A method for identifying the toxicity of haze particles after changing their physical and chemical properties and their effects on pulmonary fibrosis

Abstract

The present invention provides a method for identifying the toxicity of haze particles after the physical and chemical properties are changed and the effect on pulmonary fibrosis, belonging to haze particles PM 2.5 The present invention uses poly-L-lysine to neutralize PM 2.5 The negative charge in the smog particles is close to neutral charge. Neutral PM 2.5 , and selected Pluronic F108 as a dispersant to promote PM 2.5 Dispersion to obtain very small haze particles Size PM 2.5 , through PM 2.5 Modification of physical and chemical properties to evaluate PM 2.5 The effects of the physicochemical properties of on macrophage NLRP3 inflammasome activation and on the formation of pulmonary fibrosis in mice were investigated to identify whether the physicochemical properties (charge and size) are the source of chronic pulmonary fibrosis.

Description

Technical Field

[0001] The present invention relates to haze particles (PM 2.5 ) toxicity evaluation technology field, specifically involving a method of changing PM 2.5 PM can be identified by its charge and size 2.5 Methods of initiating toxic sources of chronic pulmonary fibrosis. Background Art

[0002] A considerable burden on human health can be attributed to environmental pollution, including air, water, soil, heavy metal and chemical pollution, of which air pollution is the main cause. 2.5 , dynamic diameter <2.5μm), as one of the main components of air pollution, it has attracted extensive attention in terms of toxicological properties because it can penetrate deep into the respiratory tract and have adverse effects on the lungs. More and more evidence shows that PM 2.5 It can cause a series of respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD), leading to lung inflammation and fibrosis. 2.5 The various physical and chemical properties of PM2.5 may be the source of the toxic effects, but there is currently no effective method to determine the correlation between these toxic effects and the physical and chemical properties. Therefore, it is necessary to develop effective identification methods to determine the toxicity of PM2.5. 2.5 The sources of physical and chemical properties that cause toxicity are of great significance for establishing effective means of protecting human health in the future. Summary of the invention

[0003] The present invention provides a method for identifying the toxicity of haze particles after the physical and chemical properties are changed and the effect on pulmonary fibrosis. 2.5 The physicochemical properties (charge and size) of PM 2.5 Whether the physicochemical properties of the substance are the source of its toxicity.

[0004] In order to achieve the above object, the technical solution of the present invention is as follows:

[0005] A method for identifying the toxicity of haze particles after the physical and chemical properties are changed and the effect on pulmonary fibrosis, comprising the following steps:

[0006] Step 1: Get PM 2.5 Particles;

[0007] Step 2: PM for Step 1 2.5 The physical and chemical properties of the particles are modified, including PM 2.5 The charge or size of the particle gives Neutral PM 2.5 or Size PM 2.5 ;

[0008] Step 3: Use Neutral PM 2.5 or Size PM 2.5 Differently differentiated THP-1 cells were treated, and the release of IL-1β was detected by Elisa to identify the activation status of inflammasomes;

[0009] Step 4: Oral inhalation of mice Neutral PM 2.5 or Size PM 2.5 , and Masson staining was performed to identify the effects of haze particles with changed physical and chemical properties on pulmonary fibrosis.

[0010] Preferably, in step 2, PM 2.5 The charge of the particles is modified, including:

[0011] PM 2.5 The aqueous suspension was mixed with polylysine and stirred, the resulting mixture was centrifuged to remove excess PLL, the precipitate was freeze-dried and stored, and the charge-modified Neutral PM 2.5 .

[0012] Preferably, the PM 2.5 The concentration of the aqueous suspension was 250 μg mL -1 , the concentration of poly-lysine was 2.5 μg mL -1 .

[0013] Preferably, the PM 2.5 The volume ratio of the water suspension to polylysine was 4:1.

[0014] Preferably, in step 2, PM 2.5 The particle size is modified, including:

[0015] PM 2.5 The aqueous suspension was mixed with a Pluronic F108 (PF108) solution and sonicated in a water bath. The suspension was then centrifuged to remove excess PF108, and the precipitate was freeze-dried and stored to obtain a size-changed Size PM 2.5 .

[0016] Preferably, the PM 2.5 The concentration of the aqueous suspension is 1 mg mL -1 The concentration of Pluronic F108 solution was 375 μg mL -1 .

[0017] Preferably, the PM 2.5 The volume ratio of the water suspension to the Pluronic F108 (PF108) solution was 1:4.

[0018] Preferably, the ultrasonic temperature is 25° C. and the ultrasonic time is 30-60 min.

[0019] Preferably, the step three is specifically:

[0020] Step 1: Cell culture:

[0021] THP-1 cells were cultured in a medium containing 10% fetal bovine serum, 100 U mL -1 of penicillin and 100 mg mL -1 The culture conditions were 37°C and 5% CO2 in RPMI1640 medium containing streptomycin, and the medium was changed every other day;

[0022] Step 2: Elisa to detect the release of IL-1β:

[0023] THP-1 cells were plated at 3 × 10 4 The cells were placed in a 96-well plate at a density of 100 μL and incubated for 16 h with 100 μL of cell culture medium containing 1 μg mL -1 THP-1 cells were induced to differentiate with phorbol 12-myristic acid 13-acetate (PMA) and then treated with Neutral PM 2.5 or Size PM 2.5 Differentiated THP-1 cells were treated with lipopolysaccharide (LPS, 10 ng mL -1 ) for another 24 hours, the cell supernatant was collected, and IL-1β was detected by Elisa.

[0024] Preferably, the step 4 is specifically as follows:

[0025] Eight-week-old female Balb / c mice were raised under standard laboratory conditions and exposed to PM 2.5 It was performed by oropharyngeal aspiration as described by NIOSH, with 50 μL of PBS containing 400 μg mL -1 Neutral PM 2.5 or Size PM 2.5 The drug was dropped behind the tongue of the mouse, and the nasal cavity was pinched to allow it to enter the lungs. The mice were killed after 21 days, and the lung tissues were collected for Masson staining.

[0026] Beneficial effects of the present invention

[0027] The present invention provides a method for identifying the toxicity of haze particles after the physical and chemical properties are changed and the effect on pulmonary fibrosis, PM 2.5 With high negative charge and large aggregation size, 0.1% poly-L-lysine, as a positively charged polymer, has good biocompatibility and high affinity for negatively charged substrates. The present invention uses poly-L-lysine to neutralize PM 2.5 The negative charge in the smog particles is close to neutral charge. Neutral PM 2.5 In addition, PF108 is a nonionic triblock copolymer composed of two hydrophilic polyethylene oxide (PEO) chains and a dispersed hydrophobic polypropylene oxide (PPO) domain. Due to its amphiphilicity and steric hindrance effect, it can play a good dispersing role. Therefore, the present invention selects Pluronic F108 (PF108) as a dispersant to promote PM 2.5 Dispersion to obtain very small haze particles Size PM 2.5 , through PM 2.5 Modification of physical and chemical properties to evaluate PM 2.5 The effects of the physicochemical properties of on NLRP3 inflammasome activation in macrophages (such as THP-1 cells) and on the formation of pulmonary fibrosis in mice were investigated to identify whether the physicochemical properties (charge and size) are the source of chronic pulmonary fibrosis.

[0028] The experimental results showed that, as determined by incubation with macrophages and enzyme-linked immunosorbent assay, the 2.5 (negative charge), Neutral PM 2.5 It can significantly increase the release level of interleukin-1β, indicating that its ability to activate inflammasomes is enhanced; Size PM 2.5 The release level of interleukin-1β was reduced, indicating that its ability to activate inflammasomes was reduced. The mice were further allowed to inhale modified or unmodified haze particles by the oropharyngeal method. After 21 days, lung tissues were collected and mass stained. Neutral PM 2.5 Leading to more severe levels of pulmonary fibrosis, Size PM 2.5 The fibrosis level was reduced. The present invention identified PM by comparing the toxicity levels before and after the modification of the physical and chemical properties of haze particles. 2.5 PM, the source of chronic pulmonary fibrosis 2.5 The identification of the sources of toxic physical and chemical properties has important guiding significance for establishing effective human health protection measures and the treatment of chronic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 For PM 2.5 SEM (A) and TEM (B) characterization photos;

[0030] Figure 2 For PM 2.5 Particle size analysis diagram;

[0031] Figure 3 For PM 2.5 Histogram of charge and particle size changes before and after PLL transformation;

[0032] Figure 4 For PM 2.5 Particle size change curve before and after PF108 transformation;

[0033] Figure 5 For PM 2.5 , Neutral PM 2.5 and Positive PM 2.5 Bar graph of induced IL-1β production;

[0034] Figure 6 For PM 2.5 and Size PM 2.5 Bar graph of induced IL-1β production;

[0035] Figure 7 For PM 2.5 , Neutral PM 2.5 and Size PM 2.5 Photographs of the formation of induced pulmonary fibrosis in mice. DETAILED DESCRIPTION

[0036] A method for identifying the toxicity of haze particles after the physical and chemical properties are changed and the effect on pulmonary fibrosis, comprising the following steps:

[0037] Step 1: Get PM 2.5 Particles;

[0038] Step 2: PM for Step 1 2.5 The physical and chemical properties of the particles are modified, including PM 2.5 The charge or size of the particle gives Neutral PM 2.5 or Size PM 2.5 ;

[0039] Step 3: Use Neutral PM 2.5 or Size PM 2.5Differently differentiated THP-1 cells were treated, and the release of IL-1β was detected by Elisa to identify the activation status of inflammasomes;

[0040] Step 4: Oral inhalation of mice Neutral PM 2.5 or Size PM 2.5 , and Masson staining was performed to identify the effects of haze particles with changed physical and chemical properties on pulmonary fibrosis.

[0041] According to the present invention, the PM 2.5 The acquisition is as follows:

[0042] Anderson G1200 sampler was used to sample nitrocellulose at 16.7 L min -1 The flow rate of PM 2.5 The sampler was set up on the roof of a building about 20m high, and PM was carefully scraped from the filter membrane using a scraper. 2.5 Particles.

[0043] According to the present invention, in step 2, PM 2.5 Modification of the physical and chemical properties of particles includes 2.5 The charge or size of the particles is modified, and the PM 2.5 The transformation of the charge includes:

[0044] PM 2.5 The aqueous suspension is mixed with polylysine (PLL) and stirred, and the stirring time is preferably 30-60 minutes. The obtained mixture is centrifuged, and the centrifugal speed is preferably 10000-12000 rpm, and the time is preferably 20-60 minutes to remove excess PLL. The precipitate is freeze-dried and stored to obtain a charge-changed Neutral PM 2.5 The PM 2.5 The concentration of the aqueous suspension is preferably 250 μg mL -1 The concentration of poly-lysine is preferably 2.5 μg mL -1 , the PM 2.5 The volume ratio of the aqueous suspension to polylysine is preferably 4:1.

[0045] The present invention gradually neutralizes PM by controlling the concentration of L-lysine. 2.5 The negative charge (-31.5mV) of the haze fine particles is obtained with a charge close to neutral (-2.9mV) Neutral PM 2.5 ).

[0046] According to the present invention, the PM 2.5The particle size is modified, including:

[0047] PM 2.5 The aqueous suspension is mixed with a Pluronic F108 (PF108) solution and sonicated in a water bath at a temperature of 25° C. for preferably 30-60 min. The suspension is then centrifuged at 10,000-32,000 g for 30-60 min to remove excess PF108. The precipitate is freeze-dried and stored to obtain a size-changed Size PM 2.5 The PM 2.5 The concentration of the aqueous suspension is preferably 1 mg mL -1 The concentration of Pluronic F108 solution is preferably 375 μg mL -1 , the PM 2.5 The volume ratio of the aqueous suspension to the Pluronic F108 (PF108) solution is preferably 1:4.

[0048] According to the present invention, PM is dispersed by Pluronic F108 2.5 , so that the size is reduced from 849.5nm to 448.3nm, and small-sized haze fine particles are obtained ( Size PM 2.5 ).

[0049] According to the present invention, the step three is specifically as follows:

[0050] Step 1: Cell culture:

[0051] THP-1 cells were cultured in a medium containing 10% fetal bovine serum, 100 U mL -1 of penicillin and 100 mg mL -1 The culture conditions were 37°C and 5% CO2 in RPMI1640 medium containing streptomycin, and the medium was changed every other day;

[0052] Step 2: Elisa to detect the release of IL-1β:

[0053] THP-1 cells were plated at 3 × 10 4 The cells were placed in a 96-well plate at a density of 100 μL and incubated for 16 h with 100 μL of cell culture medium containing 1 μg mL -1 THP-1 cells were induced to differentiate with phorbol 12-myristic acid 13-acetate (PMA) and then treated with Neutral PM 2.5 , Size PM 2.5 Differentiated THP-1 cells were treated with lipopolysaccharide (LPS, 10 ng mL-1 ) for another 24 h, and PM 2.5 The supernatant of exposed cells was used to detect IL-1β by Elisa.

[0054] According to the present invention, the step 4 is specifically as follows:

[0055] Eight-week-old female Balb / c mice were purchased from Beijing Weitonglihua Animal Technology Co., Ltd. All animals were raised under standard laboratory conditions. 2.5 It was performed by oropharyngeal aspiration as described by NIOSH, with 50 μL of PBS containing 400 μg mL -1 Neutral PM 2.5 or Size PM 2.5 The drug was dropped behind the tongue of the mouse, and the nasal cavity was pinched to allow it to enter the lungs. The mice were killed after 21 days, and the lung tissues were collected for Masson staining.

[0056] The present invention is described in detail below with reference to the accompanying drawings.

[0057] Example 1

[0058] A method for identifying the toxicity of haze particles after changing their physical and chemical properties and their effects on pulmonary fibrosis, the specific steps are as follows:

[0059] 1.PM 2.5 Obtaining:

[0060] In Changchun, China, PM was collected on nitrocellulose filters using an Anderson G1200 sampler at a flow rate of 16.7 L min-1. 2.5 The sampler was set up on the roof of a building about 20 m high, which was considered to be a representative area integrating residential, transportation, construction and industry. PM was carefully scraped from the filter membrane using a scraper. 2.5 particles and analyzed by SEM ( Figure 1 A) TEM Figure 1 B) and DLS to characterize the physicochemical properties of PM2.5, such as Figure 1 and Figure 2 shown.

[0061] 2.PM 2.5 Charge transformation

[0062] 4 mL 250 μg mL -1 PM 2.5 Aqueous suspension (charge: -31.5 mV) with 1 mL 2.5 μg mL -1Polylysine (PLL) was mixed and stirred for 30 min. The resulting mixture was centrifuged at 12000 rpm for 20 min to remove excess PLL. The precipitate was freeze-dried and stored at 4°C. This sample was named Neutral PM 2.5 (charge: -2.9 mV) Similarly, 1 mL 100 μg mL -1 PLL is used to prepare positively charged haze particles, named Positive PM 2.5 (charge: +29.8mV), charge before and after PLL transformation ( Figure 3 A) and particle size ( Figure 3 B) Changes such as Figure 3 shown.

[0063] 3.PM 2.5 Dispersion of particles

[0064] 11.6 mL 1 mg mL -1 PM 2.5 Aqueous suspension (size: 849.5 nm) with 46.4 mL 375 μg mL -1 The Pluronic F108 (PF108) solution was mixed and sonicated in a water bath for 30 min. The suspension was then centrifuged at 32,000 g for 30 min to remove excess PF108. The precipitate was lyophilized and stored at 4 °C. This sample was named Size PM 2.5 (Size: 448.3nm), the particle dispersion effect is as follows Figure 4 .

[0065] 4. Cell culture:

[0066] THP-1 cells were cultured in a medium containing 10% fetal bovine serum, 100 U mL-1 penicillin, and 100 mg mL-1 -1 The cells were cultured in RPMI1640 medium containing streptomycin at 37°C and 5% CO2, and the medium was replaced every other day.

[0067] 5. Elisa to detect the release of IL-1β:

[0068] THP-1 cells were plated at 3 × 10 4 The cells were placed in a 96-well plate at a density of 100 μL and incubated for 16 h with 100 μL of cell culture medium containing 1 μg mL -1 THP-1 cells were induced to differentiate by 12-myristic acid 13-acetate (PMA). Neutral PM 2.5 , Size PM 2.5 and PM2.5 Differentiated THP-1 cells were treated with lipopolysaccharide (LPS, 10 ng mL -1 ) for another 24 hours. 2.5 The supernatant of exposed cells was used to detect IL-1β by Elisa method to explore its effect on IL-1β release. Figure 5 and Figure 6 When the significant difference p < 0.05 was shown, it was considered to significantly reduce or increase PM 2.5 Inflammasome activation ability, Figure 5 and 6 Compared with the unmodified PM 2.5 (negative charge), Neutral PM 2.5 It can significantly increase the release level of interleukin-1β, indicating that its ability to activate inflammasomes is enhanced; Size PM 2.5 This reduced the release of interleukin-1β, indicating a reduced ability to activate inflammasomes.

[0069] 6. Masson staining to detect the formation of pulmonary fibrosis in mice:

[0070] Eight-week-old female Balb / c mice were purchased from Beijing Weitonglihua Animal Technology Co., Ltd. All animals were raised under standard laboratory conditions. 2.5 It was performed by oropharyngeal aspiration as described by NIOSH, with 50 μL of PBS containing 400 μg mL -1 PM 2.5 , Neutral PM 2.5 or Size PM 2.5 The solution was dripped behind the tongue of the mouse, and the nasal cavity was pinched to allow it to enter the lungs. The mice were killed 21 days later, and the lung tissues were collected and stained with Masson staining. Figure 7 As shown, mice Neutral PM 2.5 The level of lung fibrosis after exposure was defined as bridging perisinusoidal fibrosis. Size PM 2.5 The level of lung fibrosis after exposure was defined as perisinusoidal fibrosis, and the PM 2.5 The level of lung fibrosis after exposure was identified as periportal fibrosis, indicating Neutral PM 2.5 Leading to more severe levels of pulmonary fibrosis, Size PM 2.5 Reduced the level of fibrosis.

Claims

1. A method for identifying the toxicity of haze particles after changing their physical and chemical properties and their effects on pulmonary fibrosis, characterized in that: The following steps are involved: Step 1: Get PM 2.5 Particles; Step 2: PM for Step 1 2.5 The physical and chemical properties of the particles are modified, including PM 2.5 The charge or size of the particle gives Neutral PM 2.5 or Size PM 2.5 ; Step 3: Use Neutral PM 2.5 or Size PM 2.5 Differently differentiated THP-1 cells were treated, and the release of IL-1β was detected by Elisa to identify the activation status of inflammasomes; Step 4: Oral inhalation of mice Neutral PM 2.5 or Size PM 2.5 , and Masson staining was performed to identify the effects of haze particles with changed physical and chemical properties on pulmonary fibrosis.

2. The method for identifying the toxicity of haze particles after changing their physical and chemical properties and their effects on pulmonary fibrosis according to claim 1, characterized in that: In step 2, PM 2.5 The charge of the particles is modified, including: PM 2.5 The aqueous suspension was mixed with polylysine and stirred, the resulting mixture was centrifuged to remove excess polylysine, the precipitate was freeze-dried and stored, and the charge-changed Neutral PM 2.5 .

3. The method for identifying the toxicity of haze particles after the physical and chemical properties are changed and the effect on pulmonary fibrosis according to claim 2, characterized in that: The PM 2.5 The concentration of the aqueous suspension is 250 μg mL -1 The concentration of poly-lysine was 2.5 μg mL -1 .

4. The method for identifying the toxicity of haze particles after the physical and chemical properties are changed and the effect on pulmonary fibrosis according to claim 2, characterized in that: The PM 2.5 The volume ratio of the water suspension to polylysine was 4:

1.

5. The method for identifying the toxicity of haze particles after changing their physical and chemical properties and their effects on pulmonary fibrosis according to claim 1, characterized in that: In step 2, PM 2.5 The particle size is modified, including: PM 2.5 The aqueous suspension was mixed with a Pluronic F108 solution and sonicated in a water bath. The suspension was then centrifuged to remove excess Pluronic F108, and the precipitate was freeze-dried and stored to obtain a size-changed Size PM 2.5 .

6. The method for identifying the toxicity of haze particles after the physical and chemical properties are changed and the effect on pulmonary fibrosis according to claim 5, characterized in that: The PM 2.5 The concentration of the aqueous suspension is 1 mg mL -1 The concentration of Pluronic F108 solution was 375 μg mL -1 .

7. The method for identifying the toxicity of haze particles after the physical and chemical properties are changed and the effect on pulmonary fibrosis according to claim 5, characterized in that: The PM 2.5 The volume ratio of the water suspension to the Pluronic F108 solution was 1:

4.

8. The method for identifying the toxicity of haze particles after the physical and chemical properties are changed and the effect on pulmonary fibrosis according to claim 5, characterized in that: The ultrasonic temperature is 25°C, and the ultrasonic time is 30-60 minutes.

9. The method for identifying the toxicity of haze particles after changing their physical and chemical properties and their effects on pulmonary fibrosis according to claim 1, characterized in that: The step three is specifically as follows: Step 1: Cell culture: THP-1 cells were cultured in a medium containing 10% fetal bovine serum, 100 U mL -1 of penicillin and 100 mg mL -1 The culture conditions were 37°C and 5% CO2 in RPMI1640 medium containing streptomycin, and the medium was changed every other day; Step 2: Elisa to detect the release of IL-1β: THP-1 cells were plated at 3 × 10 4 The cells were placed in a 96-well plate at a density of 100 μL and incubated for 16 h with 100 μL of cell culture medium containing 1 μg mL -1 Phobor 12-myristic acid 13-acetate was used to induce THP-1 cell differentiation, and then, Neutral PM 2.5 or Size PM 2.5 Differentiated THP-1 cells were treated with lipopolysaccharide (LPS), 10 ng mL -1 The cells were treated with 5% paraformaldehyde for 24 h in the presence of 5% paraformaldehyde, and the cell supernatant was collected and IL-1β was detected by Elisa.

10. The method for identifying the toxicity of haze particles after the physical and chemical properties are changed and the effect on pulmonary fibrosis according to claim 1, characterized in that: The step 4 is specifically as follows: Eight-week-old female Balb / c mice were raised under standard laboratory conditions and exposed to PM 2.5 It was performed by oropharyngeal aspiration as described by NIOSH, with 50 μL of PBS containing 400 μg mL -1 Neutral PM 2.5 or Size PM 2.5 The drug was dropped behind the tongue of the mouse, and the nasal cavity was pinched to allow it to enter the lungs. The mice were killed after 21 days, and the lung tissues were collected for Masson staining.

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