An alveolar microresponse system for PM2.5 carrying non-water-soluble particles and a model for assessing its relative bioavailability.

Abstract

This invention belongs to the field of environmental and human health technology, and discloses a PM... 2.5 An alveolar microresponsive system carrying insoluble particles and a model for evaluating its relative bioefficacy. This alveolar microresponsive system establishes an alveolar microresponsive system by carrying insoluble PM... 2.5 A model for the bioaccumulation and relative bioavailability of -M, derived from lung surfactant extraction and PM2.5. 2.5 The process of incubating and extracting adsorbed substances from M, screening adsorbent layer lipids and proteomes, and assembling microreaction system models revealed the properties of water-insoluble PM. 2.5 The accumulation and absorption characteristics of PM2.5 particles in the alveoli; lipidomics and proteomics analyses clarified the composition of insoluble PM2.5 particles in the alveoli. 2.5 A degradation and transformation microreaction system consisting of -M and alveolar fluid was constructed, and a non-water-soluble PM2.5 was first constructed based on the microreaction system. 2.5 - Model for calculating alveolar turnover rate after M respiratory exposure and the amount of water-insoluble PM in alveoli 2.5 -M bioefficacy assessment model.

Description

Technical Field

[0001] This invention belongs to the field of environmental and human health technology, specifically, it relates to a PM... 2.5 An alveolar microresponse system carrying non-water-soluble particles and an evaluation model for its relative bioavailability. Background Technology

[0002] Fine particulate matter (PM2.5) 2.5 Pollution is one of the world's most pressing environmental issues. Data from the World Health Organization shows that over 90% of the world's population breathes air containing PM2.5. 2.5 Concentrations all exceeded their guidance limits, with exposure levels being particularly severe in low- and middle-income countries, where high concentrations of PM2.5 were observed. 2.5 Pollution can cause more than 7 million premature deaths worldwide each year. PM 2.5 Its chemical composition is complex, and heavy metals are a significant toxic component. Heavy metals themselves possess toxicity to various tissues and organs, while PM2.5... 2.5 Carrier non-water-soluble particles (PM) 2.5 PM2.5 (PM2.5) is characterized by its diverse chemical forms, abundant content, wide spread, and high absorption rate by the body, potentially causing greater health hazards. However, in toxicological effect studies, PM2.5... 2.5 The bioaccumulation, absorption characteristics, and mechanisms of -M have not yet been clarified.

[0003] Respiratory exposure has been proven to be PM2.5 2.5 -M enters the human body primarily through absorption. During respiratory exposure, lung tissue plays a crucial role in the body's absorption and transport of PM2.5. 2.5 -M is crucial. Existing research focuses on PM. 2.5 The main findings of -M lung exposure revolve around the following three aspects: 1) water-soluble PM2.5 2.5 - Characteristics and in vitro models of pulmonary degradation and absorption of PM2.5; 2) Water-soluble PM2.5 2.5 - Characteristics and mechanisms of M transpulmonary transport; 3) In vitro PM testing using simulated lung fluid 2.5 -M bioavailability study analysis. Meanwhile, non-water-soluble PM2.5... 2.5 -M is often considered to have extremely low bioavailability, thus no studies have been conducted specifically targeting non-water-soluble PM2.5. 2.5 -M is conducting research on the characteristics and mechanisms of transpulmonary absorption. Although existing research in PM 2.5 Significant breakthroughs have been achieved in the transport and absorption of PM2.5 via the respiratory exposure pathway, but non-water-soluble PM2.5 remains a concern. 2.5 The chemical reaction process, system, and bioavailability assessment model of -M in lung tissue still need to be explored. Summary of the Invention

[0004] Based on the problems existing in the above-mentioned background technology, the primary objective of this invention is to provide a PM2.5 An alveolar microreaction system carrying non-water-soluble particles, based on the real-world environment's non-water-soluble PM... 2.5 -M pollution concentration and composition, using animal models, revealed the concentration and composition of non-water-soluble PM2.5. 2.5 The accumulation and absorption characteristics of -M particles in the alveoli; lipidomics and proteomics analyses clarified the characteristics of insoluble PM in the alveoli. 2.5 Chemical composition and reaction process of the degradation and transformation microreaction system composed of M particles and alveolar fluid.

[0005] Another objective of this invention is to provide a method for constructing PM in alveoli based on the above-mentioned microreaction system. 2.5 -M is a model for assessing relative bioavailability.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A type of PM 2.5 The construction of an alveolar microreaction system carrying non-water-soluble particles includes the following specific steps:

[0008] S1. Establish non-water-soluble PM 2.5 -M particle bioaccumulation and relative bioavailability model

[0009] ① Constructing animal exposure models

[0010] (c) Collect atmospheric PM2.5 2.5 -M sample was ball-milled to below 2.5 μm as a standard for compound M to obtain PM. 2.5 -M exposure material, where M is a non-water-soluble particle, is added to physiological saline to prepare non-water-soluble PM. 2.5 -M suspension;

[0011] (d) Mice were exposed to non-water-soluble PM2.5 via inhalation. 2.5 - Inhalation exposure to M suspension and water-soluble M solution, as shown in formula (1):

[0012]

[0013] In the formula: The average daily PM2.5 inhalation rate of mice 2.5 -M exposure, mg / kg / d;

[0014] ADD M溶液 The average daily exposure to M solution in mice is expressed in mg / kg / d.

[0015] PM2.5 2.5 -Total concentration of M in sample M, μg / m 3 ;

[0016] InhR mouse The respiratory rate of mice, m 3 / d;

[0017] EF mouse Mice inhaled PM 2.5 -M exposure frequency, d / w;

[0018] EF mouse Mice inhaled PM 2.5 -M exposure period, w;

[0019] BW mouse The average weight of the mice is in kg;

[0020] AT is a method for mice to inhale PM. 2.5 -M's average exposure time, d;

[0021] (c) Monitor the concentration of M ions in mouse urine during exposure. End exposure when the M ion concentration reaches equilibrium, and detect insoluble PM2.5. 2.5 -M accumulation concentration in tissues other than lung tissue in mice after exposure to M suspension and water-soluble M solution (Tissue (PM) 2.5 -M) j And Tissue (M solution) j and blood M accumulation concentration (PM) 2.5 -M) and Blood (M solution); PM was calculated 2.5 -M particles are a relative bioavailability indicator (RBA) for accumulation in biological tissues. T As shown in equations (2)-(4):

[0022]

[0023]

[0024] In the formula: RBA T For PM 2.5 -M Overall relative bioavailability index, %;

[0025] RBA Tissue Mice inhaled PM 2.5 -M, relative bioavailability index of tissue j, %;

[0026] RBA Blood Mice inhaled PM 2.5 -M, a blood relative bioavailability index, %;

[0027] Tissue (PM) 2.5 -M) j and Blood (PM)2.5 -M) represents the amount of PM inhaled by mice. 2.5 -M accumulation concentrations in tissues and blood after exposure to M, mg / kg and mg / mL;

[0028] Tissue (M solution) j Blood (M solution) represents the concentrations of M accumulated in tissues and blood of mice after exposure to M solution via inhalation, in mg / kg and mg / mL, respectively.

[0029] S2. Constructing water-insoluble PM 2.5 -M particle alveolar microreaction system model

[0030] ①PM 2.5 -M incubation and extraction of adsorbed substances from M

[0031] (c) Extract rat lung surfactant, freeze-dry it into powder, and redissolve it to prepare lung surfactant incubation solution;

[0032] (b) PM 2.5 PM made by adding physiological saline to -M exposure material 2.5 -M suspension was added to the pulmonary surfactant incubation solution, and after isothermal shaking incubation at 36-38°C, the mixture was centrifuged and washed to collect PM containing adsorbed lipids and proteins. 2.5 -M precipitate particles, add organic phase chloroform-methanol solution to precipitate particles and centrifuge for the first extraction, collect the organic phase containing lipids and proteins; then add aqueous phase SDS solution to precipitate particles, heat at 90-95℃ and centrifuge again to collect the aqueous phase containing proteins;

[0033] (c) The organic phase and aqueous phase were mixed and centrifuged at 0-4℃ for a second extraction to obtain an organic phase containing lipids and an aqueous phase containing proteins; the organic phase was dried by nitrogen blowing to obtain lipids adsorbed on M; the aqueous phase was mixed with acetone and precipitated overnight at -80℃ and then centrifuged to obtain proteins adsorbed on M.

[0034] ② Screening lipids and proteins

[0035] Based on whether the protein has a metal-binding site, the screened proteins are divided into metal-related proteins and ordinary proteins. Differentiation is used as the screening index for proteins, and K types (K=1,2…n) of metal-related proteins with differential FC ≥15 and L types (L=1,2…n) of ordinary proteins with differential FC ≥120 are selected. Content is used as the screening index for lipids, and N types (N=1,2…n) of lipids with PP content ≥60% are selected.

[0036] ③ Assemble the microreaction system

[0037] The proteins and lipids selected in step ② were used as the protein and lipid components of the microreaction system, and Gamble's solution was used as the simulated lung fluid, along with the PM obtained in step S1. 2.5 -M exposed materials were mixed and incubated at a constant temperature of 36–38°C with shaking to assemble a microreaction system.

[0038] Preferably, the non-water-soluble M particles in step S1①(a) are lead, cadmium, nickel, gold, silver, silicon, microplastics, or aerosols.

[0039] Preferably, in step S2①(b), the volume ratio of chloroform to methanol in the organic phase chloroform-methanol solution is (1-3):1; in step S2①(c), the volume ratio of the organic phase to the aqueous phase is (3-5):1, and the volume ratio of the aqueous phase to acetone is 1:(2-3).

[0040] Preferably, the oscillation incubation time in step S2③ is 0.5 to 1 hour.

[0041] A PM based on the above 2.5 An assessment model for the relative bioavailability of alveolar microresponse systems carrying non-water-soluble particles was constructed, comprising the following steps:

[0042] ① Model building

[0043] (a) After incubating the microreaction system assembled according to claim 1 at a constant temperature of 36–38°C with shaking, centrifuge to obtain supernatant and M particles, and detect the total amount of M ions in the supernatant. MRS and M particle morphology conversion rate T MRS Calculate PM in micro-reaction systems 2.5 -M Conversion Rate TP MRS As shown in equations (5)-(6):

[0044]

[0045] In the formula: TP MRS PM for micro-reaction systems 2.5 -M conversion rate, %;

[0046] M origin-M The total amount of M particles in the microreaction system before incubation, in mg;

[0047] M MRS The total amount of M in the supernatant after incubation of the microreaction system, in mg;

[0048] T MRS The morphological conversion rate of M particles after incubation in the microreaction system is expressed as %, %.

[0049] M MRS-new The total amount of the new form of M particles after incubation in the microreaction system, in mg;

[0050] (d) Atmospheric PM2.5 collected 2.5 -M sample was ball-milled to below 2.5 μm as a standard for compound M to obtain PM. 2.5 -M Exposure, PM 2.5 -M exposed material was added to a lung surfactant incubation solution and incubated with constant temperature shaking at 36-38℃. After centrifugation, the supernatant and M particles were obtained, and the total M ion content in the supernatant was detected. PS and M particle morphology conversion rate T PS Calculate PM of pulmonary surfactant 2.5 -M conversion rate TR PS As shown in equations (7)-(8):

[0051]

[0052] In the formula: TR PS PM as a pulmonary surfactant 2.5 -M conversion rate, %;

[0053] M origin-P The total amount of M particles before incubation with the lung surfactant incubation solution, in mg;

[0054] M PS The total amount of M in the supernatant after incubation with the lung surfactant incubation solution, in mg;

[0055] T PS The morphological conversion rate (%) of M particles after incubation with the lung surfactant incubation solution;

[0056] M PS-new The total amount of the new morphology of M particles after incubation with the lung surfactant incubation solution, in mg;

[0057] (c) Based on the PM of the microreaction system 2.5 -M Conversion Rate TP MRS PM and lung surfactant 2.5 -M conversion rate TR PS Calculation standard PM 2.5 -M Relative Conversion Rate RTR S As shown in equation (9):

[0058]

[0059] Where: RTR S Standard PM 2.5 -M relative conversion rate, %;

[0060] TR PS PM as a pulmonary surfactant 2.5 -M conversion rate, %;

[0061] TP MRS PM for micro-reaction systems 2.5 -M conversion rate, %;

[0062] (d) Based on standard PM 2.5 -M Relative Conversion Rate RTR S Calculate PM 2.5 The relative bioavailability (RBA) of -M is shown in equations (10)-(12):

[0063]

[0064] In the formula: RBA represents the PM to be measured. 2.5 -M relative bioavailability, %;

[0065] TR represents the PM to be tested. 2.5 -M conversion rate in the microreaction system, %;

[0066] PM to be tested 2.5 -M Total inhaled exposure, mg / kg;

[0067] RBA T PM to be tested 2.5 -M Overall relative bioavailability index, %;

[0068] RTR S Standard PM 2.5 -M relative conversion rate, %;

[0069] ω is the conversion factor = 0.02;

[0070] M supernatant PM to be tested 2.5 The total amount of M ions in the supernatant after incubation of the microreaction system composed of M components, in mg;

[0071] T Particle PM to be tested 2.5 - The morphological conversion rate of M particles after incubation in the M-component microreaction system, %;

[0072] M origin PM to be tested 2.5 -Total M content of M particles in the microreaction system before incubation, mg;

[0073] ADD M PM to be tested 2.5 -M Daily Average Inhalation Exposure, mg / kg / d;

[0074] AT M PM to be tested 2.5 -M Exposure time, d;

[0075] ② Model Evaluation

[0076] Collect atmospheric PM2.5 samples 2.5 -M sample, total amount of M origin PM to be tested 2.5 -M is a microreaction system according to claim 1, and is continuously incubated for 24-48 hours. The supernatant and M particles are obtained by centrifugation, and the total M ion content in the supernatant is detected. supernatant and M particle morphology conversion rate T Particle The PM2.5 concentration in the atmosphere to be measured is calculated according to equations (10)-(12). 2.5 -M sample relative bioavailability (RBA); when RBA < 20%, PM is considered... 2.5 -M does not have relative bioavailability; when 20% ≤ RBA, PM is judged. 2.5 -Pb has relative bioavailability.

[0077] This invention is based on existing research theories that the water solubility of chemical pollutants is positively correlated with their bioavailability. However, the applicant's previous research found that the body is exposed to non-water-soluble PM2.5. 2.5 Long-term respiratory exposure to Pb showed high tissue bioavailability (62.1%), resulting in the accumulation of large amounts of Pb particles in the alveoli. This finding expands and deepens our understanding of the role of non-water-soluble PM2.5 in respiratory tract infections. 2.5 Our understanding of the bioaccumulation and absorption of -Pb through respiratory exposure has clarified its potential reaction processes, chemical composition, and mechanisms.

[0078] Compared with the prior art, the present invention has the following beneficial effects:

[0079] 1. This invention utilizes pulmonary surfactant extraction and PM... 2.5 The process of incubating and extracting the adsorption layer, screening adsorbed lipids and proteomes, and assembling microreaction system models led to the first construction of a PM2.5 adsorption molecule. 2.5 Intraalveolar microreaction system carrying non-water-soluble particles. Non-water-soluble PM 2.5 -M particles, after entering the alveoli through respiration, adsorb pulmonary surfactant to form an encapsulation layer, and interact with lipid proteins within this layer to form a microreaction system. This is based on the observation of non-water-soluble PM2.5 in real-world environments. 2.5 -M pollution concentration and composition, using animal models, revealed the concentration and composition of non-water-soluble PM2.5. 2.5 The accumulation and absorption characteristics of -M particles in the alveoli; lipidomics and proteomics analyses clarified the characteristics of insoluble PM in the alveoli. 2.5 A microreaction system for the degradation and transformation of -M particles and alveolar fluid was constructed for the first time based on the microreaction system model, demonstrating the first successful construction of a non-water-soluble PM2.5 degradation and transformation system. 2.5A model for calculating alveolar conversion efficiency after exposure to M-particles was developed, and based on this model, the alveolar conversion efficiency of insoluble PM2.5 in the alveoli was constructed for the first time. 2.5 -M bioavailability assessment model.

[0080] 2. This invention, based on an animal model method, reveals the presence of non-water-soluble PM. 2.5 -M bioaccumulation and relative bioavailability characteristics after respiratory exposure; this method can also be applied to atmospheric PM2.5. 2.5 The model also applies to the microreaction processes formed by other non-water-soluble substances carried by the carrier in other scenarios; it can also be applied to other proteins and PM. 2.5 -M compound; a water-insoluble PM compound was constructed for the first time based on a microreaction system. 2.5 -M model for calculating alveolar conversion efficiency after respiratory exposure; this model is also applicable to atmospheric PM2.5. 2.5 Calculation of the conversion rate of other non-water-soluble substances carried on the carrier. Attached Figure Description

[0081] Figure 1 For the present invention PM 2.5 Flowchart of the alveolar microreaction system carrying non-water-soluble particulate matter.

[0082] Figure 2 This is a graph showing the lipidomics content in the non-water-soluble particle adsorption layer.

[0083] Figure 3 This is a graph showing the differences in common proteins in the adsorption layer of insoluble particles.

[0084] Figure 4 This is a graph showing the differences in metal-binding proteins in the adsorption layer of insoluble particles. Detailed Implementation

[0085] The present invention will be further described below with reference to specific embodiments, but these should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0086] Example 1

[0087] A type of PM 2.5 Intraalveolar microreaction systems carrying non-water-soluble particles, such as Figure 1 As shown, its construction includes the following specific steps:

[0088] 1. Establish non-water-soluble PM 2.5 -Pb particle bioaccumulation and relative bioavailability model

[0089] ① Constructing animal exposure models

[0090] (a) Atmospheric PM2.5 samples were collected using a medium-flow intelligent TSP sampler. 2.5 -M sample was ball-milled to below 2.5 μm as a standard for compound M to obtain PM. 2.5 -M exposure, weigh the collected filter membrane, and detect PM on the filter membrane using an X-ray diffractometer. 2.5 -M form and content, detecting atmospheric PM2.5 2.5 -M mainly consists of non-water-soluble lead particulate matter (PM2.5). 2.5 The composition and proportions of Pb3O4 (45.88%), PbS (35.69%), PbSO4 (12.59%), and PbO2 (5.84%) were mixed with physiological saline (0.9%) to prepare PM2.5. 2.5 -Pb suspension. Lead acetate was selected as the water-soluble control lead, and a suspension was prepared with PM2.5. 2.5 -Pb suspension is equivalent to a water-soluble Pb(Ac)2 solution of the same concentration.

[0091] (b) Mice were exposed to PM via inhalation. 2.5 - Inhalation exposure to Pb suspension and lead acetate solution, as shown in formula (1):

[0092]

[0093] In the formula: The average daily PM2.5 inhalation rate of mice 2.5 -M exposure, mg / kg / d;

[0094] ADD M溶液 The average daily exposure to M solution in mice is expressed in mg / kg / d.

[0095] PM2.5 2.5 -Total concentration of M in sample M, μg / m 3 ;

[0096] InhR mouse The respiratory rate of mice, m 3 / d;

[0097] EF mouse Mice inhaled PM 2.5 -M exposure frequency, d / w;

[0098] ED mouse Mice inhaled PM 2.5 -M exposure period, w;

[0099] BW mouse The average weight of the mice is in kg;

[0100] AT is a method for mice to inhale PM. 2.5 -M's average exposure time, d;

[0101] (c) Monitor Pb ion concentration in mouse urine during exposure. Pb ion concentration reached equilibrium at week 8 post-exposure, at which point exposure was terminated. PM2.5 concentration was then measured using an inductively coupled plasma analyzer. 2.5 Lead accumulation concentration in tissues (excluding lung tissue) of mice after exposure to Pb and Pb(Ac)2 (PM2.5) 2.5 -Pb) j and Tissue(Pb(Ac)2) j and blood lead concentration (PM) 2.5 -Pb) and Blood((Pb(Ac)2), the results are shown in Table 1:

[0102] Table 1 Lead accumulation concentrations in mouse tissues and blood after exposure.

[0103]

[0104] PM was calculated 2.5 -Pb is a relative bioavailability indicator (RBA) for accumulation in biological tissues. T As shown in equations (2)-(4):

[0105]

[0106] In the formula: RBA T For PM 2.5 -Pb overall relative bioavailability index, %;

[0107] RBA Tissue Mice inhaled PM 2.5 -Pb, a relative bioavailability index of tissue j, %;

[0108] RBA Blood Mice inhaled PM 2.5 -Pb, a relative bioavailability index in blood, %;

[0109] Tissue (PM2.5-Pb) j Blood (PM2.5-Pb) was used to treat inhaled PM2.5 in mice. 2.5 -Pb accumulation concentrations in tissues and blood after Pb exposure, mg / kg and mg / mL;

[0110] Tissue(Pb(Ac)2) jBlood(Pb(Ac)2) represents the concentrations of Pb accumulated in tissues and blood of mice after exposure to Pb(Ac)2 solution, in mg / kg and mg / mL, respectively.

[0111] The calculation result is RBA Tissue =47.2%, RBA Blood =76.9%, EBA T =62.1%.

[0112] 2. Constructing water-insoluble PM 2.5 -Pb alveolar microreaction system model

[0113] Non-water-soluble PM 2.5 - After Pb enters the alveoli through inhalation, it adsorbs lung surfactants to form a coating layer and interacts with lipid proteins in the coating layer to form a microreaction system. The specific steps are as follows:

[0114] ①PM 2.5 -Pb incubation and extraction of Pb adsorbates

[0115] (a) Eight-week-old SD rats were selected and anesthetized by intraperitoneal injection of sodium pentobarbital. Lung lavage fluid was collected by lavage with physiological saline and then freeze-dried to prepare lung surfactant powder (lung surfactant is a mixture of lipids and proteins secreted by epithelial cells in the alveolar cavity). The lipid and protein contents of the lung surfactant powder were detected using a lipid assay and a BCA protein kit, and the protein and lipid contents were found to be 14.49 mg / g and 174.99 mg / g, respectively. The lung surfactant powder was then reconstituted with pure water to prepare a lung surfactant incubation solution (lipid content 1 mg / mL).

[0116] (b) PM 2.5 -Pb suspension was added to lung surfactant incubation solution at a solid-liquid ratio of 1 g: 5000 mL. After incubation at 37°C and 120 rpm for 24 h with constant temperature shaking, an equal volume of 30% sucrose solution was added, and the mixture was centrifuged at 4°C and 8000 g for 15 min. The precipitate particles were washed three times with pure water. 1 mL of chloroform-methanol solution (organic phase) with a volume ratio of 2:1 was added to the precipitate particles, vortexed for 10 min, and centrifuged at 4°C and 8000 g for 10 min. This process was repeated three times, and the organic phase containing lipids and proteins was collected. Subsequently, 1 mL of 4% SDS solution (aqueous phase) was added to the precipitate particles, heated at 95°C for 10 min, and then vortexed for 5 min. This process was repeated twice under the same conditions, and the aqueous phase containing proteins was collected.

[0117] (c) Mix the organic phase and the aqueous phase at a volume ratio of 5:1, vortex for 30 min, and then centrifuge at 4 °C and 2400 rpm for 10 min to separate the lipid-containing organic phase and the protein-containing aqueous phase. Blow the organic phase dry with nitrogen to obtain the lipids adsorbed on Pb. Mix the aqueous phase at a volume ratio of 1:3 with acetone and precipitate overnight at -80 °C. Then centrifuge at 4 °C and 20000 g for 15 min to obtain the protein adsorbed on Pb.

[0118] ② Screening lipids and proteins

[0119] (a) Lipids and proteins adsorbed on Pb were detected using high-performance liquid chromatography-tandem triple quadrupole mass spectrometry (UPLC-MS / MS). Based on whether the proteins possessed metal-binding sites, the screened proteins were classified into metal-associated proteins and ordinary proteins. The results are as follows: Figure 2-4 As shown. Figure 2 This is a lipidomics diagram showing the lipid content in the non-water-soluble particle adsorption layer. From... Figure 2 It can be seen that lipids with a PP content ≥ 60% are PC, and the representative lipid in PC is DPPC; Figure 3 This is a graph showing the differential pharmacological composition of common proteins in the adsorption layer of insoluble particles. From... Figure 3 It can be seen that the proteins with differential FC ≥ 120 are Clathrin heavy chain 1, Ras-related protein Rab-5C, Histone H2B type 2-B, and Actin-related protein 2; Figure 4 This is a graph showing the chemical differences in metal-binding proteins within the adsorption layer of insoluble particles. From... Figure 4 It can be seen that proteins with differential FC ≥ 15 are Catalase, Ras GTPase-activating-like protein, Fibrinogen gamma chain, Annexin A7, Pigment epithelium-derived factor, and Serine / threonine-protein phosphatase 5. From... Figure 2-4 The results show the types, degree of difference, and content of lipids and proteins adsorbed on Pb compared to those in the pulmonary surfactant incubation solution, indicating that PM 2.5 -Pb can selectively adsorb lipids and proteins in pulmonary surfactants.

[0120] (b) Using difference as the screening index for proteins, Catalase, Fibrinogen gammachain, Serine / threonine-protein phosphatase-5, and Ras-related protein Rab-5C were selected; using content as the screening index for lipids, DPPC lipids were selected.

[0121] ③ Assemble the microreaction system

[0122] (a) The protein selected in step ② was used as the protein component of the microreaction system, DPPC as the lipid component, and Gamble's solution as the simulated lung fluid. The protein component (each protein concentration was determined by 100-fold UPLC-MS / MS), lipid component (lipid concentration was 0.1 mg / mL), simulated lung fluid, and PM2.5 were compared. 2.5 -Pb suspension (total amount of M) origin The four components were mixed at a solid-liquid ratio of 1g:5000mL and incubated at 37℃ and 120rpm for 1h under constant temperature shaking to assemble a microreaction system. The composition of this system is shown in Tables 2 and 3.

[0123] Table 2PM 2.5 -Pb microreaction system composition

[0124]

[0125] Table 3 Gamble's solution formulation table

[0126]

[0127]

[0128] Example 2: Constructing PM 2.5 -Pb alveolar microresponse system relative bioefficacy assessment model

[0129] ① Model building

[0130] (a)PM 2.5 Carrier PM 2.5 The relative bioavailability assessment model of the -Pb alveolar microresponse system is based on the assembly of the microresponse system, using the PM composed of Example 1. 2.5 The Pb alveolar microreaction system was incubated at 37±1℃ with constant temperature shaking for 24 h, and then centrifuged to obtain the supernatant and Pb particles. The total Pb ion content (M) in the supernatant was measured. MRS Pb particle morphology conversion rate T MRS Calculate PM in micro-reaction systems 2.5 -Pb conversion rate TP MRSAs shown in equations (5)-(6):

[0131]

[0132] In the formula: TP MRS PM for micro-reaction systems 2.5 -Pb conversion rate, %;

[0133] M origin-Pb The total amount of Pb in the Pb particles before incubation of the microreaction system, in mg;

[0134] M MRS The total Pb content in the supernatant after incubation of the microreaction system, in mg;

[0135] T MRS The morphological conversion rate of Pb particles after incubation in the microreaction system is expressed as %, in percentage.

[0136] M MRS-new The total amount of the new Pb particle morphology after incubation in the microreaction system, in mg;

[0137] (b) Collect atmospheric PM 2.5 -Pb samples were ball-milled to below 2.5 μm to obtain PM. 2.5 -Pb exposure will PM 2.5 Pb exposure was added to a pulmonary surfactant incubation solution and incubated at 37±1℃ with constant temperature shaking. After centrifugation, the supernatant and Pb particles were obtained, and the total Pb ion content (M) in the supernatant was determined. PS Pb particle morphology conversion rate T PS Calculate PM of pulmonary surfactant 2.5 -Pb conversion rate TR PS As shown in equations (7)-(8):

[0138]

[0139] In the formula: TR PS PM as a pulmonary surfactant 2.5 -Pb conversion rate, %;

[0140] M origin-P The total amount of Pb in Pb particles before incubation with the lung surfactant incubation solution, in mg;

[0141] M PS The total Pb content (mg) in the supernatant after incubation with the lung surfactant incubation solution;

[0142] T PS The morphological conversion rate of Pb particles after incubation with lung surfactant incubation solution, in %;

[0143] M PS-newThe total amount of new Pb particle morphology after incubation with lung surfactant incubation solution, in mg;

[0144] (c) Based on the PM of the microreaction system 2.5 -Pb conversion rate TP MRS PM and lung surfactant 2.5 -Pb conversion rate TR PS Calculation standard PM 2.5 -Pb relative conversion rate RTR S As shown in equation (9):

[0145]

[0146] Where: RTR S Standard PM 2.5 -Pb relative conversion rate, %;

[0147] TR PS PM as a pulmonary surfactant 2.5 -Pb conversion rate, %;

[0148] TP MRS PM for micro-reaction systems 2.5 -Pb conversion rate, %;

[0149] (d) Based on standard PM 2.5 -Pb relative conversion rate RTR S Calculate PM 2.5 The relative bioavailability (RBA) of -Pb is shown in equations (10)-(12):

[0150]

[0151] In the formula: RBA represents the PM to be measured. 2.5 -Pb relative bioavailability, %;

[0152] TP represents the PM to be tested. 2.5 -Pb conversion rate in the microreaction system, %;

[0153] PM to be tested 2.5 -Total inhaled exposure to Pb, mg / kg;

[0154] RBA T PM to be tested 2.5 -Pb overall relative bioavailability index, %;

[0155] RTR S Standard PM 2.5 -Pb relative conversion rate, %;

[0156] v is the conversion factor = 0.02;

[0157] M supernatant PM to be tested 2.5 The total amount of Pb ions in the supernatant after incubation of the micro-reaction system composed of Pb, in mg;

[0158] T Particle PM to be tested 2.5 - The morphological conversion rate of Pb particles after incubation in the Pb-based microreaction system, %;

[0159] M origin PM to be tested 2.5 -Total Pb content of Pb particles in the Pb-composed microreaction system before incubation, mg;

[0160] ADD Pb PM to be tested 2.5 - Daily average inhaled exposure to Pb, mg / kg / d;

[0161] AT Pb PM to be tested 2.5 -Pb exposure time, d;

[0162] ② Model Evaluation

[0163] (a) A medium-flow intelligent TSP sampler was used to collect PM2.5 samples from an atmospheric location. 2.5 -Pb sample and its concentration were detected. The total amount is m origin PM 2.5 -Pb was used to form a microreaction system according to steps 2-③ and incubated for 24 hours. After centrifugation, the supernatant and Pb particles were obtained, and the total Pb ion content M in the supernatant was detected. supernatant Pb particle morphology conversion rate T Particle PM is calculated according to equations (10)-(12). 2.5 The relative bioavailability (RBA) of Pb was 42.6% (≥20%), indicating that the PM2.5 concentration at this location was... 2.5 -Pb has relative bioavailability. Example 3: Non-water-soluble PM 2.5 The reaction process and mechanism of -Pb particles in the alveolar microreaction system

[0164] 1. Add four proteins—Catalase, Fibrinogen gamma chain, threonine-protein phosphatase-5, and Ras-related protein Rab-5C—to Gamble's solution (containing 0.1 mg / mL DDPC) to prepare protein incubation solutions.

[0165] 2. Using a solid-liquid ratio of 1g:5000mL, the total amount of M...origin PM 2.5 -Pb was added to each protein incubation solution and incubated at 4°C and 120 rpm for 24 h with constant temperature shaking.

[0166] 3. Add the same volume of 30% sucrose solution to the incubation solution, and centrifuge at 4℃ and 8000g for 15 min to separate the supernatant and precipitate particles;

[0167] 4. The total lead ion content (M) in the supernatant was determined using inductively coupled plasma mass spectrometry (ICP-MS). supernatant X-ray diffraction was used to detect PM in particulate precipitates. 2.5 -Pb new form, calculating the morphological transformation rate T particle ;

[0168] 5. Calculate PM according to formula (10) 2.5 The conversion rates of -Pb in various protein incubation solutions are shown in Table 4.

[0169] Table 4 PM of each protein in the microreaction system 2.5 -Pb conversion rate

[0170] Protein names <![CDATA[PM 2.5 -Pb conversion rate]]> Catalase 2.34％ Fibrinogengammachain 1.03％ Threonine-proteinphosphatase-5 0.23％ Ras-related protein Rab-5C 0.10％

[0171] Select PM 2.5 Quantum chemical calculations were performed on Catalase, the protein with the highest Pb conversion rate (2.34%). Catalase is a metalloprotein that naturally carries Fe. 2+ Ions. Isopropanol dehydrogenase (NADP+) is an important coenzyme for maintaining cellular redox homeostasis and plays a crucial role in electron transport within the function of catalase. With NADP+ assistance, the PM of the catalase protein... 2.5 -Pb conversion was significantly improved (from 2.34% to 6.60%), indicating that Catalase may be able to convert its Fe²⁺ to Fe³⁺. 2+ The active site is replaced by Pb 2+ This approach, in turn, promoted PM 2.5 -Pb conversion.

[0172] PM 2.5 - Pb contains four types of Pb compounds (Pb3O4, PbS, PbSO4, and PbO2). Each of the four Pb compounds was added to Catalase protein incubation solution and incubated. Steps 2-5 were repeated, and the conversion rates of the four Pb compounds in the Catalase protein incubation solution were calculated. The results are shown in Table 5.

[0173] Table 5. Conversion rates of four Pb compounds in Catalase protein

[0174] Pb compound types and names Conversion rate <![CDATA[Pb3O4]]> 0.14％ PbS 0.14％ <![CDATA[PbSO4]]> 51.43％ <![CDATA[PbO2]]> 0.01％

[0175] Based on this, select PM 2.5 - Among Pb, PbSO4 showed the best conversion efficiency (51.43%) as PM2.5. 2.5 -Pb represents the compound, which established the interaction between Catalase protein and PM with NADP+ assistance. 2.5 The reaction model for -Pb was established, and quantum chemical calculations were performed. The model is as follows:

[0176]

[0177] In the formula: The active center is Fe 2+ Catalase protein;

[0178] The active site is Pb 2+ Catalase protein;

[0179] NADP + It is isopropanol dehydrogenase;

[0180] H + It is a hydrogen ion;

[0181] NADPH is a reducing coenzyme II.

[0182] Quantum chemical calculations yielded a Gibbs free energy of ΔG = -317.76 Kcal / mol for this reaction model, indicating that the reaction can occur spontaneously. This demonstrates that Catalase can displace Fe... 2+ The active site is replaced by Pb 2+ Promoted PM 2.5 -Pb conversion.

[0183] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A type of PM 2.5 An alveolar microreaction system carrying non-water-soluble particles, characterized in that, Its construction includes the following specific steps: S1. Establish non-water-soluble PM 2.5 -M particle bioaccumulation and relative bioavailability model ① Constructing animal exposure models (a) Collect atmospheric PM2.

5. 2.5 -M sample was ball-milled to below 2.5 μm as a standard for compound M to obtain PM. 2.5 -M exposure material, where M is a non-water-soluble particle, is added to physiological saline to prepare non-water-soluble PM. 2.5 -M suspension; (b) Mice were exposed to non-water-soluble PM2.5 via inhalation. 2.5 - Inhalation exposure to M suspension and water-soluble M solution, as shown in formula (1): In the formula: ADD PM2.5-M The average daily PM2.5 inhalation of mice 2.5 -M exposure, mg / kg / d; ADD M溶液 The average daily exposure to M solution in mice is expressed in mg / kg / d. C PM2.5-M PM2.5 2.5 -Total concentration of M in sample M, μg / m 3 ; InhR mouse The respiratory rate of mice, m 3 / d; EF mouse Mice inhaled PM 2.5 -M exposure frequency, d / w; ED mouse Mice inhaled PM 2.5 -M exposure period, w; BW mouse The average weight of the mice is in kg; AT is a method for mice to inhale PM. 2.5 -M's average exposure time, d; (c) Monitor the concentration of M ions in mouse urine during exposure. End exposure when the M ion concentration reaches equilibrium, and detect insoluble PM2.

5. 2.5 -M accumulation concentration in tissues other than lung tissue in mice after exposure to M suspension and water-soluble M solution (Tissue (PM) 2.5 -M) j And Tissue (M solution) j and blood M accumulation concentration (PM) 2.5 -M) and Blood (M solution); PM was calculated 2.5 -M particles are a relative bioavailability indicator (RBA) for accumulation in biological tissues. T As shown in equations (2)-(4): In the formula: RBA T For PM 2.5 -M Overall relative bioavailability index, %; RBA Tissue Mice inhaled PM 2.5 -M, relative bioavailability index of tissue j, %; RBA Blood Mice inhaled PM 2.5 -M, a blood relative bioavailability index, %; Tissue (PM) 2.5 -M) j and Blood (PM) 2.5 -M) represents the amount of PM inhaled by mice. 2.5 -M accumulation concentrations in tissues and blood after exposure to M, mg / kg and mg / mL; Tissue (M solution) j Blood (M solution) represents the concentrations of M accumulated in tissues and blood of mice after exposure to M solution via inhalation, in mg / kg and mg / mL, respectively. S2. Constructing an intraalveolar microreaction system model for non-water-soluble PM2.5-M particles. ①Incubation of PM2.5-M and extraction of adsorbed substances from M (a) Extract rat lung surfactant, freeze-dry it into powder, and redissolve it to prepare lung surfactant incubation solution; (b) A PM2.5-M suspension prepared by adding physiological saline to PM2.5-M exposure material was added to a lung surfactant incubation solution. After constant temperature shaking incubation at 36–38 °C, the mixture was centrifuged and washed to collect PM2.5-M precipitates containing adsorbed lipids and proteins. An organic phase chloroform-methanol solution was added to the precipitates and centrifuged for the first extraction to collect the organic phase containing lipids and proteins. Then, an aqueous phase SDS solution was added to the precipitates, and the mixture was heated at 90–95 °C and centrifuged again to collect the aqueous phase containing proteins. (c) The organic phase and aqueous phase were mixed and centrifuged at 0-4℃ for a second extraction to obtain an organic phase containing lipids and an aqueous phase containing proteins; the organic phase was dried by nitrogen blowing to obtain lipids adsorbed on M; the aqueous phase was mixed with acetone and precipitated overnight at -80℃ and then centrifuged to obtain proteins adsorbed on M. ② Screening lipids and proteins Based on whether the protein has a metal-binding site, the screened proteins are divided into metal-related proteins and ordinary proteins. Differentiality is used as the screening index for proteins, and K types (K = 1, 2…n) of metal-related proteins with differential FC ≥ 15 and L types (L = i, 2…n) of ordinary proteins with differential FC > 120 are selected. Content is used as the screening index for lipids, and N types (N = 1, 2…n) of lipids with PP content > 60% are selected. ③ Assemble the microreaction system The proteins and lipids selected in step ② were used as the protein and lipid components of the microreaction system, and Gamble's solution was used as the simulated lung fluid, along with the PM obtained in step S1. 2.5 -M exposed materials were mixed and incubated at a constant temperature of 36–38°C with shaking to assemble a microreaction system.

2. The PM according to claim 1 2.5 An alveolar microreaction system carrying non-water-soluble particles, characterized in that, The non-water-soluble M particles mentioned in step S1①(a) are lead, cadmium, nickel, gold, silver, silicon, microplastics or aerosols.

3. The PM according to claim 1 2.5 An alveolar microreaction system carrying non-water-soluble particles, characterized in that, In step S2①(b), the volume ratio of chloroform to methanol in the organic phase chloroform-methanol solution is (1-3):1; in step S2①(c), the volume ratio of the organic phase to the aqueous phase is (3-5):1, and the volume ratio of the aqueous phase to acetone is 1:(2-3).

4. The PM according to claim 1 2.5 An alveolar microreaction system carrying non-water-soluble particles, characterized in that, The oscillation incubation time described in step S2③ is 0.5 to 1 hour.

5. A PM based on any one of claims 1-4 25 A model for assessing the relative bioavailability of alveolar microresponse systems carrying non-water-soluble particles, characterized in that... The construction of the evaluation model includes the following steps: ① Model building (a) After incubating the microreaction system assembled according to claim 1 at a constant temperature of 36–38°C with shaking, centrifuge to obtain supernatant and M particles, and detect the total amount of M ions in the supernatant. MRS and M particle morphology conversion rate T MRS Calculate PM in micro-reaction systems 2.5 -M Conversion Rate TP MRS As shown in equations (5)-(6): In the formula: TP MRS PM for micro-reaction systems 2.5 -M conversion rate, %; M origin-M The total amount of M particles in the microreaction system before incubation, in mg; M MRS The total amount of M in the supernatant after incubation of the microreaction system, in mg; T MRS The morphological conversion rate of M particles after incubation in the microreaction system is expressed as %, %. M MRS-new The total amount of the new form of M particles after incubation in the microreaction system, in mg; (b) Collected atmospheric PM2.5 2.5 -M sample was ball-milled to below 2.5 μm as a standard for compound M to obtain PM. 2.5 -M Exposure, PM 2.5 -M exposed material was added to a lung surfactant incubation solution and incubated with constant temperature shaking at 36-38℃. After centrifugation, the supernatant and M particles were obtained, and the total M ion content in the supernatant was detected. PS and M particle morphology conversion rate T PS Calculate PM of pulmonary surfactant 2.5 -M conversion rate TR PS As shown in equations (7)-(8): In the formula: TR PS PM as a pulmonary surfactant 2.5 -M conversion rate, %; M origin-P The total amount of M particles before incubation with the lung surfactant incubation solution, in mg; M PS The total amount of M in the supernatant after incubation with the lung surfactant incubation solution, in mg; T PS The morphological conversion rate (%) of M particles after incubation with the lung surfactant incubation solution; M PS-new The total amount of the new morphology of M particles after incubation with the lung surfactant incubation solution, in mg; (c) Based on the PM of the microreaction system 2.5 -M Conversion Rate TP MRS PM and lung surfactant 2.5 -M conversion rate TR PS Calculation standard PM 2.5 -M Relative Conversion Rate RTR S As shown in equation (9): Where: RTR S For standard PM 2.5 -M relative conversion rate, %; TR PS PM as a pulmonary surfactant 2.5 -M conversion rate, %; TP MRS PM for micro-reaction systems 2.5 -M conversion rate, %; (d) Based on standard PM 2.5 -M Relative Conversion Rate RTR S Calculate PM 2.5 The relative bioavailability (RBA) of -M is shown in equations (10)-(12): TDD PM2.5-M =ADD M ×AT M (12) In the formula: RBA represents the PM to be measured. 2.5 -M relative bioavailability, %; TR represents the PM to be tested. 2.5 -M conversion rate in the microreaction system, %; TDD PM2.5-M PM to be tested 2.5 -M Total inhaled exposure, mg / kg; RBA T PM to be tested 2.5 -M Overall relative bioavailability index, %; RTR S For standard PM 2.5 -M relative conversion rate, %; ω is the conversion factor = 0.02; M supernatant PM to be tested 2.5 The total amount of M ions in the supernatant after incubation of the microreaction system composed of M components, in mg; T Particle PM to be tested 2.5 - The morphological conversion rate of M particles after incubation in the M-component microreaction system, %; M origin PM to be tested 2.5 -Total M content of M particles in the microreaction system before incubation, mg; ADD M PM to be tested 2.5 -M Daily Average Inhalation Exposure, mg / kg / d; AT M PM to be tested 2.5 -M Exposure time, d; ② Model Evaluation Collect atmospheric PM2.5 samples 2.5 -M sample, total amount of M origin PM to be tested 2.5 -M is a microreaction system according to claim 1, and is continuously incubated for 24-48 hours. The supernatant and M particles are obtained by centrifugation, and the total M ion content in the supernatant is detected. supernatant and M particle morphology conversion rate T Particle The PM2.5 concentration in the atmosphere to be measured is calculated according to equations (10)-(12). 2.5 -M sample relative bioavailability (RBA); when RBA < 20%, PM is considered... 2.5 -M does not have relative bioavailability; when 20% ≤ RBA, PM is judged. 2.5 -Pb has relative bioavailability.

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