CN117243977A - Application of isoxazole-9 combined mesenchymal stem cells in preparation of medicines for preventing or treating acute lung injury - Google Patents
Application of isoxazole-9 combined mesenchymal stem cells in preparation of medicines for preventing or treating acute lung injury Download PDFInfo
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- CN117243977A CN117243977A CN202311318623.9A CN202311318623A CN117243977A CN 117243977 A CN117243977 A CN 117243977A CN 202311318623 A CN202311318623 A CN 202311318623A CN 117243977 A CN117243977 A CN 117243977A
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Abstract
The invention provides an application of isoxazole-9 combined mesenchymal stem cells in preparing a medicament for preventing or treating acute lung injury, which is used for preventing or treating acute lung injury. The invention also provides application of the composition comprising isoxazole-9 and mesenchymal stem cells in preparing medicines for preventing or treating acute lung injury. The invention discovers that ISX-9 can improve the capacity of MSCs for treating ALI for the first time, and proves that ISX-9 can improve the duration and strength of the action of mesenchymal stem cells in lung and enhance the capacity of the mesenchymal stem cells for treating ALI. The proposal is verified in cell and mouse ALI models, and has potential clinical application value.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to application of isoxazole-9 combined mesenchymal stem cells in preparation of a medicine for preventing or treating acute lung injury.
Background
At present, the acute lung injury (Acute lung injury, ALI) and the acute respiratory distress syndrome (Acute respiratory distress syndrome, ARDS) are clinically treated by mainly adopting a lung protective ventilation means and assisting with medicaments such as diuretics and the like, and no specific treatment method exists.
ALI and ARDS are typically characterized by diffuse alveolar lesions, manifested by elevated levels of inflammatory cells and inflammatory factors, increased alveolar exudates, normal epithelial-endothelial barrier dysfunction, the formation of a hyaline membrane within the alveolar space, and ultimately leading to hyaline membrane fibrosis.
Mesenchymal stem cells (Mesenchymal stem cell, MSCs) are cells that have self-renewing ability and can be induced to differentiate. Research shows that MSCs mediate immune regulation by paracrine of various cytokines such as IL-1 receptor antagonists, IL-10, VEGF and the like, thereby stabilizing the internal immune environment, playing an anti-inflammatory role and promoting lung injury repair. MSCs are relatively easy to culture, amplify and assess viability in vitro, and several studies have found that different types of MSCs have potential for use in a variety of diseases after transplantation or co-culture.
However, the multidirectional differentiation and targeting instability of MSCs makes it difficult to maintain a stable phenotype, and the paracrine capacity of MSCs is also inhibited by inflammatory environments or anti-inflammatory stimuli.
Keratinocyte growth factor (Keratinocyte growth factor, KGF) has a high level of receptor specificity and plays an important role in tissue repair and maintenance of mucosal barrier integrity. The research shows that KGF can be used as a paracrine medium of MSCs to mediate the interaction of mesenchymal and epithelial and promote the proliferation and transformation of epithelial cells. KGF can also increase alveolar clearance by affecting the activities of ENaC and Na, K-ATPase, alleviate pulmonary edema and alleviate inflammation of acute lung injury. The expression of KGF was reduced at ALI, but the application of exogenous KGF to the treatment of ALI in clinical therapy was unsuccessful (Sadeghi S, kalhor H, panahi M, abolhasani H, rahimi B, kalhor R, mehrabi A, vahdatimia M, rahimi H.Keratinoche growth factor in focus: A comprehensive review from structural and functional aspects to therapeutic applications of palifemin.int J Biol macromol.2021Nov30; 191:1175-1190.doi:10.1016/j.ijboom.2021.09.151.Epub 2021Oct 1.PMID:34606789).
Recent studies have found that isoxazole-based small molecule N-cyclopopyl-5- (thiophen-2-yl) -isoxazole-3-carboxamide (ISX) selectively affects specific stem cell differentiation and proliferation. ISX compounds can change the stability of mRNA and gene expression by combining RNA and RNA binding protein, so as to promote gene expression and induce stem cell differentiation. Among ISX compounds, isoxazole 9 (ISX-9) has the highest neurogenic activity and sufficient water solubility, and can enhance the neuron differentiation of human cerebral cortex neuron cells, adult mouse whole brain and subventricular zone progenitor cells and P19 embryonic cancer cells. Meanwhile, through establishing a KGF-hCMSCs (human chorionic mesenchymal stem cells) compound screening model for promoting KGF expression, the fact that ISX-9 activates hCMSCs to express KGF and Ang-1 to a greater extent is found.
The prior literature already reports that MSCs or ISX-9 are singly applied to diseases such as neuronal injury, pancreas, cardiovascular diseases and the like, but no related report of combined application of MSCs and ISX-9 in ALI and ARDS treatment exists.
Disclosure of Invention
The inventor discovers that the isoxazole-9 can regulate and control a downstream ERK-TAU-beta-catenin signal shaft through a membrane receptor protein NGFR, promote the paracrine KGF function of mesenchymal stem cells and strengthen the effect of MSCs in repairing tissue injury.
The invention aims to promote MSCs to secrete KGF, relieve inflammatory reaction and repair tissue injury through isoxazole-9.
The invention discloses application of isoxazole-9 combined mesenchymal stem cells in preparation of medicines for preventing or treating acute lung injury.
The invention discloses a use of a composition comprising isoxazole-9 and mesenchymal stem cells in preparing a medicament for preventing or treating acute lung injury.
Preferably, the isoxazole-9 has the chemical formula:
preferably, the mesenchymal stem cells are one of human placental chorion-derived mesenchymal stem cells and rat bone marrow mesenchymal stem cells.
Preferably, the use is to reduce pulmonary oedema occurring in acute lung injury.
Preferably, the use is to alleviate inflammatory responses in acute lung injuries.
Preferably, the use is to promote repair of the lung barrier following acute lung injury.
Preferably, the use is to increase the expression of KGF gene.
Compared with the prior art, the invention has at least the following beneficial effects:
1. the invention applies ISX-9 and MSCs to treat acute lung injury, wherein ISX-9 is a small molecular compound which is determined by a compound screening model and can promote MSCs to secrete KGF, and in the microenvironment of inflammation burst in vivo, the paracrine capacity of MSCs is recovered, the anti-inflammatory effect is promoted and the lung injury is repaired. MSCs are candidates for cell therapies whose immunomodulatory and inflammatory inhibition functions can rapidly alleviate lung injury and promote lung tissue repair. The ability of KGF to promote alveolar epithelial cell proliferation makes it of great potential for the treatment of ALI. ISX-9 not only regulates and controls the differentiation and proliferation of mesenchymal stem cells, but also targets the promoter of KGF gene, promotes MSCs to express KGF highly, can improve the effectiveness of paracrine of MSCs, and enhances the therapeutic effect of clinical transformation of MSCs.
2. The invention discovers that ISX-9 can improve the capacity of MSCs for treating ALI for the first time, and proves that ISX-9 can improve the duration and strength of the action of mesenchymal stem cells in lung and enhance the capacity of the mesenchymal stem cells for treating ALI. The proposal is verified in cell and mouse ALI models, and has potential clinical application value.
3. The invention can be applied to a pharmaceutical composition for treating acute lung injury, the pharmaceutical composition comprises mesenchymal stem cells and ISX-9, and in experiments, sprague-Dawley rats (6-8 weeks old, weight 220+ -20 g) recommended dose of rat BMSCs (bone marrow mesenchymal stem cells) (Sai-Biotech Co.) is 1×10 6 Once every other day, ISX-9 (MCE Biotech Co.) was 25mg/kg once daily.
4. The pharmaceutical dosage forms formed by the invention can be intravenous injection agents and/or intraperitoneal injection agents, and can also be other pharmaceutically acceptable carriers.
Drawings
FIG. 1 shows the construction of CV-123-KGF-master-Luc recombinant plasmid according to the present invention.
FIG. 2 shows how the results of the dual luciferase reporter gene reflect KGF activation in the examples of the invention.
FIG. 3 shows that qPCR experiments in examples of the present invention reflect that ISX-9 significantly promotes the expression of KGF and Ang-1 in MSCs at the mRNA level.
FIG. 4 shows the optimum acting time and concentration of the ISX-9 for promoting BMSCs to secrete KGF by Western blot and RT-qPCR in the embodiment of the invention.
FIG. 5 is an in vivo experiment in rats reflecting that ISX-9 promotes BMSCs transplantation to alleviate ALI inflammation in an embodiment of the present invention.
FIG. 6 is a graph showing that HE staining of rat lung tissue sections reflects ISX-9 promoting improvement of ALI lung pathology by BMSCs in an embodiment of the present invention.
FIG. 7 shows that the rat lung tissue RT-qPCR experiment in the example of the present invention reflects that ISX-9 promotes BMSCs to reduce inflammatory factor expression in lung tissue.
FIG. 8 is a graph showing the effects of ISX-9 enhanced BMSCs in lung tissue as reflected in live rat imaging in an example of the present invention.
FIG. 9 shows Western blot detection of lung tissue of rats to promote expression of KGF in lung tissue after BMSCs transplantation by ISX-9 in the examples of the present invention.
FIG. 10 shows that immunofluorescence of rat lung tissue sections in the examples of the present invention reflects the promotion of KGF expression in lung tissue by treating ALI with ISX-9 in combination with BMSCs.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention.
Embodiment one: in vitro drug effect study of isoxazole-9 combined mesenchymal stem cells
1. Establishment of stable transgenic MSCs strain for stable expression of KGF gene promoter
1.1 amplification of KGF Gene promoter sequences
The promoter genomic sequence of KGF Gene was found from NCBI Gene library, and KGF-promoter was searched for, which contained 3000bp of the promoter sequence of KGF Gene as the target promoter. And designing a primer by using primer primer5.0 primer design software, designing a primer of a KGF promoter, performing PCR (polymerase chain reaction) by taking an artificially synthesized promoter sequence KGF as a template, amplifying to obtain a promoter sequence of a target fragment KGF, and identifying a PCR product by using agarose gel electrophoresis to obtain a target product with PacI and BamHI enzyme cutting sites at two ends of about 3000bp.
Primer design:
KGF-F and KGF-R are consistent with the section [0024] of the specification of the screening model for treating acute lung injury by targeting mesenchymal stem cells of KGF promoter (publication No. CN 113403280A) in the reference patent document.
1.2 preparation of Carrier CV123
CV123 vector (purchased from Ji Kai gene) containing the double luciferase reporter gene was subjected to PacI and BamHI double cleavage treatment, and then purified and recovered to obtain CV123-Luc.
1.3 construction of recombinant plasmid of luciferase reporter Gene carrying KGF Gene promoter
After PacI and BamHI double enzyme digestion treatment, purifying and recovering the sequence of the KGF gene promoter, connecting with CV123-Luc, then transforming into E.coli DH5 alpha competence, inversely culturing for 16-20h on LB solid culture medium at 37 ℃, picking ampicillin resistance positive monoclonal, extracting plasmid DNA, and obtaining recombinant plasmid CV 123-KGF-precursor-Luc. Construction of the recombinant plasmid is shown in FIG. 1.
1.4 preparation of lentiviral solution containing KGF Gene promoter
The recombinant plasmid CV123-KGF-promoter-Luc was co-transfected with the lentiviral packaging helper plasmids Helper1.0 and Helper2.0 in 293T cells. The virus was harvested 72h after the completion of transfection, and concentrated and purified to obtain a lentiviral solution LV-KGF with a titer of 2E+8 TU/ml.
1.5 screening of mesenchymal Stem cells stably expressing KGF promoter
The lentivirus solution was transfected into human hCMSCs (supplied by the China Hospital, university of double denier), cultured at 37℃for 12-16 hours, and replaced with complete medium, and the culture was continued. After 48-72h of infection, the cells were continuously cultured in puromycin-containing 2ug/ml medium, and puromycin-containing medium was changed every 2-3 days until uninfected control cells were all killed by puromycin, while no cells in the infected virus group died. Finally obtaining the mesenchymal stem cell stable transgenic strain hCMSCs-KGF for stably expressing the KGF promoter.
2. Double-luciferase reporter gene experiment for detecting influence of ISX-9 on activation degree of KGF in hCMSCs
hCMSCs stable transgenic strain expressing KGF promoter every 2×10 4 The individual cells were plated in 96-well plates and added with ISX-9 (available from Tao Shu organisms) and 8 other compounds related to the biological function of stem cells, cardiomyopathy C (Cardiogenol C), 1-METHYL-N- [2-METHYL-4- [2- (2-tolyl) diazenyl]phenyl-1H-pyrazole-5-carboxamide (CH-223191), 2- (2-amino-3-methoxyphenyl) -4H-1-benzopyran-4-one (PD-98059), 4- [4- [3- (pyridin-2-yl) -1H-pyrazol-4-yl]Pyridin-2-yl]-N- (tetrahydropyran-4-yl) benzamide (GW-788388), 1- (4-methoxyphenyl) -2-methyl-3-nitro-1H-indol-6-ol (ID-8), kenparone, 6-bromoindirubin-3' -acetoxime (BIO-acetime), sodium valproate (all of the above 8 compoundsPurchased from Tao Shu biosystems), the blank control is culture medium added with DMSO, 4 holes are formed in each group, 100 mu L of luciferase detection reagent is added after 72 hours, the mixture is kept stand at room temperature for 15 minutes, renin fluorescence intensity is detected by an enzyme-labeled instrument, then 100 mu L of double luciferase detection working solution is added, the mixture is incubated at room temperature for 15 minutes, and the fluorescence intensity of firefly reporter genes is detected by the enzyme-labeled instrument. According to the formula: degree of activation of the target gene = RLU measured by firefly luciferase reporter/RLU measured by renilla luciferase reporter, the degree of activation of compound-targeted KGF to promote luciferase expression was calculated.
The result shows that: referring to FIG. 2, the relative increase in ISX-9 luciferase activity was 3.18-fold, and the expression of KGF was maximally activated compared to other compounds.
3. RT-qPCR experiment detects influence of ISX-9 on hCMSCs expressing KGF mRNA and Ang-1mRNA 3.1 and extracts total RNA of cells
Inoculating hCMSCs in a 6-hole plate, adding ISX-9 with different concentrations into the 6-hole plate after 12 hours, discarding the culture medium when the cell fusion degree reaches 90%, washing the cells with PBS, adding 500 mu L of lysate, shaking for 1-2 minutes at room temperature, adding equal volume of absolute ethyl alcohol, fully and uniformly mixing, adding a centrifugal column, centrifuging for 2 minutes at 4000g, discarding the liquid, adding 500 mu L of washing liquid, centrifuging for 2 minutes at 12000g, and taking out the centrifugal column into a new RNase-free EP tube. 25. Mu.L of eluent is added to the column, the column is left to stand for 3 minutes with the lid open, and the column is centrifuged at 12000g for 2 minutes, and the obtained liquid is the total RNA extracted.
3.2 Total RNA reverse transcription
TABLE 1 reverse transcription System
Reverse transcription reaction conditions: the reaction was carried out at 42℃for 15 minutes and at 95℃for 30 seconds, and cDNA was obtained after the completion of the reverse transcription step.
3.3, RT-qPCR assay
The cDNA obtained in the above 3.2 was subjected to a sterilized ddH 2 O is diluted 5 times, and qPCR reaction system is configured：
TABLE 2 reaction System for configuring qPCR
TABLE 3qPCR reaction procedure
qPCR plates were centrifuged in a centrifuge for 2 min and then checked on-machine.
3.4 analysis of Experimental results
The calculation formula is as follows: delta ct=ct (target gene) -Ct (reference gene)
ΔΔct= Δct (experimental group) - Δct (control group)
RQ＝2 -△△Ct
The result shows that: referring to FIG. 3, ISX-9 significantly promoted hCMCs to express KGF mRNA and Ang-1mRNA at the mRNA level. KGF mRNA increased more than 20 fold and Ang-1mRNA increased 28 fold after 10. Mu.M ISX-9 was applied to hCMCs. The effect of ISX-9 concentration less than 10 mu M on BMSCs is most remarkable, and KGF mRNA is increased by 2.3 times compared with the control group. It is proved that ISX-9 can promote mesenchymal stem cells to secrete KGF and Ang-1 cytokines beside, thereby repairing damaged lung tissues.
4. Western-blot and qPCR experiments detect influence of ISX-9 on BMSCs to secrete KGF and Ang-1 4.1, and extract total cell proteins
BMSCs were inoculated in six-well plates, and after cell attachment, different concentrations of ISX-9 diluted with medium were applied to BMSCs for 24 hours, the medium was discarded, and washed three times with PBS. To 1mLRIPA cell lysate was added 10. Mu.L of leupeptin and 1. Mu.LDTT, and then 5. Mu.L of PMSF was added and mixed. To the cell wells, 150. Mu.L of total protein lysate was added and allowed to act by gentle tapping, and reacted at-20℃for 15 minutes. The cell is blown by sucking the lysate with a gun head, transferring the lysate containing the cell into a 1.5mL centrifuge tube, centrifuging at 12000g for 25 minutes at 4 ℃, and collecting the supernatant to obtain the total cell protein.
4.2 protein quantification
The concentration of BSA standard was diluted to a final concentration of 1. Mu.g/. Mu.L, 0.5. Mu.g/. Mu.L, 0.25. Mu.g/. Mu.L, 0.125. Mu.g/. Mu.L, 0.0625. Mu.g/. Mu.L, 0.03125. Mu.g/. Mu.L, and the required BCA working fluid volumes were prepared as indicated by the BCA protein quantification kit. Adding 25 mu L of diluted BSA standard substance and protein sample into a well of a 96-well plate with a mark, carrying out 3 parallel reactions on each sample, adding BCA working solution, uniformly mixing, incubating for 30 minutes at 37 ℃, cooling to room temperature, measuring the absorbance value of each sample at 562nm and the BSA standard substance by an enzyme-labeled instrument, drawing a protein concentration standard curve, and calculating to obtain the protein sample concentration. Adding a loading buffer solution into a protein sample, mixing uniformly by vortex, putting the mixture at 95 ℃ for 15 minutes to denature the protein, and preserving the mixture at-20 ℃ for standby after restoring to room temperature.
4.3 Western-blot experiments
Protein samples were mixed and loaded according to experimental groups and electrophoresed in Tris-Glycine electrophoresis buffer at a voltage of 70V for 120 min. The transfer fluid was prepared according to the instructions of the fast transfer fluid manufacturer. PVDF membrane was activated with methanol for 2 minutes and transferred to the transfer solution. Cutting out corresponding strip glue from the taken gel according to the size and the grouping of the protein, wetting the gel with a film transfer liquid, placing the gel on a sandwich film transfer clamping plate, pouring the film transfer liquid, setting a constant current of 400mA, and adjusting the film transfer time. And taking out the membrane after membrane transfer, soaking in TBST solution, and cleaning for 3 times with shaking for 5 minutes each time. After the cleaning is finished, the mixture is sealed in a quick sealing liquid for 20min. And preparing primary antibody and secondary antibody incubation liquid by using an antibody diluent. After blocking, the membranes were washed 3 times with TBST for 10 minutes each and incubated overnight at 4℃in a refrigerator. The next day after membrane recovery to room temperature, the membranes were washed 3 times with TBST for 5 minutes each and the secondary antibody was incubated for 2 hours. After the end, the membrane was washed 3 times with TBST for 5 minutes each, the strips were exposed using ECL hypersensitivity color development solution, and the strip pictures were imported into imageJ software for quantitative analysis.
4.4, RT-qPCR experiments
RNA reverse transcription and RT-qPCR were performed in the same manner as in example 3.
The result shows that: referring to FIG. 4, ISX-9 promoted the expression of KGF and Ang-1 by BMSCs at the protein level, and at an action concentration of 5. Mu.M, an action time of 24 hours promoted the secretion of KGF by BMSCs to the maximum.
EXAMPLE two in vivo efficacy study of ISX-9 in combination with BMSCs for ALI
1. Establishment of BMSC cell line for stabilizing low NGFR Gene expression
Day 1: BMSCs were digested and a cell suspension was prepared and seeded into six well plates. The culture was carried out at 37℃for 16-24 hours, and the inoculation density was 20% after 24 hours.
Day 2: infection, adding a suitable volume of a viral liquid (shNGFR lentiviral stock solution (available from Shanghai Ji Kai Gene science and technology Co., ltd.) to the size of the inoculated well plate, the number of cells per well of the six well plate was about 2X10 5 10. Mu.L of viral fluid was added according to the appropriate cell MO values. After 16 hours of culture in the incubator, the virus solution was removed and replaced with a normal medium.
Day 3-4: continuing to culture, changing the liquid of the cells in the middle, and keeping the activity of the cells.
Day 5: about 72 hours after infection, the infection efficiency was observed.
Day 6: after confirming the infection efficiency, stable transformants were selected, and after 72 hours of lentivirus infection (70-80% fusion), puromycin was added to the medium at a suitable concentration, and after 48 hours, the empty cells and puromycin groups all died, leaving all positive cells in the infected virus group. During the subsequent culture, puromycin with low concentration is maintained for continuous culture, and cells with stably knocked down NGFR are frozen.
2. Establishing an experimental animal model
Acquisition of BMSCs for use in the experiments can be obtained in the manner described in example one. 36 adult male Sprague-Dawley rats were randomly divided into 6 groups:
control group: n=6;
ALI group: 5mg/kgLPS, n=6;
ISX-9 treatment of ALI group alone: 5mg/kg LPS,25mg/kg ISX-9, n=6;
BMSCs alone treatment ALI group: 5mg/kg LPS,1 x10 6 BMSCs，n＝6；
BMSCs and ISX-9 combination therapy ALI group: 5mg/kg LPS,25mg/kg ISX-9, 1X 10 6 BMSCs，n＝6；
BMSCs (shNGFR-BMSCs) knocked down nerve growth factor receptor gene in combination with ISX-9 for ALI group: 5mg/kg LPS,25mg/kg ISX-9, 1X 10 6 shNGFR-BMSCs，n＝6。
After the rats were anesthetized with pentobarbital sodium, LPS (O55: B5, purchased from Sigma Co.) at a concentration of 5mg/kg was dissolved in 10. Mu. PBS, instilled into the rat trachea, and induced to establish the rat ALI model. After 4 hours, the groups were grouped as described above, with 1 x10 injections per day tail vein 6 BMSCs (available from Saiko Biotech Co., ltd.) were intraperitoneally injected with 25mg/kg of ISX-9 (available from MCE Co.) daily for one week, and rats were euthanized by intraperitoneal injection of excess chloral hydrate, and BALF and lung tissue were collected and stored at-80 ℃.
2. Detection of rat lung tissue Dry to Wet ratio, superoxide dismutase (SOD) Activity in lung tissue and protein content in alveolar lavage fluid
Right lung of 3 rats was randomly taken from each group, wet weight thereof was weighed using a precision electronic balance, then left lung tissue was put into an oven at 65 ℃ for baking for 48-72 hours to obtain constant weight (dry weight), W/D ratio was calculated to reflect pulmonary edema, and average value and SD value thereof were calculated.
Samples were processed according to the instructions of the superoxide dismutase (SOD) activity detection kit manufacturer. About 0.1g of right lung tissue was weighed randomly in each group of rats, n=3, 1mL of the extract was added, ice bath homogenization was performed, centrifugation was performed at 8000g at 4 ℃ for 15min, and the homogenized supernatant was placed on ice. Adding corresponding reagents according to the instruction, detecting the absorbance of the sample at the wavelength of 560nm, and calculating the activity of SOD in the tissue according to a formula.
The calculation formula is as follows:
percent inhibition = (Δa blank- Δa assay) +.Δa blank x 100%
SOD activity (U/g mass) = [ percent inhibition
=11.11×percent inhibition ≡ (1-percent inhibition) ≡w×f
3 rat lung tissues were randomly taken from each group, and the right bronchi were ligated with suture lines, and left lung cannulation was performed. 3mL of physiological saline was gently injected into the left lung, lavage fluid was withdrawn, each rat was repeatedly subjected to this procedure 3 times, the collected alveolar lavage fluid was centrifuged at 1500rpm for 10 minutes at 4℃and the supernatant was taken and the protein concentration in the supernatant was detected by BCA protein quantification kit.
The result shows that: referring to FIG. 5, the dry-wet ratio and the BALF protein concentration of ISX-9 injected lung tissue alone did not significantly change, and could not alleviate pulmonary edema and inflammatory degree, and the dry-wet ratio and the protein concentration of BMSCs injected by BMSCs alone decreased, and the dry-wet ratio and the BALF protein content of lung tissue decreased compared with BMSCs alone after ISX-9 was combined with BMSCs, and the SOD content of lung tissue increased significantly, which showed that ALI effect of BMSCs combined with ISX-9 was better than that of BMSCs injected by BMSCs alone, and ISX-9 promoted the efficacy of BMSCs. And compared with an ALI model group, the combination of ISX-9 and shNGFR-BMSC has certain capabilities of reducing pulmonary edema and reducing inflammatory, but is obviously inferior to the combination treatment group of ISX-9 and BMSCs, which shows that the influence of ISX-9 on BMSCs is related to NGFR genes.
3. Rat lung tissue section HE staining
After one week of treatment of the 6 groups of rats according to the protocol described above, lung tissue from one of the right lung of one rat was randomly collected for each group to assess the severity of lung injury. Lung lobes were fixed with 4% paraformaldehyde and embedded in paraffin and cut into 4 μm sections, which were dewaxed and stained with hematoxylin and eosin (H & E), observed under a microscope and photographed, and severity assessed according to internationally accepted lung injury scoring criteria.
The result shows that: referring to fig. 6, ali rats were treated with ISX-9 alone with no significant relief in the extent of lung injury; the alveolar space of BMSCs injected alone is obviously improved, and inflammatory cell infiltration is reduced; the effect of the combined treatment of ISX-9 and BMSCs on restoring ALI alveolar structure is most obvious, and inflammatory cell accumulation in lung cavities is remarkably relieved; the combination of ISX-9 with shNGFR-BMSCs can partially reduce inflammatory cell infiltration, but the ability to repair alveoli is still significantly different from that of normal BMSCs. ISX-may therefore mediate the repair of the lung barrier by mesenchymal stem cells by acting on NGFR. The lung injury score indicates that the BMSCs treatment group can reduce the lung injury degree, but the lung injury score is the lowest when being combined with ISX-9, and the ALI relieving effect is better.
4. RT-qPCR experiment for detecting inflammatory factor content in lung tissue
4.1 extraction of tissue RNA
One euthanized right lung tissue is randomly taken from each of the 6 groups of rats, the tissue sample is weighed, the sample is ground in a lysate, the mixture is kept stand for 5 minutes at room temperature, and the equal volume of absolute ethyl alcohol is added for full and uniform blowing. The sample mixture was loaded into a centrifuge column and centrifuged at 4000g for 1 minute at room temperature. 500. Mu.L of the washing solution was added to the column, and the column was centrifuged at 12000g for 1 minute at room temperature. Transferring the centrifugal column into a new EP tube, uncovering and airing for 2 minutes, adding 20-30 mu L of washing liquid into the centrifugal column, centrifuging for 1 minute at the room temperature by 12000g, discarding the centrifugal column, and obtaining the centrifugal product, namely RNA.
4.2、RT-qPCR
RNA reverse transcription and RT-qPCR were performed in the same manner as in example 3.
TABLE 4 primers for detection of inflammatory factors
The result shows that: referring to FIG. 7, the mRNA levels of pro-inflammatory factors IL-12, TNF- α, IL-1β and IL-6 were inhibited in lung tissue of BMSCs treated rats, but ISX-9 combined treatment with BMSCs resulted in the most significant decrease in pro-inflammatory factors, indicating that ISX-9 combined with BMSCs was more effective in reducing inflammatory responses, repairing lung tissue.
5. In vivo imaging to observe the duration and intensity of action of BMSC in rats
8 adult male Sprague-Dawley rats (6-8 weeks old, 220+ -20 g body weight) were used. The random groups were 2 groups, the experimental groups were:
1) ALI and BMSCs groups;
2) ALI and BMSCs, group ISX-9.
Part of the same embodiment as 1Similarly, 4 hours after ALI model establishment, tail vein injection BMSCs1×10 6 ISX-925mg/kg is injected into the abdominal cavity once, the distribution and action intensity of BMSCs in the body are observed through a PE IVIS Spectrum CT-hour animal Living body imager 2 hours, 24 hours, 48 hours and 72 hours after injection, and the data quantitative analysis is carried out through the live Image software.
The result shows that: referring to fig. 8, tail vein injected BMSCs would target the site of inflammation to the lungs through blood circulation, suggesting that mesenchymal stem cell therapy targets inflammation. On the sixth day after injection, the average fluorescence intensity of the BMSCs alone treatment group was 15.03%, the average fluorescence intensity of the combination treatment group with ISX-9 was 6.79%, and the combination application of ISX-9 showed an increase in the average fluorescence intensity of the BMSCs compared with the cell alone treatment, indicating that ISX-9 not only promoted the action intensity of the BMSCs, but also promoted the residence of the BMSCs in the target tissue, prolonging the action effect thereof.
6. Western blot experiment for detecting expression of KGF in lung tissue
One euthanized rat from each of the 6 groups was randomly harvested and any left lung tissue was frozen in-80 refrigerator for subsequent tissue protein extraction. Tissue proteins were extracted according to the instructions of the kit (tissue total protein extraction kit) manufacturer. The subsequent Western blot experiment operations such as protein quantification and electrophoresis are the same as those in the first embodiment 5.
The result shows that: referring to FIG. 9, the significant decrease in KGF expression in lung tissue in the ALI model group, and the increase in KGF protein expression in lung tissue in both BMSCs alone and ISX-9 in combination with BMSCs for 7 days, showed that the effect of ISX-9 on BMSCs to reduce inflammation in vivo was most significant by increasing KGF secretion. The capability of cells with the combination of ISX-9 and the cells with the knockdown NGFR to promote KGF secretion is lower than that of normal BMSCs, which indicates that the capability of ISX-9 to promote the mesenchymal stem cells to secrete KGF in vivo is probably regulated by NGFR.
7. Detection of KGF expression in lung tissue by tissue section immunofluorescence
One euthanized rat from each of the 6 groups was randomly harvested and 4% paraformaldehyde was used to randomly collect any left lung tissueFixing, embedding paraffin, cutting into paraffin sections of 4mm, baking in an oven at 65 ℃ for 60 minutes, soaking in xylene for 10 minutes, repeating the steps once, and putting the sections into gradient ethanol for hydration (100% -95% -85% -75%), wherein the sections are soaked in ethanol of each concentration for 5 minutes. With ddH 2 O-washing for 5min was repeated 3 times. 10mmol/L citrate buffer (pH 6.0) was used and heated to boiling for antigen retrieval. Repeatedly boiling the slice in repairing solution for 5 times, cooling to room temperature, taking out the slice, and using ddH 2 O-washing was repeated 3 times. Then, 50. Mu.L of 3% BSA-PBST was added dropwise to each slice, and the mixture was blocked in a wet box at room temperature for 30 minutes, and 50. Mu.L of primary antibody was added according to the instructions of the target protein antibody, and incubated overnight in a wet box at 4 ℃. The next day was taken out and rewarmed at room temperature for 45 minutes, PBST soaked for 5 minutes, washed 3 times, and the Cy 3-labeled goat anti-rabbit IgG (H+L) added dropwise to each slice was 25. Mu.L, and the secondary antibody was incubated at room temperature in a dark place for 2 hours. PBST was soaked for 5 minutes and washed 3 times. DAPI staining nuclei, incubation for 5min at room temperature, cover slip covering, fluorescent microscopy.
The result shows that: referring to fig. 10, the ali model group showed a sudden drop in KGF expression level, indicating that acute lung injury attenuated KGF expression in lung tissue. After ISX-9, BMSCs and ISX-9 are combined with BMSCs, the KGF content expressed by lung tissues is obviously increased. The level of KGF expression in rat lung tissue in the treatment group with ISX-9 in combination with BMSCs was even higher than that in the control group, whereas the ability to restore KGF expression in lung tissue in the BMSCs group alone or in the group with ISX-9 in combination with shNGFR-BMSCs was significantly lower than that in ISX-9 in combination with BMSCs, indicating that ISX-9 in combination with BMSCs exhibited the best effect of promoting KGF expression in lung tissue and that KGF expression could be regulated by acting on cellular NGFR receptor proteins.
Claims (8)
1. Application of isoxazole-9 combined mesenchymal stem cells in preparing medicaments for preventing or treating acute lung injury.
2. Use of a composition comprising isoxazole-9 and mesenchymal stem cells in the preparation of a medicament for preventing or treating acute lung injury.
3. Use according to claim 1 or 2, characterized in that:
the chemical structural formula of the isoxazole-9 is as follows:
4. use according to claim 1 or 2, characterized in that: the mesenchymal stem cells are one of human placenta chorion-derived mesenchymal stem cells and rat bone marrow mesenchymal stem cells.
5. The use according to claim 1 or 2, characterized in that: the use is to reduce pulmonary oedema occurring in acute lung injury.
6. The use according to claim 1 or 2, characterized in that: the use is to alleviate inflammatory response in acute lung injury.
7. The use according to claim 1 or 2, characterized in that: the use is to promote repair of the lung barrier following acute lung injury.
8. The use according to claim 1 or 2, characterized in that: the purpose is to increase the expression of KGF genes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311318623.9A CN117243977A (en) | 2023-10-12 | 2023-10-12 | Application of isoxazole-9 combined mesenchymal stem cells in preparation of medicines for preventing or treating acute lung injury |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311318623.9A CN117243977A (en) | 2023-10-12 | 2023-10-12 | Application of isoxazole-9 combined mesenchymal stem cells in preparation of medicines for preventing or treating acute lung injury |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117243977A true CN117243977A (en) | 2023-12-19 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311318623.9A Pending CN117243977A (en) | 2023-10-12 | 2023-10-12 | Application of isoxazole-9 combined mesenchymal stem cells in preparation of medicines for preventing or treating acute lung injury |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117243977A (en) |
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2023
- 2023-10-12 CN CN202311318623.9A patent/CN117243977A/en active Pending
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