Application of dimethyl fumarate in preparation of alveolar cell regeneration medicine

Abstract

The invention belongs to the field of drug activity, and particularly relates to application of dimethyl fumarate in preparation of alveolar cell regeneration drugs. The invention systematically evaluates the effect of dimethyl fumarate in the process of promoting pulmonary alveolar regeneration by constructing a pulmonary alveolar organ model and a bleomycin-induced lung injury mouse model. Research results show that dimethyl fumarate can significantly promote regeneration of alveolar epithelium AT2 cells, so that alveolar epithelium structure repair is accelerated.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical activity, specifically relating to the application of dimethyl fumarate in the preparation of alveolar cell regeneration drugs. Background Technology

[0002] Pulmonary epithelial mucosal injury is a common pathological process in various disease states (such as infection, inflammation, drug toxicity, or mechanical ventilation), mainly manifested as the disruption of the alveolar epithelial barrier and impaired gas exchange function. The integrity of the alveolar epithelium is crucial for maintaining lung tissue homeostasis; if damage is not repaired promptly and effectively, it can lead to serious consequences such as pulmonary edema, persistent inflammatory response, and even pulmonary fibrosis. Studies have shown that lung tissue possesses a certain self-repair capacity. Type II alveolar epithelial cells (AT2 cells), as stem cells in the alveolar region, can proliferate and differentiate into type I alveolar epithelial cells (AT1 cells), thereby achieving the regeneration and reconstruction of alveolar epithelial structure and function. Therefore, activating the function of AT2 cells is considered a key step in achieving lung tissue repair.

[0003] Dimethyl fumarate is a drug with anti-inflammatory and antioxidant activities, approved for the treatment of immune-related diseases such as multiple sclerosis. Studies have reported its use in alleviating lung inflammation and inhibiting fibrosis progression, but its applications mainly focus on immunomodulation and anti-inflammatory effects, lacking systematic research on its role in AT2 cell activation and epithelial structure repair. Currently available technologies do not demonstrate that dimethyl fumarate significantly promotes AT2 cell proliferation and differentiation, nor have they verified its specific role in alveolar structural reconstruction through organoids and animal models. Furthermore, relevant literature does not address its application in AT2 cell-driven tissue repair processes. Therefore, exploring the retargeting applications of dimethyl fumarate in enhancing AT2 cell function and promoting alveolar structural reconstruction constitutes a research direction not addressed in existing technologies, possessing considerable innovation and application potential. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide an application of dimethyl fumarate in the preparation of alveolar cell regeneration drugs.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] The use of dimethyl fumarate in the preparation of alveolar cell regeneration drugs.

[0007] Preferably, it is used to promote the regeneration and repair of damaged alveolar epithelial mucosa.

[0008] Compared with the prior art, the beneficial effects of the present invention are:

[0009] This invention systematically evaluated the role of dimethyl fumarate in promoting alveolar regeneration by constructing alveolar organoid models and bleomycin-induced lung injury mouse models. The results showed that dimethyl fumarate significantly promoted the regeneration of alveolar epithelial AT2 cells, thereby accelerating alveolar epithelial structural repair. This application reveals for the first time the potential application of dimethyl fumarate in alveolar AT2 cell-driven tissue regeneration, expanding its indications in the treatment of lung diseases and providing a new strategic basis for drug repositioning. It provides important theoretical and experimental support for the "drug repurposing" of existing drugs in the treatment of lung injury. Compared with new drug development, the drug repositioning strategy proposed in this invention can significantly reduce R&D costs, shorten the development cycle, and has good prospects for clinical translation. Attached Figure Description

[0010] Figure 1 These are the experimental results of dimethyl fumarate stimulating alveolar organoids in vitro. Figure A shows a schematic diagram of alveolar organoid culture; Figure B shows a representative image of alveolar organoids cultured to day 7; Figure C shows the average diameter of alveolar organoids with a diameter greater than 100 μm; Figure D shows the formation rate of alveolar organoids with a diameter greater than 100 μm; Figure E shows the expression level of SPC in alveolar organoids cultured to day 10; Figure F shows the expression level of T1α in alveolar organoids cultured to day 10.

[0011] Figure 2 This is a diagram showing the experimental results of dimethyl fumarate treatment for bleomycin-induced lung injury in mice. Figure A is a schematic diagram of the experimental design; Figure B is a representative image of lung tissue immunofluorescence staining, T1α (green), SPC (red), scale bar = 50 μm. Detailed Implementation

[0012] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and preferred embodiments.

[0013] Experimental animals: The experimental animals used in this study were C57BL / 6J mice, purchased from Beijing Spefol Biotechnology Co., Ltd., aged 8 weeks and weighing 25±2g. They were caged for 7 days before the experiment to acclimatize to the environment.

[0014] Example 1: Constructing an alveolar organoid culture model to study the effect of dimethyl fumarate on the regenerative capacity of alveolar AT2 cells:

[0015] (1) Isolation and culture of primary lung fibroblasts

[0016] ① Anesthetize mice: Anesthetize mice by intraperitoneal injection of 7.5% chloral hydrate solution;

[0017] ② Fix the mouse: Fix the mouse’s head and limbs to the operating table to fully expose its chest and abdomen, and disinfect the skin of the chest and abdomen with 75% ethanol.

[0018] ③ Dissect the mouse: Open the abdominal cavity, cut the inferior mesenteric artery to kill the mouse, and open the thoracic cavity to expose the lungs and trachea;

[0019] ④ Endotracheal intubation: Insert the endotracheal tube above the cartilage ring and fix it with surgical sutures;

[0020] ⑤ Cardiac perfusion: 1×PBS is injected into the right ventricle and flows into the lungs through the cardiopulmonary circulation system to remove blood from the lungs;

[0021] ⑥ Bronchoalveolar lavage: 1×EGTA is injected into the alveoli through the endotracheal tube, and after two aspirations, the EGTA is discarded to remove inflammatory cells in the lungs;

[0022] ⑦ Separate the lung lobes: Cut off all the lung lobes and discard the heart and trachea;

[0023] ⑧ Washing lung lobes: Place the cut lung lobes in sterile 1×PBS and wash 3 times, 5 min each time;

[0024] ⑨ Shred lung tissue: Place the cleaned lung lobes into a tube and cut them into small pieces with scissors;

[0025] ⑩ Lyse red blood cells: Add 500 μL of red blood cell lysis buffer and time for 90 seconds;

[0026] To terminate the reaction, add 1 mL of 1×PBS and transfer to a centrifuge tube.

[0027] Centrifuge and discard the supernatant: 4℃, 600g, 5min;

[0028] Digestion of lung tissue: Collagenase 4 and Dispase II were dissolved in 1×PBS to prepare a digestion solution with a concentration of 2 mg / mL. The solution was filtered through a 0.2 μm filter to remove bacteria. 5 mL of the digestion solution was added to the lung tissue precipitate, mixed well, and then heated in a 37°C water bath for 30 min, mixing every 10 min.

[0029] Termination of digestion: Add 5 mL of DMEM cell culture medium (DMEM containing 10% FBS and 1% PS) to terminate digestion;

[0030] Centrifuge and discard the supernatant: 4℃, 3000rpm, 10min;

[0031] Wash cells: Add 7 mL of DMEM cell culture medium to wash cells;

[0032] Centrifuge and discard the supernatant: 4℃, 600g, 5min;

[0033] Repeat the steps of washing cells and centrifuging to discard the supernatant twice;

[0034] Cell seeding: Add 10 mL of DMEM cell culture medium, disperse the cells, transfer them to a P100 cell culture dish, and incubate them in a 37°C, 5% CO2 cell culture incubator.

[0035] After the P0 generation cells have reached full growth, the culture medium is removed.

[0036] Add 3 mL of Typsin-EDTA and incubate in a 37°C cell culture incubator for 3 min.

[0037] Gently tap the culture dish to detach the cells, then add 7 mL of DMEM cell culture medium to stop the digestion.

[0038] Transfer the cell suspension to a centrifuge tube and centrifuge at 600g for 5 min at 4°C;

[0039] Discard the supernatant, add 3 mL of DMEM cell culture medium to resuspend the cells, and take 1 mL of the cell suspension to add to a P100 cell culture dish containing 9 mL of DMEM cell culture medium.

[0040] Shake the culture dish to distribute the cells evenly, then incubate in a 37°C, 5% CO2 cell culture incubator.

[0041] P1 generation cells are ready for use after reaching confluence.

[0042] (2) Flow cytometry sorting of AT2 cells:

[0043] ① Anesthetize mice: Anesthetize mice by intraperitoneal injection of 7.5% chloral hydrate solution;

[0044] ② Fix the mouse: Fix the mouse’s head and limbs to the operating table to fully expose its chest and abdomen, and disinfect the skin of the chest and abdomen with 75% ethanol.

[0045] ③ Dissect the mouse: Open the abdominal cavity, cut the inferior mesenteric artery to kill the mouse, and open the thoracic cavity to expose the lungs and trachea;

[0046] ④ Endotracheal intubation: Insert the endotracheal tube above the cartilage ring and fix it with surgical sutures;

[0047] ⑤ Cardiac perfusion: 1×PBS is injected into the right ventricle and flows into the lungs through the cardiopulmonary circulation system to remove blood from the lungs;

[0048] ⑥ Bronchoalveolar lavage: 1×EGTA is injected into the alveoli through the endotracheal tube, and after two aspirations, the EGTA is discarded to remove inflammatory cells in the lungs;

[0049] ⑦ Separate lung tissue: Cut the trachea above the endotracheal intubation port, separate the entire lung and heart along the trachea and place them in a centrifuge tube containing 1×PBS;

[0050] ⑧ Preheat lung tissue: Place centrifuge tubes in a 37°C water bath for 30 minutes to preheat;

[0051] ⑨ Digest lung tissue: Inject 3 mL of Elastase (4 u / mL) into the alveoli via endotracheal intubation. First, inject 1 mL, and then inject the remaining 2 mL in 4 divided doses, with each dose 5 min apart.

[0052] ⑩ Shred the lung tissue: Place the digested lung tissue on absorbent paper to remove excess water, cut off the lung lobes, discard the heart and trachea, place the lung lobes in a petri dish and shred them with a blade;

[0053] Digestion: Add 5 mL DNase I (500 μg / mL), mix by pipetting, and incubate at 37°C for 15 min;

[0054] To terminate digestion: Add 10 mL of ice-cold Hank's+ buffer to terminate digestion;

[0055] Filtration: After filtration through a 70μm filter, the sample was collected into centrifuge tubes;

[0056] Centrifuge and discard the supernatant: 4℃, 600g, 5min;

[0057] Lysis of red blood cells: Add 1 mL of red blood cell lysis buffer, mix well by pipetting and aspiration, and time for 90 seconds;

[0058] To terminate the reaction, add 20 mL of ice-cold Hank's+ buffer.

[0059] Centrifuge and discard the supernatant: 4℃, 700g, 8min;

[0060] Resuspending cells: Resuspend the cells in 500 μL of Hank's+ buffer and transfer the cell suspension to a flow cytometry tube. Add another 500 μL of Hank's+ buffer to dilute the remaining cell suspension in the centrifuge tube. Mix well and divide evenly into 5 flow cytometry tubes to prepare blank tubes and single positive tubes.

[0061] Add primary antibody incubation: Add the antibody to the sample tube and the single positive tube (CD31, CD34 and CD45 are added to the same single positive tube and named APC-Cy7), mix well, and incubate at 4°C in the dark for 45 min; as shown in Table 1;

[0062] Table 1

[0063]

[0064] To terminate the reaction, add 1 mL of Hank's+ buffer.

[0065] Centrifuge and discard the supernatant: 4℃, 600g, 5min;

[0066] Resuspend cells: Resuspend cells in 500 μL of Hank's+ buffer;

[0067] Secondary antibody incubation: Add 5 μL Streptavidin APC-eFluor 780 to the sample tube and APC-Cy7 single positive tube, mix well by pipetting, and incubate in a 4°C refrigerator in the dark for 30 min.

[0068] To terminate the reaction, add 1 mL of Hank's+ buffer.

[0069] Centrifuge and discard the supernatant: 4℃, 600g, 5min;

[0070] Resuspend cells: Resuspend cells in 500 μL of Hank's+ buffer;

[0071] Filtration: Filter the cell suspension through a 70μm filter screen;

[0072] AT2 cells were sorted: CD31-CD34-CD45-EpCAM cells were analyzed and sorted by flow cytometry. + CD24-Sca-1- AT2 cells.

[0073] (3) Constructing an alveolar organoid culture model:

[0074] ① Primary lung fibroblasts were prepared into a cell suspension, the cells were counted, and the suspension was diluted to 1×10⁻⁶. 6 Units / mL available for use;

[0075] ② Mix all the sorted AT2 cells into one centrifuge tube (4×10⁻⁶ cells / mL). 4 AT2 / hole);

[0076] ③ Centrifuge and discard the supernatant: 4℃, 600g, 5min;

[0077] ④ Add diluted primary lung fibroblast suspension, 200 μL / well;

[0078] ⑤ Centrifuge and discard the supernatant: 4℃, 600g, 5min;

[0079] ⑥ Resuspend cells in stem cell culture medium (DMEM / F12 containing 10% FBS, 1% ITS and 1% PS), 50 μL / well;

[0080] ⑦ Add an equal volume of Matrigel base gel and mix thoroughly by blowing and sucking (Note: This process requires using an ice gun on ice to prevent the Matrigel base gel from solidifying).

[0081] ⑧ Use an ice gun tip to gently drip 95 μL of the mixture to the bottom of the chamber (Note: avoid generating air bubbles); ⑨ Place the culture plate in a 37°C incubator for 30 minutes to allow the mixture to solidify;

[0082] ⑩ Add 410 μL of stem cell culture medium (containing Y27632 and SB431542) to the outside of the small cell and incubate it in a 37℃, 5% CO2 cell culture incubator;

[0083] The growth status of organoids was observed daily using a microscope, and the culture medium was changed every other day (Y27632 was removed from the stem cell culture medium on the second day, and SB431542 was removed from the stem cell culture medium on the seventh day).

[0084] On day 7 of culture, organoids were photographed using a microscope, and the organoid formation rate and diameter were counted. The organoid formation rate = number of organoids with a diameter greater than 100 μm / number of AT2 cells inoculated × 100%.

[0085] On day 10 of culture, 1 mL of Trizol was added to lyse alveolar organoids. RNA was then extracted and PCR was used to detect the proliferation (SPC) and differentiation markers (T1α) of AT2 cells.

[0086] (4) RNA extraction and real-time quantitative PCR:

[0087] ① Add 400 μL of chloroform to the sample tube, shake to mix, and let stand at room temperature for 3 min;

[0088] ② Centrifugation: 4℃, 10000rpm, 20min;

[0089] ③ After centrifugation, the mixture separates into three layers. Take 400 μL of the supernatant into a new EP tube.

[0090] ④ Add 400 μL of isopropanol to the supernatant, shake to mix, and let stand at room temperature for 10 min;

[0091] ⑤ Centrifugation: 4℃, 10000rpm, 10min;

[0092] ⑥ Discard the supernatant;

[0093] ⑦ Add 1 mL of 75% alcohol and shake to mix.

[0094] ⑧ Centrifugation: 4℃, 7000rpm, 10min;

[0095] 9. Discard the supernatant;

[0096] ⑩ Add 300 μL of 75% alcohol and shake to mix.

[0097] Centrifugation: 4℃, 7000 rpm, 5 min;

[0098] Discard the supernatant and invert the EP tube onto absorbent paper to air dry the RNA for 30 minutes to 1 hour;

[0099] Add 12 μL of Nuclease-Free Water to dissolve the RNA and incubate at 55°C for 20 min.

[0100] Take 2 μL of the liquid and test the concentration and purity of the RNA using an ELISA reader;

[0101] Dilute the RNA concentration to 80 ng / μL;

[0102] Preparation of reaction system: 8 μL RNA + 8 μL 4×gDNA Wiper Mix + 16 μL Nuclease-Free Water;

[0103] After mixing, place the mixture into a PCR instrument and run the genomic DNA removal reaction program (42℃: 2min; 25℃: ∞).

[0104] After the procedure is complete, remove the sample and add 8 μL of 5×HiScript III RT SuperMix;

[0105] After mixing, place the mixture into a PCR instrument and run the reverse transcription reaction program (37℃: 15min; 85℃: 5s; 4℃: ∞).

[0106] Preparation of amplification system: 10μL ChamQ Universal SYBR qPCR Master Mix + 0.5μL Forward Primer + 0.5μL Reverse Primer + 6.5μL Nuclease-Free Water + 2.5μL cDNA, primer concentration is 5μM;

[0107] After mixing, place the mixture into a PCR instrument and execute the amplification reaction program, in which the three processes of preheating, denaturation and annealing are performed for 50 cycles (preheating 95℃: 3min; denaturation 95℃: 15s; annealing 60℃: 45s; extension 72℃: 10s; 95℃: 10s; 65℃: 1min; 97℃: 1s; 37℃: 30s);

[0108] Using β-actin as an internal reference gene, the relative expression level of the target gene at the RNA level was calculated. Results are expressed as mean ± standard deviation. Independent samples t-tests were used to compare the differences between the experimental and control groups. P < 0.05 indicated statistical significance, *P < 0.05, **P < 0.01, ***P. Table 2 shows the primer sequences.

[0109] Table 2

[0110]

[0111] (5) Results:

[0112] To investigate the effects of dimethyl fumarate on the proliferation and differentiation capacity of AT2 cells, this study constructed an alveolar organoid culture model and set up a control group and an experimental group. Figure 1 A). An equal volume of DMSO was added to the control group as a solvent, while dimethyl fumarate was added to the experimental group at a final concentration of 2 μg / mL. On day 7 of culture, the growth of alveolar organoids was photographed and recorded, and the organoid formation rate and average diameter were calculated. On day 10, organoids were collected, total RNA was extracted, and the expression levels of molecular markers related to AT2 cell proliferation and differentiation were detected using real-time quantitative PCR. The results showed that the organoid formation rate was significantly increased in the experimental group, and the average diameter was significantly larger (…). Figure 1B–D). PCR results further showed that the expression levels of SPC, an AT2 cell proliferation marker, were significantly higher in the experimental group than in the control group, and the expression level of T1α, a differentiation marker, also showed an increasing trend. Figure 1 (E–F). The above results indicate that dimethyl fumarate can effectively promote the proliferation and differentiation of AT2 cells in vitro, and contribute to the regeneration of alveolar epithelium, thus possessing potential value as a candidate drug for the treatment of lung injury.

[0113] Example 2: Establishing a bleomycin-induced lung injury model to study the effect of dimethyl fumarate on epithelial regeneration and repair.

[0114] (1) Constructing a mouse model of lung injury:

[0115] ① Anesthetize mice: Anesthetize mice by intraperitoneal injection of 7.5% chloral hydrate solution;

[0116] ② Expose the trachea: Make an incision in the skin of the neck along the direction of the trachea to expose the trachea;

[0117] ③ Injection administration: Based on the mouse's weight, inject 2 U / kg of bleomycin into the trachea using an insulin syringe;

[0118] ④ Suture the skin on the mouse's neck and gently press on the heart area to promote the even distribution of bleomycin in the lungs;

[0119] ⑤ Place the mouse under an infrared heating lamp to keep it warm and wait for it to wake up;

[0120] ⑥ Mice were randomly divided into two groups and treated during days 11 to 20 after modeling. The control group received daily intraperitoneal injections of PBS containing 10% hydroxypropyl-β-cyclodextrin as a solvent control, while the experimental group received intraperitoneal injections of dimethyl fumarate solution at a dose of 90 mg / kg. Mice were sacrificed on day 21, and lung tissue was collected to prepare paraffin sections for subsequent immunofluorescence staining analysis.

[0121] (2) Preparation of paraffin sections of lung tissue samples:

[0122] ① Formaldehyde fixation of the left lung: 4% paraformaldehyde was injected into the alveoli through endotracheal intubation and fixed for 15 minutes. The lung lobe was cut off and placed in an embedding box, which was then soaked in a container containing 4% paraformaldehyde for more than 6 hours.

[0123] ② Dehydration: The lung tissue was placed in an automatic dehydrator and the following dehydration program was executed: 30% alcohol (1h), 50% alcohol (1h), 75% alcohol (1h), 95% alcohol I (1h), 95% alcohol II (1h), anhydrous ethanol I (1h), anhydrous ethanol II (1h), xylene I (1h), xylene II (1h), paraffin I (1h), paraffin II (1h);

[0124] ③ Embedding: Turn on the embedding machine to melt the wax, put the lung lobe into the mold, slightly cool it to fix the position of the lung lobe, add paraffin wax to completely cover the lung lobe, cover the embedding box, and wait for the paraffin wax to solidify before demolding;

[0125] ④ Sectioning: Use a paraffin microtome to cut lung tissue into 5μm thin slices, place them in a slide spreader, place 2-3 lung tissue slices on a glass slide, and let them dry before use for subsequent immunofluorescence staining.

[0126] (3) Immunofluorescence staining:

[0127] ① Baked slides: Place lung tissue slices on a baked slide machine and heat at 60℃ for 40 minutes;

[0128] ② Dewaxing: The product was dewaxed sequentially in xylene (twice, 20 min each time), anhydrous ethanol (twice, 20 min each time), 95% ethanol (twice, 5 min each time), 85% ethanol (5 min), 75% ethanol (5 min), distilled water (5 min), and 1×PBS (10 min).

[0129] ③ Antigen repair: Dissolve the citric acid antigen repair powder in 2 liters of distilled water, heat it to boiling in a pressure cooker, put the dewaxed lung tissue slices into it, cover the pot and continue heating. Start timing after the pressure valve turns, stop heating after 2 minutes, open the pot lid and let it cool to room temperature naturally.

[0130] ④ Washing: Immerse the lung tissue sections in 1×PBS 3 times, 5 min each time;

[0131] ⑤ Use absorbent paper to blot away any remaining fluid around the lung tissue;

[0132] ⑥ Perforate the membrane: Add 0.2% Triton to the lung tissue and punch holes at room temperature for 1 hour;

[0133] ⑦ Washing: Immerse the lung tissue sections in 1×PBS 3 times, 5 min each time;

[0134] ⑧ Serum blocking: Add 5% NGS to lung tissue and block for 1 hour at room temperature;

[0135] 9. Use absorbent paper to remove the serum;

[0136] ⑩ Add primary antibody: Add the diluted antibody to lung tissue and incubate overnight at 4°C;

[0137] Washing: Immerse lung tissue sections in 1×PBS 3 times, 5 min each time;

[0138] Use absorbent paper to blot away any remaining fluid around the lung tissue;

[0139] Add secondary antibody: Add the diluted antibody to the lung tissue and incubate at room temperature in the dark for 2 hours (this step requires protection from light).

[0140] Washing: Immerse lung tissue sections in 1×PBS 3 times, 5 min each time;

[0141] Use absorbent paper to blot away any remaining fluid around the lung tissue;

[0142] Add diluted DAPI to lung tissue and incubate at room temperature for 20 minutes.

[0143] Washing: Immerse lung tissue sections in 1×PBS for 5 min;

[0144] Use absorbent paper to blot away any remaining fluid around the lung tissue;

[0145] Mounting: Apply an appropriate amount of fluorescent mounting medium to the lung tissue and cover with a coverslip;

[0146] The staining results were observed and photographed using an inverted fluorescence microscope.

[0147] (4) Results:

[0148] To investigate the effects of dimethyl fumarate on alveolar cell regeneration, a bleomycin-induced lung injury mouse model was established. After modeling, mice were randomly divided into two groups and received intervention treatment from day 11 to day 20. Figure 2 A). Mice were sacrificed on day 21, and lung tissue was collected for paraffin section preparation. Immunofluorescence staining was used to detect alveolar epithelial regeneration. Results showed that the alveolar damage area was significantly reduced in the dimethyl fumarate-treated group. Figure 2 B) suggests that dimethyl fumarate can effectively promote the regeneration of damaged alveolar epithelium.

[0149] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of dimethyl fumarate in the preparation of alveolar cell regeneration drugs.

2. The application according to claim 1, characterized in that, It is used to promote the regeneration and repair of damaged alveolar epithelial mucosa.

Images