Method for evaluating intestinal toxicity of nano polypropylene plastic

Abstract

The invention provides a method for evaluating the intestinal toxicity of nano polypropylene plastic, which comprises the following steps of: 1, exposing macrophages in a culture medium containing polypropylene nano particles with different concentrations and / or different particle sizes for culturing, and detecting any one or more of cell survival rate, glucose consumption level, lactic acid production level and glycolysis level; the method 2 comprises the following steps: exposing an enteritis model mouse in the polypropylene nano-plastic, recording the fecal state, hematochezia degree and weight during administration, and / or calculating a disease activity index, and / or detecting the pathological state of the intestinal tract of the mouse. The invention focuses on analyzing the influence of the polypropylene nano-plastic on glycolysis of macrophages, reveals the potential effect of the polypropylene nano-plastic on enteritis progression, and provides theoretical basis and technical support for prevention and treatment of nano-plastic related diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology and environmental toxicology evaluation, and particularly relates to a method for evaluating the influence of polypropylene nanoplastics on intestinal inflammation in a laboratory. BACKGROUND

[0002] With the widespread use of plastic products, plastic pollution has become a global environmental and health problem. Plastic waste is decomposed into microplastics (1-5 mm in diameter) and nanoplastics (5 mm-1 μm in diameter) under the action of physics, chemistry and biology. These tiny plastic particles can enter the human body through food, air, skin contact and other ways. Due to its small size, large specific surface area, strong permeability, easy penetration of biological barriers, and easy absorption by the human body, the potential risk of nanoplastics to human health far exceeds that of microplastics, which has attracted widespread attention.

[0003] The intestine is one of the target organs for the accumulation of nanoplastics, and is also the largest immune organ in the human body. Studies have shown that nanoplastics can trigger oxidative stress, inflammation and other reactions, and damage the intestinal barrier. The destruction of the intestinal barrier function and the abnormal activation of the immune system are considered to be important inducers of intestinal inflammation. Macrophages play a key role in intestinal immunity, can remove pathogens and foreign substances through phagocytosis, and regulate immune responses by secreting cytokines. Glycolysis, as the main pathway of energy metabolism in macrophages, plays an important role in immune response. Under stress, macrophages enhance glycolysis to meet energy demand, and regulate immune response through metabolic products such as lactic acid. However, exposure to nanoplastics may interfere with the metabolic pathways of macrophages, leading to abnormal glycolysis and exacerbating inflammatory response.

[0004] Although there have been studies on the potential impact of nanoplastics on health, research on how nanoplastics affect the energy metabolism status of macrophages and how they affect the progression of intestinal inflammation is still limited, and the research and analysis methods have many shortcomings, making it difficult to systematically analyze the potential threat of nanoplastics to patients with intestinal inflammation. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a method for evaluating the intestinal toxicity of nanopolypropylene plastics.

[0006] To solve the above technical problems, the present application discloses the following technical solutions;

[0007] A method for evaluating the influence of polypropylene nano-plastics on intestinal inflammation, first, the influence of polypropylene nanoparticles of different particle sizes on the survival rate of macrophages, the level of glucose consumption, the level of lactic acid production, and the level of sugar metabolism is evaluated at the cellular level. Then, the intestinal inflammation model mice are made to ingest polypropylene nanoparticles through gavage, and the body weight and fecal state of the mice are recorded. After the administration is completed, the pathological condition of the intestinal tissue of the mice is analyzed.

[0008] Specifically, the method comprises:

[0009] Method 1: exposing macrophages to culture medium containing polypropylene nanoparticles of different concentrations and / or different particle sizes, and detecting any one or several of the levels of cell survival rate, glucose consumption level, lactic acid production level, and glycolysis level;

[0010] Method 2: exposing intestinal inflammation model mice to polypropylene nano-plastics, recording the fecal state, degree of hematochezia, and body weight during administration, and / or calculating the disease activity index, and / or detecting the pathological state of the intestinal tract of the mice.

[0011] In method 1, the macrophages are mouse monocyte macrophage leukemia cell line RAW264.7.

[0012] In method 1, the particle size of the polypropylene nanoparticles is 50-1000 nm, such as 100-500 nm; and the concentration of the polypropylene nanoparticles in the culture medium is 0-160 μg / mL.

[0013] In method 1, the detection of the cell survival rate, glucose consumption level, and lactic acid production level is by colorimetric method.

[0014] In method 1, the indicators of the glycolysis level include glycolytic capacity, maximum glycolytic capacity, and glycolytic volume.

[0015] In method 1, the glycolysis level is detected by seahorse cell energy metabolism analyzer.

[0016] In method 2, the intestinal inflammation model mice are intestinal inflammation model mice constructed by dextran sulfate sodium.

[0017] In method 2, the intestinal inflammation model mice are 5-6-week-old male Balb / c mice.

[0018] In method 2, the mice are randomly divided into negative control group, model group, and experimental group, with 4-15 mice in each group; the negative control group is administered with saline, the model group is intestinal inflammation model mice, and the experimental group is intestinal inflammation model mice exposed to polypropylene nanoparticles through gavage; the particle size of the polypropylene nanoparticles is 100-500 nm.

[0019] In method 2, the disease activity index is a standard for evaluating the severity of intestinal inflammation, and the disease activity index is the sum of each item of stool form, degree of hematochezia and decrease in body weight change;

[0020] wherein,

[0021] Stool form: normal score 0, soft formed score 1, soft unformed score 2, and diarrhea score 3;

[0022] Degree of hematochezia: no occult blood score 0, occult blood score 1, small amount of hematochezia score 2, and severe hematochezia score 3;

[0023] Body weight change: decrease of 0% score 0, decrease of 1%-5% score 1, decrease of 5%-10% score 2, and decrease of 10%-15% score 3.

[0024] In method 2, the pathological state of the mouse intestinal tract is the pathological condition of the mouse intestinal tissue, and the tissue sample is taken from the colon of the mouse.

[0025] In the present application, the morphology of the polypropylene nanoparticles is spherical, and the particle size of the polypropylene nanoparticles is the diameter of the nanoparticles.

[0026] The present application provides a method for comprehensively evaluating the influence of polypropylene nano-plastic on the energy metabolism of macrophages and its potential role in the progression of intestinal inflammation.

[0027] Compared with the prior art, the present application has the following advantages:

[0028] (1) The present application takes polypropylene widely used in life as the research object, and has important reference significance for evaluating its toxic effect.

[0029] (2) The present application takes the particle size of polypropylene nano-plastic as a variable to analyze the relationship between the particle size and the toxicity of nano-plastic, which is targeted.

[0030] (3) The present application focuses on the influence of polypropylene nano-plastic on the glucose metabolism of macrophages, and reveals its potential role in the progression of intestinal inflammation. The angle is novel, which provides theoretical basis and technical support for the prevention and treatment of nano-plastic related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0031] The above and / or other aspects of the present application will become apparent from the following detailed description of the application taken in conjunction with the accompanying drawings.

[0032] Figure 1 The survival rate of macrophages affected by polypropylene nano-plastic.

[0033] Figure 2 The colorimetric method is used to detect the glucose consumption level of cells.

[0034] Figure 3 For colorimetric method to detect the level of cell lactic acid production.

[0035] Figure 4 For energy metabolism analyzer to detect the level of cell glycolysis.

[0036] Figure 5 For intestinal inflammation disease index chart of mice during drug administration.

[0037] Figure 6 For mouse colon pathological section. DETAILED DESCRIPTION

[0038] The present application can be better understood according to the following examples. However, it is readily apparent to a person skilled in the art that the content described in the examples is only for illustrating the present application, and should not and will not limit the present application as described in detail in the claims.

[0039] The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0040] s100 and s500 in the following examples respectively refer to polypropylene with particle sizes of 100 nm and 500 nm.

[0041] Example 1:

[0042] Colorimetric method to detect the effect of polypropylene nano-plastic on the survival rate of macrophages.

[0043] Logarithmic growth RAW264.7 cells were made into cell suspension and cell counting was performed, and the cell concentration was adjusted to 5×10 4 μL per well in a 96-well plate, and after the cells adhered, the medium was aspirated and 100 μL of complete medium containing s100, s500 was added. Five parallel experimental wells were set for each material, and after incubation, the medium was aspirated and 0.5 mg / mL 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl bromide tetrazolium thiazole blue (MTT) containing serum-free medium was added to incubate the cells for 4 h. The wells with only MTT were set as the reference group, and the wells with only cells without incubation of the material were set as the negative control group. The MTT solution was aspirated and 100 μL of DMSO was added and continuously shaken to completely dissolve the MTT. The absorbance of the plate at 490 nm was detected by a microplate reader, and the survival rate of the cells was calculated using the following formula:

[0044] Cell survival rate (%) = [(Ax-Ab) / (Ac-Ab)]x100%

[0045] Wherein Ax is the average absorbance of the experimental group; Ab is the average absorbance of the reference group; Ac is the average absorbance of the negative group.

[0046] The experimental results are shown in Table 1. Figure 1 As shown in Table 1, when the concentration of s100 and s500 is as high as 80 μg / mL, the cell survival rate is 95% and 90%, respectively. When the concentration of s100 and s500 is as high as 160 μg / mL, the cell survival rate decreases slightly, indicating that the toxicity produced is concentration-dependent.

[0047] Example 2

[0048] The effect of polypropylene nanoplastics with different particle sizes and morphologies on the glucose consumption level of macrophages was detected by colorimetry.

[0049] Logarithmic growth RAW264.7 cells were prepared into a cell suspension, and cell counting was performed to adjust the cell concentration to 5×10 4 The cells were inoculated in a 96-well plate at 100 μL per well, and after the cells were attached for 24 h, the culture medium was aspirated and 100 μL of complete culture medium containing 75 μg / mL of s100 or 100 μL of complete culture medium containing 75 μg / mL of s500 was added. Each group of materials was set up in 5 parallel experimental wells, and the wells with only cells incubated without materials were set up as the negative control group, and the materials were incubated with the cells for 24 h. After incubation, the culture medium was aspirated, and the glucose consumption level of the cells was evaluated using a glucose detection kit.

[0050] The experimental results are shown in Table 2. Figure 2 As shown in Table 2, compared with the control group, s100 and s500 both led to different degrees of increase in glucose consumption, indicating that polypropylene nanoparticles affected the glucose metabolism state of macrophages. The glucose consumption of the s100 group was the highest, followed by the s500 group, indicating that the glucose consumption of the cells decreased with the increase of particle size.

[0051] Example 3

[0052] The effect of polypropylene nanoplastics with different particle sizes and morphologies on the lactic acid production level of macrophages was detected by colorimetry.

[0053] Logarithmic growth RAW264.7 cells were prepared into a cell suspension, and cell counting was performed to adjust the cell concentration to 5×10 4The cells were seeded in 96-well plates at a concentration of 10,000 cells / mL, 100 μL / well, and after the cells adhered, 100 μL of complete medium containing 75 μg / mL of s100 or 100 μL of complete medium containing 75 μg / mL of s500 was added to each well, and 5 parallel experimental wells were set for each material. A well with only cells and no incubation material was set as a negative control group. Then, the material and the cells were incubated for 24 h. After the incubation, the medium was aspirated, and the lactic acid content in the medium was detected using a lactic acid concentration detection kit.

[0054] The experimental results are shown in Table 1. Figure 3 As shown in Table 1, the lactic acid production levels of the cells treated with polypropylene nanoplastic were increased to varying degrees, and the s100 group > the s500 group, indicating that the lactic acid production level of the cells decreased with the increase of the particle size.

[0055] Example 4

[0056] The sugar metabolism of the cells was detected by a seahorse cell energy metabolism analyzer.

[0057] (1) The seahorse XF calibration solution was added to the 96-well hydration plate matched with the instrument, and the hydration was performed overnight in a 37°C CO2-free incubator.

[0058] (2) The RAW264.7 cells in the logarithmic growth phase were seeded in the cell culture plate matched with the seahorse energy analyzer, and after the cells adhered, complete medium containing 75 μg / mL of s100 or s500 was added, and the cells were incubated for 24 h. Five parallel experimental wells were set for each material.

[0059] (3) According to the glucose oxidation pressure test kit instructions, the buffer solution, glucose, oligomycin, and 2-deoxyglucose solution were prepared.

[0060] (4) The experimental template was designed, and the instrument was detected according to the instrument prompt.

[0061] The experimental results are shown in Table 2. Figure 4 As shown in Table 2, the glycolysis rate, glycolysis capacity, and glycolysis reserve of the cells treated with polypropylene nanoplastic were increased. Compared with the control group, the glycolysis indexes of the cells treated with s100 were most obviously increased, indicating that the level of cell glycolysis induced by polypropylene nanoplastic decreased with the increase of the particle size.

[0062] Example 5

[0063] 1. Model administration

[0064] 5-6 weeks old male Balb / c mice were selected to construct dextran sulfate sodium (DSS) enteritis model. After the model was successfully constructed, the mice were divided into 4 groups: saline, DSS, DSS+s100, DSS+s500. The saline group of mice was the negative control, and the DSS group of mice was the enteritis group control. The DSS+s100, DSS+s500 group of mice were given polypropylene nano-plastic by gavage, once a day, and the polypropylene dose was 10 mg / kg, for 28 days.

[0065] 2. Effects of s100 and s500 on body weight and activity index

[0066] During the administration period, the body weight, fecal status, and occult blood of the mice were recorded, and the obtained scores were added according to the following scoring rules: fecal form: normal (score 0), soft and shaped (score 1), soft and unshaped (score 2), and diarrhea (score 3). The degree of occult blood: no occult blood (score 0), occult blood (score 1), small amount of occult blood (score 2), and severe occult blood (score 3). Body weight change: 0% decrease (score 0), 1%-5% decrease (score 1), 5%-10% decrease (score 2), and 10%-15% decrease (score 3).

[0067] Experimental results: as shown in Figure 5 The body weight of the mice orally administered with DSS decreased significantly in the first 7 days. After the administration of DSS was completed, the body weight of the DSS group began to recover slowly, but the body weight of the s100 and s500 groups decreased to varying degrees, and their DAI also increased significantly. The body weight of the s100 group decreased the most, and the mice died during the administration period. The s500 group was second, and the cumulative body weight decreased by 14% after the administration was completed.

[0068] 3. Effects of s100 and s500 on the pathological state of the mouse intestine

[0069] After the administration was completed, the mice were euthanized, and the colon of the mice was taken and fixed with a polyethylene glycol solution. After fixation, the paraffin embedding, slicing, and immunofluorescence staining steps were as follows:

[0070] (1) Slice deparaffinization to water: xylene I 5 min; xylene II 5 min; xylene III 5 min; 100% ethanol I 5 min; 100% ethanol II 5 min; 95% ethanol I 5 min; 95% ethanol II 5 min; 90% ethanol 5 min; 80% ethanol 5 min; 70% ethanol 5 min; 50% ethanol 5 min; PBS I 10 min; PBS II 10 min.

[0071] (2) Peroxidase elimination: The cut tissue sections were treated with 3% hydrogen peroxide for 30 min and then washed with PBS three times, each time for 5 min.

[0072] (3) Antigen retrieval: The tissue sections were boiled in Tris-EDTA buffer in a microwave oven for 20 min to complete antigen retrieval, and then washed with PBS three times, 5 min each time.

[0073] (4) Blocking: Shake off the liquid around the sliced ​​tissue, add 10% goat serum, and incubate at 37°C for 30 minutes. Add primary antibody: After drying the blocking solution, directly add the diluted primary antibody and incubate in an antibody incubation box at 4°C overnight.

[0074] (5) Add secondary antibody: Shake dry and wipe away the liquid around the tissue, add Cy3 goat anti-rabbit IgG (1:400) (for immunofluorescence double staining, add Cy3 goat anti-rabbit IgG and FITC goat anti-mouse IgG at the same time), incubate at 37°C for 1 hour, wash three times with PBS, 10 minutes each time. Add DAPI (1:3000), incubate at room temperature for 10 minutes, wash three times with PBS, 10 minutes each time. Seal the slides with anti-fluorescence quenching mounting medium and observe under a fluorescence microscope.

[0075] Immunohistochemistry procedures are as follows: Prepared paraffin sections are washed twice in xylene for 5 minutes each. Dewax the sections in water using a sequential alcohol concentration of 100%-95%-90%-80%-70% for 3 minutes each, followed by two 5-minute washes in PBS. Treat the sections with working solution for 20 minutes, followed by 8 minutes in cell permeabilization solution. After rinsing twice with PBS, incubate the sections in the MPO reaction mixture for 60 minutes. After rinsing three times with PBS, examine the sections under a microscope for MPO expression.

[0076] The steps of hematoxylin-eosin (H&E) are similar to those described above and will not be repeated here.

[0077] Experimental results: Figure 5 As shown, H&E sections in the DSS group showed a disruption of the colonic epithelial barrier, with decreased expression of tight junction proteins ZO-1 and OC-1 between intestinal epithelial cells. Disruption of the colonic epithelial barrier in the s100 group was more severe than in the DSS group, with the most severe damage in the PP nanoparticle group. The number of goblet cells in colonic tissue and the expression of ZO-1 and OC-1 all gradually and significantly decreased.

[0078] The above embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the patent scope of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are all within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A method of assessing the effect of polypropylene nanoplastics on intestinal inflammation, characterized in that, The application relates to a method for detecting the effect of polypropylene nanoparticles on intestinal inflammation. The method comprises the following steps: In method 1, the polypropylene nanoparticles have a particle size of 100-500 nm; and the concentration of the polypropylene nanoparticles in the culture medium is 0-160 mu g / mL.

2. The method of claim 1, wherein, In method 1, the detection of the cell survival rate, the glucose consumption level and the lactic acid production level is carried out by colorimetry.

3. The method of claim 1, wherein, In method 1, the index of the glycolysis level comprises the glycolysis ability, the maximum glycolysis ability and the glycolysis capacity.

4. The method of claim 1, wherein, In method 1, the glycolysis level of the cells is detected by a seahorse cell energy metabolism analyzer.

5. The method of claim 1, wherein, In method 2, the intestinal inflammation model mice are constructed by using dextran sulfate sodium.

6. The method of claim 1, wherein, In method 2, the intestinal inflammation model mice are constructed by using 5-6-week-old male Balb / c mice.

7. The method of claim 1, wherein, In method 2, the mice are randomly divided into a negative control group, a model group and an experimental group, and each group contains 4-15 mice; the negative control group is given saline, the model group is the intestinal inflammation model mice, and the experimental group is the intestinal inflammation model mice exposed to polypropylene nanoparticles by gavage; and the polypropylene nanoparticles have a particle size of 100-500 nm.

8. The method of claim 1, wherein, In method 2, the disease activity index is a standard for evaluating the severity of intestinal inflammation, and the disease activity index is the sum of the stool form, the degree of hematochezia and the weight change.

9. The method of claim 1, wherein, In method 2, the pathological state of the intestinal tract of the mice refers to the pathological condition of the intestinal tract tissue of the mice, and the tissue sample is taken from the colon of the mice. ​ ​ ​ ​ 10. The method of claim 1, wherein, ​

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