CN111135300A - Application of protein inhibitor in preparation of medicine for relieving pneumonia caused by haze - Google Patents
Application of protein inhibitor in preparation of medicine for relieving pneumonia caused by haze Download PDFInfo
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- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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Abstract
The invention discloses an application of a protein inhibitor in preparing a medicine for relieving pneumonia caused by haze, wherein the protein inhibitor is adopted to inhibit the activity of heat shock protein 90kDa α B1 to achieve the aim of relieving the pneumonia caused by haze.
Description
Technical Field
The invention relates to an application of a protein inhibitor in preparation of a medicine for relieving pneumonia caused by haze, and belongs to the field of nano toxicology.
Background
Epidemiological studies have shown that the development of pulmonary disease may be associated with prolonged excessive inhalation of air pollution particles such as PM 2.5. PM2.5 refers to particles having an aerodynamic equivalent diameter of less than or equal to 2.5 microns in the atmosphere, also known as accessible lung particles. The PM2.5 entering the body can cause cytotoxicity or cell injury, such as apoptosis, inflammation activation, oxidative stress and the like, and the pneumonia can be caused after long-term accumulation in the body.
The main causes of atmospheric haze pollution include industrial production waste gas emission, automobile waste gas emission, coal burning, climate change and the like. Related departments issue a series of prevention and control measures for atmospheric haze pollution: the method has the advantages that a sound and complete related environmental protection legal system is established, the scientific research investigation related to the haze is developed, the economic development mode is quickened to be changed, green trips are advocated, the haze prevention and control is vigorously publicized, and the like. However, most of the existing measures emphasize prevention, so that the situation that the haze causes pneumonia high incidence cannot be improved in a short period of time after the way of changing from the environment is too long. Meanwhile, the pathological changes and treatment caused by particle inhalation in the aspect of disease treatment have not been studied in a targeted way, and medicines considered from the direction of nanoparticles are lacked.
The protein corona formed by the adsorption of the particles determines the biological activity in vivo and is the main reason of the biological toxicity of the nanoparticles, and through proteomics, biochemical analysis and research in cell models and animal models, the denatured protein in the protein corona mediates the reaction of the cell misfolded protein through the heat shock protein 90kDa α b1, activates the downstream response of the cell and induces the pneumonia.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides the application of a protein inhibitor in preparing a medicament for relieving pneumonia caused by haze.
In order to achieve the purpose, the protein inhibitor is used for preparing the medicine for relieving the pneumonia caused by haze, and the protein inhibitor is used for inhibiting the activity of the heat shock protein 90kDa α B1, so that the purpose of relieving the pneumonia caused by haze is achieved.
As an improvement, the protein inhibitor adopts any one of geldanamycin, geldanamycin derivatives, radicicol, neomycin and purine structure-based compounds.
As an improvement, the haze mainly refers to haze particles, namely PM 2.5.
As an improvement, the medicament is a medicament prepared by taking a protein inhibitor as an active ingredient and adding pharmaceutically acceptable auxiliary materials.
As an improvement, the medicament is an injection.
As an improvement, the injection dosage of the protein inhibitor is 1.0-3.0mg/kg, and the concentration of the protein inhibitor in the medicament is 0.1-0.3 mg/ml.
The principle of the invention is as follows:
the protein expression profiling analysis of various cells shows that the cellular misfolded protein closely related to the heat shock protein 90kDa α b1, an inflammatory response and an epithelial cell-mesenchymal cell transformation pathway are activated, the protein immunoblot analysis, immunofluorescence detection and pathology analysis show that the heat shock protein 90kDa α b1 mediates a pulmonary inflammatory response related to PM2.5, the heat shock protein 90kDa α b1 inhibitor geldanamycin and the like can reduce the pulmonary inflammatory response caused by PM2.5, the heat shock protein 90kDa α b1 combined with denatured protein can cause dissociation and multimerization of heat shock factor 1, then enters a cell nucleus and enhances the expression of molecular chaperone 90aa1 and heat shock protein 90 α b1, the process is called as a protein response, the protein response is a part of the intracellular cellular misfolded protein process and is also a key pathway for causing the inflammatory response, and the heat shock protein 4690 b 84 inhibitor can reduce the action of the heat shock protein 465, thereby providing a remarkable idea for treating PM2 diseases.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention researches the pathogenic principle of haze particles, analyzes particle adsorption protein crowns from the field of nano toxicology, and systematically recognizes the biological response and related pathways of pneumonia caused by PM2.5 particles.
(2) The invention finds a heat shock protein 90kDa α b1 which is a key target point of causing pneumonia by PM 2.5.
(3) According to the invention, the heat shock protein 90kDa α b1 inhibitor is used to relieve the pneumonia reaction by geldanamycin, so that a new treatment scheme is provided for the current situation that the pneumonia high incidence cannot be relieved in a short period of haze prevention and treatment.
Drawings
FIG. 1 is a graph showing polyacrylamide gel electrophoresis and staining of a particle-protein corona complex according to example 1 of the present invention;
FIG. 2 is a histological analysis test chart in example 3 of the present invention.
Detailed Description
The following examples are further illustrative of the present invention as to the technical content of the present invention, but the essence of the present invention is not limited to the following examples, and one of ordinary skill in the art can and should understand that any simple changes or substitutions based on the essence of the present invention should fall within the protection scope of the present invention.
Example 1
The method for rapidly detecting the denaturation of PM2.5 induced by protein adsorption comprises the following steps:
1) collecting and separating PM 2.5:
PM2.5 is collected in the inhabited Xixia area of Nanjing city, and the parameters of the atmospheric particulate matter sampler are uniformly set as: the flow rate is 100L/min, the collection time of each filter membrane is 72 hours, and after the collection is finished, the glass fiber filter membrane loaded with PM2.5 particles is cut into pieces and soaked in phosphate buffer solution for the next preparation; soaking a glass fiber filter membrane loaded with particles for 72 hours, performing ultrasonic overnight to promote the particles to be fully separated from the filter membrane, centrifuging for many times and removing precipitated glass fibers, taking a small amount of supernatant containing the particles for characterization and analysis of the particles, performing next-step centrifugation on the residual supernatant to obtain particle precipitates, and performing vacuum freeze-drying for later use;
2) extraction of non-denatured cytoplasmic protein
The protein solution was extracted from RAW264.7 macrophages (cell bank of chinese academy of sciences) using a non-denaturing buffer, as follows: collecting cell clusters by centrifugation at 350Xg for 5 minutes at 4 ℃ and washing three times with pre-cooled phosphate buffer, then suspending and dispersing 10^6 cells in pre-cooled 400 μ l buffer A, and standing on ice for 20 minutes; then adding 10% (v/v) NP-40 to 0.6% (v/v) and vortexing and shaking for 20 seconds to obtain a cell extract; centrifuging at 500Xg for 10 minutes at 4 ℃ to precipitate nuclei and collecting the supernatant, centrifuging at 12,000Xg for 10 minutes at 4 ℃ to collect the supernatant and filtering through a filter to obtain a protein solution;
3) preparation and isolation of protein crowns
Incubating the particles with the protein solution extracted under non-denaturing conditions at a concentration of 0.1mg/ml (1 mg/ml for PM 2.5) in a volume of 5ml for 4 hours at room temperature with shaking, and then centrifuging the protein solution at 15,300Xg for 20 minutes at 4 ℃ using 0.7M sucrose density buffer to separate the particlized protein corona complexes; washing 6 times with precooled phosphate buffer, adding lysis solution into the precipitate, placing on ice for lysis for 30 minutes, heating at 95 ℃ for 5 minutes, centrifuging at 4 ℃ for 10 minutes at 12,000rpm, collecting supernatant, and storing in a refrigerator for later use;
4) polyacrylamide gel electrophoresis and staining of particle-protein corona complexes
With reference to fig. 1, PM 2.5-protein corona complex precipitates were obtained by incubation of PM2.5 with protein, sucrose density centrifugation and washing, and two different electrophoretic analyses were performed to compare the protein adsorption capacities of different particles;
4.1) adding a denatured protein buffer solution containing SDS to the precipitate, heating at 95 ℃ for 5 minutes to lyse the proteins adsorbed to the particles, centrifuging at 12,000rpm at 4 ℃ for 10 minutes, collecting the supernatant and performing denatured polyacrylamide gel electrophoresis;
4.2) adding a non-denatured protein buffer solution without SDS, carrying out non-denatured polyacrylamide gel electrophoresis on the protein solution containing the PM2.5 protein crown complex, then carrying out Coomassie brilliant blue staining on the gel, observing the migration condition of the protein, carrying out gel imaging by using a multifunctional chemiluminescence imager, and carrying out gray scale analysis and relative quantification by using Image J software.
The results show that: PM2.5 and protein solution extracted from RAW264.7 macrophage are adsorbed to form protein crown after incubation.
Circular dichroism is a technique which can quickly evaluate the secondary structure of protein, the folding property of protein and the interaction of protein with a small amount of protein, and has been widely used for evaluating the folding state and structural stability of recombinant protein and purified protein, although the method cannot provide specific information of protein residue like X-ray and nuclear magnetic resonance, the method is often used for evaluating the influence of nanoparticles on the secondary structure of protein, especially on α spiral structure.
The specific operation is as follows: first, commercial proteins including albumin (20. mu.M), ferritin (1.25mg/ml), myoglobin (20. mu.M), lysozyme (20. mu.M), cytochrome C (20. mu.M) and hemoglobin (20. mu.M) were dissolved in protein buffer at various concentrations, and they were stored at 4 ℃ for several days after filtration. The particles were dispersed in a protein solution at a concentration of 0.01mg/ml (PM2.5 at 0.1mg/ml), incubated at room temperature for 4 hours with shaking, and then the proteins were separately diluted to a certain concentration and tested on the machine, and a Tris-HCl stock solution containing no protein but containing the particles was used as a calibration baseline.
The results show that PM2.5 denatures adsorbed proteins after incubation with the extracted protein solution from RAW264.7 macrophages.
Example 2
Protein immunoblotting was used to verify that PM2.5 adsorbed protein induces its denaturation and recruits the chaperone heat shock protein 90kDa α B1:
the PM 2.5-protein crown complex treated by the denaturant (guanidine hydrochloride 6M GdHCL) can adsorb more heat shock protein 90kDa α B1 (the denatured protein promotes the recruitment of heat shock protein 90kDa α B1), the PM 2.5-protein crown complex treated by the renaturator (0.5M Arg,0.4M Gly,0.4M sucrose, 0.5M Tris,0.5M NaCl and pH 7.0) has reduced capability of adsorbing heat shock protein 90kDa α B1, the molecular chaperone can recognize and bind to the denatured protein to promote the proper folding or degradation of the protein, and therefore, the level of the PM 2.5-protein crown complex adsorbing the heat shock protein 90kDa α B1 can be used as an index for evaluating the capability of PM2.5 to induce the protein denaturation.
The specific operation is as follows: PM2.5 was incubated at a concentration of 1mg/ml with 1ml of a protein solution (20. mu.M albumin, 25mg/ml ferritin, 20. mu.M myoglobin, 20. mu.M lysozyme C, 18. mu.M cytochrome C and 0.4. mu.M hemoglobin), a particle system containing no protein was set as a control group, incubated at room temperature for 4 hours with shaking, and washed 3 times with phosphate buffer;
then 500. mu.l of phosphate buffer solution is added to resuspend the complex, and 0.5ug of recombinant human heat shock protein 90kDa α B1 protein (Abcam company) is added and incubated for 1 hour at room temperature with shaking;
and finally, washing the protein by using phosphate buffer solution for 3 times, adding lysis solution for lysis to obtain a protein crown component, storing the protein crown component, then carrying out western blotting and exposure, carrying out gray value analysis, comparing the level of the heat shock protein 90kDa α B1 adsorbed by PM2.5 by using albumin or other proteins in the protein crown as an internal reference, and simultaneously carrying out polyacrylamide gel electrophoresis on the lysed protein and carrying out gel staining by using a commercial high-sensitivity silver staining kit.
The result shows that the adsorption of the heat shock protein 90kDa α B1 by the PM 2.5-protein crown complex can be detected by performing western blotting, performing SDS-PAGE electrophoresis, membrane transfer, blocking the PVDF membrane with 5% milk for 1h, incubating the blocked PVDF membrane with a primary antibody (heat shock protein 90kDa α B1 antibody, BOSTER company, dilution ratio 1:1000), incubating overnight at 4 ℃, incubating a secondary antibody (I125-labeled ProteinA) at room temperature for 1h, dropwise adding a developing solution, and developing the PVDF membrane under a developer.
Example 3
The heat shock protein 90kDa α B1 inhibitor geldanamycin is used for inhibiting the occurrence of lung inflammation:
1) building of pneumonia model
Female mice (20 ± 2g) with the same genetic background were purchased and housed in a ventilated, thermostated and 50% -60% relative humidity room, and provided sufficient food and water following a 12 hour light on and 12 hour light off environmental profile.
The specific experiment is as follows:
1.1) preparing materials, namely preparing 2mg/ml PM2.5 particle dispersion liquid for later use, dissolving geldanamycin to 5mg/ml by using dimethyl sulfoxide, and then diluting the geldanamycin to 0.2mg/ml by using β -cyclodextrin solution of 0.2g/ml for later use, preparing 10mg/ml pentobarbital sodium by using phosphate buffer solution as an anesthetic, and preparing a 1ml syringe, a surgical instrument, a suture and erythromycin ointment;
1.2)1 day model: the mice were randomly divided into 4 groups (each group containing at least 7 mice), respectively a phosphate buffer group, a geldanamycin group, a PM2.5 group, and a PM2.5+ geldanamycin group, and were intraperitoneally injected 6 hours prior to infusion of the granules for mice requiring geldanamycin treatment (2 mg/kg); before surgery, each mouse was intraperitoneally injected with sodium pentobarbital (45mg/kg), the trachea was slowly injected with 50 μ l of the particle dispersion using a syringe (sonication before use), bronchial lavage fluid was collected 24 hours after the mice were perfused with particles, and lung tissue was collected for subsequent biological and histological analysis.
2) Histological analysis and detection
The mice are sacrificed and the lungs are taken out, paraffin embedding, slicing and H & E staining are carried out, as shown in figure 2, the H & E staining result shows that a large number of inflammatory cells are infiltrated around the bronchus of the lung tissue and a granuloma structure is formed, and geldanamycin injection can obviously relieve the pathological process, in figure 2, the upper left graph and the right graph are compared to show that PM2.5 (namely a key protein heat shock protein 90kDa α B1) causes the bronchitis of the lung tissue, and the upper graph and the lower graph are compared to show that the granuloma structure added with GA is reduced and the inflammation is relieved, namely the GA can relieve the pneumonia.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (6)
1. An application of a protein inhibitor in preparing a medicine for relieving pneumonia caused by haze is characterized in that the protein inhibitor is adopted to inhibit the activity of heat shock protein 90kDa α B1, so that the aim of relieving the pneumonia caused by haze is fulfilled.
2. The use of a protein inhibitor according to claim 1 in the preparation of a medicament for reducing haze-induced pneumonia, wherein the protein inhibitor is any one of geldanamycin, geldanamycin derivatives, radicicol, neomycin, purine structure based compounds.
3. The use of the protein inhibitor in the preparation of a medicament for alleviating the pneumonia caused by haze according to claim 1, wherein the medicament is a medicament prepared by using the protein inhibitor as an active ingredient and adding pharmaceutically acceptable auxiliary materials.
4. The use of the protein inhibitor according to claim 3 in the preparation of a medicament for reducing the pneumonia caused by haze, wherein the medicament is an injection.
5. The use of a protein inhibitor in the preparation of a medicament for reducing haze-induced pneumonia according to claim 3 or 4, wherein the concentration of the protein inhibitor in the medicament is 0.1-0.3 mg/ml.
6. The use of a protein inhibitor in the preparation of a medicament for reducing haze-induced pneumonia according to claim 3 or 4, wherein the protein inhibitor is injected in an amount of 1.0-3.0 mg/kg.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003159087A (en) * | 2001-09-13 | 2003-06-03 | Japan Science & Technology Corp | Tissue fibrosing inhibitory oligonucleotide |
| CN1460474A (en) * | 2003-07-08 | 2003-12-10 | 中国医学科学院医药生物技术研究所 | Application of geldanamycin in preparation of medicine for curing SARS |
| CN102741410A (en) * | 2009-12-09 | 2012-10-17 | 日东电工株式会社 | Modulation of HSP47 expression |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003159087A (en) * | 2001-09-13 | 2003-06-03 | Japan Science & Technology Corp | Tissue fibrosing inhibitory oligonucleotide |
| CN1460474A (en) * | 2003-07-08 | 2003-12-10 | 中国医学科学院医药生物技术研究所 | Application of geldanamycin in preparation of medicine for curing SARS |
| CN102741410A (en) * | 2009-12-09 | 2012-10-17 | 日东电工株式会社 | Modulation of HSP47 expression |
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| ANURAN CHATTERJEE ET AL: ""Heat Shock Protein 90 Inhibitors Prolong Survival，Attenuate Inflammation, and Reduce Lung Injury in Murine Sepsis"", 《AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE》 * |
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