CN114487432B - A method for identifying the toxicity of haze particles after changing their physical and chemical properties and their effects on pulmonary fibrosis - Google Patents

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, and belongs to the technical field of toxicity evaluation of haze particles PM _2.5 . The present invention adopts poly-L-lysine to neutralize the negative charge in PM _2.5 to obtain haze particles _Neutral PM _2.5 with a charge close to neutrality, and selects Pluronic F108 as a dispersant to promote the dispersion of PM _2.5 , and obtains haze particles _Size PM _2.5 with a very small size. By modifying the physicochemical properties of PM _2.5 , the effects of the physicochemical properties of PM _2.5 on the activation of macrophage NLRP3 inflammasome and the formation of mouse pulmonary fibrosis are evaluated, thereby identifying whether the physicochemical properties (charge and size) are the source of chronic pulmonary fibrosis.

Description

Method for identifying toxicity of haze particles with changed physical properties and influence on pulmonary fibrosis

Technical Field

The invention relates to the technical field of toxicity evaluation of haze particles (PM _2.5), in particular to a method for identifying a toxicity source of PM _2.5 induced pulmonary chronic fibrosis by changing the charge and the size of PM _2.5.

Background

Considerable human health burden can be attributed to environmental pollution, including air, water, soil, heavy metals, and chemical pollution, with air pollution being the leading cause. Fine particulate matter (PM _2.5, kinetic diameter <2.5 μm) in air, which is one of the main components of atmospheric pollution, is of great interest in toxicological properties because it can penetrate deep into the respiratory tract, adversely affecting the lungs. There is growing evidence that PM _2.5 can cause a range of respiratory diseases, such as asthma, chronic Obstructive Pulmonary Disease (COPD), and the like, leading to pulmonary inflammation and fibrosis. Various physicochemical properties of PM _2.5 are likely sources of the toxic effects that are caused, however, there is currently no effective method to determine the correlation of these toxic effects with physicochemical properties. Therefore, developing an effective identification method to determine the physicochemical property source of PM _2.5 -induced toxicity is of great significance for establishing effective human health protection means in the future.

Disclosure of Invention

The invention improves a method for identifying the toxicity of haze particles after changing physical properties and the influence on pulmonary fibrosis, evaluates the influence of PM _2.5 on chronic pulmonary fibrosis by modifying the physical and chemical properties (charge and size) of PM _2.5, and identifies whether the physical and chemical properties of PM _2.5 are toxic sources.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

A method of identifying haze particle toxicity and effects on pulmonary fibrosis after changing physical properties comprising the steps of:

Step one, PM _2.5 particles are obtained;

modifying the physical properties of the PM _2.5 particles in the first step, wherein the physical properties comprise the charge or the size of the PM _2.5 particles, so as to obtain _NeutralPM_2.5 or _SizePM_2.5;

Treating different differentiated THP-1 cells with _NeutralPM_2.5 or _SizePM_2.5, detecting the release of IL-1 beta by using an Elisa method, and identifying the activation state of an inflammatory body;

And fourthly, inhaling _NeutralPM_2.5 or _SizePM_2.5 into the mice through an oropharynx method, and dyeing Massion to identify the influence of haze particles with changed physical properties on pulmonary fibrosis.

Preferably, the modifying the charge of the PM _2.5 particles in the second step specifically includes:

The aqueous PM _2.5 suspension was mixed with polylysine and stirred, the resulting mixture was centrifuged to remove excess PLL, the precipitate was lyophilized and stored to give charge-altered _NeutralPM_2.5.

Preferably, the PM _2.5 aqueous suspension has a concentration of 250 μg mL ^-1 and polylysine has a concentration of 2.5 μg mL ^-1.

Preferably, the volume ratio of the PM _2.5 aqueous suspension to the polylysine is 4:1.

Preferably, the modifying the size of the PM _2.5 particles in the second step specifically includes:

the PM _2.5 aqueous suspension was mixed with Pluronic F108 (PF 108) solution and sonicated in a water bath, and the suspension was centrifuged to remove excess PF108, and the pellet was lyophilized and stored to give a size-changed _SizePM_2.5.

Preferably, the PM _2.5 aqueous suspension has a concentration of 1mg mL ^-1 and the Pluronic F108 solution has a concentration of 375 μg mL ^-1.

Preferably, the volume ratio of the PM _2.5 aqueous suspension to the Pluronic F108 (PF 108) solution is 1:4.

Preferably, the ultrasonic temperature is 25 ℃, and the ultrasonic time is 30-60min.

Preferably, the third step specifically comprises:

step 1, cell culture:

THP-1 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum, 100U mL ^-1 of penicillin and 100mg mL ^-1 of streptomycin, under culture conditions of 37 ℃ and 5% CO ₂ and medium was changed every other day;

step 2, elisa detection of IL-1β release:

THP-1 cells were incubated at a density of 3X 10 ⁴ cells per well in 96-well plates for 16h with 100. Mu.L of cell culture medium containing 1. Mu.g of mL ^-1 phorbol 12-myristate 13-acetate (PMA) to induce THP-1 cell differentiation, then the differently differentiated THP-1 cells were treated with _NeutralPM_2.5 or _SizePM_2.5, further treated for 24h in the presence of lipopolysaccharide (LPS, 10ng mL ^-1), cell supernatants were collected and assayed for IL-1β using the Elisa method.

Preferably, the fourth step specifically comprises:

Female Balb/c mice of 8 weeks of age were incubated under standard laboratory conditions, animals exposed to PM _2.5 by oropharyngeal aspiration as described by NIOSH, 50 μl of PBS containing 400 μg mL ^-1 _NeutralPM_2.5 or _SizePM_2.5 was dropped behind the tongue of the mice, the nasal cavity was pinched into the lungs, the mice were sacrificed after 21 days, and lung tissue was collected for Masson staining.

The beneficial effects of the invention are that

The invention provides a method for identifying haze particle toxicity and influence on pulmonary fibrosis after changing physical properties, PM _2.5 has high negative charge and large aggregation size, 0.1% poly-L-lysine is used as a positively charged polymer, good biocompatibility and high affinity are achieved for negatively charged substrates, and poly-L-lysine is used for neutralizing negative charge in PM _2.5 to obtain haze particle _NeutralPM_2.5 with nearly neutral charge. In addition, PF108 is a nonionic triblock copolymer consisting of two hydrophilic polyethylene oxide (PEO) chains and a dispersed hydrophobic polypropylene oxide (PPO) domain, which can perform a good dispersing effect due to its amphiphilic and steric effects, so that the present invention selects Pluronic F108 (PF 108) as a dispersing agent to promote the dispersion of PM _2.5, to obtain haze particles _SizePM_2.5 with small size, and evaluates the effect of the physicochemical properties of PM _2.5 on the activation of macrophages (such as THP-1 cells) NLRP3 inflammasome and on the formation of pulmonary fibrosis by modifying the physicochemical properties of PM _2.5, thereby identifying whether the physicochemical properties (charge and size) are the source of chronic fibrosis in the lung.

Experimental results show that compared with unmodified PM _2.5 (negative charge), _NeutralPM_2.5 can significantly improve the release level of the interleukin 1 beta, which shows that the release level of the interleukin 1 beta is reduced, and _SizePM_2.5 can improve the capability of the interleukin 1 beta to activate the inflammatory body, which is confirmed by incubation with macrophages and detection of an enzyme-linked immune reaction. Further, by means of an oropharynx method, a mouse is inhaled with or without reconstruction of haze particles, lung tissues are collected and Massion dyed after 21 days, _NeutralPM_2.5 is found to cause more serious pulmonary fibrosis level, _SizePM_2.5 weakens the pulmonary fibrosis level, a source of PM _2.5 for inducing pulmonary chronic fibrosis is identified through comparison of toxicity levels before and after reconstruction of physical properties of the haze particles, and identification of the source of PM _2.5 toxic physicochemical properties has important guiding significance for establishing effective human health protection measures and treatment of chronic diseases.

Drawings

FIG. 1 is a photograph of SEM (A) and TEM (B) characterization of PM _2.5;

FIG. 2 is a graph of particle size analysis of PM _2.5;

FIG. 3 is a bar graph of charge and particle size change before and after modification of PM _2.5 by a PLL;

FIG. 4 is a graph showing the particle size change of PM _2.5 before and after modification by PF 108;

FIG. 5 is a bar graph of IL-1β production induced by PM _2.5、_NeutralPM_2.5 and _PositivePM_2.5;

FIG. 6 is a bar graph of IL-1β production induced by PM _2.5 and _SizePM_2.5;

Fig. 7 is a photograph of the formation of PM _2.5、_NeutralPM_2.5 and _SizePM_2.5 induced pulmonary fibrosis in mice.

Detailed Description

A method of identifying haze particle toxicity and effects on pulmonary fibrosis after changing physical properties comprising the steps of:

Step one, PM _2.5 particles are obtained;

modifying the physical properties of the PM _2.5 particles in the first step, wherein the physical properties comprise the charge or the size of the PM _2.5 particles, so as to obtain _NeutralPM_2.5 or _SizePM_2.5;

Treating different differentiated THP-1 cells with _NeutralPM_2.5 or _SizePM_2.5, detecting the release of IL-1 beta by using an Elisa method, and identifying the activation state of an inflammatory body;

And fourthly, inhaling _NeutralPM_2.5 or _SizePM_2.5 into the mice through an oropharynx method, and dyeing Massion to identify the influence of haze particles with changed physical properties on pulmonary fibrosis.

According to the invention, the PM _2.5 is obtained by:

PM _2.5 was collected on a nitrocellulose filter using an Anderson G1200 sampler at a flow rate of 16.7L min ^-1. The sampler was placed on a building roof about 20m high and PM _2.5 particles were carefully scraped from the filter by a spatula.

According to the present invention, the modification of the physical properties of the PM _2.5 particles in the second step includes modifying the charge or size of the PM _2.5 particles, and the modification of the PM _2.5 charge specifically includes:

Mixing PM _2.5 aqueous suspension with Polylysine (PLL) for 30-60min, centrifuging the obtained mixture at 10000-12000rpm for 20-60min to remove excessive PLL, lyophilizing the precipitate, and preserving to obtain _NeutralPM_2.5 with changed charge. The concentration of PM _2.5 aqueous suspension is preferably 250 mug mL ^-1, the concentration of polylysine is preferably 2.5 mug mL ^-1, and the volume ratio of PM _2.5 aqueous suspension to polylysine is preferably 4:1.

According to the invention, by controlling the concentration of L-lysine, negative charges (-31.5 mV) of PM _2.5 are gradually neutralized, and haze fine particles (_NeutralPM_2.5) with charges close to neutrality (-2.9 mV) are obtained.

According to the invention, the modification of the size of PM _2.5 particles specifically comprises:

mixing PM _2.5 aqueous suspension with Pluronic F108 (PF 108) solution, and performing ultrasonic treatment in water bath at 25deg.C for 30-60min, centrifuging the suspension at 10000-32000g for 30-60min to remove excessive PF108, lyophilizing the precipitate, and storing to obtain _SizePM_2.5 with changed size. The concentration of PM _2.5 aqueous suspension is preferably 1mg mL ^-1, the concentration of Pluronic F108 solution is preferably 375 μg mL ^-1, and the volume ratio of PM _2.5 aqueous suspension to Pluronic F108 (PF 108) solution is preferably 1:4.

According to the present invention, PM _2.5 was dispersed by Pluronic F108 such that the size was reduced from 849.5nm to 448.3nm, resulting in small-sized haze fine particles (_SizePM_2.5).

According to the invention, the third step is specifically as follows:

step 1, cell culture:

THP-1 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum, 100U mL ^-1 of penicillin and 100mg mL ^-1 of streptomycin under conditions of 37℃and 5% CO ₂ and medium was changed every other day;

step 2, elisa detection of IL-1β release:

THP-1 cells were placed in 96-well plates at a density of 3X 10 ⁴ cells per well, incubated for 16h with 100. Mu.L of cell culture medium containing 1. Mu.g of mL ^-1 phorbol 12-myristate 13-acetate (PMA) to induce THP-1 cell differentiation, then the differently differentiated THP-1 cells were treated with _NeutralPM_2.5,_SizePM_2.5 and treated for an additional 24 h in the presence of lipopolysaccharide (LPS, 10ng mL ^-1), PM _2.5 exposed cell supernatants were collected and IL-1β was detected using the Elisa method.

According to the invention, the step four specifically comprises the following steps:

Female Balb/c mice of 8 weeks old were purchased from bevertolihua animal technologies, inc. Of beijing, all animals were cultured under standard laboratory conditions, animals were exposed to PM _2.5 by oropharyngeal aspiration as described by NIOSH, 50 μl PBS containing 400 μg mL ^-1 _NeutralPM_2.5 or _SizePM_2.5 was dropped behind the tongue of the mice, the nasal cavity was pinched into the lungs, the mice were sacrificed 21 days later, lung tissue was collected, and Masson staining was performed.

The present invention will be described in detail with reference to the accompanying drawings.

Example 1

A method for identifying haze particle toxicity and effects on pulmonary fibrosis after changing physical properties, comprising the steps of:

Acquisition of pm _2.5:

PM _2.5 was collected on nitrocellulose filter membrane using an Anderson G1200 sampler at a flow rate of 16.7Lmin-1 in vinca, china. The sampler is placed on the roof of a building about 20m high, which is considered to be a representative area integrating housing, traffic, construction and industry. PM _2.5 particles were carefully scraped from the filter by a doctor blade and the physicochemical properties of PM2.5 were characterized by SEM (FIG. 1A), TEM (FIG. 1B) and DLS, as shown in FIGS. 1 and 2.

Charge modification of PM _2.5

4ML of 250. Mu.g mL ^-1PM_2.5 aqueous suspension (charge: -31.5 mV) was mixed with 1mL of 2.5. Mu.g mL ^-1 Polylysine (PLL) and stirred for 30min. The resulting mixture was centrifuged at 12000rpm for 20min to remove excess PLL. The pellet was lyophilized and stored at 4 ℃. This sample was designated _NeutralPM_2.5 (charge: -2.9 mV) and similarly 1mL 100 μg mL ^-1 PLL was used to prepare haze particles with positive charge, designated _PositivePM_2.5 (charge: +29.8 mV), and the charge (FIG. 3A) and particle size (FIG. 3B) changes before and after PLL modification are shown in FIG. 3.

Dispersion of particulate matter in PM _2.5

11.6ML of 1mg mL ^-1PM_2.5 aqueous suspension (size: 849.5 nm) was mixed with 46.4mL 375. Mu.g mL ^-1 Pluronic F108 (PF 108) solution and sonicated in a water bath for 30min. The suspension was then centrifuged at 32000g for 30min to remove excess PF108. The pellet was lyophilized and stored at 4 ℃. This example is designated _SizePM_2.5 (size: 448.3 nm) and its particle dispersion effect is shown in FIG. 4.

4. Cell culture:

THP-1 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum and 100U mL-1 penicillin and 100mg mL ^-1 streptomycin. The culture conditions were 37 ℃ and 5% CO ₂ and medium was changed every other day.

Elisa assay for IL-1β release:

THP-1 cells were plated in 96-well plates at a density of 3X 10 ⁴ cells per well and incubated with 100. Mu.L of cell culture medium for 16h. The medium contained 1. Mu.g of Mug mL ^-1 of phorbol 12-myristate 13-acetate (PMA) to induce THP-1 cell differentiation. The differently differentiated THP-1 cells were then treated with _NeutralPM_2.5、_SizePM_2.5 and PM _2.5, and treated for an additional 24 hours in the presence of lipopolysaccharide (LPS, 10ng mL ^-1). Cell supernatants exposed to PM _2.5 were collected and assayed for IL-1β by the Elisa method to investigate its effect on IL-1β release. As shown in fig. 5 and 6, when the significance difference p <0.05, it was considered that the inflammatory body activation capacity of PM _2.5 was significantly reduced or increased, and fig. 5 and 6 illustrate that _NeutralPM_2.5 was able to significantly increase the level of release of interleukin 1β, indicating that it had an increased capacity to activate inflammatory bodies, and _SizePM_2.5 was able to decrease the level of release of interleukin 1β, indicating that it had a decreased capacity to activate inflammatory bodies, compared to unmodified PM _2.5 (negative charge).

Masson staining to detect the formation of pulmonary fibrosis in mice:

Female Balb/c mice at 8 weeks of age were purchased from Beijing Vitrehua animal technologies Inc., and all animals were cultured under standard laboratory conditions. Animal exposure to PM _2.5 was performed by oropharyngeal aspiration as described by NIOSH, 50 μ LPBS containing 400 μg mL ^-1PM_2.5、_NeutralPM_2.5 or _SizePM_2.5 was instilled behind the tongue of the mice, pinching the nasal cavity into the lungs. Mice were sacrificed 21 days later and lung tissue was collected and Masson stained. As shown in fig. 7, the pulmonary fibrosis level of mice exposed to _NeutralPM_2.5 was identified as bridging Dou Zhou fibrosis, the pulmonary fibrosis level exposed to _SizePM_2.5 was identified as Dou Zhou fibrosis, the pulmonary fibrosis level exposed to PM _2.5 was identified as portal Dou Zhou fibrosis, indicating that _NeutralPM_2.5 resulted in a more severe pulmonary fibrosis level, and _SizePM_2.5 attenuated this fibrosis level.

Claims (10)

1. A method for identifying haze particle toxicity and effects on pulmonary fibrosis after changing physicochemical properties comprising the steps of:

Step one, PM _2.5 particles are obtained;

modifying the physical properties of the PM _2.5 particles in the first step, wherein the physical properties comprise the charge or the size of the PM _2.5 particles, so as to obtain _NeutralPM_2.5 or _SizePM_2.5;

Treating different differentiated THP-1 cells with _NeutralPM_2.5 or _SizePM_2.5, detecting the release of IL-1 beta by using an Elisa method, and identifying the activation state of an inflammatory body;

and fourthly, inhaling _NeutralPM_2.5 or _SizePM_2.5 into the mice through an oropharynx method, and carrying out Masson dyeing to identify the influence of haze particles with changed physical properties on pulmonary fibrosis.

2. A method according to claim 1 for identifying haze particle toxicity and effects on pulmonary fibrosis after changing physicochemical properties, the method is characterized in that in the second step, the charges of PM _2.5 particles are modified, and the method specifically comprises the following steps:

Mixing PM _2.5 water suspension with polylysine, stirring, centrifuging the obtained mixture to remove excessive polylysine, lyophilizing the precipitate, and preserving to obtain _NeutralPM_2.5 with changed charge.

3. The method of claim 2, wherein the aqueous suspension of PM _2.5 has a concentration of 250 μg mL ^-1 and the concentration of polylysine has a concentration of 2.5 μg mL ^-1.

4. A method according to claim 2 for identifying haze particle toxicity and effects on pulmonary fibrosis after changing physicochemical properties, the preparation method is characterized in that the volume ratio of the PM _2.5 aqueous suspension to the polylysine is 4:1.

5. A method according to claim 1 for identifying haze particle toxicity and effects on pulmonary fibrosis after changing physicochemical properties, the method is characterized in that the modification of the size of PM _2.5 particles in the second step specifically comprises the following steps:

The aqueous PM _2.5 suspension was mixed with the Pluronic F108 solution and sonicated in a water bath, and the suspension was centrifuged to remove excess Pluronic F108, and the pellet was lyophilized and stored to give _SizePM_2.5 with a modified size.

6. The method of claim 5, wherein the aqueous suspension of PM _2.5 has a concentration of 1 mg/mL ^-1 and the Pluronic F108 solution has a concentration of 375 μg/mL ^-1.

7. A method according to claim 5 for identifying haze particle toxicity and effects on pulmonary fibrosis after changing physicochemical properties, the preparation method is characterized in that the volume ratio of the PM _2.5 aqueous suspension to the Pluronic F108 solution is 1:4.

8. A method according to claim 5 for identifying haze particle toxicity and effects on pulmonary fibrosis after changing physicochemical properties, the ultrasonic temperature is 25 ℃, and the ultrasonic time is 30-60min.

9. A method according to claim 1 for identifying haze particle toxicity and effects on pulmonary fibrosis after changing physicochemical properties, the method is characterized by comprising the following steps:

step 1, cell culture:

THP-1 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum, 100U mL ^-1 of penicillin and 100mg mL ^-1 of streptomycin, under culture conditions of 37 ℃ and 5% CO ₂ and medium was changed every other day;

step 2, elisa detection of IL-1β release:

THP-1 cells were incubated at a density of 3X 10 ⁴ cells per well in 96-well plates for 16h with 100. Mu.L of cell culture medium containing 1. Mu.g of mL ^-1 phorbol 12-myristate 13-acetate to induce THP-1 cell differentiation, then the differently differentiated THP-1 cells were treated with _NeutralPM_2.5 or _SizePM_2.5, further treated for 24 h in the presence of lipopolysaccharide LPS,10ng mL ^-1, cell supernatants were collected and assayed for IL-1β by Elisa method.

10. A method according to claim 1 for identifying haze particle toxicity and effects on pulmonary fibrosis after changing physicochemical properties, the method is characterized by comprising the following steps:

Female Balb/c mice of 8 weeks of age were incubated under standard laboratory conditions, animals exposed to PM _2.5 by oropharyngeal aspiration as described by NIOSH, 50 μl of PBS containing 400 μg mL ^-1 _NeutralPM_2.5 or _SizePM_2.5 was dropped behind the tongue of the mice, the nasal cavity was pinched into the lungs, the mice were sacrificed after 21 days, and lung tissue was collected for Masson staining.