CN114681438B - Use of metformin and other guanidine-containing compounds for reversing the crystallization tendency of Gal-10 and for alleviating the associated diseases - Google Patents

Use of metformin and other guanidine-containing compounds for reversing the crystallization tendency of Gal-10 and for alleviating the associated diseases Download PDF

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CN114681438B
CN114681438B CN202210534642.4A CN202210534642A CN114681438B CN 114681438 B CN114681438 B CN 114681438B CN 202210534642 A CN202210534642 A CN 202210534642A CN 114681438 B CN114681438 B CN 114681438B
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guanidine
gal
crystals
metformin
clcs
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CN114681438A (en
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王晨轩
张文博
王洋
李淑媛
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Institute of Basic Medical Sciences of CAMS
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/10—Drugs for disorders of the urinary system of the bladder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C279/04—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C279/20—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups containing any of the groups, X being a hetero atom, Y being any atom, e.g. acylguanidines
    • C07C279/24—Y being a hetero atom
    • C07C279/26—X and Y being nitrogen atoms, i.e. biguanides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/22—Cyclohexane rings, substituted by nitrogen atoms
    • C07H15/238—Cyclohexane rings substituted by two guanidine radicals, e.g. streptomycins

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

Use of metformin and other guanidine-containing compounds for reversing the crystallization tendency of Gal-10 and for alleviating the associated diseases
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to application of guanidine-containing compounds in reversing Gal-10 crystallization trend and relieving related diseases.
Background
Often, various functions within the human body require different proteins to perform. These proteins either dissolve in body fluids or bind to membrane structures such as cell membranes, but rarely crystallize. Abnormal protein crystals produced in human cells are often characterized pathologically.
In 1853, charcot and Robin found a morphologically diverse crystal deposit in the blood and spleen of a leukemia patient, and subsequently in 1872 Leyden found the same crystal in the sputum of an asthmatic patient, which crystal was also called Charcot-Leyden crystal (CLCs, charcco-Leyden crystal). The crystal is rhombic colorless transparent compass, has long tips at two ends, different sizes and strong refraction, and is formed by mutually fusing eosinophilic particles after eosinophil rupture. Initially CLCs were considered inorganic crystals and were not confirmed to consist of protein crystals until 1950. Studies have shown that the major component in CLCs is galectin-10 (Gal-10). Gal-10 is a very abundant protein in eosinophils and basophils, whose formation is closely related to the release of extracellular traps from eosinophils, and is also closely related to a variety of diseases including, but not limited to, infectious diseases: suppurative lymphadenitis, eosinophilic cystitis, liver abscess, etc., and inflammatory diseases: asthma, allergic rhinitis, eosinophilic colitis, and the like. The treatment of these diseases generally has different treatment methods for different lesions, and there are few therapeutic drugs targeting the markers CLCs/Gal-10 protein.
At present, only one artificial monoclonal antibody exists for the target regulatory molecule of Gal-10, and the monoclonal antibody of the crystal can dissolve the crystal in vitro and inhibit the natural immune response of the lung of the mice caused by CLCs. However, the subsequent development of the monoclonal antibody drug is difficult in consideration of the problems of high cost, poor stability, difficult subsequent drug transportation, severe storage conditions and the like of the monoclonal antibody drug.
Compared with monoclonal antibody medicines, the organic micromolecular medicines have the advantages of simple structure, good patentability and easy industrial production. It is important to find small molecules that regulate Gal-10 assembly and inhibit its pathological effects.
Disclosure of Invention
The invention aims to screen small molecule inhibitors targeting CLCs, and further develop potential drugs capable of regulating and controlling CLCs assembly and inhibiting related inflammation.
In a first aspect, the invention provides the use of a guanidine-containing compound for dissolving Gal-10 crystals, for the preparation of a medicament for the treatment of diseases associated with Gal-10 crystals.
Preferably, the guanidine-containing compound is represented by the following general formula:
wherein R is 1 、R 2 、R 3 、R 4 、R 5 、R 6 Each independently may be a hydrogen atom or a substituent.
Preferably, the substituents include, but are not limited to, the following substituents and the substituents containing heteroatoms as are common: hydrocarbyl, guanidino, diazo, carboxyl, sulfonic, hydrocarbyloxycarbonyl, formyl, haloformyl, oxo, carbamoyl, cyano, phenolic hydrocarbyl, phenolic hydroxyl, alcoholic hydroxyl, amino, hydrocarbyloxy, nitro, nitroso, mercapto, amino, nitro, acyl, silicon, acyloxy, oxyacyl, dihydroxyboron, hydroxyamino, nitroso, disilyl; the substituents may also include C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Cycloalkyl, aryl, heteroaryl, heterocyclyl- (CH 2) n-, aryl-C 1-6 Alkyl-, heteroaryl-C 1-6 Alkyl-, aryl- (CH) 2 ) n-O-, heteroaryl- (CH) 2 )n-O-、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 ) n-O-, heteroaryl- (CH) 2 )n-O-、C 3-8 cycloalkyl-C (O) -, heterocyclyl-C (O) -, aryl-C (O) -.
Preferably, the substituent contains a carbon atom, the number of which is not particularly limited;
preferably, the substituents contain 1 to 20 carbon atoms; specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 carbon atoms.
Preferably, the hydrocarbyl 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 ) Amyl (-C) 5 H 11 )。
Preferably, the guanidine-containing compound comprises metformin, 1-methyl guanidine, 1-dimethyl guanidine, 1, 3-tetramethyl guanidine, 1-ethyl guanidine, 1-phenyl biguanide, 1- (o-tolyl) biguanide, streptomycin, cimetidine.
Preferably, the guanidine-containing compound is metformin, which has the following structural formula:
preferably, the guanidine-containing compound is 1-methylguanidine, and the 1-methylguanidine has the following structural formula:
preferably, the guanidine-containing compound is 1, 1-dimethylguanidine, and the 1, 1-dimethylguanidine has the following structural formula:
preferably, the guanidine-containing compound is 1, 3-tetramethylguanidine, the structure of the 1, 3-tetramethylguanidine
The formula is as follows:
preferably, the guanidine-containing compound is 1-ethylguanidine, and the 1-ethylguanidine has the following structural formula:
preferably, the guanidine-containing compound is 1-phenyl biguanide, the structural formula of the 1-phenyl biguanide being as follows:
preferably, the guanidine-containing compound is 1- (o-tolyl) biguanide, the 1- (o-tolyl) biguanide having the formula:
preferably, the guanidine-containing compound is streptomycin, and the streptomycin has the following structural formula:
preferably, the guanidine-containing compound is cimetidine, which is of the formula:
the terms "Gal-10 crystals", "galectin-10", "Leiden crystals", "CLCs", "CLC crystals" as used herein are used interchangeably to refer to crystals formed from galectin-10 (Gal-10). The crystals formed from galectin-10 are typically biconic hexagonal crystals, having a length of about 20-40 μm and a width of about 2-4 μm. The term "Gal-10 crystal" is broad enough to encompass human proteins and homologs of any species.
The invention discloses a method for dissolving Gal-10 crystals, which comprises reversing the crystallization trend of Gal-10, reducing the crystallization speed of Gal-10 and improving the dissolution rate of Gal-10.
Preferably, the Gal-10 crystal-related disease includes an infectious disease, an inflammatory disease.
Preferably, the pathogens of the infectious diseases include bacteria, mycoplasma, chlamydia, mycobacteria, fungi, viruses, parasites, and the like. Illustratively, the infectious diseases include suppurative lymphadenitis, eosinophilic cystitis, liver abscess, and the like.
Preferably, the inflammation comprises any inflammation known in the art, including inflammation that causes an increase in IL-1β, IL-6, TNF- α, CCL-2 at the genetic level upon stimulation of CLCs (Gal-10 crystals), as demonstrated by specific embodiments of the invention, in particular, such as asthma, rhinitis, colitis, and the like.
Preferably, the inflammation may be allergy-induced.
Preferably, the colitis is eosinophilic colitis.
In another aspect, the invention provides a pharmaceutical composition comprising a guanidine-containing compound as described above.
More specifically, the guanidine-containing compound is used as an active ingredient in a pharmaceutical composition.
The pharmaceutical composition of the present invention may be administered by any of the following means: oral, spray inhalation, rectal, nasal, buccal, parenteral, e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intracardiac, intrasternal or intravenous administration.
Preferably, the pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, lozenges, syrups, liquids (solutions), emulsions, suspensions, controlled release formulations, aerosols, films, injections, intravenous drip agents, transdermal absorption formulations, ointments, lotions, adhesive formulations, suppositories, pellets, nasal formulations, pulmonary formulations, eye drops and the like, oral or parenteral formulations.
As described in the embodiments of the present invention, the pharmaceutical composition may be a liquid, the solvent is PBS (phosphate buffer saline, also written as PBS in the embodiments), and the guanidine-containing compound may be formulated into various concentrations of solutions according to its own solubility, specifically, the concentrations are expressed in molar concentrations. The liquid may also include any pharmaceutically acceptable solvent, and may also contain other pharmaceutically common ingredients.
The invention discloses that the Pbs are phosphate buffer salt solution which is usually used as solvent to play a role of dissolving protective reagent, is a buffer solution which is most widely used in biochemical research, and comprises the main component of Na 2 HPO 4 、KH 2 PO 4 NaCl and KCl due to Na 2 HPO 4 And KH 2 PO 4 They have secondary dissociation and buffer pH ranges are very broad;whereas NaCl and KCl act primarily to increase salt ion concentration. The buffered pH of the PBS was in a wide range, and as used in the present invention, the PBS had a pH of 7.4.
Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.
Preferably, the pharmaceutically acceptable carrier, diluent or excipient includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant or emulsifier that has been approved by the U.S. food and drug administration or the Chinese food and drug administration for use in humans or livestock.
The term "pharmaceutically acceptable" means that the molecular entity and composition do not produce adverse, allergic or other untoward reactions when properly administered to an animal or human.
In another aspect, the invention provides a method of treating a Gal-10 crystal-related disease comprising administering to a subject the guanidine-containing compound of the invention.
Preferably, the Gal-10 crystal-related disease includes an infectious disease, an inflammatory disease.
Preferably, the pathogens of the infectious diseases include bacteria, mycoplasma, chlamydia, mycobacteria, fungi, viruses, parasites, and the like. Illustratively, the infectious diseases include suppurative lymphadenitis, eosinophilic cystitis, liver abscess, and the like.
Preferably, the inflammation includes asthma, rhinitis, colitis, and the like.
Preferably, the inflammation may be allergy-induced.
Preferably, the colitis is eosinophilic colitis.
Preferably, the subject of the present invention comprises a human or non-human animal, illustratively comprising: mice, 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 disorder refers to curing the disease or disorder and/or alleviating or eradicating symptoms associated with the disease or disorder, thereby alleviating the suffering of the patient.
In another aspect, the invention provides a method of dissolving Gal-10 crystals, said method comprising contacting Gal-10 crystals with a guanidine-containing compound of the invention.
Preferably, the method is performed in vitro.
Preferably, the method is non-therapeutic.
More specifically, the dissolution of Gal-10 crystals according to the invention may also be referred to as accelerating the dissolution of Gal-10 crystals.
Drawings
FIG. 1 is a graph showing the results of detection of CLC crystal morphology changes in different salt solutions under a light microscope.
Fig. 2 is a graph of statistical results of the relative area of CLC crystals in salt solutions of different concentrations over time, a: (CH) 3 ) 4 NCl，B：KCl，C：NaCl，D：GdmCl。
Figure 3 is a graph of statistical results of the inherent initial rates of dissolution of CLC crystals in different salt solutions.
FIG. 4 is a graph showing the results of detection of the morphological changes of CLC crystals in metformin solutions of different concentrations under a light microscope.
Fig. 5 is a graph of statistical results of the relative area of CLC crystals in different cimetidine concentrations over time.
FIG. 6 is a graph showing the statistical results of the relative area of CLC crystals in different concentrations of streptomycin over time.
Fig. 7 is a graph of statistical results of the relative area of CLC crystals in different concentrations of metformin over time.
FIG. 8 is a graph of statistical results of the inherent initial rates of dissolution of CLC crystals in different guanidine-containing compound solutions. (this cannot be referred to as "salt")
FIG. 9 is a graph showing the results of detection of IL-1β, IL-6, TNF-. Alpha.and CCL-2qPCR in mice treated with tracheal injection, A: IL-1 beta, B: IL-6, C: TNF- α, D: CCL-2.
FIG. 10 is a graph showing the results of detection of IL-1β, IL-6, TNF- α, CCL-2qPCR in mice treated with oral administration, A: IL-1 beta, B: IL-6, C: TNF- α, D: CCL-2.
FIG. 11 is a graph showing the results of ELISA detection of the expression levels of IL-1β, IL-6, TNF- α, and CCL-2 in mice treated with tracheal injection, A: IL-1 beta, B: IL-6, C: TNF- α, D: CCL-2.
FIG. 12 is a graph showing the results of ELISA for detecting the expression levels of IL-1β, IL-6, TNF- α and CCL-2 in mice treated with oral administration, A: IL-1 beta, B: IL-6, C: TNF- α, D: CCL-2.
Fig. 13 is a staining result of lung tissue pathology sample, a:6 hours, B: and 12 hours.
FIG. 14 is a graph showing the statistical results of the relative area of CLC crystals in solutions of different guanidine-containing compounds at different concentrations over time, A: 1-ethylguanidine, B:1, 1-dimethylguanidine, C: 1-methylguanidine, D:1, 3-tetramethylguanidine, E: 1-phenyl biguanide, F:1- (o-tolyl) biguanide.
FIG. 15 is a graph of statistical results of the initial rate of dissolution of CLC crystals in different guanidine-containing compound solutions.
Detailed Description
The present invention is further described in terms of the following examples, which are given by way of illustration only, and not by way of limitation, of the present invention, and any person skilled in the art may make any modifications to the equivalent examples using the teachings disclosed above. Any simple modification or equivalent variation of the following embodiments according to the technical substance of the present invention falls within the scope of the present invention.
Example 1 preparation of CLCs and in vitro characterization experiments of salt-solubilized CLCs
1. Experimental materials
1.1 plasmid: after humanized codon optimization of Gal-10 protein sequence, the N terminal (MASTTHHHHHDTDIPTTGGGSRPDDDDDKENLYFQGHM) is added to clone the sequence on a pET-28a vector plasmid through NcoI/XhoI double cleavage sites to form pET-28a-6His-TEV-Gal10.
1.2 reagent materials and instrumentation
1.2.1 chemical reagents:
kanamycin (Kanamycin) and Ampicillin (Ampicillin) were purchased from kazaku biotechnology limited; isopropyl- β -D-thiogalactoside (IPTG) and escherichia coli BL21 (DE 3) were purchased from beijing holomorphic gold company; SDS, trizol and imidazole were purchased from Sigma company; TEV enzyme was purchased from Beijing Yiqiao Shenzhou Co., ltd; coomassie brilliant blue dye solution (homemade); cDNA reverse transcription reagents were purchased from Takara corporation; real-time fluorescent quantitative PCR reagents were purchased from shanghai roche limited; elisa kit was purchased from R&D Systems Inc.; naCl was purchased from the Tianjin metallocene chemical reagent plant; (CH) 3 ) 4 NCl, KCl, gdmCl, kac, KBr, streptomycin, cimetidine, dexamethasone, 1-methylguanidine, 1, 3-tetramethylguanidine, 1-dimethylguanidine, 1-ethylguanidine, 1-phenylbiguanide, 1- (o-tolyl) biguanide were all available from Shanghai Meilin Biochemical technologies Co., ltd; DEPC water, ELISA stop solution, TMB single component color development solution was purchased from Soy Corp.
1.2.2 consumables and instrumentation:
Ni-NTA affinity chromatography column was purchased from GE company; 10kDa concentration tube was purchased from Millipore company; an electrothermal incubator (XMTD HH. B11-600); vertical pressure steam sterilizing pot (Shanghai Boxun, china); constant temperature bacterial incubator (Shanghai Boqun, china); a PCR instrument (Biometra Tgradient); 384 well plate real-time fluorescent quantitative PCR instrument (Roche 480 II); bench Centrifuge (eppendorf, centrafuge 5415D); an electric heating constant temperature water tank (SHH W21 600); a micro ultraviolet spectrophotometer (NanoDrop 2000); electronic balances (Sartorius 2000S); an optical inverted microscope (XDS-1B); a micropipette (eppendorf Research plus); -80 ℃ ultra low temperature refrigerator (SANYO, MDF-382E); high speed refrigerated centrifuges (Beckman, germany); pH meter (Thermo Orion 868); magnetic stirrer (IKA RH-KT/C); an ultrasonic breaker;pure 25 protein purification System (Superdex 75, GE Healthcare); SDS-PAGE electrophoresis apparatus (Bio-Rad); gel imager (tan); 1mL disposable sterile syringe (available from Bidi medical instruments Co., ltd.).
2. Experimental method
2.1 expression, purification and Condition of Gal10 recombinant proteinsCLCPreparation of crystals
2.1.1 conversion:
1) Melting BL21 (DE 3) competent cells and pET-28a-6His-TEV-gal10 recombinant plasmid on ice;
2) Adding 0.5 mu L (0.2 mu g/. Mu.L) of recombinant plasmid into BL21 (DE 3) competent cells, and incubating on ice for 15-20min;
3) Heat shock in 42 deg.c water bath for 90s;
4) Rapidly placing on ice, and ice-bathing for 5min;
5) Adding 500 μl of non-resistant LB, and culturing at 37deg.C in a constant temperature incubator for 40-50min;
6) 40. Mu.L of the bacterial liquid was inoculated onto LB solid medium containing Kanamycin (25. Mu.g/mL), and cultured overnight at 37℃in a constant temperature incubator.
2.1.2 bacterial culture:
positive monoclonal BL21 (DE 3)/pET-28 a-gal10-TEV-6His on the solid medium in the previous step was picked up using Kanamycin (25. Mu.g/mL) as a selection marker, inoculated in 20mL of LB liquid medium containing Kanamycin resistance, and shake-cultured at 37℃for 8h with a 210r/min shaker. The bacterial liquid is inoculated into 1L of LB liquid medium containing Kanamycin resistance according to the proportion of 1:100, and cultured under the shaking condition of 37 ℃ and 210 r/min.
2.1And 3, induced expression and acquisition of target proteins:
1) When the optical density (OD 600) of the escherichia coli at 600nm is 0.6-0.8, adding IPTG with the final concentration of 1mM, culturing for 12-16h under the oscillation condition of 28 ℃ and 210r/min, and inducing the target protein to express;
2) Enrichment of overnight expressed bacterial culture solution, centrifugation at 6000g and 4 ℃ for 20min, and discarding the supernatant. Buffer Lysis Buffer (50 mM NaH) 2 PO 4 The method comprises the steps of carrying out a first treatment on the surface of the 300mM NaCl pH 7.4), and concentrating in a beaker with a volume of about 60-80mL, and adding PMSF (final concentration of 1 mM) at a ratio of 1:100 to prevent the target protein from being degraded;
3) Ultrasonic disruption of bacteria: placing the beaker on ice for ultrasonic treatment, wherein the power is 25%, the working time is 25min, the ultrasonic on time is 3s, and the ultrasonic off time is 9s;
4) Collecting protein: cell debris was removed by centrifugation (4 ℃,12000 g, 30 min), and nucleic acid released after cell disruption was broken up, and the supernatant was collected, and the soluble target protein was present in the supernatant.
2.1.4 purification of Gal-10 protein:
2.1.4.1Ni-NTA affinity column chromatography:
1) Balance Ni column: the Lysis Buffer is used for penetrating out the volume capacity of about 2-3 column volumes of the Ni column, and then protein elution can be started;
2) Loading the supernatant obtained by centrifugation, and passing through a Ni column for 2-3 times;
3) The column was washed with Lysis Buffer containing 20mM imidazole and 0.1% Empigen detergent, with the aim of eluting the hetero protein;
4) Washing the column with a Lysis Buffer containing 500mM imidazole to remove the combination of the target protein and the nickel column;
5) Concentrating: and (3) placing the target protein collected by eluting in the step (4) into a 10kDa concentration tube, centrifuging at 4 ℃ and 3000 Xg for 10min, and adding a small amount of Lysis Buffer or PBS to dilute imidazole in the concentration process so as to prevent the target protein from aggregating and precipitating.
2.1.4.2 pure 25 protein purification:
1) Cleaning the chromatographic column: the column was washed with sterile water, approximately 8mL. Then the pump head is placed in PBS solution, the steps are repeated and executed, and the pump head is cleaned by about 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: washing the sample loading ring with PBS for 2-3 times, pumping the concentrated sample with about 1.5mL into the sample loading ring, and slightly sucking PBS and pumping into the sample loading ring to avoid sample residue. The collection tube was set up and the object valve was selected: an object;
4) And (3) collecting: when the UV280 mark line representing the protein content rises, collecting target protein by using a sample automatic collector, and setting the collection amount of each tube to be 0.3mL;
5) Cleaning the chromatographic column: after the sample is collected, the pump head is placed in PBS, and is continuously operated until 20mL of the sample passes through, the sample is replaced by sterile water for cleaning, 10mL of the sample is operated, and then 20mL of the sample is replaced by 20% ethanol solution for operation, so that the machine can be turned off;
6) Protein treatment: and measuring the concentration of each tube of the collected protein, marking, quick-freezing in liquid nitrogen, and freezing and preserving in a refrigerator at the temperature of minus 80 ℃. And standing at room temperature to slowly melt before the experiment.
2.1.4.3SDS-PAGE identification of expression products:
1) Sample preparation: the whole bacteria sample, supernatant sample after centrifugation, protein supernatant sample after passing through a chromatographic column, 20mM imidazole sample, 500mM imidazole sample, protein sample collected at three positions of peak head, peak tip and peak tail in a molecular sieve;
2) And (3) glue preparation: 5% of concentrated glue; selecting 15% of separation gel according to the size of the protein;
3) Sample preparation: mixing the 2X Loading Buffer with the sample 1:1;
4) Loading: 5 mu L of whole bacteria sample, 3 mu L of marker and 10 mu L of other samples;
5) Running glue: constant pressure 80V running concentrated gel; constant pressure 120V running separating glue;
6) Dyeing: placing the protein glue in coomassie brilliant blue dye solution, and carrying out microwave medium and high fire for 2min;
7) Decoloring: placing the dyed albumin glue into tap water, and heating with microwave for 20-40min.
Generation and concentration determination of 2.1.5CLC crystals:
1) Mixing TEV enzyme and pET-28a-gal10-TEV-6His recombinant protein according to the mass ratio of 1:10, and carrying out shaking table enzyme digestion at 4 ℃ for overnight incubation;
2) Centrifuging at 4deg.C for 10min at 600g, sucking supernatant after centrifuging, adding appropriate amount of PBS, and repeating for three times to obtain pure CLCs;
3) The CLCs were disassembled with 6M guanidine hydrochloride and the concentration of CLCs was measured using a spectrophotometer One-Drop.
2.2 in vitro characterization experiments of salt-solubilized CLCs
2.2.1 dissolving CLCs in PBS (pH 7.4), and adjusting the concentration to 0.6mg/ml;
2.2.2 taking into account solubility and safety factors, selecting (CH 3 ) 4 NCl, KCl, naCl, gdmCl, KAc, KBr, preparing a salt solution with PBS (phosphate buffer solution) with a molar concentration of 4M and a pH value of 7.4;
2.2.3 mixing CLCs and salt solutions in proportions, diluting them separately to working concentrations:
1) The CLCs are diluted, and the working concentration is 0.15mg/ml;
2) Dilution (CH) 3 ) 4 NCl, molar concentration gradient 1.0m,1.5m,2.0m,3.0m;
3) KCl was diluted with a molar concentration gradient of 1.0M,1.5M,2.0M,3.0M;
4) The molar concentration gradient of diluted NaCl is 1.0M,1.5M,2.0M and 3.0M;
5) Diluted GdmCl with a molar concentration gradient of 0.2M,0.4M,0.5M,0.6M;
2.2.4 dripping the mixed liquid on a crystal plate, observing by using an optical microscope, and photographing every interval of time (the detection results of the CLC crystal morphology changes in different salt solutions under the optical lens are shown in figure 1);
2.2.5 processing the obtained image by using Photoshop, intercepting the same area, calculating the intercepted area by using imageJ, and normalizing the data obtained at the first time point for 10min (the statistical result of the relative area change of CLC crystals in salt solutions with different concentrations along with time is shown in figure 2);
2.2.6 according to R (t) =k.A (t) · [1-exp (βΔμ (c))]Find the initial reaction Rate R int (intrinsic onset Rate of dissolution of CLC crystals in different anionic and cationic solutions)The statistics of (2) are shown in FIG. 3).
+ The above experimental results prove that Gal-10 crystals have selective response to the above selected salt ions, wherein Gdm shows It is known that Gal-10 crystals are dissolved, and that the dissolution of Gal-10 crystals is time-dependent.
2.3 in vitro characterization experiments of dissolution of guanidine-containing Compounds in CLCs
2.3.1 dissolving CLCs in PBS (pH 7.4), and adjusting the concentration to 0.3mg/ml;
2.3.2 dissolving metformin in PBS buffer at a molar concentration gradient of 0.2mM,2.0mM,10.0mM,20.0mM,100.0mM,200.0mM,300.0mM,400mM,800mM;
the structural formula of the metformin is as follows:
2.3.3 dissolving streptomycin in PBS buffer at molar concentration gradients of 0.2mM,2.0mM,10.0mM,20.0mM;
the structural formula of the streptomycin is as follows:
2.3.4 dissolution of cimetidine Ding Yu in PBS buffer with a molar concentration gradient of 0.2mM,2.0mM,10.0mM,20.0mM;
the structural formula of cimetidine is as follows:
2.3.5 mixing the salt solution with the CLCs in a volume ratio of 1:1, dripping on a crystal plate, observing with an optical microscope, and photographing at intervals; (the results of detection of CLC crystal morphology changes in different guanidine-containing compound solutions under the light microscope are shown in FIG. 4)
2.3.6 processing the obtained image by using Photoshop, intercepting the same area, calculating the intercepted area by using imageJ, and taking the first time point for 10min for normalization; (statistical results of the relative area of CLC crystals in different guanidine-containing compound solutions over time are shown in FIGS. 5-7)
2.3.7 according to R (t) =k.A (t) · [1-exp (βΔμ (c))]Find the initial reaction Rate R int . (statistical results of the intrinsic initial rate of dissolution of CLC crystals in different guanidine-containing compound solutions are shown in FIG. 8)
The above results show that the metformin dissolves Gal-10 crystals with better solubility in three small molecule drugs.
EXAMPLE 2 preparation of CLCs and salt dissolutionCLCsIn vitro characterization experiments of (2)
Experimental method
1 tracheal injection administration
1.1 laboratory animals
The C57BL/6 strain mice are purchased from animal resource institute of laboratory of Chinese food and drug administration. Mice (wild type, male, 6 week old, C57BL/6 strain mice, body weight 20+ -2 g) were randomly divided into five groups of 6 mice each; and preparing the following groups of perfusion medicaments:
1) PBS (negative control),
2)CLCs(1.5mg/ml)，
3) Metformin (0.45 mg/kg),
4) CLCs plus metformin (CLCs: 1.5mg/ml, metformin: 0.45 mg/kg),
5) CLCs plus dexamethasone (0.5 mg/kg) (positive control);
1.2 dosing phase:
1) The mice were intraperitoneally injected with a sterile syringe with a tribromoethanol anesthetic at the following anesthetic dose: mice body weight (g) ×15 μl/mouse, mice were fixed after observing that the mice were fully anesthetized (negative for foot compression reflex);
2) Maintaining the airway of the mouse vertical, exposing a longitudinal incision with a length of 0.5-0.8cm at 1.5cm of the anterior mandible of the cervical skin with an ophthalmic scissors, and blunt separating subcutaneous tissue, connective tissue and cervical muscle along the midline with an ophthalmic forceps until the trachea is fully exposed;
3) Tilting the mouse fixing device by about 30 degrees, sucking 50 mu L of air by using a syringe, then sucking 70 mu L of prepared medicine, inserting a needle below the median thyroid cartilage of the trachea parallel to the longitudinal axis of the trachea by using the syringe, wherein the depth of the needle insertion is about 1cm, and rapidly instilling the air and the perfusion medicine into the trachea of the mouse at the position;
4) After the operation is completed, the respiratory rate of the mice is observed to be suddenly increased, and the mice fixing device is placed perpendicular to the operation table for about 1min to help the medicine distribution; the mouse fixing device is reset, the neck tissues of the mouse are clamped layer by layer according to anatomical layering by using ophthalmic curved forceps, the fixation of the mouse is relieved, the mouse is placed in a lateral position, and the state of the mouse is observed until the mouse returns to an autonomous activity.
2 oral administration
2.1 laboratory animals
Mice (wild type, male, 6 week old, C57BL/6 strain mice, body weight 20+ -2 g) were randomly divided into 6 groups of 3; and preparing the following groups of perfusion medicaments:
1) PBS (negative control),
2)CLCs(1.5mg/ml)，
3) Metformin (0.45 mg/kg),
4) CLCs plus metformin 1× (clc: 1.5mg/ml, metformin: 0.45 mg/kg),
5) CLCs plus metformin 3× (CLCs: 1.5mg/ml, metformin: 1.35 mg/kg),
6) CLCs plus metformin 10× (CLCs: 1.5mg/ml, metformin: 4.5 mg/kg).
2.2 dosing phase:
the mice were grasped so that their head, neck and body were in alignment. Using a syringe equipped with a gastric lavage needle, the needle was advanced from the mouse's horn, pressed against the tongue, pushed gently inward against the palate, advanced slightly deeper into the esophagus and infused with the drug solution.
And 3, sample collection:
sampling was started after 6h and 12h of tracheal injection administration, and sampling was started after 6h of oral administration.
1) The mice were intraperitoneally injected with a sterile syringe with a tribromoethanol anesthetic at the following anesthetic dose: mice body weight (g) ×15 μl/mouse, after observing that mice were fully anesthetized (negative for foot compression reflex), fixed after sacrificing mice;
2) Exposing the mouse trachea again along the trachea instillation incision, and cutting an inverted V-shaped incision below the thyroid cartilage, wherein the operation is gentle to avoid separating the mouse trachea;
3) Drawing 600 mu L of sterile PBS, allowing the pipette to extend into the incision parallel to the longitudinal axis of the trachea until the PBS is completely attached to the inner wall of the mouse trachea, injecting the PBS into the mouse bronchus through the mouse trachea at a constant speed, and then sucking back at a constant speed, wherein a large amount of bubbles are found in the sucking back process and are an indicator of successful operation, and after the injection-sucking back process is repeated for 4-5 times, placing the recovery liquid into a 1.5mL centrifuge tube, and blowing and uniformly mixing;
4) Repeating the step (3), putting the recovery liquid into the same 1.5mL sterile EP tube, and preserving at-80 ℃;
5) The mouse chest was opened, the sternum was cut off, the heart was extracted to cut off connective tissue and fascia between the lung tissue and chest, the lung tissue was removed, rinsed with sterile PBS, 1ml Trizol was added for cleavage and stored at-80 ℃.
Treatment effect detection
1. RNA extraction and cDNA reverse transcription
1.1RNA extraction:
1) Placing the lysate stored in Trizol on ice for slow thawing;
2) After the RNA sample is completely thawed, the sample is taken to room temperature and placed for 10min, so that the RNA is fully dissolved, chloroform is added according to the proportion of Trizol to chloroform=5:1 (v/v), and the mixture is fully and evenly mixed by forced oscillation and placed for 10-15min at room temperature;
3) Centrifuging at 4 ℃ for 15min at 12000 r/min;
4) Transferring the upper aqueous phase into a new RNase-free 1.5mL centrifuge tube;
5) Adding 0.6-0.8 times of pre-cooled isopropanol, mixing, and standing at-20deg.C for more than 30 min;
6) Centrifuging at 4 ℃ for 15min at 12000r/min, and removing the supernatant to ensure that RNA precipitate exists at the bottom of the centrifuge tube;
7) Adding 1mL of 75% ethanol prepared from DEPC treated water or enzyme-free sterile water, gently shaking the centrifuge tube, and resuspending the pellet;
8) Centrifuging at 12000r/min for 15min at 4deg.C, discarding supernatant as much as possible, and repeating steps 7-8;
9) Airing at room temperature, and evaporating 75% ethanol;
10 Dissolving the RNA precipitate with DEPC treated water or enzyme-free sterile water;
11 2. Mu.L of RNA sample is taken, the concentration of RNA is measured by a micro ultraviolet spectrophotometer, and a proper amount of DEPC treated water or enzyme-free sterile water is added to adjust the concentration of RNA solution to 200 ng/. Mu.L, and the RNA solution is frozen at-80 ℃.
1.2 reverse transcription of RNA into cDNA:
reverse transcription of cDNA was performed according to the instructions of Takara company cDNA Reverse Transcription Kits (cat.no RR 036A);
1)5×PrimeScript RT Master Mix(Perfect Real Time):2μL
1×Total RNA：0.5μg
RNase Free dH 2 O up to 10μL
2) After gentle mixing, reverse transcription was performed under the following conditions:
15min at 37℃C (reverse transcription reaction)
85 ℃ for 5sec (reverse transcriptase inactivation reaction)
And (5) preserving at 4 ℃ for a short time.
2. Real-time fluorescent quantitative PCR (RT-qPCR) detection of gene expression changes
According toInstructions for 480SYBR Green I Master perform a real-time fluorescent quantitative PCR experiment;
1) The working fluid added in each reaction system is as follows:
2) Adding the working solution into a 384-well plate, adding 1 mu L of cDNA obtained by reversing the previous steps into each well, flatly attaching a sealing film on the 384-well plate, and instantaneously separating for 90 seconds;
3) Placing a 384-well plate on a reaction bracket according to the instruction of an instrument, and setting the following reaction program;
4) After the reaction is finished, use480 software calculates a reaction dissolution curve (Melt), observes whether the amplification curve is a single peak to exclude non-specific amplification, and automatically calculates the relative expression amount of the detected gene according to the Ct values of the target gene and the reference gene, wherein the algorithm is as follows: gene relative expression magnitude=2 (- (Ct target gene-Ct reference gene)) ；
5) The differences were plotted and analyzed for significance in Graphpad Prism 8.0 software.
The results of the tracheal injection qPCR are shown in FIG. 9, and the stimulation of CLCs causes IL-1 beta, IL-6, TNF-alpha, CCL-2 to be in genes Elevated levels, while metformin treated groups significantly reduced inflammatory factor expression in lung tissue and were statistically significant.
The qPCR results of the oral administration group are shown in FIG. 10, in which the CLCs were stimulated to cause IL-1β, IL-6, TNF- α, CCL-2 on the base The figure shows the detection result of 6 hours of administration due to the rise of the level, and the oral metformin treatment group1X can obviously reduce inflammation in lung tissue The effect of reducing inflammation is not 1x better due to the toxicity effect on cells after the concentration of the expressed factors is increased.
3. Enzyme-linked immunosorbent assay (ELISA) for detecting protein changes
3.1 coating ELISA 96 well plates: diluting the capture antibody by using sterile PBS according to the working concentration, adding 100 mu L/hole of the diluted capture antibody into a 96-hole ELISA plate with high binding force, completely covering the upper surface by using a sealing film, and incubating overnight at room temperature;
3.2 detection process:
1) Removing the capture antibody, adding 200 mu L of washing liquid into each hole, washing, removing the washing liquid, and sucking the residual liquid with filter paper for 3 times;
2) mu.L of diluent (PBS containing 1% BSA) was added to each well and incubated at room temperature for at least 1h;
3) Discarding the dilution and repeating step 1);
4) Preparing standard substances by using new diluent, adding 100 mu L of prepared standard substances or samples into each hole, carrying out double-hole mixing on each sample, slightly shaking, pasting a cover plate, and incubating at 37 ℃ for 2 hours;
5) Discard the sample and repeat step 1);
6) Diluting the detection antibody to the working concentration by using a new diluent, adding 100 mu L of detection antibody working solution into each hole, laminating a cover plate, and incubating for 2 hours at room temperature;
7) Discarding the detection antibody and repeating step 1);
8) Diluting strepavidin-HRP with new diluent to obtain working solution concentration according to the ratio of 1:40, adding 100 mu L of each hole, laminating, and incubating for 20min at room temperature in a dark place;
9) Discarding strepitavidin-HRP working fluid and repeating step 1);
10 Adding 100 mu L of TMB single-component color development liquid into each hole, laminating, and incubating for 10-20min at room temperature in a dark place;
11 Adding 50 mu L of a chromogenic stop solution into each hole, shaking and uniformly mixing, detecting OD values of 450nm and 570nm by using a full-function microplate detector, and subtracting the OD value of 570nm from the OD value of 450 nm;
12 Diagramming in Graphpad Prism 8.0 software and performing significant difference analysis. (FIG. 11, FIG. 12)
The results of the tracheal injection ELISA are shown in FIG. 11, and the CLCs are stimulated to cause IL-1 beta, IL-6, NF-alpha, CCL-2 in protein Elevated levels, while the metformin-treated group significantly reduced inflammatory factors in bronchoalveolar lavage fluid and had statistics Meaning of science.
ELISA results of the oral administration group are shown in FIG. 12, in which the CLCs were stimulated to cause IL-1β, IL-6, TNF- α, CCL-2 in Elevated protein levels, oral metformin-treated group 1X significantly reduced inflammatory factors in bronchoalveolar lavage fluid.
4. Pulmonary tissue pathological sample H of mice in tracheal injection administration group&E staining
4.1 air-tube injection administration groups 6h and 12h later left lung tissue of mice (n=3) was carefully harvested, washed in PBS and placed in a 10mL tube containing 8mL paraformaldehyde (4%) which was then left to stand at room temperature for 48-72h;
4.2 dewatering, embedding, dewaxing, dyeing, dewatering sealing sheets (the work of this step is done by Wohan Seville Biotechnology Co., ltd.)
The results are shown in fig. 13, and the inflammatory cell infiltration of lung tissue is significantly relieved after the mice are subjected to tracheal injection of metformin.
EXAMPLE 3 Structure-Activity relationship (other guanidine-containing Compounds)
1. Dilution of CLC crystals to a concentration of 0.3mg/ml
2. Dissolving 1-methyl guanidine in PBS buffer with a molar concentration gradient of 2mM;20mM;100mM;200mM;400mM.
3. Dissolving 1, 3-tetramethylguanidine in PBS buffer with a molar concentration gradient of 0.2mM;1.0mM;4.0;10.0mM.
4.1, 1-dimethylguanidine was dissolved in PBS buffer with a molar concentration gradient of 2mM;20mM;100mM;200mM;400mM.
5. Dissolving 1-ethylguanidine in PBS buffer with a molar concentration gradient of 2mM;20mM;100mM;200mM;400mM.
6. Dissolving 1-phenyl biguanide in PBS buffer with a molar concentration gradient of 0.2mM;2.0mM;4.0mM;10.0mM;20.0mM.
7. Dissolving 1- (o-tolyl) biguanide in PBS buffer with a molar concentration gradient of 0.2mM;2.0mM;4.0mM;10.0mM.
8. Mixing the salt solution and the CLC crystal in a volume ratio of 1:1, observing on a crystal plate, and photographing every time interval;
9. intercepting the same area by using PhotoShop, calculating the intercepting area by using imageJ, and taking the first time point for 10min for normalization;
10. the initial reaction rate Rint was determined from R (t) =k·a (t) · [1-exp (βΔμ (c)) ].
Statistical results of the relative area of CLC crystals in solutions of different guanidine-containing compounds at different concentrations over time are shown in the figure 14, the statistical results of the initial rate of dissolution of CLC crystals in different guanidine-containing compound solutions are shown in fig. 15.
The above experimental results prove that the quantity of nonpolar functional groups and the capacity 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.
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半乳糖凝集素-3及其抑制剂的研究进展;张文博;中国药学杂志;第44卷(第03期);第165-169页 *
大剂量链霉素治疗仔猪急性支气管肺炎;李有业;兽药与饲料添加剂;第1999卷(第05期);摘要 *
海马内注射5-HT3受体激动剂后c-fos在不同脑区的表达;刘社兰，等;细胞与分子免疫学杂志;第2003卷(第05期);第454-455+458页 *
西咪替丁治疗轮状病毒肠炎患儿前后血清细胞因子的变化;贾秀红，等;中国儿童保健杂志;第17卷(第06期);摘要 *
贾秀红，等，.西咪替丁治疗轮状病毒肠炎患儿前后血清细胞因子的变化.中国儿童保健杂志.2009,第17卷(第06期),摘要. *
钟薇，等.二甲双胍对哮喘小鼠的气道炎症的影响.广西医科大学学报.2016,第33卷(第04期),摘要、讨论第1段. *

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