CN116870025A - Pulmonary fibrosis therapeutic cow milk exosome-siTGF-beta 1 medicine - Google Patents
Pulmonary fibrosis therapeutic cow milk exosome-siTGF-beta 1 medicine Download PDFInfo
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- CN116870025A CN116870025A CN202310898871.9A CN202310898871A CN116870025A CN 116870025 A CN116870025 A CN 116870025A CN 202310898871 A CN202310898871 A CN 202310898871A CN 116870025 A CN116870025 A CN 116870025A
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Abstract
The invention belongs to the technical field of medicines, and particularly relates to a lung fibrosis therapeutic cow milk exosome-siTGF-beta 1 medicine. The efficacy of M-siTGF- β1 was demonstrated in both the bias-2 b cell level and bleomycin-induced mouse lung fibrosis model. M-siTGF- β1 was found to be effective in delivering target siRNA to the lung by tracheal nebulization, resulting in selective gene silencing, inhibition of TGF- β1 protein expression, and epithelial-mesenchymal transition pathway (EMT). The pharmacological and pharmacodynamic results in vitro and in vivo show that M-siTGF-beta 1 reduces infiltration of inflammatory cells, reduces deposition of extracellular matrix and improves survival rate of pulmonary fibrosis mice. M-siTGF-. Beta.1 may represent a potential strategy for treating pulmonary fibrosis.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a lung fibrosis therapeutic cow milk exosome-siTGF-beta 1 medicine.
Background
Pulmonary fibrosis (Pulmonary fibrosis, PF) is a severe diffuse interstitial lung disease, and PF can be caused by inflammatory lung disease, inhalation of particulate matter, and the use of certain drugs. PF is pathologically characterized by inflammatory cell infiltration, fibroblast proliferation, massive deposition of extracellular matrix (Extra cellular matrix, ECM), ultimately leading to irreversible deformation of the lung structure. The pathological process is almost irreversible, and once the disease is generated, the survival time median of the patient is only 3-5 years. In recent years, the incidence of PF has increased year by year due to factors such as air pollution and specific pathogens. The main therapeutic drugs of PF include pirfenidone and nidanib, but their therapeutic effects are very limited. At present, an effective means for treating the pulmonary fibrosis is lung transplantation, but organ resources are deficient, the supply of a donation mechanism is limited, risks such as infection, rejection and complications exist later, the median survival time after transplantation is only 4.5 years, the high cost and the long-term need of immunosuppressant treatment limit the clinical popularization and use of the traditional Chinese medicine. In the treatment process of patients with pulmonary fibrosis, if the development of pulmonary injury can be timely inhibited, the progress of pulmonary fibrosis is blocked, and great help is provided for reducing the degree of fibrosis injury and improving the pulmonary function of patients, so that the pathogenesis of PF is discussed, an effective treatment target is determined, and a treatment drug with stable curative effect and small side effect is sought to reduce the death rate and improve prognosis of PF patients, so that the method has profound significance for the important problems to be solved in the clinical medical community and scientific research work at present.
One of the most significant features of PF is the excessive deposition of ECM proteins, resulting in replacement of normal tissues with nonfunctional scar tissue. Epithelial-mesenchymal transition (EMT) is considered to be the most important process leading to the development of IPF, which is the major source of pulmonary myofibroblast formation. During pulmonary fibrosis, alveolar epithelial cells differentiate into mesenchymal-like cells under a variety of fibrogenic stimuli and further proliferate and differentiate into myofibroblasts, expressing the markers of myofibroblasts, α -smooth muscle actin (α -SMA), releasing a large number of ECMs, mainly comprising type I collagen. Activated transformation of lung fibroblasts is a key step in the EMT pathway, transforming growth factor- β1 (TGF- β1) is considered to be the most critical cell growth factor involved in this process. In addition, TGF-. Beta.1 can directly stimulate receptor production of various growth factors, such as EGFR, PDGF, CTGF, which play an important role in fibroblast transformation and ECM production.
In the TGF-beta superfamily, three subtypes have been identified: namely, TGF-beta 1 subtype, TGF-beta 2 subtype and TGF-beta 3 subtype, wherein TGF-beta 1 plays a dominant role in promoting fibrotic diseases, is a key fibroblast activating factor and has a strong fibrosis promoting effect. In the activation of TGF-beta, mature TGF-beta homodimers bind first to the homodimers of TGF-beta RII with the aid of TGF-beta RIII, and then phosphorylate the TGF-beta RI homodimers (TGF-beta RI, ALK 5) under the action of the hexamer complexes of TGF-beta, TGF-beta RII and TGF-beta I homodimers. Then, TGF-. Beta.RI phosphorylates Smad2 protein in cytosol and phosphorylates Smad3 protein, and finally Smad2/3 translocates to nucleus and becomes transcriptionally active under tight binding with Smad4 protein.
Bleomycin (BLM) is a broad-spectrum chemotherapeutic drug that has found application in many tumors, but clinical studies have found that BLM can cause multiple organ fibrosis, with PF being the most common, the mechanism of which may be associated with the lack of BLM hydrolase in the lung. Researchers have found that administration of BLM by tracheal instillation to animals stably causes PF in animals, and that its pathological processes are similar to human PF, both acute/chronic inflammation leading to damage of lung epithelial cells, proliferation of lung fibroblasts, collagen deposition, and eventually alveolar structure loss, pulmonary remodeling, loss of pulmonary ventilation/ventilation functions, and even death.
Exosomes (exosomes, exo) refer to extracellular vesicles (Extracellular vesicles, EVs) secreted by cells, about 30-150nm in diameter, which are vesicles that are targeted to the plasma membrane by the budding of multiple vesicles into the cell, a process that is related to the endosomal sorting complex pathway, the ceramide dependent pathway, required for transport. A variety of cells secrete exosomes under normal and pathological conditions, and exosomes are naturally found in body fluids, including blood, saliva, urine, cerebrospinal fluid and milk.
The exosomes are formed by lipid bilayer membranes comprising proteins, nucleic acids and lipids and derivatives thereof, etc. Proteins contained in exosomes include endoluminal proteins and transmembrane proteins, such as heat shock proteins, tetra-transmembrane proteins, etc., wherein tetra-transmembrane proteins, particularly leukocyte differentiation antigen 63 (CD 63) and leukocyte differentiation antigen 81 (CD 81), are common specific exosome markers. Nucleic acids contained in exosomes include messenger RNAs, micrornas (mirnas), long non-coding RNAs, and the like. Research has shown that nucleic acids in exosomes are involved in many disease processes, such as tumors, cardiovascular diseases, autoimmune diseases, etc. The exosome-rich lipids mainly comprise cholesterol, glycosphingolipids, sphingomyelin, phosphatidylserine, arachidonic acid and the like, and the lipids are involved not only in the constitution of the exosome but also in the physiological and pathological processes of the cells. It is thought that communication with neighboring cells through cell-cell contact is the primary role of exosomes, which can communicate with distant cells through secreted soluble factors (e.g., hormones, cytokines) and can also be involved in signal transduction through electronic, chemical signals (e.g., nucleotides, lipids, and short peptides); in addition, exosomes play a key role in both maintaining tissue cell function and regulating intracellular environmental homeostasis, and can modulate immune cell function, thereby mediating immune responses.
According to the source of exosomes, the exosomes have unique biological characteristics, milk exosomes (M-EXO) are obtained by separating and purifying fresh milk, are rich in phospholipid bilayer membrane structures which are the same as cell membranes, tightly wrap specific proteins, lipids, miRNA (micro ribonucleic acid), DNA (deoxyribonucleic acid) fragments such as cytokines, growth factors and the like, can rapidly open cell biomembrane barriers, and accurately identify and locate cell injuries and the like. M-EXO is an evolutionarily conserved unique class of microbubbles that maintain the integrity of their contained nucleic acids and proteins en route through the stomach and gastrointestinal tract, which can function locally or be transported into the circulatory system. Furthermore, milk exosomes are more stable than other naturally occurring exosomes, which exhibit excellent stability under acidic conditions and other harsh conditions.
Disclosure of Invention
The invention aims to provide a lung fibrosis therapeutic bovine milk exosome-siTGF-beta 1 drug.
Another object of the invention is to provide the use of a bovine milk exosome-siTGF- β1 drug in the treatment of pulmonary fibrosis.
Further, the preparation method of the cow milk exosome-siTGF-beta 1 drug comprises the following steps: introducing siRNA mediating TGF-beta 1 low expression into exosomes by taking bovine milk exosomes as RNAi delivery system to form M-siTGF-beta 1 (bovine milk exosomes-siTGF-beta 1) medicaments, wherein the sense strand of the siRNA sequence mediating TGF-beta 1 low expression is shown as SEQ ID. NO. 1: CCCAAGGGCUACCAUGCCAACUUCU, the antisense strand is shown in SEQ ID No. 2: AGAAGUUGGCAUGGUAGCCCUUGGG.
Further, the preparation method of the cow milk exosome-siTGF-beta 1 drug is an electrotransformation method, and comprises the following steps: bovine milk exosomes were mixed with siRNA in a ratio in 500 μl of PBS solution, i.e. 500 μl system. Incubation was performed for 30min at 4℃after mixing, electrotransformation was performed as follows: 220V, pulse time 10ms, pulse 3 times, interval 2s. After electrotransformation, the treated samples were removed and incubated at 37℃for 1h to promote recovery of the exosome membrane.
Further, the ratio of the milk exosomes to the siRNA is 1:5.
Further, the preparation method of the cow milk exosome-siTGF-beta 1 drug is an ultrasonic method and comprises the following steps: bovine milk exosomes and siRNA were mixed in PBS at a ratio of 1:5 (mass/mass), 30w,6 on/off cycles of 30 seconds, with a cooling time of 2 minutes between each cycle. After sonication, the solution was incubated at 37 ℃ for 30 minutes to restore the exosome membrane.
Further, the preparation method of the cow milk exosome-siTGF-beta 1 drug is modified CaCl 2 A method comprising the steps of: mixing siRNA with cow milk exosomes in PBS, and adding CaCl 2 (final concentration 100 mM). The final volume was adjusted to 500 μl using sterile PBS. The mixture was placed on ice for 30 minutes. After thermal shock at 42 ℃ for 60 seconds, the mixture was left on ice for an additional 5 minutes.
The invention has the beneficial effects that: the efficacy of M-siTGF- β1 was demonstrated in both the bias-2 b cell level and bleomycin-induced mouse lung fibrosis model. M-siTGF- β1 was found to be effective in delivering target siRNA to the lung by tracheal nebulization, resulting in selective gene silencing, inhibition of TGF- β1 protein expression, and epithelial-mesenchymal transition pathway (EMT). The pharmacological and pharmacodynamic results in vitro and in vivo show that M-siTGF-beta 1 reduces infiltration of inflammatory cells, reduces deposition of extracellular matrix and improves survival rate of pulmonary fibrosis mice. M-siTGF-. Beta.1 may represent a potential strategy for treating pulmonary fibrosis.
Drawings
Fig. 1: A. electroporation, sonication and modified CaCl 2 Transfection method M-EXO loaded siRNA encapsulation efficiency, B. Electric transfer method M-EXO and siRNA encapsulation efficiency in different proportion, C. Encapsulation efficiency under different voltage of electric transfer method;
fig. 2: B. particle size change of Exo before and after atomization, difference of Zeta potential before and after atomization, and encapsulation rate of siRNA entrapped by Exo before and after atomization;
fig. 3: stability experiments of M-EXO under different pH conditions;
fig. 4: A. cell viability at different BLM concentrations b. expression of tgfβ1 at different BLM concentrations;
fig. 5: uptake of M-EXO by Beas-2b cells
Fig. 6: bleomycin-induced Beas-2b cell viability in different drug-treated groups
Fig. 7: profile of M-EXO in mouse plot;
fig. 8: different treatments of lung tissue detect TGF-beta 1 expression;
fig. 9: status and time to survival profiles of different treated mice;
fig. 10: differential treatment of lung tissue status and lung weight map
Fig. 11: differential treatment of lung histopathological staining HE staining and Masson staining
Fig. 12: expression levels of IL-6 in different treatments
Fig. 13: A. expression level of E-cadherein in different treatments, B. expression level of Vimentin different treatments;
fig. 14: different treatments of the expression levels of MMP2 and MMP 9;
fig. 15: the ECM proteins Collagen I (involved in the formation and remodelling of fibrotic ECM in the lung), fibrinectin, α -SMA, expression levels of CTGF were treated differently.
Detailed Description
Example 1
Preparation and characterization of M-siTGF-beta 1
1.1M-siTGF-beta 1 preparation method investigation
Acquisition of milk exosomes
Purchased fromUR53202 milk exosome extract
Milk exosome extract concentration 1ug/uL
TGF-. Beta.1 sequences (5 '-3'):
sense strand: CCCAAGGGCUACCAUGCCAACUUCU
Antisense strand: AGAAGUUGGCAUGGUAGCCCUUGGG
1) Electric conversion method
Bovine milk exosomes were mixed with siRNA in a ratio in 500 μl of PBS solution, i.e. 500 μl system. Incubation was performed for 30min at 4℃after mixing, electrotransformation was performed as follows: 220V, pulse time 10ms, pulse 3 times, interval 2s. After electrotransformation, the treated samples were removed and incubated at 37℃for 1h to promote recovery of the exosome membrane.
2) Ultrasonic method
Bovine milk exosomes and siRNA were mixed in PBS at a ratio of 1:5 (mass/mass), 30w,6 on/off cycles of 30 seconds, with a cooling time of 2 minutes between each cycle. After sonication, the solution was incubated at 37 ℃ for 30 minutes to restore the exosome membrane.
3) Improved CaCl plate 2 Method of
For improved CaCl 2 The method comprises mixing siRNA with milk exosomes in PBS, and adding CaCl 2 (final concentration 100 mM). The final volume was adjusted to 500 μl using sterile PBS. The mixture was placed on ice for 30 minutes. After thermal shock at 42 ℃ for 60 seconds, the mixture was left on ice for an additional 5 minutes.
1.2M-siTGF-. Beta.1 characterization
1.2.1 exosome particle size and concentration
The nanoparticle tracking analyzer (Nanosight 3000, ns 300) irradiates the nanoparticle suspension with a laser light source, forms a completely black background by means of a metal coating, and clearly observes brownian motion of the particles with scattered light. For a wide distribution system, NTA can simultaneously carry out direct observation, automatic tracking and particle size calculation on each particle, and meanwhile obtain the particle size distribution information of the whole system. By combining the scattering intensity of the particles, NTA software can draw a three-dimensional map of the particle size, the distribution intensity of the corresponding quantity and the scattering intensity, and can clearly distinguish samples with the same particle size but different materials.
1.2.2zeta potential
The zeta potential of the exosomes was detected using a nanoparticle potentiometric analyzer. Approximately 800. Mu.L of PBS resuspended exosomes were added to the sample cell for on-board detection of zeta potential.
Encapsulation efficiency detection of 1.2.3M-siTGF-beta 1 before and after atomization
The total RNA content and the free RNA content were determined from samples after electrotransformation using the RiboGreen kit.
Total RNA was obtained by "demulsification" of the exosome samples with an equal volume of 2% TritonX100 solution.
1.3 experiments on stability of M-EXO under different pH conditions
To examine the stability of M-Exo in weak acid environment, M-Exo was incubated in 10% FBS-containing PBS at pH 7.4 or 6.8 at 37℃for 0h, 6h, 12h, 24h and 48h, respectively. Particle size measurements were performed on M-Exo at different pH and time points using NTA.
Results
Preparation process and characterization of M-siTGF-beta 1
1.1M-siTGF-beta 1 preparation method investigation
First, we used 3 different methods to load siRNA into M-EXO: electroporation, sonication and modified CaCl, respectively 2 Transfection method. As shown in FIG. 1A, the encapsulation efficiencies of the M-EXO after siRNA loading were 34.99%, 16.28% and 4.68%, respectively. The encapsulation efficiency of the electrotransformation method is higher than that of the other two methods, and the technological conditions of the electrotransformation method for encapsulating siRNA are continuously examined.
The investigation is carried out according to the preparation process of an electrotransformation method, and the encapsulation efficiency is used as an evaluation index. As shown in FIG. 1B, other factors were fixed, and only the ratio of M-EXO to siRNA was changed to 1:1,1:5,1:10,1:15, the encapsulation efficiency is 1:5, so that the ratio of M-EXO to siRNA was chosen to be 1:5.
as shown in fig. 1C, other factors are fixed, and when the voltage is changed to 100V, 160V, 220V, 280V, the encapsulation efficiency increases with the increase of the voltage, and when the voltage increases to 220V, it is found that the voltage continues to increase, and the encapsulation efficiency tends to decrease, so that the electric conversion voltage is selected to be 220V.
1.2M-siTGF-. Beta.1 characterization
siRNA was first electroporated into M-Exo. Nebulization was performed for drug loaded (Exo-siRNA) and drug unloaded (Exo-Con) Exo using nebulization. Measuring the particle size change of Exo by using NTA; measuring the change in Exo potential using Zeta; by means of Quant-iT TM RiboGreen TM The RNA Assay Kit measures the change in encapsulation efficiency.
As shown in fig. 2B, particle size: the particle sizes of Exo before and after atomization are not different, and the atomization has no influence on the particle sizes of Exo; as shown in fig. 2C, zeta potential: the potential measured after siRNA is loaded at the self point position of the exosome around-5 is around-20. The free siRNA was negatively charged, the absolute value of the potential after nebulization was slightly reduced but there was no difference, and nebulization had no effect on the potential of Exo. As shown in fig. 2D, there was a slight decrease in the encapsulation efficiency of the Exo-entrapped siRNA before and after nebulization, but no difference, and nebulization had no effect on the encapsulation efficiency.
1.3 experiments on stability of M-EXO under different pH conditions
As shown in FIG. 3, there was no significant change in particle size when M-EXO was incubated in Phosphate Buffer (PBS) containing 10% Fetal Bovine Serum (FBS) at pH 7.4 or 6.8 over 48 hours, indicating that M-EXO can maintain an intact structure in the slightly acidic extracellular pH of IPF lung tissue.
Example 2
2. Cell experiment
2.1 cellular uptake of M-Exo
Beas-2b cells were grown at 3X 10 4 The density of each hole is inoculated into a 24-hole plate, and the mixture is placed into an incubator for culturing for 24 hours, and fluorescence is usedThe light marked cow milk exosome suspension replaces the original culture solution, and is incubated with the co-cultured cells for 1h and 4h, and after treatment, the cells are washed 3 times by PBS, and the nuclei are marked by DAPI staining. Subsequently, 500. Mu.L of 4% paraformaldehyde was added and incubated for 15min to fix the cells. Cells were washed 3 times with PBS and imaged with a high resolution live cell imaging system (station TM BioTek, US).
2.2 construction of BLM-induced cell fibrosis model
The optimal dose and time for BLM to induce cellular fibrosis was screened using the MTT assay method for cell viability. To study the effect of BLM on fibrosis at the cellular level, 96-well plates were selected and the cells were plated at 5X 10 3 Cell density of cells/well cell density was seeded in well plates and incubated in an incubator for 24 hours. Beas-2b cells were treated with BLM (0, 0.5, 1 and 2. Mu.g/mL) at various concentrations and then maintained in 100. Mu.L of culture broth for 24 hours with the cells. Three groups of replicates were established for each concentration, while a blank group was established, and the activity of this group was taken as 100% cell viability. Each group was directly added with 10. Mu.L of MTT solution, and after 4 hours in the incubator, was reduced by living cells. The liquid was aspirated off, 100-200. Mu.L of DMSO was added, and the well plate was placed in a shaker for 10min at 490nm to determine absorbance.
2.3 determination of cell viability
Cells were seeded in 96-well plates at a Beas-2b cell density of 5000 cells/well, and when the cells had proliferated to 70% -80%, BLM (diluted with conditioned medium) at a concentration of 1. Mu.g/mL was added, ctrl groups (no BLM added), BLM groups, M-EXO groups, BLM-EXO groups were set, and after 24 hours of action, cell viability was examined with MTT.
Results
2.1 Effect of BLM on Beas-2b cell proliferation
To investigate the molecular mechanisms by which exosomes alleviate pulmonary fibrosis, we constructed a fibrosis model at the cellular level using BLM. Tgfβ1 plays a key driving role in the development and progression of fibrosis. As shown in FIG. 4, we first screened the optimum concentration of BLM for induction of cellular fibrosis using TGF-beta 1 expression, and BLM induced high expression of TGF-beta 1 at both 1 μg/ml and 2 μg/ml, so we selected a concentration of BLM of 1 μg/ml to induce Beas-2b cell fibrosis in subsequent experiments.
2.2M-EXO accessible to target cells
As shown in the experimental results of FIG. 5, using DIR to label bovine milk exosomes, red coated exosomes were observed to be efficiently absorbed by Beas-2b cells. The presence of BLM increases the uptake of bovine milk exosomes by the bias-2 b cells. And uptake for 4h was greater than 1h, as was cells treated with BLM.
2.3M-siTGF-beta 1 enhances the resistance of Beas-2b cells to the action of BLM
To study the effect of M-siTGF-beta 1 on IPF cell model inflammation and fibrosis related molecule expression, first 1 μg/mL BLM was used to act on Beas-2b cells for 24h, induced to build up a cell fibrosis model, after treatment with different drugs, cell viability was determined.
As shown in the experimental results of FIG. 6, the BLM can obviously reduce the cell viability of the Beas-2b, the M-EXO, naked siTGF-beta 1 and M-siNC are added, the cell growth state cannot be influenced, and the viability of the cell is obviously improved after the M-siTGF-beta 1 is added, so that the resistance of the Beas-2b cell to the BLM is enhanced by the M-siTGF-beta 1.
Example 3
3. Animal experiment
3.1M-Exo in vivo distribution and uptake
After the DIR is used for marking the cow milk exosomes and the cow milk exosomes are atomized and injected into the cow milk exosomes marked, the in-vivo images are shot by using a small animal imaging system at the time points of 0h, 4h, 1day, 3day, 5day and 7day respectively, tissues such as heart, liver, lung, kidney and spleen are collected, and the retention condition of the exosomes in the lung is observed according to the biological distribution of the cow milk exosomes marked by the DIR.
3.2 establishment of animal model for pulmonary fibrosis
The majority of intrapulmonary administration in mice involved in the past studies was post-incision administration, but the biggest adverse effect of this approach was: animal self-control behavior is poor, the animal is difficult to tolerate to the operation wound, the animal is easy to scratch and bite, and wound healing is affected; meanwhile, the tracheotomy wound surface is large, the infection risk is high, the death rate is high, and the accuracy of experimental results is seriously affected. Therefore, after examining a large number of documents and a number of experimental attempts, the present subject decided to establish an in vivo model by means of the administration of a mouse by tracheal atomization.
Male C57BL/6 mice at 8 weeks of age were randomly divided into 6 groups, control group (5) and Bleomycin (BLM) treated group (8), BLM+M-EXO group (8), BLM+Naked siTGF-. Beta.1 (8), BLM+M-siNC (8) and BLM+M-siTGF-. Beta.1 (8), respectively. On day 0, control mice were injected with 50. Mu.l of an equal volume of saline by intratracheal injection of 1.5mg/kg of BLM solution in saline (50. Mu.L/mouse). Beginning 14 days of molding, the mice in the BLM+M-EXO group are subjected to tracheal atomization M-Exo, the mice in the BLM+Naked siTGF-beta 1 group are subjected to tracheal atomization Naked siTGF-beta 1, the mice in the BLM+M-siNC group are subjected to tracheal atomization M-siNC, the mice in the BLM+M-siTGF-beta 1 group are subjected to tracheal atomization M-siTGF-beta 1, and the same amount of nonfunctional physiological saline is atomized in the BLM group. Body weight and mortality of mice were recorded during the experiment. Mice were sacrificed on day 28 for subsequent experiments with lung tissue harvested from mice by cervical dislocation. 3.3 extraction of RNA
1. And (3) cells: after cells of the 6-well plate were grown, the medium was removed by pipetting, washing with PBS twice, adding 500. Mu.l of trizol to each well, blowing the cells down by up and down pipetting, and adding to a clean 1.5ml RNase-free centrifuge tube.
Tissue: 0.1g of tissue was weighed, placed in a 1.5ml centrifuge tube, 200. Mu.l of trizol was added, the tissue was cut into small pieces with scissors, the tissue was ground with a grinding rod, and 800. Mu.l of trizol was added to make up to 1ml.
2. The tissue was added with 200. Mu.l of chloroform (halving the cells), vigorously shaken for 15s, and left at room temperature for 3 minutes.
3. Centrifugation at 12000rpm at 4℃for 15min separated into 3 layers, the upper RNA layer, the middle layer and the lower protein layer, and the supernatant (about 500. Mu.l) was placed in a clean 1.5ml RNase-free centrifuge tube.
4. RNA was precipitated by adding 500. Mu.l of isopropanol (halving the cells), mixed well and left at room temperature for 10min.
5. Centrifuge at 12000rpm,4℃for 10min.
6. The supernatant was discarded, 1ml of 80% ethanol was added, and the RNA was washed upside down.
7. Centrifuge at 12000rpm,4℃for 5min.
8. The supernatant was decanted, the remaining ethanol was removed as much as possible, left at room temperature for 3min until the remaining ethanol evaporated, and an appropriate amount of DEPC water was added to dissolve RNA (20-40. Mu.l of tissue/10. Mu.l of cells).
9. The integrity of the extracted RNA samples was checked by agarose gel electrophoresis.
10. The concentration and quality of the extracted RNA samples were checked using a NanoDrop 2000.
3.4RT reaction
Total RNA was extracted by Trizol according to the manufacturer's instructions. Evo M-MLV RT Kit with gDNA Clean for qPCRII kit used in reverse transcription experiments was from the Ai Kerui organism (Accurate Biotechnology).
(1) Genomic DNA was removed as follows:
after 10. Mu.l of the solution was mixed, the mixture was allowed to stand at 42℃for 2 minutes in a PCR instrument, and then stored at 4℃for a while.
(2) Reverse transcription, the system is as follows:
after 20. Mu.l of the solution was mixed, the mixture was placed in a PCR apparatus and reacted at 37℃for 15 minutes and at 85℃for 5 seconds, followed by temporary storage at 4 ℃. The cDNA sample after reverse transcription was stored at-20 ℃.
3.5qPCR
(1)
Primer sequences for qRT-PCR
(2) The fluorescent quantitative PCR reaction system is as follows:
(3) Fluorescent quantitative PCR reaction procedure
1)95℃10min
Relative expression, subsequent processing and analysis of the data was performed using Excel and GraphPad Prism. The relative expression of the gene is calculated by a 2 (-delta CT) method.
3.6 extraction of Total protein
(1) PMSF (99:1) was added to RIPA lysate and ready-to-use.
(2) Tissue sample: a suitable amount of tissue (15-20 mg) was added to a 1.5ml EP tube on ice, 300 μl RIPA was added, the sample was sheared using scissors and then ground on ice with a homogenizing rod;
(3) After placing the EP tube on ice for 20min, it was centrifuged at 12000rpm at 4℃for 10min.
(4) The centrifuged supernatant was carefully transferred to a new EP tube and added to a loading buffer for mixing.
(5) Protein denaturation by boiling the sample in boiling water for 10min
(6) After fully reversing and uniformly mixing, carrying out moderate centrifugation at room temperature, and storing the mixture into a refrigerator at the temperature of minus 80 ℃ for standby.
3.7Western Blot
(1) And (3) glue preparation: preparing 10% polyacrylamide gel separating gel, pouring the gel separating gel into the middle of a gel plate, and adding absolute ethyl alcohol for gel pressing. Pouring the absolute ethyl alcohol after the separation gel is solidified, adding the concentrated gel, and inserting into a comb.
(2) Electrophoresis: after concentration and gel fixation, 10 mug of protein sample is added into the sample loading hole, and electrophoresis is carried out at constant current of 30 mA.
(3) Transferring: and taking out gel after electrophoresis, placing the PVDF film into absolute methanol for activation, placing the PVDF film on a film transfer clamp according to the sequence of blackboard-sponge-filter paper-gel-PVDF film-filter paper-sponge-whiteboard, inserting the film transfer clamp into a film transfer groove, and transferring the film for one hour in ice at constant current of 300 mA.
(5) Closing: after transfer, the PVDF membrane was placed in a blocking solution (5% nonfat dry milk) and blocked for one hour.
(6) Incubation resistance: the blocked PVDF membrane was rinsed with TBST, the approximate band of the protein was cut off according to the position of the marker, and the membrane was placed in a diluted primary antibody and incubated overnight at 4 ℃.
(7) The PVDF membrane was rinsed three times for 10 minutes each using TBST.
(8) Secondary antibody incubation: PVDF membranes were placed in diluted secondary antibodies and incubated for one hour.
(7) The PVDF membrane was rinsed again with TBST three times for 15 minutes each.
(8) An appropriate amount of ECL luminescent liquid (a: b=1:1) was added dropwise to the film, exposure was performed on a chemiluminescent imager, and the subsequent results were analyzed and processed using ImageJ and Excel.
3.8 tissue specimens were obtained
Each group of mice was sacrificed 28 days after surgery and tissue specimens were obtained.
(1) The mice were weighed and recorded and sacrificed by cervical dislocation.
(2) Fixing the mouse on an animal experiment operating table in a supine position, shearing off the skin and hair of the chest wall of the mouse along the front midline, extracting the surface muscle of the chest wall by forceps, shearing off diaphragm muscle and two side ribs by scissors along the gesture, and exposing the two sides of the lung;
(3) Drawing materials: completely peeling the lung tissue of the mouse by using an ophthalmic scissors, putting the mouse into a culture dish filled with normal saline, washing the residual blood on the surface, and recording the lung weight;
(6) And (3) sample collection: the pathogenically stained specimens were stored in 1.5ml centrifuge tubes containing 4% paraformaldehyde; placing the RNA tissue sample into a centrifuge tube with the capacity of 1.5ml RNase-free; western Blot specimens were placed in a conventional centrifuge tube with a capacity of 1.5ml, and the experimental tools were sorted.
(7) And (3) storing a specimen: the pathological staining specimen is preserved at normal temperature or 4 ℃; the RNA tissue sample and the Western Blot sample are taken and then put into-80 ℃ for preservation.
3.9HE staining
Fixing the left lung of the mouse in 4% paraformaldehyde for 24 hours, taking out 1/3 tissue in the middle of the left leaf of the fixed mouse, placing the tissue into a tissue embedding box, washing the tissue embedding box with tap water, dehydrating and immersing the left lung of the mouse in wax through a cylinder in sequence, and finishing embedding; then, the sheet was sliced to a thickness of 5. Mu.m. Dewaxing and rehydrating the prepared paraffin sections sequentially through a cylinder; and finally, carrying out corresponding tissue staining on the sections after rehydration.
Paraffin sections were routinely dewaxed into water: the dyeing method comprises the steps of respectively extracting xylene I, xylene II and xylene III for 10min, 100% alcohol I and xylene II for 5min, 95% alcohol I and xylene II for 5min, 80% alcohol for 5min, 70% alcohol for 5min, and immersing the mixture in PBS for washing for about 15 s.
Hematoxylin staining: adding hematoxylin dye into a staining jar, soaking for 5-15min (7 min), and washing with water for 1min.
Differentiation and blue returning: after hematoxylin staining, the solution was immersed in 1% hydrochloric acid alcohol (1 mL hydrochloric acid+99 mL 75% alcohol) for 2-5s. Differentiation to tissues appeared red, and washing with running water until the dripping water drops were clear.
Eosin staining: immersing the glass slide after bluing into an eosin staining jar for staining for 2-3min. Flushing with running water until clear.
And (3) dehydration and transparency: washing with water, 95% alcohol 10s, 100% alcohol 10s, and xylene 3 times, 30s each time.
Sealing piece: a drop of neutral gum was added dropwise to the dehydrated and transparent slide, then covered with a cover slip, air-dried in a fume hood for one day, and scanned by microscopic examination.
3.10Masson staining
Conventional dewaxing of paraffin sections to water, putting the sections into Bouin liquid, mordant dyeing for 2 hours, and then flushing under running water until the yellow color of the samples on the sections disappears; soaking in azure blue dyeing liquid for 3min, and slightly washing with water; soaking in hematoxylin staining solution for 3min, and slightly washing with water; acid ethanol is differentiated for about 30s, and the acid ethanol is washed by running water to differentiate and return to blue for 10min; dip-dyeing in ponceau dyeing liquid for about 10min, and slightly adding water for washing; placing the slices in a phosphomolybdic acid solution for about 10min; dip-dyeing in aniline blue dyeing liquid for 5min; treating in weak acid solution for 2min; quick dehydration of 95% ethanol, dehydration of absolute ethanol for 3 times, 10s each time; the xylene is transparent for 3 times, each time for 1min; and (5) performing lens inspection on the neutral resin sealing piece.
Analysis of results
Therapeutic action and mechanism of M-siTGF-beta 1 on PF
3.1 tracheal nebulization of M-EXO delivery to the lung
As shown in FIG. 7, we further followed the distribution of M-EXO labeled with a lipophilic tracer DiR, which showed the ideal fluorescent signal cell membrane by incorporation into in vivo imaging. Mice were anesthetized and then DiR-labeled M-EXO was delivered by nebulization. Images of the whole body and major organs were captured at the indicated time points, diR fluorescence was only observed in the lungs after nebulization of M-EXO, and the fluorescent signal was gradually diminished after day 3, so the following experiment was dosed twice a week.
3.2 establishment of PF mouse model
To study the therapeutic role of M-siTGF- β1 in BLM-induced idiopathic pulmonary fibrosis, we induced the mouse PF model by intratracheal injection of BLM (1.5 mg/kg) and nebulized M-siTGF- β1 administration on day 14, mice were sacrificed on day 28 and their lung tissues were taken for detection of TGF- β1 expression.
As shown in FIG. 8, the expression level of TGF-. Beta.1 in Control group, BLM group, BLM+M-EXO group, BLM+Naked siTGF-. Beta.1 group, BLM+M-siNC group, BLM+M-siTGF-. Beta.1 treatment group was examined by qRT-PCR method. The qRT-PCR result shows that BLM can rapidly induce high expression of TGF-beta 1, and after M-siTGF-beta 1 treatment, the expression of TGF-beta 1 is obviously reduced. BLM successfully induced pulmonary fibrosis in mice and PF mice model M-simf- β1 was successfully established.
3.3 influence of M-siTGF-. Beta.1 on the general status of PF model mice
As shown in FIG. 9, to further observe whether M-siTGF-. Beta.1 has a PF effect for treatment of BLM induction, we observed the general status of mice, showing that Control group mice were normal in activity, hair gloss and respiratory rate; BLM mice showed reduced activity, loss of gloss and increased respiratory rate of hair relative to Control. The status of the mice in the BLM+M-EXO group, the BLM+Naked siTGF-. Beta.1 group, the BLM+M-siNC group was similar to that in the BLM group, and the BLM+M-siTGF-. Beta.1 treatment group was slowly improved, but still worse than that in the Control group.
As shown by the weight change results of the mice, the weight of the mice in the Control group keeps stably rising; BLM group mice had a drop in body weight due to the aerosolized bleomycin in the trachea; the blm+m-siTGF- β1 group mice began to gain weight slowly after administration of M-siTGF- β1, but were lighter relative to the Control group mice, compared to the BLM group, blm+m-EXO group, blm+naked siTGF- β1 group, blm+m-siNC group. At the end of the experiment, the body weight of the blm+m-siTGF- β1 group of mice was reduced minimally compared to the initial body weight, suggesting that BLM may inhibit the increase in body weight of mice, while M-siTGF- β1 may improve the body weight inhibition of mice caused by BLM.
Bleomycin-induced fibrosis mice died early in the PBS-treated group. In contrast, mice treated with 1.5mg/kg siRNA had the latest death and highest final survival rate throughout the experiment, indicating that M-siTGF- β1 increased overall survival of bleomycin-induced fibrotic mice.
As shown in fig. 10, the mouse model was sacrificed by cervical removal, the lung tissue of the mouse was isolated, and the volume and color of the lung tissue were visually observed, and the results showed that the two lung tissue of the Control group mice were more normal in volume and light pink in color; the BLM mice had severe hyperemia seen in both lungs, and the volume was slightly smaller than that of the Control group; compared with the BLM group, the BLM+M-EXO group, the BLM+Naked siTGF-beta 1 group and the BLM+M-siNC group, the hyperemia state of the mice in the BLM+M-siTGF-beta 1 group is obviously improved, and the volume of the hyperemia state is between that of the Control group and the BLM group. In addition, we also measured lung weight and found that after 28 days of BLM induction, the weight of blm+m-simf- β1 group of lungs was significantly reduced compared to BLM group, suggesting that M-simf- β1 could improve the damaging congestion of BLM-induced PF model murine lung tissue, but if the effect exerted by inhibiting the pulmonary fibrosis process would need further investigation.
3.4M-siTGF- β1 Effect on lung histopathology in PF model mice
To assess whether M-siTGF- β1 could ameliorate BLM-induced idiopathic pulmonary fibrosis in mice, C57BL/6 mice were aerosolized with M-siTGF- β1 on day 14 after tracheal injection of bleomycin, and the lung tissue of mice sampled on day 28 was observed for histopathological changes in the lungs by H & E, masson and sirius red staining.
As shown in fig. 11, in the Control group, the alveolar tissue of the mice was substantially normal, and no pathological change was seen under the light microscope; BLM group showed morphological lesions characterized by partial intra-lobular, intra-alveolar hemorrhage, interstitial edema, and intra-lobular inflammatory cell infiltration; the BLM+M-EXO group, the BLM+Naked siTGF-. Beta.1 group, the BLM+M-siNC group are similar to the BLM group; bleomycin-induced tissue damage was significantly reduced in the blm+m-siTGF- β1 group with little or no infiltration of inflammatory cells. The results show that M-siTGF-beta 1 can reduce bleomycin-induced pathological damage to lung tissue.
3.5M-siTGF-beta 1 Effect on PF model murine inflammatory factors
The above results show that M-siTGF-beta 1 can alleviate the fibrosis of the PF mice induced by bleomycin and improve the pathological lesions of lung tissues, but the action mechanism is not clear, and the pathological lesions of lung tissues of PF are mainly mediated by pro-inflammatory cytokine infiltration, so in order to know whether M-siTGF-beta 1 can influence the inflammatory cytokine production of PF model mice, we have conducted the following study.
As shown in FIG. 12, the lung tissue of the mice was isolated, and the expression level of IL-6 was detected by q RT-PCR. The results showed that the expression level of BLM group IL-6 was significantly increased as compared to Control; the expression level of IL-6 was slightly decreased in the BLM+M-EXO group, the BLM+Naked siTGF-. Beta.1 group, and the BLM+M-siNC group, and significantly decreased in the BLM+M-siTGF-. Beta.1 group, as compared with the BLM group. The results indicate that M-siTGF-beta 1 can reduce the secretion of mouse inflammatory cytokines induced by BLM, thereby reducing inflammatory response of lung tissues and improving pulmonary fibrosis.
3.6 influence of M-siTGF-beta 1 on the expression of PF model murine EMT and fibrosis related molecules
In vitro studies we have found that M-siTGF- β1 is able to affect BLM-induced secretion of mouse pro-inflammatory cytokines, thereby reducing the inflammatory response of lung tissue and improving pulmonary fibrosis. And in vitro studies demonstrated that M-siTGF- β1 was able to affect EMT and pulmonary fibrosis-related molecule expression in PF cell models, we examined the expression of epithelial cell markers (E-cadherein), mesenchymal cell markers (Vimentin) and fibrosis-related molecules Collagen I, fibrinecin, a-SMA, CTGF at the mrna level in order to further investigate the EMT effect of TGF- β1siRNA silencing on BLM-induced pulmonary fibrosis in mice.
As shown in FIG. 13, first, we examined the expression level of the epithelial cell marker E-cadherin, and the results showed that BLM effectively reduced E-cadherin expression in mouse lung tissue, while silencing of TGF-. Beta.1 siRNA gene restored most of E-cadherin expression. This suggests that BLM may induce activation of epithelial cells, while silencing of TGF- β1 gene may inhibit epithelial cell activation induced by BLM.
Furthermore, we examined the expression level of the mesenchymal cell marker Vimentin. The expression level of the vector is detected by q RT-PCR on the mRNA level, and the result shows that BLM effectively increases the expression of Vimentin in lung tissues of mice, and M-siTGF-beta 1 can reduce the expression of Vimentin. This suggests that BLM may induce the production of mesenchymal cells, while M-siTGF- β1 may inhibit the production of mesenchymal cells induced by BLM.
It is described that during fibrosis MMP2 and MMP9, known as gelatinase a and gelatinase B, respectively, are normally upregulated, which can lead to pulmonary fibrosis by degrading type IV collagen to disrupt basement membrane and induce pulmonary tissue remodeling and alveolar thickening. Here, the expression levels of MMP2, MMP9 were assessed by q PCR analysis. As shown in FIG. 14, the expression of MMP2 and MMP9 was significantly up-regulated in BLM treated mice compared to control mice. Treatment with M-siTGF-. Beta.1 significantly inhibited BLM-mediated MMP2 and MMP9 expression.
Finally we examined the expression levels of the fibrosis-associated molecular ECM protein Collagen I (involved in the formation and remodelling of fibrotic ECM in the lung), fibrinectin, α -SMA, CTGF. First, the expression level was measured at the mRNA level using qRT-PCR, as shown in FIG. 15, and the results showed that BLM group effectively increased the expression of collagen I, fn, α -SMA, CTGF in mouse lung tissue, while M-siTGF- β1 could decrease the expression of collagen I, fn, α -SMA, CTGF. This suggests that BLM may induce ECM formation, while M-siTGF- β1 may inhibit ECM formation that may be induced by BLM.
Claims (7)
1. A pharmaceutical composition for treating pulmonary fibrosis, SITGF-beta 1.
2. Use of a bovine milk exosome-siTGF- β1 medicament according to claim 1 in the treatment of pulmonary fibrosis.
3. A method for preparing a bovine milk exosome-siTGF- β1 medicament according to any one of claims 1 to 2, wherein: and (3) introducing siRNA mediating TGF-beta 1 low expression into an exosome by taking the bovine milk exosome as an RNAi delivery system to form M-siTGF-beta 1, namely a bovine milk exosome-siTGF-beta 1 drug, wherein the sense strand of the siRNA sequence mediating TGF-beta 1 low expression is shown as SEQ.ID.NO.1, and the antisense strand is shown as SEQ.ID.NO. 2.
4. A method for preparing a bovine milk exosome-siTGF- β1 drug as claimed in claim 3, wherein: the preparation method of the cow milk exosome-siTGF-beta 1 drug is an electrotransformation method, and comprises the following steps: bovine milk exosomes were mixed with siRNA in a ratio in 500 μl of PBS solution, i.e. 500 μl system. Incubation was performed for 30min at 4℃after mixing, electrotransformation was performed as follows: 220V, pulse time 10ms, pulse 3 times, interval 2s. After electrotransformation, the treated samples were removed and incubated at 37℃for 1h to promote recovery of the exosome membrane.
5. The method for preparing the bovine milk exosome-siTGF-beta 1 drug according to claim 4, wherein: the ratio of bovine milk exosomes to siRNA is 1:1,1:5,1:10 or 1:15, preferably 1:5.
6. A method for preparing a bovine milk exosome-siTGF- β1 drug as claimed in claim 3, wherein: the preparation method of the cow milk exosome-siTGF-beta 1 drug is an ultrasonic method and comprises the following steps: bovine milk exosomes and siRNA were mixed in PBS at a ratio of 1:5 (mass/mass), 30w,6 on/off cycles of 30 seconds, with a cooling time of 2 minutes between each cycle. After sonication, the solution was incubated at 37 ℃ for 30 minutes to restore the exosome membrane.
7. A method for preparing a bovine milk exosome-siTGF- β1 drug as claimed in claim 3, wherein: the preparation method of the cow milk exosome-siTGF-beta 1 medicine is an improved CaCl 2 A method comprising the steps of:
mixing siRNA with cow milk exosomes in PBS, and adding CaCl 2 (final concentration 100 mM).
The final volume was adjusted to 500 μl using sterile PBS. The mixture was placed on ice for 30 minutes. After thermal shock at 42 ℃ for 60 seconds, the mixture was left on ice for an additional 5 minutes.
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