CN114681438B - Metformin and other guanidine-containing compounds are used to reverse the tendency of Gal-10 crystallization and alleviate related diseases. - Google Patents

Abstract

The invention belongs to the field of biological medicine, and in particular relates to application of metformin and other guanidine-containing compounds in reversing Gal-10 crystallization trend and relieving related diseases. The Gal-10 crystal-related diseases include infectious diseases and inflammatory diseases.

Description

Metformin and other guanidine-containing compounds reverse the tendency of Gal-10 crystallization and alleviate the Application in related diseases

Technical field

The invention belongs to the field of biomedicine, and specifically relates to the application of guanidine-containing compounds in reversing the crystallization tendency of Gal-10 and alleviating related diseases.

Background technique

Various functions in the human body often require different proteins to complete. These proteins are either dissolved in body fluids or bound to membrane structures such as cell membranes, but rarely form crystals. Abnormal protein crystals produced in human cells often have pathological characteristics.

In 1853, Charcot and Robin discovered a variety of crystal deposits in the blood and spleen of a leukemia patient. Subsequently, in 1872, Leyden also discovered the same crystal in the sputum of an asthmatic patient. The crystals are also called Charcot-Leyden crystals (CLCs). Charcot-Leyden crystals are colorless and transparent rhombic compass-like crystals with long pointed ends, varying sizes, and strong refractive properties. They are formed by the fusion of eosinophilic granules after the rupture of eosinophils. CLCs were initially thought to be inorganic crystals, but it was not confirmed until 1950 that they were composed of protein crystals. Research shows that the main component in CLCs is galectin-10 (Gal-10). Gal-10 is a protein that is very abundant in eosinophils and basophils. Its formation is closely related to the release of eosinophil extracellular traps and is closely related to a variety of diseases, including but not limited to Not limited to infectious diseases: suppurative lymphadenitis, eosinophilic cystitis, liver abscess, etc., inflammatory diseases: asthma, allergic rhinitis, eosinophilic colitis, etc. The treatment of these diseases usually has different treatments for different lesions, and there are few therapeutic drugs targeting the marker CLCs/Gal-10 protein.

Currently, there is only one artificial monoclonal antibody targeting Gal-10. The monoclonal antibody of this crystal can dissolve the crystal in vitro and inhibit the natural immune response in the lungs of mice caused by CLCs. However, considering the high cost of monoclonal antibody drugs, poor stability, difficulty in subsequent drug transportation, harsh storage conditions and other difficult-to-solve problems, the subsequent development of this kind of drug is more difficult.

Compared with monoclonal antibody drugs, organic small molecule drugs have the advantages of simple structure, good druggability, and easy industrial production. It is crucial to find small molecules that regulate Gal-10 assembly and inhibit its pathological effects.

Contents of the invention

The purpose of the present invention is to screen small molecule inhibitors targeting CLCs and then develop potential drugs that can regulate the assembly of CLCs and inhibit related inflammation.

In a first aspect, the present invention provides the use of guanidine-containing compounds in dissolving Gal-10 crystals and preparing drugs for diseases related to Gal-10 crystals.

Preferably, the guanidine-containing compound is represented by the following general formula:

Among them, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ can each independently be a hydrogen atom or a substituent.

Preferably, the substituents include but are not limited to the following common substituents and the substituents containing heteroatoms: hydrocarbyl, guanidine group, diazo group, carboxyl group, sulfonic acid group, hydrocarbonoxycarbonyl group, formyl group, halomethyl group Acyl, oxo, carbamoyl, cyano, phenolic alkyl, phenolic hydroxyl, alcoholic hydroxyl, amino, alkoxy, nitro, nitroso, mercapto, amino, nitro, acyl, silicon, acyloxy , oxyacyl, dihydroxyboryl, hydroxylamino, nitroso, disilyl; the substituents may also include C _1-6 alkyl, C _1-6 alkoxy, C _1-6 cycloalkyl , aryl, heteroaryl, heterocyclyl, heterocyclyl-(CH2)n-, aryl-C _1-6 alkyl-, heteroaryl-C _1-6 alkyl-, aryl-(CH ₂ )nO-, heteroaryl-(CH ₂ )nO-, C _3-8 cycloalkyl-C(O)-, heterocyclyl-C(O)-, aryl-C(O)-, or Heteroaryl-C(O), wherein C _1-6 alkyl, C _1-6 alkoxy, C _3-8 cycloalkyl, aryl, heterocyclyl-(CH ₂ )n-, aryl- C _1-6 alkyl-, heteroaryl-C _1-6 alkyl-, aryl-(CH ₂ )nO-, heteroaryl-(CH ₂ )nO-, C _3-8 cycloalkyl-C (O)-, heterocyclyl-C(O)-, aryl-C(O)-.

Preferably, the substituent contains carbon atoms, and the number of carbon atoms is not particularly limited;

Preferably, the substituent contains 1 to 20 carbon atoms; specifically including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20 carbon atoms.

Preferably, the hydrocarbon group includes but is not limited to methyl (-CH ₃ ), ethyl (-C ₂ H ₅ ), propyl (-C ₃ H ₇ ), butyl (-C ₄ H ₉ ), pentyl (-C ₅ H ₁₁ ).

Preferably, the guanidine-containing compound includes metformin, 1-methylguanidine, 1,1-dimethylguanidine, 1,1,3,3-tetramethylguanidine, 1-ethylguanidine, 1-phenylbiguanide , 1-(o-tolyl)biguanide, streptomycin, cimetidine.

Preferably, the guanidine-containing compound is metformin, and the structural formula of metformin is as follows:

Preferably, the guanidine-containing compound is 1-methylguanidine, and the structural formula of the 1-methylguanidine is as follows:

Preferably, the guanidine-containing compound is 1,1-dimethylguanidine, and the structural formula of the 1,1-dimethylguanidine is as follows:

Preferably, the guanidine-containing compound is 1,1,3,3-tetramethylguanidine, and the structure of the 1,1,3,3-tetramethylguanidine

The formula is as follows:

Preferably, the guanidine-containing compound is 1-ethylguanidine, and the structural formula of the 1-ethylguanidine is as follows:

Preferably, the guanidine-containing compound is 1-phenyl biguanide, and the structural formula of the 1-phenyl biguanide is as follows:

Preferably, the guanidine-containing compound is 1-(o-tolyl)biguanide, and the structural formula of the 1-(o-tolyl)biguanide is as follows:

Preferably, the guanidine-containing compound is streptomycin, and the structural formula of streptomycin is as follows:

Preferably, the guanidine-containing compound is cimetidine, and the structural formula of cimetidine is as follows:

The terms "Gal-10 crystals", "galectin-10", "Charcot-Leyden crystals", "CLCs" and "CLC crystals" used herein are interchangeable and refer to semi- Crystals formed from lactose lectin-10 (Gal-10). Crystals formed from galectin-10 are typically bipyramidal hexagonal crystals with a length of approximately 20-40 μm and a width of approximately 2-4 μm. The term "Gal-10 crystal" is broad enough to encompass the human protein and any species homolog.

"Dissolving Gal-10 crystals" in the present invention includes reversing the crystallization trend of Gal-10, reducing the crystallization rate of Gal-10, and increasing the dissolution rate of Gal-10.

Preferably, the Gal-10 crystal-related diseases include infectious diseases and inflammatory diseases.

Preferably, the pathogens of the infectious diseases include bacteria, mycoplasma, chlamydia, mycobacteria, fungi, viruses, parasites, etc. Exemplarily, the infectious diseases include suppurative lymphadenitis, eosinophilic cystitis, liver abscess, etc.

Preferably, the inflammation includes any inflammation well known in the art. As verified by the specific embodiments of the present invention, the inflammation includes IL-1β, IL-6, TNF-α caused by stimulation of CLCs (Gal-10 crystals), Inflammation in which CCL-2 is elevated at the gene level, specifically, such inflammation as asthma, rhinitis, colitis, etc.

Preferably, the inflammation may be caused by allergies.

Preferably, the colitis is eosinophilic colitis.

In another aspect, the present invention provides pharmaceutical compositions comprising a guanidine-containing compound as described above.

More specifically, the guanidine-containing compound is used as an active ingredient in pharmaceutical compositions.

The pharmaceutical composition of the present invention can be administered in any of the following ways: oral administration, spray inhalation, rectal administration, nasal administration, buccal administration, parenteral administration, such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, cardiac Intraventricular, intrasternal or intravenous administration.

Preferably, the pharmaceutical composition can be tablets, pills, powders, granules, capsules, lozenges, syrups, liquids (solutions), emulsions, suspensions, controlled release preparations, aerosols, and films. , injections, intravenous drips, transdermal absorption preparations, ointments, lotions, adhesive preparations, suppositories, small pills, nasal preparations, pulmonary preparations, eye drops, etc., oral or parenteral preparations.

As recorded in the specific embodiments of the present invention, the pharmaceutical composition can be liquid, and the solvent is PBS (phosphate buffer saline, also written as pbs in specific embodiments). The guanidine-containing compound can be configured according to its own solubility: Solutions of various concentrations, specifically, the concentrations are expressed as molar concentrations. The liquid may also include any pharmaceutically acceptable solvent, and the liquid may also contain other common pharmaceutical ingredients.

As described in the present invention, PBS is a phosphate buffered saline solution. It is usually used as a solvent to dissolve protective reagents. It is the most widely used buffer in biochemical research. Its main components are Na ₂ HPO ₄ , KH ₂ PO ₄ , NaCl and KCl, because Na ₂ HPO ₄ and KH ₂ PO ₄ have secondary dissociation, the pH value range of the buffer is very wide; and the main function of NaCl and KCl is to increase the concentration of salt ions. The PBS buffer has a wide range of pH values. As used in the present invention, the pH of the PBS is 7.4.

Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier, diluent or excipient.

Preferably, the pharmaceutically acceptable carrier, diluent or excipient includes, but is not limited to, any adjuvant, carrier, Excipients, glidants, sweeteners, diluents, preservatives, dyes/colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, Surfactants or emulsifiers.

The term "pharmaceutically acceptable" means that molecular entities and compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.

In another aspect, the invention provides a method of treating a Gal-10 crystal-related disease, the method comprising administering to a subject a guanidine-containing compound of the invention.

Preferably, the Gal-10 crystal-related diseases include infectious diseases and inflammatory diseases.

Preferably, the pathogens of the infectious diseases include bacteria, mycoplasma, chlamydia, mycobacteria, fungi, viruses, parasites, etc. Exemplarily, the infectious diseases include suppurative lymphadenitis, eosinophilic cystitis, liver abscess, etc.

Preferably, the inflammation includes asthma, rhinitis, colitis, etc.

Preferably, the inflammation may be caused by allergies.

Preferably, the colitis is eosinophilic colitis.

Preferably, the subjects of the present invention include humans or non-human animals. Examples of the non-human animals include: rats, pigs, cattle, horses, sheep, monkeys, rabbits, etc.

Preferably, the subject of the invention is a human.

As used herein, a method of "treating" a disease or condition means curing the disease or condition and/or reducing or eradicating symptoms associated with the disease or condition, thereby alleviating the suffering of the patient.

In another aspect, the present invention provides a method for dissolving Gal-10 crystals, the method comprising contacting the Gal-10 crystals with the guanidine-containing compound of the present invention.

Preferably, the method is performed in vitro.

Preferably, the method is non-therapeutic.

More specifically, dissolving Gal-10 crystals according to the present invention may also be referred to as accelerating the dissolution of Gal-10 crystals.

Description of the drawings

Figure 1 shows the detection results of CLC crystal morphology changes in different salt solutions under a light microscope.

Figure 2 is a statistical result chart showing the relative area of CLC crystals in salt solutions of different concentrations changing with time, A: (CH ₃ ) ₄ NCl, B: KCl, C: NaCl, D: GdmCl.

Figure 3 is a graph of statistical results of the intrinsic initial rate of dissolution of CLC crystals in different salt solutions.

Figure 4 is a graph showing the detection results of CLC crystal morphology changes in metformin solutions with different concentrations under a light microscope.

Figure 5 is a statistical graph showing the relative area changes of CLC crystals in different concentrations of cimetidine over time.

Figure 6 is a statistical result chart showing the change of the relative area of CLC crystals with time in different concentrations of streptomycin.

Figure 7 is a statistical graph showing the relative area changes of CLC crystals in different concentrations of metformin over time.

Figure 8 is a statistical result diagram of the intrinsic initial rate of dissolution of CLC crystals in different guanidine compound solutions. (This cannot be called "salt")

Figure 9 is a graph showing the detection results of IL-1β, IL-6, TNF-α, and CCL-2 qPCR in mice treated with tracheal injection. A: IL-1β, B: IL-6, C: TNF-α, D :CCL-2.

Figure 10 is a graph showing the detection results of IL-1β, IL-6, TNF-α, and CCL-2 qPCR in mice treated with oral administration. A: IL-1β, B: IL-6, C: TNF-α. D: CCL-2.

Figure 11 is a graph showing the results of ELISA detection of IL-1β, IL-6, TNF-α, and CCL-2 expression in mice treated with tracheal injection. A: IL-1β, B: IL-6, C: TNF- α, D: CCL-2.

Figure 12 is a graph showing the results of ELISA detection of IL-1β, IL-6, TNF-α, and CCL-2 expression in mice treated with oral administration. A: IL-1β, B: IL-6, C: TNF. -α, D: CCL-2.

Figure 13 shows the staining results of lung tissue pathological samples, A: 6 hours, B: 12 hours.

Figure 14 is a statistical graph showing the relative area changes of CLC crystals in solutions of different guanidine-containing compounds with different concentrations over time. A: 1-ethylguanidine, B: 1,1-dimethylguanidine, C: 1-methylguanidine , D: 1,1,3,3-tetramethylguanidine, E: 1-phenylbiguanide, F: 1-(o-tolyl)biguanide.

Figure 15 is a statistical result diagram of the inherent initial rate of dissolution of CLC crystals in different guanidine-containing compound solutions.

Detailed ways

The present invention will be further described below in conjunction with the examples. The following descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Any skilled person familiar with the art may make use of the technical content disclosed above. Changes to equivalent embodiments of equivalent variations. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention fall within the protection scope of the present invention.

Example 1. Preparation of CLCs and in vitro characterization experiment of salt-dissolved CLCs

1. Experimental materials

1.1 Plasmid: After humanized codon optimization of the Gal-10 protein sequence, add its N terminus (MASTTHHHHHDTDIPTTGGGSRPDDDDDKENLYFQGHM) and clone it into the pET-28a vector plasmid through the NcoI/XhoI double restriction site to form pET-28a-6His- TEV-Gal10.

1.2 Reagents, materials, instruments and equipment

1.2.1 Chemical reagents:

Kanamycin and ampicillin were purchased from Tiangen Biotechnology Co., Ltd.; isopropyl-beta-D-thiogalactoside (IPTG) and E. coli BL21 (DE3) was purchased from Beijing Quanzhijin Company; SDS, Trizol and imidazole were purchased from Sigma Company; TEV enzyme was purchased from Beijing Yiqiao Shenzhou Co., Ltd.; Coomassie Brilliant Blue dye (homemade); cDNA reverse transcription reagent was purchased from Takara Company; Real-time fluorescence quantitative PCR reagents were purchased from Shanghai Roche Co., Ltd.; Elisa kits were purchased from R&D Systems; NaCl was purchased from Tianjin Damao Chemical Reagent Factory; (CH ₃ ) ₄ NCl, KCl, GdmCl, Kac, KBr, streptomycin , cimetidine, dexamethasone, 1-methylguanidine, 1,1,3,3-tetramethylguanidine, 1,1-dimethylguanidine, 1-ethylguanidine, 1-phenylbiguanide, 1-(O-tolyl)biguanide was purchased from Shanghai McLean Biochemical Technology Co., Ltd.; DEPC water, ELISA stop solution, and TMB single-component chromogenic solution were purchased from Solebao Company.

1.2.2 Consumables and equipment:

Ni-NTA affinity chromatography column was purchased from GE Company; 10kDa concentrator tube was purchased from Millipore Company; electric thermostatic incubator (XMTD HH.B11-600); vertical pressure steam sterilizer (Shanghai Boxun, China); thermostatic bacteria Incubator (Shanghai Boxun, China); PCR instrument (Biometra Tgradient); 384-well plate real-time fluorescence quantitative PCR instrument (Roche 480Ⅱ); desktop centrifuge (eppendorf, Centrifuge 5415D); electric thermostatic water tank (SHH W21 600); trace UV Spectrophotometer (NanoDrop 2000); electronic balance (Sartorius 2000S); optical inverted microscope (XDS-1B); micropipette (eppendorf Research plus); -80°C ultra-low temperature refrigerator (SANYO, MDF-382E); high-speed refrigerated centrifuge Machine (Beckman, Germany); pH meter (Thermo Orion 868); Magnetic stirrer (IKA RH-KT/C); Ultrasonic crusher; pure 25 protein purification system (superdex75, GE Healthcare); SDS-PAGE electrophoresis instrument (Bio-Rad); gel imager (Tanon); 1mL disposable sterile syringe (purchased from BiDi Medical Instruments Co., Ltd.).

2. Experimental methods

2.1 Expression, purification and preparation of CLC crystals of Gal10 recombinant protein

2.1.1 Conversion:

1) Thaw BL21(DE3) competent cells and pET-28a-6His-TEV-gal10 recombinant plasmid on ice;

2) Add 0.5 μL (0.2 μg/μL) of the recombinant plasmid into BL21 (DE3) competent cells and incubate on ice for 15-20 minutes;

3) Heat shock in 42℃ water bath for 90 seconds;

4) Quickly place on ice and keep in ice bath for 5 minutes;

5) Add 500 μL of non-resistant LB and incubate in a 37°C constant temperature incubator for 40-50 minutes;

6) Take 40 μL of bacterial solution, inoculate it on LB solid medium containing Kanamycin (25 μg/mL), and culture it in a 37°C constant-temperature incubator overnight.

2.1.2 Bacterial culture:

Using Kanamycin (25 μg/mL) as a selection marker, pick the positive monoclonal BL21(DE3)/pET-28a-gal10-TEV-6His on the solid medium in the previous step and inoculate it into 20 mL of LB liquid culture containing Kanamycin resistance. The culture medium was shaken at 37°C and 210r/min for 8 hours. The bacterial solution was inoculated into 1L LB liquid medium containing Kanamycin resistance at a ratio of 1:100, and cultured under shaking conditions of 37°C and 210r/min.

2.1.3 Induced expression and acquisition of target protein:

1) When the optical density (OD600) of E. coli at 600nm is 0.6-0.8, add IPTG with a final concentration of 1mM, and culture it for 12-16h under shaking conditions of 28°C and 210r/min to induce the expression of the target protein;

2) Enrich the bacterial culture fluid expressed overnight, centrifuge at 6000g and 4°C for 20 minutes, and discard the supernatant. Resuspend in LysisBuffer (50mM NaH ₂ PO ₄ ; 300mM NaCl pH7.4), concentrate it in a beaker, the volume is about 60-80mL, add PMSF at a ratio of 1:100 (final concentration is 1mM), Prevent the target protein from being degraded;

3) Ultrasonic breakage of bacteria: Place the beaker on ice for ultrasonic use, with a power of 25%, a working time of 25 minutes, an ultrasonic on time of 3 seconds, and an ultrasonic off time of 9 seconds;

4) Collect protein: Centrifuge (4°C, 12000g, 30min) to remove cell debris, and at the same time break the nucleic acid released after cell disruption, collect the supernatant, and the soluble target protein will be present in the supernatant.

2.1.4 Purification of Gal-10 protein:

2.1.4.1Ni-NTA affinity column chromatography:

1) Equilibrate the Ni column: Use Lysis Buffer to fill the Ni column with about 2-3 column volumes, and then start elution of the protein;

2) Load the supernatant obtained by centrifugation and pass it through the Ni column 2-3 times;

3) Wash the column with Lysis Buffer containing 20mM imidazole and 0.1% Empigen detergent in order to elute impurity proteins;

4) Wash the column with Lysis Buffer containing 500mM imidazole to debind the target protein from the nickel column;

5) Concentration: Place the target protein eluted and collected in step (4) into a 10kDa concentration tube, and centrifuge at 3000×g for 10 minutes at 4°C. During the concentration process, add a small amount of Lysis Buffer or PBS to dilute the imidazole to prevent the target protein from aggregating and precipitating. .

2.1.4.2 pure 25 protein purification:

1) Clean the chromatography column: Use sterile water to clean the chromatography column, about 8 mL. Then place the pump head in the PBS solution, repeat the above steps and execute, cleaning approximately 36mL;

2) Parameters: System flow: 0.4mL/min; Column position: 2; Alarm delta column pressure enabled: 3.0; Alarm pre column pressure enabled: 5.0;

3) Loading the sample: First rinse the sample loop 2-3 times with PBS, then pour approximately 1.5mL of the concentrated sample into the sample loop, and finally absorb a little PBS before pumping it into the sample loop to avoid sample residue. Arrange the collection tube and select inject valve: inject;

4) Collection: When the UV280 mark line representing the protein content rises, use the automatic sample collector to collect the target protein, and set the collection volume of each tube to 0.3mL;

5) Clean the chromatography column: After collecting the samples, place the pump head in PBS, continue to run until 20mL passes, replace with sterile water for cleaning, run 10ml, and finally replace with 20% ethanol solution to run 20mL, that is Can be shut down;

6) Protein processing: Measure the concentration of each tube of collected protein and mark it, quickly freeze it in liquid nitrogen, and store it in a -80°C refrigerator. Let it sit at room temperature until it melts slowly before experimenting.

2.1.4.3 SDS-PAGE identification of expression products:

1) Sample preparation: whole bacteria sample, supernatant sample after centrifugation, protein supernatant sample after passing through the chromatography column, 20mM imidazole passing sample, 500mM imidazole passing sample, and collect the peak head, peak tip and peak tail of the molecular sieve protein sample;

2) Gel preparation: 5% stacking gel; select 15% separating gel according to the size of the protein;

3) Sample preparation: Mix 2X Loading Buffer and sample 1:1;

4) Loading samples: 5 μL of whole bacterial sample, 3 μL of marker, and 10 μL of remaining samples;

5) Gel running: 80V constant voltage running for stacking gel; 120V constant voltage running for separating gel;

6) Staining: Place the protein gel in Coomassie Brilliant Blue dye solution and microwave at medium-high heat for 2 minutes;

7) Decolorization: Place the dyed protein gel in tap water and microwave at high heat for 20-40 minutes.

2.1.5 Generation and concentration determination of CLC crystals:

1) Mix TEV enzyme and pET-28a-gal10-TEV-6His recombinant protein at a mass ratio of 1:10, and incubate for enzyme digestion on a shaking table at 4°C overnight;

2) Centrifuge at 600 g and 4°C for 10 minutes. After centrifugation, aspirate the supernatant and add an appropriate amount of PBS to resuspend. Repeat three times to obtain pure CLCs;

3) Use 6M guanidine hydrochloride to deassemble CLCs, and use a spectrophotometer One-Drop to measure the concentration of CLCs.

2.2 In vitro characterization experiments of salt-dissolved CLCs

2.2.1 Dissolve CLCs in PBS (pH 7.4), and adjust the concentration to make the CLCs concentration 0.6mg/ml;

2.2.2 Considering factors such as solubility and safety, select six salts (CH ₃ ) ₄ NCl, KCl, NaCl, GdmCl, KAc, and KBr. Use PBS to prepare a salt solution with a molar concentration of 4M and pH 7.4;

2.2.3 Mix CLCs and salt solution in proportion and dilute them to working concentrations:

1) Dilute CLCs to a working concentration of 0.15mg/ml;

2) Dilute (CH ₃ ) ₄ NCl, the molar concentration gradient is 1.0M, 1.5M, 2.0M, 3.0M;

3) Dilute KCl, the molar concentration gradient is 1.0M, 1.5M, 2.0M, 3.0M;

4) Dilute NaCl, the molar concentration gradient is 1.0M, 1.5M, 2.0M, 3.0M;

5) Dilute GdmCl, the molar concentration gradient is 0.2M, 0.4M, 0.5M, 0.6M;

2.2.4 Drop the mixed liquid on the crystal plate and observe it with a light microscope, and take pictures at intervals (the results of the detection results of CLC crystal morphology changes in different salt solutions under a light microscope are shown in Figure 1);

2.2.5 Use PhotoShop to process the image obtained, intercept the same area, use ImageJ to calculate the intercepted area, and normalize the data obtained at the first time point 10 minutes (the statistical results of the relative area of CLC crystals in salt solutions of different concentrations changing with time are as follows figure 2);

2.2.6 Calculate the initial reaction rate R _int according to R(t)=k·A(t)·[1-exp(βΔμ(c))] (statistics of the inherent initial rate of dissolution of CLC crystals in different anion and cation solutions The results are shown in Figure 3).

The above experimental results prove that Gal-10 crystal has a selective response to the above selected salt ions, among which Gdm ⁺ shows Gal-10 crystals are significantly dissolved, and the dissolution of Gal-10 crystals is time-dependent.

2.3 In vitro characterization experiments on dissolution of CLCs by guanidine-containing compounds

2.3.1 Dissolve CLCs in PBS (pH 7.4), and adjust the concentration to make the CLCs concentration 0.3 mg/ml;

2.3.2 Dissolve metformin in PBS buffer, the molar concentration gradient is 0.2mM, 2.0mM, 10.0mM, 20.0mM, 100.0mM, 200.0mM, 300.0mM, 400mM, 800mM;

The structural formula of metformin is as follows:

2.3.3 Dissolve streptomycin in PBS buffer, the molar concentration gradient is 0.2mM, 2.0mM, 10.0mM, 20.0mM;

The structural formula of streptomycin is as follows:

2.3.4 Dissolve cimetidine in PBS buffer, the molar concentration gradient is 0.2mM, 2.0mM, 10.0mM, 20.0mM;

The structural formula of cimetidine is as follows:

2.3.5 Mix the salt solution and CLCs at a volume ratio of 1:1, drop them on the crystal plate for observation with a light microscope, and take photos at regular intervals; (Changes in CLC crystal morphology in different guanidine compound solutions under a light microscope The test results are shown in Figure 4)

2.3.6 Use PhotoShop to process the image obtained, intercept the same area, use ImageJ to calculate the intercepted area, and take the first time point 10 minutes for normalization; (The statistical results of the relative area change of CLC crystals in different guanidine-containing compound solutions over time are as follows Figure 5-7)

2.3.7 Calculate the initial reaction rate R _int according to R(t)=k·A(t)·[1-exp(βΔμ(c))]. (The statistical results of the intrinsic initial rate of dissolution of CLC crystals in different guanidine-containing compound solutions are shown in Figure 8)

The above results show that among the three small molecule drugs, metformin has better solubility in dissolving Gal-10 crystals.

Example 2. Preparation of CLCs and in vitro characterization experiment of salt-dissolved CLCs

experimental method

1. Administration by intratracheal injection

1.1 Experimental animals

C57BL/6 strain mice were purchased from the Laboratory Animal Resources Institute of the China Institute of Food and Drug Control. Mice (wild-type, male, 6-week-old, C57BL/6 strain mice, weight 20±2g) were randomly divided into five groups, 6 mice in each group; and the following perfusion drugs were prepared for each group:

1) PBS (negative control),

2)CLCs(1.5mg/ml),

3) Metformin (0.45mg/kg),

4) CLCs plus metformin (CLCs: 1.5mg/ml, metformin: 0.45mg/kg),

5) CLCs plus dexamethasone (0.5mg/kg) (positive control);

1.2 Administration phase:

1) Use a sterile syringe to intraperitoneally inject tribromoethanol anesthetic into the mice. The anesthetic dose is: mouse body weight (g) × 15 μL/mouse. Observe that the mice are fully anesthetized (the foot squeeze reflex is negative) and then fix the mice;

2) Keep the mouse's airway vertical, use ophthalmic scissors to expose a longitudinal incision with a length of 0.5-0.8cm in the ventral skin of the neck 1.5cm above the chin, and use ophthalmic curved forceps to bluntly separate the subcutaneous tissue and connective tissue along the midline. tissue, neck muscles until the trachea is fully exposed;

3) Tilt the mouse fixation device at about 30°. The syringe absorbs 50 μL of air and then inhales 70 μL of the pre-prepared drug. Use a syringe under the thyroid cartilage in the middle of the trachea to insert a needle parallel to the longitudinal axis of the trachea. The depth of the needle insertion is about 1cm. Keep this Position to rapidly instill air and perfusion drugs into the trachea of mice;

4) After the operation is completed, a sudden increase in the respiratory rate of the mouse can be observed. Place the mouse fixation device vertically on the operating table for about 1 minute to help distribute the drug; reset the mouse fixation device and use ophthalmic curved forceps to clamp the mouse layer by layer according to the anatomical layers. Close the neck tissue of the mouse, release the mouse from immobilization, place the mouse in a side-lying position, and observe its status until it returns to autonomous activities.

2 Oral administration

2.1 Experimental animals

Mice (wild-type, male, 6-week-old, C57BL/6 strain mice, weight 20±2g) were randomly divided into 6 groups, 3 mice in each group; and the following perfusion drugs were prepared for each group:

1) PBS (negative control),

2)CLCs(1.5mg/ml),

3) Metformin (0.45mg/kg),

4) CLCs plus metformin 1× (CLCs: 1.5mg/ml, metformin: 0.45mg/kg),

5) CLCs plus metformin 3× (CLCs: 1.5mg/ml, metformin: 1.35mg/kg),

6) CLCs plus metformin 10× (CLCs: 1.5mg/ml, metformin: 4.5mg/kg).

2.2 Administration phase:

Hold the mouse so that its head, neck and body are in a straight line. Use a syringe equipped with a gastric gavage needle. The needle enters from the corner of the mouse's mouth. Press the tongue against the roof of the mouth and gently push it inward. After entering the esophagus, it goes slightly deep and injects the drug solution.

3. Sample collection stage:

Samples were collected 6h and 12h after tracheal injection, and samples were collected 6h after oral administration.

1) Use a sterile syringe to intraperitoneally inject tribromoethanol anesthetic into the mice. The anesthetic dose is: mouse body weight (g) × 15 μL/mouse. Observe that the mice are fully anesthetized (foot squeeze reflex is negative) and then sacrifice the mice and fix them;

2) Expose the mouse trachea again along the tracheal drip incision, and cut an inverted "V"-shaped incision under the thyroid cartilage. Pay attention to gentle operation to avoid severing the mouse trachea;

3) After absorbing 600 μL of sterile PBS, extend the pipette parallel to the longitudinal axis of the trachea into the incision until it completely fits the inner wall of the mouse trachea. Inject the PBS through the mouse trachea into the mouse bronchus at a uniform speed, and then suck back at a uniform speed. A large number of bubbles will be found during the suction process, which is a sign of the success of the operation. After repeating the injection-back-aspiration process 4-5 times, put the recovery solution into a 1.5mL centrifuge tube, and pipe and mix;

4) Repeat step (3), put the recovery solution into the same 1.5mL sterile EP tube, and store it at -80°C;

5) Open the chest of the mouse, cut off the sternum, extract the heart, cut off the connective tissue and fascia between the lung tissue and the chest, remove the lung tissue and rinse it with sterile PBS, add 1 ml Trizol to lyse it and store it at -80°C.

Treatment effect testing

1. RNA extraction and cDNA reverse transcription

1.1RNA extraction:

1) Thaw the lysate stored in Trizol slowly on ice;

2) After the RNA sample is completely thawed, bring the sample to room temperature for 10 minutes to fully dissolve the RNA. Add chloroform according to the ratio of Trizol:chloroform = 5:1 (v/v), and shake vigorously to mix thoroughly at room temperature. Leave for 10-15min;

3) 4℃, 12000r/min, centrifuge for 15min;

4) Transfer the upper aqueous phase to a new RNase-free 1.5mL centrifuge tube;

5) Add 0.6-0.8 times the volume of pre-cooled isopropyl alcohol, mix thoroughly, and place at -20°C for more than 30 minutes;

6) Centrifuge at 12000r/min for 15min at 4°C. After discarding the supernatant, RNA precipitate can be seen at the bottom of the centrifuge tube;

7) Add 1 mL of 75% ethanol prepared with DEPC-treated water or enzyme-free sterile water, shake the centrifuge tube gently to resuspend the pellet;

8) Centrifuge at 12000r/min for 15min at 4℃, discard the supernatant as much as possible, and repeat steps 7-8;

9) Dry at room temperature until 75% ethanol has evaporated;

10) Use DEPC-treated water or enzyme-free sterile water to dissolve the RNA pellet;

11) Take 2 μL RNA sample and measure the RNA concentration with a micro-UV spectrophotometer, and add an appropriate amount of DEPC-treated water or enzyme-free sterile water to adjust the RNA solution concentration to 200 ng/μL, and freeze it at -80°C.

1.2 Reverse transcription of RNA into cDNA:

Carry out reverse transcription of cDNA according to the instructions of Takara's cDNA Reverse Transcription Kits (cat.no RR036A);

1)5×PrimeScript RT Master Mix(Perfect Real Time):2μL

1×Total RNA: 0.5μg

RNase Free dH ₂ O up to 10μL

2) Mix gently and then perform reverse transcription reaction. The conditions are as follows:

37℃15min (reverse transcription reaction)

85℃5sec (reverse transcriptase inactivation reaction)

Short term storage at 4℃.

2. Real-time fluorescence quantitative PCR (RT-qPCR) to detect changes in gene expression

according to Perform real-time fluorescence quantitative PCR experiments according to the instructions of 480SYBR Green I Master;

1) The working solution added to each reaction system is as follows:

2) Add the working solution to the 384-well plate, and add 1 μL of the cDNA obtained by reversing the above step into each well, and place the sealing film flatly on the 384-well plate, and briefly remove it for 90 seconds;

3) Place the 384-well plate into the reaction rack according to the instrument's instructions, and set the following reaction program;

4) After the reaction is completed, use 480 software calculates the reaction melt curve (Melt curve), observes whether the amplification curve is a single peak to exclude non-specific amplification, and automatically calculates the relative expression of the detected gene based on the Ct values of the target gene and the internal reference gene. The algorithm is: Gene Relative expression value=2 ^{(-(Ct target gene-Ct internal reference gene))} ;

5) Draw graphs and perform significant difference analysis in Graphpad Prism 8.0 software.

The results of tracheal injection qPCR are shown in Figure 9. CLCs stimulation caused IL-1β, IL-6, TNF-α, and CCL-2 to express genes in genes. The metformin treatment group could significantly reduce the expression of inflammatory factors in lung tissue with statistical significance.

The qPCR results of the oral administration group are shown in Figure 10. After CLCs stimulation, IL-1β, IL-6, TNF-α, and CCL-2 were induced in the base. Due to the increase in levels, the picture shows the test results 6 hours after administration. Oral metformin treatment group 1× can significantly reduce inflammation in lung tissue. The expression of factors and the increased concentration may have a toxic effect on cells, and the anti-inflammatory effect is not as good as 1×.

3. Enzyme-linked immunosorbent assay (ELISA) to detect protein changes

3.1 Coated ELISA 96-well plate: Dilute the capture antibody with sterile PBS according to the working concentration. Add 100 μL/well of the diluted capture antibody into the high-binding 96-well enzyme plate. Cover the upper surface completely with a sealing film and keep at room temperature. Overnight incubation;

3.2 Detection process:

1) Discard the capture antibody, add 200 μL washing solution to each well for cleaning, discard the washing solution and absorb the remaining liquid with filter paper, repeat 3 times;

2) Add 300 μL diluent (PBS containing 1% BSA) to each well and incubate at room temperature for at least 1 hour;

3) Discard the diluent and repeat step 1);

4) Use new diluent to prepare the standard, add 100 μL of the prepared standard or sample to each well, make two duplicate wells for each sample, shake gently, cover the plate, and incubate at 37°C for 2 hours;

5) Discard the sample and repeat step 1);

6) Dilute the detection antibody to the working concentration with new diluent, add 100 μL of detection antibody working solution to each well, cover the plate, and incubate at room temperature for 2 hours;

7) Discard the detection antibody and repeat step 1);

8) Dilute Streptavidin-HRP with new diluent to the working solution concentration at a ratio of 1:40, add 100 μL to each well, cover the plate, and incubate at room temperature in the dark for 20 minutes;

9) Discard the Streptavidin-HRP working solution and repeat step 1);

10) Add 100 μL TMB single-component chromogenic solution to each well, cover the plate, and incubate at room temperature in the dark for 10-20 minutes;

11) Add 50 μL of color stop solution to each well, shake and mix, use a full-function microplate detector to detect the OD values at 450nm and 570nm, and subtract the OD value at 570nm from the OD value at 450nm;

12) Draw graphs and perform significant difference analysis in Graphpad Prism 8.0 software. (Figure 11, Figure 12)

The results of tracheal injection ELISA are shown in Figure 11. CLCs stimulation induced IL-1β, IL-6, NF-α, and CCL-2 in protein The level of inflammatory factors in the bronchoalveolar lavage fluid increased significantly, and the metformin treatment group could significantly reduce the inflammatory factors in the bronchoalveolar lavage fluid with statistical learning meaning.

The ELISA results of the oral administration group are shown in Figure 12. CLCs stimulation caused IL-1β, IL-6, TNF-α, and CCL-2 to With the increase in protein levels, oral metformin treatment group 1X could significantly reduce inflammatory factors in bronchoalveolar lavage fluid.

4. H&E staining of lung tissue pathological samples of mice in the tracheal injection group

4.1 After 6h and 12h from the tracheal injection group, carefully collect the left lung tissue of mice (n=3), wash it in PBS and put it into a 10mL tube containing 8mL of paraformaldehyde (4%), and then place it in Fixed at room temperature for 48-72h;

4.2 Dehydration, embedding, dewaxing, staining, dehydration and sealing (this step is completed by Wuhan Sewell Biotechnology Co., Ltd.)

The results are shown in Figure 13. The inflammatory cell infiltration in the lung tissue of mice was significantly alleviated after metformin was injected into the trachea of mice.

Example 3. Structure-activity relationship (other guanidine-containing compounds)

1. Dilute CLC crystals to a concentration of 0.3mg/ml

2. Dissolve 1-methylguanidine in PBS buffer. The molar concentration gradient is 2mM; 20mM; 100mM; 200mM; 400mM.

3. Dissolve 1,1,3,3-tetramethylguanidine in PBS buffer. The molar concentration gradient is 0.2mM; 1.0mM; 4.0; 10.0mM.

4. Dissolve 1,1-dimethylguanidine in PBS buffer. The molar concentration gradient is 2mM; 20mM; 100mM; 200mM; 400mM.

5. Dissolve 1-ethylguanidine in PBS buffer. The molar concentration gradient is 2mM; 20mM; 100mM; 200mM; 400mM.

6. Dissolve 1-phenylbiguanide in PBS buffer. The molar concentration gradient is 0.2mM; 2.0mM; 4.0mM; 10.0mM; 20.0mM.

7. Dissolve 1-(o-tolyl)biguanide in PBS buffer. The molar concentration gradient is 0.2mM; 2.0mM; 4.0mM; 10.0mM.

8. Mix the salt solution and CLC crystal at a volume ratio of 1:1, observe on the crystal plate, and take photos at intervals;

9. Use PhotoShop to intercept the same area, use ImageJ to calculate the intercepted area, and take the first time point 10 minutes for normalization;

10. Find the initial reaction rate Rint according to R(t)=k·A(t)·[1-exp(βΔμ(c))].

The statistical results of the change of the relative area of CLC crystals with time in solutions of different guanidine-containing compounds at different concentrations are as shown in the figure. As shown in 14, the statistical results of the inherent starting rate of dissolution of CLC crystals in different guanidine compound solutions are shown in Figure 15.

The above experimental results prove that the number of non-polar functional groups is related to the ability of guanidine ion derivatives to dissolve Gal-10 crystals Positive correlation.

Claims (1)

1. A method of dissolving Gal-10 crystals, said method comprising contacting Gal-10 crystals with a guanidine-containing compound that is one or more of metformin, 1-methyl guanidine, 1-dimethyl guanidine, 1, 3-tetramethyl guanidine, 1-ethyl guanidine, 1-phenyl biguanide, 1- (o-tolyl) biguanide, streptomycin, cimetidine, said method being non-therapeutic.