonly used staining method in paraffin sectioning. Hematoxylin is a basic dye that stains cell nuclei blue-purple; eosin is an acidic dye that stains cytoplasm and extracellular matrix red. Finally, the tissue morphology was observed and photographed using a metallurgical microscope.

[0087] Experimental results

[0088] To further investigate the damage caused by polystyrene plastic particles of different sizes to mouse liver tissue, histological observation of mouse liver was performed using HE staining. The experimental results are as follows: Figure 9 As shown in the diagram, the control group exhibited clear and intact liver lobule structure, with hepatocytes arranged radially along the central vein. No hepatocyte vacuolation, necrosis, or inflammatory cell infiltration was observed. However, compared to other groups, treatment with 200 and 500 nm polystyrene plastic particles resulted in significant inflammatory cell infiltration and lipid droplet accumulation in the mouse liver tissue (arrows). This demonstrates that exposure to 200 and 500 nm polystyrene plastic particles can induce pathological damage to liver tissue.

[0089] Nanoparticles of polystyrene plastic induce inflammatory responses in liver tissue.

[0090] Experimental methods

[0091] Take a portion of liver tissue and place it in a 5 ml centrifuge tube containing tissue homogenizing buffer. Cut the tissue into small pieces and transfer the small pieces to a 50 ml centrifuge tube with a 70 μm filter. Use the inner core of a syringe to homogenize the tissue, rinsing twice with PBS if necessary. Add 10 mg / ml heparin sodium and then add Percoll to bring the final Percoll concentration to 33%. Mix well and centrifuge at 2000 rpm for 20 min (with a drop-off of 0). After centrifugation, collect the supernatant, add red blood cell lysis buffer, and lyse at room temperature for 5 min. Add an appropriate amount of PBS to stop the lysis, centrifuge at 400 g for 5 min, discard the supernatant, and collect the cell pellet. Resuspend the cells in 60 μl of FACsbuffer and divide them into two tubes (20 μl / tube). One tube is used for staining liver monocytes by co-incubating the single-cell suspension with FACsbuffer containing CD11b-FITC and Ly6C-APC. The other tube is used for staining liver neutrophils by co-incubating the single-cell suspension with FACsbuffer containing CD45-PE and Ly6G-AF488. The remaining cells are mixed and used for single-staining and unstained tubes. After incubation, wash away unbound antibodies with FACsbuffer, then fix with fixative. Finally, analyze the phenotype of liver monocytes and neutrophils using flow cytometry.

[0092] Experimental results

[0093] To investigate the effect of nanoplastics on liver inflammation and determine the relationship between the degree of liver damage and the infiltration of monocytes and neutrophils, we measured the changes in the percentage of monocytes and neutrophils in the liver. Flow cytometry images of CD11b and Ly6C (inflammatory monocytes) or CD45 and Ly6G (neutrophils) expression in liver immune cells isolated from the control group and the nano-polystyrene plastic particle treatment group are shown below. Figure 10 As shown. Clearly, compared to the control group, treatment with 200 and 500 nm polystyrene plastic particles significantly reduced CD11b levels in mouse livers. + Ly6C + Cells (inflammatory monocytes) and CD45 + Ly6G + The percentage of cells (neutrophils) increased significantly, with inflammatory monocytes increasing by 19% and 34.7%, and neutrophils increasing by 27.1% and 31.5%, respectively. This indicates that exposure to 200 and 500 nm polystyrene plastic particles induced monocyte and neutrophil infiltration into liver tissue, triggering inflammatory hepatotoxicity in mice. Therefore, detecting CD11b in mouse liver... + Ly6C + Cells (inflammatory monocytes) and CD45 + Ly6G +The extent of cell (neutrophil) infiltration can indicate the degree of damage to liver tissue caused by nanoplastic particles.

[0094] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. It should be noted that for those skilled in the art, any improvements or modifications made to the technical solution and inventive concept of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A laboratory method for evaluating the hepatotoxicity of nanoplastics, characterized in that, The method includes the following steps: 1) Mice that have been acclimatized for one week were given nanoplastics of different sizes and fed continuously for 6-8 weeks. The nanoplastics were administered by gavage. The diameter of the nanoplastic particles was 20-500 nm and the concentration of the nanoplastics of different sizes was 75-100 mg / kg. 2) Detect the infiltration of CD11b+Ly6C+ cells and CD45+Ly6G+ cells in mouse liver tissue; 3) Determine the hepatotoxicity of the nanoplastics based on the test results; Liver function was measured in mouse serum, and oxidative stress and lipid metabolism were measured in mouse liver tissue. The liver function, oxidative stress, and lipid metabolism indicators were combined with the infiltration of CD11b+Ly6C+ and CD45+Ly6G+ cells to determine the degree of hepatotoxicity corresponding to different infiltration levels of CD11b+Ly6C+ and CD45+Ly6G+ cells in mouse liver tissue.

2. The experimental method for evaluating the hepatotoxicity of nanoplastics in the laboratory according to claim 1, characterized in that, The liver function test indicators include alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

3. The experimental method for evaluating the hepatotoxicity of nanoplastics in the laboratory according to claim 1, characterized in that, The mice weighed 16-18g.

4. The experimental method for evaluating the hepatotoxicity of nanoplastics in the laboratory according to claim 1, characterized in that, This method also includes experimental steps for hepatotoxicity: 4) Culturing mouse hepatocytes in culture medium containing nanoplastics of different concentrations and particle sizes; 5) Mouse hepatocytes cultured in a culture medium without nanoplastics were used as a control group; 6) Detect cell viability, apoptosis, oxidative stress, and mitochondrial membrane potential damage; 7) Determine the hepatotoxicity of nanoplastics based on the test results.

5. The method according to claim 4, characterized in that, The concentration of nanoplastics in the culture medium is 0-800 μg / ml, and the diameter of the nanoplastic particles is 20-500 nm.

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