Microreaction system in pulmonary alveoli with PM2.5 carrying water-insoluble particles and evaluation model of relative bioavailability of microreaction system

Abstract

The invention belongs to the technical field of environment and human health, and discloses an intra-alveolar micro-reaction system with PM2.5 carrying non-water-soluble particles and an evaluation model of relative bioavailability of the intra-alveolar micro-reaction system. The micro-reaction system in the pulmonary alveoli is constructed by the following procedures: establishing a biological accumulation and relative bioavailability model of water-insoluble PM 2.5-M, extracting a pulmonary surfactant, incubating PM 2.5-M, extracting an adsorption substance for adsorbing M, screening lipids and proteomes of an adsorption layer, assembling a micro-reaction system model and the like. The accumulation and absorption characteristics of the water-insoluble PM 2.5-M particles in the pulmonary alveoli are disclosed; through lipidomics and proteomics analysis, a degradation conversion micro-reaction system formed by the water-insoluble PM 2.5-M in the pulmonary alveoli and the pulmonary alveolar fluid is clarified, and a pulmonary alveolar conversion rate calculation model after breathing exposure of the water-insoluble PM 2.5-M and an evaluation model of the bioavailability of the water-insoluble PM 2.5-M in the pulmonary alveoli are constructed for the first time based on the micro-reaction system.

Description

Technical Field

[0001] The present invention belongs to the field of environment and human health technology, and specifically relates to a PM 2.5 An intraalveolar microreaction system loaded with water-insoluble particles and a model for evaluating their relative bioavailability. Background Art

[0002] Atmospheric fine particulate matter (PM 2.5 ) Pollution is one of the hot environmental issues in the world. According to the data of the World Health Organization, more than 90% of the world's population breathes air with PM 2.5 The concentrations in all countries exceeded their guidance limits, with exposure being particularly severe in low- and middle-income countries, where high concentrations of PM 2.5 Pollution causes more than 7 million premature deaths worldwide each year. PM 2.5 The chemical composition is complex, and heavy metals are its important toxic components. Heavy metals themselves are toxic to multiple tissues and organs, and PM 2.5 Carrying water-insoluble particles (PM 2.5 -M) has the characteristics of diverse chemical forms, rich content, wide range of transmission and high body absorption rate, which may cause greater health hazards. However, in toxicological effect studies, PM 2.5 The bioaccumulation, absorption characteristics and mechanisms of -M have not yet been clarified.

[0003] Respiratory exposure has been shown to be a major cause of PM 2.5 -M is the main absorption pathway for human body. During respiratory exposure, lung tissue is responsible for the absorption and transport of PM 2.5 -M is crucial. Existing research focuses on PM 2.5 -M The main results of lung exposure focus on the following three aspects: 1) water-soluble PM 2.5 -M pulmonary degradation and absorption characteristics and in vitro model; 2) water-soluble PM 2.5 -M transport characteristics and mechanisms through the lungs; 3) In vitro PM 2.5 -M bioavailability study analysis. At the same time, the water-insoluble PM 2.5 -M is often considered to have very low bioavailability, so no studies have been conducted on water-insoluble PM. 2.5 -M to study the characteristics and mechanism of pulmonary absorption. 2.5 -M has made some breakthroughs in the transport and absorption of respiratory exposure pathways, but the water-insoluble PM 2.5 The chemical reaction process, system and bioavailability evaluation model of -M in lung tissue remain to be explored. Summary of the invention

[0004] Based on the problems existing in the above background technology, the primary purpose of the present invention is to provide a PM2.5 The alveolar micro-reaction system carrying water-insoluble particles is based on the water-insoluble PM in the real environment. 2.5 -M pollution concentrations and components, using animal models to reveal water-insoluble PM 2.5 -M particles in the alveoli accumulation and absorption characteristics; lipidomics and proteomics analysis clarified the non-water-soluble PM in the alveoli 2.5 -The chemical composition and reaction process of the degradation and transformation microreaction system composed of M particles and alveolar fluid.

[0005] Another object of the present invention is to provide a method for constructing PM in alveoli based on the above micro-reaction system. 2.5 -M relative bioavailability assessment model.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A PM 2.5 The construction of the intra-alveolar micro-reaction system carrying water-insoluble particles includes the following specific steps:

[0008] S1. Establishment of water-insoluble PM 2.5 -M particle bioaccumulation and relative bioavailability model

[0009] ①Constructing animal exposure model

[0010] (c) Collected atmospheric PM 2.5 -M samples were used as M compound standard products and ball-milled to less than 2.5 μm to obtain PM 2.5 -M exposure, M is a water-insoluble particle, added with normal saline to make a water-insoluble PM 2.5 -M suspension;

[0011] (d) Through respiratory inhalation exposure, mice were exposed to non-water-soluble PM 2.5 -M suspension and water-soluble M solution inhalation exposure, as shown in formula (1):

[0012]

[0013] Where: is the average daily PM inhaled by mice 2.5 -M exposure, mg / kg / d;

[0014] ADD M溶液 is the average daily inhalation exposure of M solution in mice, mg / kg / d;

[0015] PM 2.5 -MTotal concentration of M in sample, μg / m 3 ;

[0016] IhR mouse is the respiratory rate of mice, m 3 / d;

[0017] EF mouse PM inhalation in mice 2.5 -Exposure frequency of M, d / w;

[0018] EF mouse PM inhalation in mice 2.5 -Exposure period of M, w;

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

[0020] AT is PM inhaled by mice 2.5 - Average exposure time of M, d;

[0021] (c) Monitor the M ion concentration in the mouse urine during the exposure period. When the M ion concentration reaches equilibrium, end the exposure and detect the water-insoluble PM 2.5 -M accumulation concentration in mouse tissues other than lung tissue 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 Blood (PM 2.5 -M) and Blood (M solution); PM 2.5 -RBA, a relative bioavailability index of M particles accumulated in biological tissues T , as shown in formulas (2)-(4):

[0022]

[0023]

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

[0025] RBA Tissue PM inhalation in mice 2.5 -M, relative bioavailability index of tissue j, %;

[0026] RBA Blood PM inhalation in mice 2.5 -M, blood relative bioavailability index, %;

[0027] Tissue(PM 2.5 -M) j and Blood(PM2.5 -M) is PM inhaled by mice 2.5 - M accumulation concentration in tissues and blood after M exposure, mg / kg and mg / mL;

[0028] Tissue(M solution) j and Blood (M solution) are the M accumulation concentrations in tissues and blood of mice after inhalation exposure to M solution, mg / kg and mg / mL;

[0029] S2. Construction of water-insoluble PM 2.5 -M particle intra-alveolar micro-reaction system model

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

[0031] (c) extracting rat lung surfactant, lyophilizing it into powder, and re-dissolving it to prepare lung surfactant incubation solution;

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

[0033] (c) mixing the organic phase and the aqueous phase and vortex centrifuging at 0-4°C for a second extraction to obtain an organic phase containing lipids and an aqueous phase containing proteins; blowing the organic phase to dryness with nitrogen to obtain lipids adsorbed on M; mixing the aqueous phase with acetone, precipitating at -80°C overnight, and then centrifuging to obtain proteins adsorbed on M;

[0034] ②Screening of lipids and proteins

[0035] According to whether the protein has metal binding sites, the screened proteins are divided into metal-related proteins and common proteins. The difference is used as the protein screening index, and K types (K=1,2…n) of metal-related proteins with a difference FC≥15 and L types (L=1,2…n) of common proteins with a difference FC≥120 are selected; the content is used as the lipid screening index, and N types (N=1,2…n) of lipids with a PP content ≥60% are selected;

[0036] ③Assembly of micro-reaction system

[0037] The proteins and lipids selected in step ② were used as the protein components and lipid components of the microreactor system, and Gamble's solution was used as the simulated lung fluid. 2.5 -M exposure mixture, incubated at 36-38°C with constant shaking, and assembled into a micro-reaction system.

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

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

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

[0041] A PM based on 2.5 The construction of the relative bioavailability evaluation model of the intra-alveolar micro-reaction system carrying water-insoluble particles includes the following steps:

[0042] ① Model construction

[0043] (a) incubating the micro-reaction system assembled according to claim 1 at 36-38° C. under constant temperature oscillation, centrifuging to obtain a supernatant and M particles, and detecting the total amount of M ions in the supernatant MRS and M particle morphology conversion rate T MRS , calculate the PM of the micro-reaction system 2.5 -M conversion rate TP MRS , as shown in formula (5)-(6):

[0044]

[0045] Where: TP MRS PM for micro-reactor system 2.5 -M conversion rate, %;

[0046] M origin-M is the total amount of M particles before incubation in the micro-reaction system, mg;

[0047] M MRS M is the total amount of supernatant after incubation of the micro-reaction system, mg;

[0048] T MRS is the morphological transformation rate of M particles after incubation in the micro-reaction system, %;

[0049] M MRS-new is the total amount of new forms of M particles after incubation in the micro-reaction system, mg;

[0050] (d) Collected atmospheric PM 2.5 -M samples were used as M compound standard products and ball-milled to less than 2.5 μm to obtain PM 2.5 -M exposure, PM 2.5 -M exposure was added to the lung surfactant incubation solution, incubated at 36-38°C with constant temperature shaking, and centrifuged to obtain the supernatant and M particles. The total amount of M ions in the supernatant was detected. PS and M particle morphology conversion rate T PS , calculate the PM of lung surfactant 2.5 -M conversion rate TR PS , as shown in equations (7)-(8):

[0051]

[0052] Where: TR PS PM2.5 is lung surfactant 2.5 -M conversion rate, %;

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

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

[0055] T PS is the morphological transformation rate of M particles after incubation with lung surfactant incubation solution, %;

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

[0057] (c) PM of micro-reactor system 2.5 -M conversion rate TP MRS and PM of lung surfactant 2.5 -M conversion rate TR PS , calculate the standard PM 2.5 -Relative conversion rate (RTR) S , as shown in formula (9):

[0058]

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

[0060] TR PS PM2.5 is lung surfactant 2.5 -M conversion rate, %;

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

[0062] (d) Based on standard PM 2.5 -Relative conversion rate (RTR) S , calculate PM 2.5 The relative bioavailability RBA of -M is shown in formulas (10)-(12):

[0063]

[0064] Where: RBA is the PM to be measured 2.5 -Relative bioavailability of M, %;

[0065] TR is the PM to be tested 2.5 -Conversion rate of M in the micro-reaction system, %;

[0066] PM to be tested 2.5 -M total inhalation 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 -M composition: total amount of M ions in the supernatant after incubation of the micro-reaction system, mg;

[0071] T Particle PM to be tested 2.5 -M composition micro-reaction system after incubation, morphological transformation rate of M particles, %;

[0072] M origin PM to be tested 2.5 -The total amount of M particles before incubation of the M-composition micro-reaction system, mg;

[0073] ADD M PM to be tested 2.5 -M average daily inhalation exposure, mg / kg / d;

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

[0075] ②Model evaluation

[0076] Collect the measured atmospheric PM 2.5 -M samples, the total amount is M origin PM to be tested 2.5 -M according to the micro-reaction system of claim 1 and incubate for 24 to 48 hours, centrifuge to obtain the supernatant and M particles, and detect the total amount of M ions in the supernatant supernatant and M particle morphology conversion rate T Particle , calculate the measured atmospheric PM according to equations (10)-(12) 2.5 -Relative bioavailability (RBA) of M samples; when RBA < 20%, PM 2.5 -M does not have relative bioavailability; when 20% ≤ RBA, PM 2.5 -Pb has relative bioavailability.

[0077] The present invention is based on the existing research theory that the water solubility of chemical pollutants is positively correlated with their bioavailability. 2.5 -After long-term respiratory exposure to Pb, the tissue bioavailability was high (62.1%), and a large number of Pb particles accumulated in the alveoli. This finding expands and deepens the study of non-water-soluble PM 2.5 -The understanding of the bioaccumulation and absorption of Pb by respiratory exposure clarifies its potential reaction processes, chemical composition and its mechanism.

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

[0079] 1. The present invention extracts PM through lung surfactant 2.5 -M incubation and extraction of adsorption layer, screening of adsorbed lipids and protein groups, assembly of micro-reaction system models, etc., for the first time constructed PM 2.5 Micro-reaction system in alveoli carrying water-insoluble particles. Water-insoluble PM 2.5 After the PM2.5 particles enter the alveoli through respiratory exposure, they will absorb the lung surfactant to form a coating layer and interact with the lipid proteins in the coating layer to form a micro-reaction system. 2.5 -M pollution concentrations and components, using animal models to reveal water-insoluble PM 2.5 -M particles in the alveoli accumulation and absorption characteristics; lipidomics and proteomics analysis clarified the non-water-soluble PM in the alveoli 2.5 The degradation and transformation micro-reaction system composed of -M particles and alveolar fluid was constructed for the first time based on the micro-reaction system model. 2.5A calculation model for the alveolar conversion rate of PM-M particles after respiratory exposure was developed, and based on this model, a water-insoluble PM in the alveoli was constructed for the first time. 2.5 -M Bioavailability Assessment Model.

[0080] 2. This invention is based on animal model method and reveals that water-insoluble PM 2.5 -M bioaccumulation and relative bioavailability characteristics after respiratory exposure. This method can also be applied to atmospheric PM 2.5 Micro-reaction processes formed by other water-insoluble substances carried in other scenarios; this model can also be applied to other proteins and PM 2.5 -M compounds; for the first time, a water-insoluble PM was constructed based on a micro-reaction system 2.5 -M alveolar conversion rate calculation model after respiratory exposure, which can also be applied to atmospheric PM 2.5 Calculation of conversion rate of other water-insoluble substances carried. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 PM 2.5 Composition and model flow chart of the intra-alveolar microreaction system carrying water-insoluble particles.

[0082] Figure 2 This is a diagram of the lipidomic content in the water-insoluble particle adsorption layer.

[0083] Figure 3 This is a graph showing the common proteological differences in the water-insoluble particle adsorption layer.

[0084] Figure 4 This is a graph showing the differences in metal binding proteins in the water-insoluble particle adsorption layer. DETAILED DESCRIPTION

[0085] The content of the present invention is further described below in conjunction with specific examples, but it should not be construed as limiting the present invention. If not specifically indicated, the technical means used in the examples 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 the art.

[0086] Example 1

[0087] A PM 2.5 Intra-alveolar micro-reaction system carrying water-insoluble particles, such as Figure 1 As shown, its construction includes the following specific steps:

[0088] 1. Establishment of water-insoluble PM 2.5 - Bioaccumulation and relative bioavailability model of Pb particles

[0089] ①Constructing animal exposure model

[0090] (a) Using medium flow intelligent TSP sampler to collect atmospheric PM 2.5 -M samples were used as M compound standard products and ball-milled to less than 2.5 μm to obtain PM 2.5 -M exposure, weigh the collected filter membrane, and detect the PM on the filter membrane by X-ray diffractometer 2.5 -M form and content, detect atmospheric PM 2.5 -M is mainly water-insoluble lead particles (PM 2.5 -Pb), whose forms and proportions are Pb3O4 (45.88%), PbS (35.69%), PbSO4 (12.59%), PbO2 (5.84%), which are mixed with saline (0.9%) to make PM 2.5 -Pb suspension. Lead acetate was selected as the water-soluble reference lead and prepared with PM 2.5 -Pb suspension and water-soluble Pb(Ac)2 solution of the same concentration.

[0091] (b) Through respiratory inhalation exposure method, mice were exposed to PM 2.5 -Pb suspension and lead acetate solution inhalation exposure, exposure as shown in formula (1):

[0092]

[0093] Where: is the average daily PM inhaled by mice 2.5 -M exposure, mg / kg / d;

[0094] ADD M溶液 is the average daily inhalation exposure of M solution in mice, mg / kg / d;

[0095] PM 2.5 -MTotal concentration of M in sample, μg / m 3 ;

[0096] IhR mouse is the respiratory rate of mice, m 3 / d;

[0097] EF mouse PM inhalation in mice 2.5 -Exposure frequency of M, d / w;

[0098] ED mouse PM inhalation in mice 2.5 -Exposure period of M, w;

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

[0100] AT is PM inhaled by mice 2.5 - Average exposure time of M, d;

[0101] (c) The Pb ion concentration in the mouse urine was monitored during the exposure period. The Pb ion concentration reached equilibrium in the eighth week after exposure, at which time the exposure was terminated. After the exposure, PM 2.5 Pb accumulation concentration in tissues of mice except lung tissue after exposure to -Pb and Pb(Ac)2 2.5 -Pb) j and Tissue(Pb(Ac)2) j , and blood lead accumulation concentration Blood (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] Calculate PM 2.5 -Relative bioavailability index RBA of Pb accumulation in biological tissues T , as shown in formulas (2)-(4):

[0105]

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

[0107] RBA Tissue PM inhalation in mice 2.5 -Pb, relative bioavailability index of tissue j, %;

[0108] RBA Blood PM inhalation in mice 2.5 -Pb, blood relative bioavailability index, %;

[0109] Tissue(PM2.5-Pb) j and Blood (PM2.5-Pb) are PM2.5-Pb inhaled by mice 2.5 - Pb accumulation concentration in tissues and blood after Pb exposure, mg / kg and mg / mL;

[0110] Tissue(Pb(Ac)2) jand Blood(Pb(Ac)2) are the Pb accumulation concentrations in tissues and blood of mice after inhalation exposure to Pb(Ac)2 solution, mg / kg and mg / mL;

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

[0112] 2. Construction of water-insoluble PM 2.5 -Pb alveolar micro-reaction system model

[0113] Water-insoluble PM 2.5 After Pb enters the alveoli through respiratory exposure, it will adsorb the lung surfactant to form a coating layer and interact with the lipid proteins in the coating layer to form a micro-reaction system. The specific steps are as follows:

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

[0115] (a) 8-week-old SD rats were selected and anesthetized by intraperitoneal injection of sodium pentobarbital. The lung lavage was performed with normal saline to collect the lung lavage fluid, and the lung lavage fluid was 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 assay and BCA protein kit were used to detect the lipid and protein contents in the lung surfactant powder, and the protein and lipid contents were 14.49 mg / g and 174.99 mg / g. The lung surfactant powder was then re-dissolved in pure water as a lung surfactant incubation solution (lipid content 1 mg / mL).

[0116] (b) PM 2.5 -Pb suspension was added to the lung surfactant incubation solution at a solid-liquid ratio of 1g:5000mL. After constant temperature shaking incubation at 37°C and 120rpm for 24h, the same volume of 30% sucrose solution was added, centrifuged at 4°C and 8000g for 15min, and the precipitated particles were washed 3 times with pure water. 1mL of chloroform-methanol solution (organic phase) with a volume ratio of 2:1 was added to the precipitated particles, vortexed for 10min, and centrifuged at 4°C and 8000g for 10min, repeated 3 times, and the organic phase containing lipids and proteins was collected. Subsequently, 1mL of 4% SDS solution (aqueous phase) was added to the precipitated particles, heated at 95°C for 10min, and then vortexed for 5min. Centrifugation was repeated 2 times under the same conditions to collect the aqueous phase containing protein;

[0117] (c) The organic phase and the aqueous phase were mixed in a volume ratio of 5:1, vortexed for 30 minutes, and then centrifuged at 4°C and 2400 rpm for 10 minutes to separate the lipid-containing organic phase and the protein-containing aqueous phase. The organic phase was blown dry with nitrogen to obtain the lipid adsorbed on Pb; the aqueous phase and acetone were mixed in a volume ratio of 1:3 and precipitated overnight at -80°C, and then centrifuged at 4°C and 20,000 g for 15 minutes to obtain the protein adsorbed on Pb.

[0118] ②Screening of lipids and proteins

[0119] (a) UPLC-MS / MS was used to detect lipids and proteins adsorbed on Pb. The screened proteins were divided into metal-related proteins and common proteins according to whether the proteins had metal binding sites. The results are shown in Figure 2. Figure 2-4 shown. Figure 2 Figure 2 is a lipidomics content map of the water-insoluble particle adsorption layer. Figure 2 It can be seen that lipids with PP content ≥ 60% are PC, among which the representative lipid in PC is DPPC; Figure 3 This is a graph showing the common protein differences in the water-insoluble particle adsorption layer. 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-relatedprotein 2; Figure 4 Figure 2 is a graph showing the differences in metal binding proteins in the adsorption layer of water-insoluble particles. Figure 4 It can be seen that the proteins with differential FC ≥ 15 are Catalase, Ras GTPase-activating-like protein, Fibrinogen gamma chain, AnnexinA7, Pigment epithelium-derived factor, Serine / threonine-proteinphosphatase 5. Figure 2-4 Compared with the lung surfactant incubation solution, the types, degree of difference and content of lipids and proteins adsorbed on Pb are shown, indicating that PM 2.5 -Pb can selectively adsorb lipids and proteins in lung surfactant.

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

[0121] ③Assembly of micro-reaction system

[0122] (a) The protein selected in step ② was used as the protein component of the microreactor system, DPPC was used as the lipid component of the microreactor system, and Gamble's solution was used as the simulated lung fluid. The protein component (the content of each protein was 100 times the detection content of each protein by UPLC-MS / MS), lipid component (lipid content was 0.1 mg / mL), simulated lung fluid, PM 2.5 -Pb suspension (total amount M origin ) The four were mixed at a solid-liquid ratio of 1 g: 5000 mL, incubated at 37°C and 120 rpm for 1 h, and assembled into a micro-reaction system. The composition of the system is shown in Tables 2 and 3:

[0123] Table 2PM 2.5 -Pb micro-reaction system composition

[0124]

[0125] Table 3 Gamble's solution formula

[0126]

[0127]

[0128] Example 2 Construction of PM 2.5 -Evaluation model of relative bioavailability of Pb intra-alveolar micro-reaction system

[0129] ① Model construction

[0130] (a)PM 2.5 Carrier PM 2.5 The relative bioavailability evaluation model of the Pb intra-alveolar micro-reaction system is based on the assembly of the micro-reaction system. 2.5 -Pb alveolar microreaction system was incubated at 37±1℃ for 24h, and then centrifuged to obtain supernatant and Pb particles. The total amount of Pb ions in the supernatant was detected. MRS and Pb particle morphology conversion rate T MRS , calculate the PM of the micro-reaction system 2.5 -Pb conversion rate TP MRS, as shown in formula (5)-(6):

[0131]

[0132] Where: TP MRS PM for micro-reactor system 2.5 -Pb conversion rate, %;

[0133] M origin-Pb is the total amount of Pb in the Pb particles before incubation in the micro-reactor system, mg;

[0134] M MRS is the total amount of Pb in the supernatant after incubation of the micro-reaction system, mg;

[0135] T MRS is the morphological transformation rate of Pb particles after incubation in the micro-reaction system, %;

[0136] M MRS-new is the total amount of new forms of Pb particles after incubation in the micro-reaction system, mg;

[0137] (b) Collect atmospheric PM 2.5 -Pb samples were ball-milled to less than 2.5 μm to obtain PM 2.5 -Pb exposure, PM 2.5 -Pb exposure was added to the lung surfactant incubation solution and incubated at 37±1℃ with constant temperature shaking. The supernatant and Pb particles were obtained by centrifugation. The total amount of Pb ions in the supernatant was detected. PS and Pb particle morphology conversion rate T PS , calculate the PM of lung surfactant 2.5 -Pb conversion rate TR PS , as shown in equations (7)-(8):

[0138]

[0139] Where: TR PS PM2.5 is lung surfactant 2.5 -Pb conversion rate, %;

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

[0141] M PS is the total amount of Pb in the supernatant after incubation with lung surfactant incubation solution, mg;

[0142] T PS is the morphological transformation rate of Pb particles after incubation with lung surfactant solution, %;

[0143] M PS-newis the total amount of new forms of Pb particles after incubation in lung surfactant incubation solution, mg;

[0144] (c) PM of micro-reactor system 2.5 -Pb conversion rate TP MRS and PM of lung surfactant 2.5 -Pb conversion rate TR PS , calculate the standard PM 2.5 -Pb relative conversion rate RTR S , as shown in formula (9):

[0145]

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

[0147] TR PS PM2.5 is lung surfactant 2.5 -Pb conversion rate, %;

[0148] TP MRS PM for micro-reactor system 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] Where: RBA is the PM to be measured 2.5 -Relative bioavailability of Pb, %;

[0152] TP is the PM to be tested 2.5 -Conversion rate of Pb in the micro-reaction system, %;

[0153] PM to be tested 2.5 -Total inhalation 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 Pb-composition micro-reaction system, mg;

[0158] T Particle PM to be tested 2.5 -Pb composition micro-reaction system after incubation of the morphological transformation rate of Pb particles, %;

[0159] M origin PM to be tested 2.5 -The total amount of Pb in the Pb particles before incubation of the Pb-composition micro-reaction system, mg;

[0160] ADD Pb PM to be tested 2.5 -Average daily inhalation exposure to Pb, mg / kg / d;

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

[0162] ②Model evaluation

[0163] (a) Using a medium-flow intelligent TSP sampler to collect PM in a certain location 2.5 -Pb samples and detect their concentration The total amount is m origin PM 2.5 -Pb was used to form a micro-reaction system according to step 2-③ and incubated for 24 hours. The supernatant and Pb particles were obtained by centrifugation. The total amount of Pb ions in the supernatant was detected. supernatant and Pb particle morphology conversion rate T Particle , calculate PM according to equations (10)-(12) 2.5 -Pb relative bioavailability RBA, the result is RBA = 42.6% ≥ 20%, judging that the PM 2.5 -Pb has a relative bioavailability. Example 3 Water-insoluble PM 2.5 -The reaction process and mechanism of Pb particles in the micro-reaction system in the alveoli

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

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

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

[0167] 4. Use inductively coupled plasma mass spectrometer to detect the total amount of lead ions M in the supernatant supernatant , using X-ray diffractometer to detect PM in particle sediment 2.5 -Pb new form, calculate the form conversion rate T particle ;

[0168] 5. Calculate PM according to formula (10) 2.5 The conversion rate of -Pb in each protein incubation solution is shown in Table 4:

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

[0170] Protein name <![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 The protein Catalase with the highest conversion rate of -Pb (2.34%) was used for quantum chemical calculation. Catalase is a metalloprotein that naturally carries Fe 2+ Isopropanol dehydrogenase (NADP+) is an important coenzyme to maintain the redox balance of cells and plays an important role in the electron transfer in the function of Catalase. 2.5 The conversion of -Pb was greatly improved (from 2.34% to 6.60%), indicating that Catalase may 2+ Active center replaced by Pb 2+ way, thus promoting PM 2.5 -Conversion of Pb.

[0172] PM 2.5 -Pb contains four types of Pb compounds (Pb3O4, PbS, PbSO4, PbO2). The four types of Pb compounds are added to the Catalase protein incubation solution for incubation. Steps 2-5 are repeated to calculate the conversion rates of the four types of Pb compounds in the Catalase protein incubation solution. The calculation results are shown in Table 5:

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

[0174] Pb compound type name Conversion rate <![CDATA[Pb3O4]]> 0.14％ PbS 0.14％ <![CDATA[PbSO4]]> 51.43％ <![CDATA[PbO2]]> 0.01％

[0175] Based on this, choose PM 2.5 -PbSO4, which has the best conversion effect (51.43%) among Pb, is used as PM 2.5 -Pb represents the compound, which established the interaction between Catalase protein and PM under the assistance of NADP+ 2.5 -Pb reaction model, and perform quantum chemical calculations. The model is as follows:

[0176]

[0177] Where: The active center is Fe 2+ Catalase protein;

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

[0179] NADP + isopropanol dehydrogenase;

[0180] H + is hydrogen ion;

[0181] NADPH is the reducing coenzyme II.

[0182] Through quantum chemical calculations, the Gibbs free energy of the reaction model was obtained to be ΔG = -317.76 Kcal / mol, indicating that the reaction model can occur spontaneously, thus indicating that Catalase can convert its Fe 2+ Active center replaced by Pb 2+ , which promotes PM 2.5 -Conversion of Pb.

[0183] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A PM 2.5 The intra-alveolar micro-reaction system carrying water-insoluble particles is characterized in that: Its construction includes the following specific steps: S1. Establishment of water-insoluble PM 2.5 -M particle bioaccumulation and relative bioavailability model ①Constructing animal exposure model (a) Collect atmospheric PM 2.5 -M samples were used as M compound standard products and ball-milled to less than 2.5 μm to obtain PM 2.5 -M exposure, M is a water-insoluble particle, added with normal saline to make a water-insoluble PM 2.5 -M suspension; (b) The mice were exposed to non-water-soluble PM by respiratory inhalation. 2.5 -M suspension and water-soluble M solution inhalation exposure, as shown in formula (1): Where: ADD PM2.5-M is the average daily PM inhaled by mice 2.5 -M exposure, mg / kg / d; ADD M溶液 is the average daily inhalation exposure of M solution in mice, mg / kg / d; C PM2.5-M PM 2.5 -MTotal concentration of M in sample, μg / m 3 ; IhR mouse is the respiratory rate of mice, m 3 / d; EF mouse PM inhalation in mice 2.5 -Exposure frequency of M, d / w; ED mouse PM inhalation in mice 2.5 -Exposure period of M, w; BW mouse is the average weight of mice, kg; AT is PM inhaled by mice 2.5 - Average exposure time of M, d; (c) Monitor the M ion concentration in the mouse urine during the exposure period. When the M ion concentration reaches equilibrium, end the exposure and detect the water-insoluble PM 2.5 -M accumulation concentration in mouse tissues other than lung tissue 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 Blood (PM 2.5 -M) and Blood (M solution); PM 2.5 -RBA, a relative bioavailability index of M particles accumulated in biological tissues T , as shown in formulas (2)-(4): Where: RBA T For PM 2.5 -M Overall relative bioavailability index, %; RBA Tissue PM inhalation in mice 2.5 -M, relative bioavailability index of tissue j, %; RBA Blood PM inhalation in mice 2.5 -M, blood relative bioavailability index, %; Tissue(PM 2.5 -M) j and Blood(PM 2.5 -M) is PM inhaled by mice 2.5 - M accumulation concentration in tissues and blood after M exposure, mg / kg and mg / mL; Tissue(M solution) j and Blood (M solution) are the M accumulation concentrations in tissues and blood of mice after inhalation exposure to M solution, mg / kg and mg / mL; S2. Construction of a micro-reaction system model of water-insoluble PM2.5-M particles in the alveoli ①PM2.5-M incubation and extraction of M adsorbed substances (a) extracting rat lung surfactant, lyophilizing it into powder, and re-dissolving it into lung surfactant incubation solution; (b) adding a PM2.5-M suspension prepared by adding a PM2.5-M exposure substance to physiological saline to a lung surfactant incubation solution, incubating at a constant temperature of 36-38°C with shaking, centrifuging and washing, collecting PM2.5-M precipitate particles adsorbing lipids and proteins, adding an organic phase chloroform-methanol solution to the precipitate particles and centrifuging for the first extraction, collecting the organic phase containing lipids and proteins; then adding an aqueous phase SDS solution to the precipitate particles, heating at 90-95°C, and centrifuging again to collect the aqueous phase containing proteins; (c) mixing the organic phase and the aqueous phase and vortex centrifuging at 0-4°C for a second extraction to obtain an organic phase containing lipids and an aqueous phase containing proteins; blowing the organic phase to dryness with nitrogen to obtain lipids adsorbed on M; mixing the aqueous phase with acetone, precipitating at -80°C overnight, and then centrifuging to obtain proteins adsorbed on M; ②Screening of lipids and proteins According to whether the protein has metal binding sites, the screened proteins are divided into metal-related proteins and common proteins. The difference is used as the protein screening index, and K kinds (K=1, 2...n) metal-related proteins with a difference FC≥15 and L kinds (L=i, 2...n) common proteins with a difference FC>120 are selected; the content is used as the lipid screening index, and N kinds (N=1, 2...n) lipids with a content PP>60% are selected; ③Assembly of micro-reaction system The proteins and lipids selected in step ② were used as the protein components and lipid components of the microreactor system, and Gamble's solution was used as the simulated lung fluid. 2.5 -M exposure mixture, incubated at 36-38°C with constant shaking, and assembled into a micro-reaction system.

2. The PM according to claim 1 2.5 The intra-alveolar micro-reaction system carrying water-insoluble particles is characterized in that: The water-insoluble M particles described in step S1①(a) are lead, cadmium, nickel, gold, silver, silicon, microplastics or aerosols.

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

4. The PM according to claim 1 2.5 The intra-alveolar micro-reaction system carrying water-insoluble particles is characterized in that: The shaking incubation time in step S2③ is 0.5 to 1 h.

5. A PM according to any one of claims 1 to 4 25 A model for evaluating the relative bioavailability of intra-alveolar micro-reaction systems carrying water-insoluble particles, characterized in that: The construction of the evaluation model includes the following steps: ① Model construction (a) incubating the micro-reaction system assembled according to claim 1 at 36-38° C. under constant temperature oscillation, centrifuging to obtain a supernatant and M particles, and detecting the total amount of M ions in the supernatant MRS and M particle morphology conversion rate T MRS , calculate the PM of the micro-reaction system 2.5 -M conversion rate TP MRS , as shown in formula (5)-(6): Where: TP MRS PM for micro-reactor system 2.5 -M conversion rate, %; M origin-M is the total amount of M particles before incubation in the micro-reaction system, mg; M MRS M is the total amount of supernatant after incubation of the micro-reaction system, mg; T MRS is the morphological transformation rate of M particles after incubation in the micro-reaction system, %; M MRS-new is the total amount of new forms of M particles after incubation in the micro-reaction system, mg; (b) Collected atmospheric PM 2.5 -M samples were used as M compound standard products and ball-milled to less than 2.5 μm to obtain PM 2.5 -M exposure, PM 2.5 -M exposure was added to the lung surfactant incubation solution, incubated at 36-38°C with constant temperature shaking, and centrifuged to obtain the supernatant and M particles. The total amount of M ions in the supernatant was detected. PS and M particle morphology conversion rate T PS , calculate the PM of lung surfactant 2.5 -M conversion rate TR PS , as shown in equations (7)-(8): Where: TR PS PM2.5 is lung surfactant 2.5 -M conversion rate, %; M origin-P is the total amount of M particles before incubation with lung surfactant incubation solution, mg; M PS is the total amount of M in the supernatant after incubation with lung surfactant incubation solution, mg; T PS is the morphological transformation rate of M particles after incubation with lung surfactant incubation solution, %; M PS-new is the total amount of new forms of M particles after incubation with lung surfactant incubation solution, mg; (c) PM of micro-reactor system 2.5 -M conversion rate TP MRS and PM of lung surfactant 2.5 -M conversion rate TR PS , calculate the standard PM 2.5 -Relative conversion rate (RTR) S , as shown in formula (9): Where: RTR S Standard PM 2.5 -M relative conversion rate, %; TR PS PM2.5 is lung surfactant 2.5 -M conversion rate, %; TP MRS PM for micro-reactor system 2.5 -M conversion rate, %; (d) Based on standard PM 2.5 -MRelative conversion rate RTR S , calculate PM 2.5 The relative bioavailability RBA of -M is shown in formulas (10)-(12): TDD PM2.5-M =ADD M ×AT M (12) Where: RBA is the PM to be measured 2.5 -Relative bioavailability of M, %; TR is the PM to be tested 2.5 -Conversion rate of M in the micro-reaction system, %; TDD PM2.5-M PM to be tested 2.5 -M total inhalation exposure, mg / kg; RBA T PM to be tested 2.5 -M Overall relative bioavailability index, %; RTR S Standard PM 2.5 -M relative conversion rate, %; ω is the conversion factor = 0.02; M supernatant PM to be tested 2.5 -M composition: total amount of M ions in the supernatant after incubation of the micro-reaction system, mg; T Particle PM to be tested 2.5 -M composition micro-reaction system after incubation, morphological transformation rate of M particles, %; M origin PM to be tested 2.5 -The total amount of M particles before incubation of the micro-reaction system, mg; ADD M PM to be tested 2.5 -M average daily inhalation exposure, mg / kg / d; AT M PM to be tested 2.5 -M exposure time, d; ②Model evaluation Collect the measured atmospheric PM 2.5 -M samples, the total amount is M origin PM to be tested 2.5 -M according to the micro-reaction system of claim 1 and incubate for 24 to 48 hours, centrifuge to obtain the supernatant and M particles, and detect the total amount of M ions in the supernatant supernatant and M particle morphology conversion rate T Particle , calculate the measured atmospheric PM according to equations (10)-(12) 2.5 -Relative bioavailability (RBA) of M samples; when RBA < 20%, PM 2.5 -M does not have relative bioavailability; when 20% ≤ RBA, PM 2.5 -Pb has relative bioavailability.

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