CN102240395A - Application of interleukin-1 acceptor antagonist - Google Patents
Application of interleukin-1 acceptor antagonist Download PDFInfo
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- CN102240395A CN102240395A CN2010101755621A CN201010175562A CN102240395A CN 102240395 A CN102240395 A CN 102240395A CN 2010101755621 A CN2010101755621 A CN 2010101755621A CN 201010175562 A CN201010175562 A CN 201010175562A CN 102240395 A CN102240395 A CN 102240395A
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Abstract
The invention relates to the field of biomedicine, and specifically relates to an application of an interleukin-1 acceptor antagonist. The invention discloses an application of a protein (a) or a protein (b) in preparation of drugs for treating liver injury. An amino acid sequence of the protein (a) is shown in the formula SEQ ID NO.1. The protein (b) has an amino acid sequence at least 70% homologous with that of the protein (a) and is a protein or a polypeptide with a liver protective reagent activity. The protein provided by the invention can eliminate or reduce effectively liver injury or hepatic failure development and hepatic cell death, thus provides a novel choice for a clinical treatment of liver injury from now on.
Description
Technical field
The present invention relates to biomedicine field, be specifically related to the purposes and the pharmaceutical composition thereof of interleukin-1 receptor antagonist.
Background technology
The liver of people and animal all has the unique ability of its growth of regulation and control and weight.If certain harmful substance has damaged the part liver parenchyma, then Cun Huo hepatocyte can be duplicated, thereby substitutes the essence of damage.If the hepatectomy in viral, toxicity, immunogenicity or metabolism source or the overwhelming majority that hepatocyte injury has influenced essence to such an extent as to surpass the regeneration capacity of residual hepatic tissue, then will develop into fatefulue hepatic insufficiency.
At present, without any having the liver protection and stimulating the medicine of regeneration to can be used for treating acute or chronic hepatic insufficiency, from then on the meaning, these medicines are extremely important; Be that the used medicine of hepatology should comprise these tell-tale treatment products.Liver protection reagent is a kind ofly can protect that the hepatocyte opposing is various can be caused toxicity and/or infringement and finally cause the product or the active component of the stimulation of necrosis or apoptosis hepatocyte.Therefore, no matter when induce hepatic injury, the liver protection reagent of administration suitable dose will improve the hepatocyte survival, and it is beneficial to liver regeneration, helps liver function normalization, and can save patient's life under extreme case.Hepatic injury by the perfusion (as inductive damage in the liver transplantation in the liver transplantation) of toxic agent (comprising alcohols, medicine), virus, autoimmune disease, ischemia, ischemia/again and induced by any inflammation.The development and the hepatocellular death of hepatic injury in those situations will be got rid of or reduce to a kind of good liver protection reagent.
" interleukin-1 receptor antagonist " (interleukin-1 receptor antagonist IL-1Ra), belongs to the IL-1 family member jointly with IL-1 α, IL-1 β three.IL-1Ra is IL-1 α, the common antagonist of IL-1 β, and its protein sequence is that the C-end region is significantly similar to IL-1 α, IL-1 β at receptor binding site.IL-1Ra can emulative inhibition IL-1 and its receptors bind, is arrested in the biological activity of expressing IL-1 in a plurality of tissues and the organ, is used for diseases such as rheumatoid arthritis, septicemia clinically.The IL-1Ra of people and mice has 77% homology, and the biological action of IL-1Ra does not have species specificity.The difference of the IL-1Ra molecular weight that different cells produce is mainly by due to the degree of glycosylation difference.Confirm that glycosylation does not have influence (R Daig, G Rogler, E Aschenbrenner, et al.Gut2000 (46): 350-358) to the IL-1Ra biologic activity.
Summary of the invention
Technical problem to be solved by this invention is under viral, metabolic or the etiologic cause of disease situation of toxicity, provides a kind of liver protection reagent can get rid of or reduce the development and the hepatocellular death of hepatic injury.
For this reason, the invention discloses a kind of following (a) or albumen (b) and be used for the treatment of purposes in the medicine of hepatic injury in preparation:
(a) its aminoacid sequence such as the described albumen of SEQ ID NO.1; (b) at least with (a) in aminoacid sequence 70% homology and have the albumen of liver protection agent of activity.
In some embodiments, the acute hepatic failure or acute liver damage, subacute liver failure, slow extra urgaent dispatch (subacute) liver failure, chronic liver failure or the chronic hepatic injury that cause for the toxicity nosetiology of described hepatic injury.The hepatic injury that described hepatic injury is preferably caused by toxicant, described toxicant can be interior extracellular toxin, flavacin, nitrosamine, ethanol, medicine or virus, most preferably is drug induced hepatic injury or liver failure.
In some embodiments, the preparation of described medicine is an intestinal external administration preparation, and described intestinal external administration preparation can be injection or injectable sterile powder.
On the other hand, the invention discloses above-mentioned albumen and can treat the method for etiologic acute, subacute, the explosive or chronic hepatopathy of viral, metabolic or toxicity as active component, this method comprises the above-mentioned albumen that gives effective dose.
Compared with prior art, the present invention has following beneficial effect: the development and the hepatocellular death of hepatic injury can be got rid of or reduce to albumen of the present invention effectively, and new selection may be provided for the treatment of clinical hepatic injury from now on.
Description of drawings
Fig. 1: rhIL-1Ra is to the influence of the inductive acute hepatic failure of 650mg/kg acetaminophen (APAP) (ALF) mice survival rate;
Fig. 2: rhIL-1Ra is to the influence of the inductive ALF mice of the APAP of 550mg/kg survival rate;
Fig. 3: rhIL-1Ra is to 2.6ml/kg CCl
4Induce the influence of acute liver damage model mice survival rate
Fig. 4: rhIL-1Ra has significantly reduced the ALT and the AST level of the inductive ALF mice serum of APAP of 550mg/kg.
Fig. 5: rhIL-1Ra has significantly reduced the necrosis area of the inductive ALF mouse liver of APAP of 550mg/kg
Fig. 6: rhIL-1Ra has significantly reduced the hepatocellular apoptosis quantity of the inductive ALF mice of APAP of 550mg/kg
Fig. 7: rhIL-1Ra has significantly promoted the inductive ALF mouse liver cell of the APAP of 550mg/kg regeneration speed
Fig. 8: rhIL-1Ra has significantly reduced the necrosis area of the inductive ALF mouse liver of APAP of 550mg/kg
Fig. 9: rhIL-1Ra has significantly alleviated mouse liver apoptosis after the inductive ALF modeling of APAP of 550mg/kg
Figure 10: rhIL-1Ra has significantly accelerated the inductive ALF mouse liver cell of the APAP regeneration speed of 550mg/kg
Figure 11: rhIL-1Ra has significantly suppressed the activity of the inductive ALF murine liver tissue of the APAP apoptosis signal transduction pathway of 550mg/kg
Figure 12: rhIL-1Ra is to 1ml/kg CCl
4Induce glutamic oxaloacetic transaminase, GOT level affects in the acute liver damage model mice body;
Figure 13: rhIL-1Ra is to 1ml/kg CCl
4Induce gpt level influence in the acute liver damage model mice body;
Figure 14: rhIL-1Ra is to 1ml/kg CCl
4Induce acute liver damage model mice hepatic tissue section HE dyeing hepatic necrosis area relatively.
The specific embodiment
At this paper, the described hepatic injury of term is by various poisonous chemical substances such as bacteriogenic inside and outside toxin, the damage that exists flavacin, nitrosamine, ethanol, medicine and virus etc. that liver is caused in the food.
Described drug induced hepatic injury is meant because the hepatic injury that medicine or its metabolite cause; the medicine of common caused drug induced hepatic injury: antitubercular agent (as Rimactazid, pyrazinamide, ethionamide); antibiotic (as chlortetracycline, sulfadiazine, erythromycin, ofloxacin, furazolidone); Chinese patent medicine (as GANJI SAN, KEYIN PIAN, XIAOHE PIAN, Radix Tripterygii Wilfordii tablet, detoxicating tablet of cow-bezoar, LIUSHEN WAN); Chinese herbal medicine (as Herba Sidae Rhombifoliae soup); antipyretic analgesic (as acetaminophen, aspirin, indometacin), hypolipidemic (as fenofibrate, nicotinic acid) etc.
" interleukin-1 receptor antagonist " (interleukin-1 receptor antagonist IL-1Ra), belongs to the IL-1 family member jointly with IL-1 α, IL-1 β three.IL-1Ra is IL-1 α, the common antagonist of IL-1 β, and its protein sequence is that the C-end region is significantly similar to IL-1 α, IL-1 β at receptor binding site.The aminoacid sequence of hIL-1Ra is as described in the SEQ ID NO.1, and the IL-1Ra of people and mice has 77% homology, and the biological action of IL-1Ra does not have species specificity.The difference of the IL-1Ra molecular weight that different cells produce is mainly by due to the degree of glycosylation difference.Confirm, glycosylation to the IL-1Ra biologic activity do not have influence (R Daig, G Rogler, E Aschenbrenner, et al.Gut 2000 (46): 350-358).
Described protein of the present invention preferably this material is hIL-1Ra protein and mutant thereof; Its functional activity fragment or its analog; Has for example dna vector (plasmid or virus) of proteinic carrier that the homologue of high homology and coding comprise the aminoacid sequence that SEQ ID NO.1 describes.Functional activity fragment or analog can form by one or more amino acid residue that adds, inserts, modifies, replaces or lack in the above listed aminoacid sequence.
Term " analog " also comprises precursor and other functional equivalent or the analogies of chimeric protein, fusion rotein, anti-idiotype antibody, above-claimed cpd.Also comprise the conjugated protein active compound body of Simulation with I L-1Ra.
Term " mutant " is meant the mutant of aminoacid sequence as hIL-1Ra as described in the SEQ ID NO.1.Than natural IL-1Ra albumen, this mutant is compared with their wild types, has enhanced activity and/or altered stereospecificity.The aminoacid sequence mutant of native protein can be by introducing suitable nucleotide variation or preparing by external synthetic required polypeptide in nucleotide of the present invention.These mutants comprise, for example lack, insert or replace the residue in this aminoacid sequence.Can provide final protein product by disappearance, the combination of inserting and replacing to obtain final construct.
Proteic percent homology is analyzed (GCG program) by GAP (Needleman and Wunsh, 1970) and is determined, parameter gap creation penalty=5 wherein, gap extension penalty=0.3.When analyzed sequence length was at least 15 aminoacid, GAP just analyzed and tests in 15 the amino acid whose zones that are at least of two sequences that participate in test.More preferably, when analyzed sequence length was at least 50 aminoacid, GAP just analyzed and tests in 50 the amino acid whose zones that are at least of two sequences that participate in test.More preferably, when analyzed sequence length was at least 100 aminoacid, GAP just analyzed and tests in 100 the amino acid whose zones that are at least of two sequences that participate in test.More preferably, when analyzed sequence length was at least 250 aminoacid, GAP just analyzed and tests in 250 the amino acid whose zones that are at least of two sequences that participate in test.Even more preferably, when analyzed sequence length was at least 500 aminoacid, GAP just analyzed and tests in 500 the amino acid whose zones that are at least of two sequences that participate in test.
The aspect that the present invention relates to also comprises IL-1Ra albumen analog; carrying out different modifications between their synthesis stages or after synthetic; for example, by biotinylation, benzylization, glycosylation, acetylation, phosphorylation, by known protection/blocking groups derivatization, proteoclastic dissection, be connected to antibody molecule or other cell ligand is first-class.These modifications can be used for increasing proteic stability of IL-1RA of the present invention and/or biological activity.
Proteic expression
The carrier, the transformant that the present invention includes the coding proteic DNA of IL-1Ra of the present invention and contain these DNA.
Among the present invention, the term of use " transformant " (transformant) promptly has the host cell of allogeneic dna sequence DNA molecule.
The present invention also comprises by synthetic and recombinant technique and produces proteic method of the present invention.Can separate and purification polynucleotide (DNA or RNA), carrier, transformant and organism by methods known in the art.
Being used for carrier of the present invention can be as phage, plasmid, cosmid, mini-chromosome, virus or retroviral vector.The carrier that can be used for cloning and/or express polynucleotide of the present invention is to duplicate and/or to express the carrier that duplicates and/or express polynucleotide in the host cell of polynucleotide at need.In general, polynucleotide and/or carrier can be used for any eucaryon or prokaryotic cell, comprise mammalian cell (as people (as HeLa), monkey (as Cos), rabbit (as the rabbit reticulocyte), rat, hamster (as CHO, NSO and baby hamster kidney cell) or mouse cell (as the L cell)), plant cell, yeast cells, insect cell or bacterial cell (as escherichia coli).Relevantly be applicable to that the example of the suitable carrier of polytype host cell can be referring to for example F.Ausubel et al., Current Protocols in Molecular Biology.GreenePublishing Associates and Wiley-Interscience (1992) and Sambrook etal. (1989).Can use the host cell that contains these polynucleotide to come great expression to can be used for for example protein of medicine, diagnostic reagent, vaccine and therapeutic agent.
Having developed several different methods is used for via complementary sticky end polynucleotide being operated with carrier and links to each other.For example, can add complementary by the DNA section in desire is inserted carrier DNA with the aggressiveness sequence fragment.Then by the hydrogen bond connection carrier between the complementary homopolymeric tail and DNA section to form recombinant DNA molecules.
The synthetic linker that contains one or more restriction site provides the method for another kind of connection DNA section and carrier.Handle the DNA section that produces by the endonuclease restrictive diges-tion with phage T4DNA polymerase or e. coli dna polymerase I, described two kinds of polymerases with its 3 ', 5 '-exonucleolytic activity is removed outstanding γ-strand end, and mends flat 3 '-recessed end with its polymerization activity.Therefore, these active associatings have produced flush end DNA section.Then can catalysis the enzyme that connects of flush end dna molecular, as under the existence of phage T4DNA ligase the linkers of flush end section with molar excess being incubated.Therefore, product is the DNA section that end carries the polylinker sequence.Use these DNA sections of suitable Restriction Enzyme cracking then, and be connected in the expression vector of using enzymatic lysis, described enzyme can produce and the compatible end of described DNA section.Can buy the synthetic linker that contains a plurality of restriction endonucleases site from a plurality of businessmans.
The polynucleotide insert should be operably connected to on the compatible suitable promoter of the host cell of expressing polynucleotide.Promoter can be strong promoter and/or inducible promoter.The example of some promoteres of enumerating comprises phage PL promoter, escherichia coli lac, trP, phoA, tac promoter, SV40 is early stage and late promoter and retrovirus LTR promoter.Other suitable promoter is well known by persons skilled in the art.The express recombinant carrier further contains transcription initiation, termination site, and contains the ribosome binding site that is useful on translation at transcriptional domain.The coded portion of the transcript that recombinant vector is expressed can comprise translation initiation codon that is positioned at the starting point place and the termination codon that suitably is positioned at the end that is translated polypeptide (UAA, UGA or UAG).
As mentioned above, expression vector can comprise at least one selected marker.Described labelling comprises dihydrofolate reductase, G418, glutamine synthase or the neomycin resistance for eukaryotic cell culture; And the tetracycline, kanamycin or the ampicillin resistance gene that are used for escherichia coli and other antibacterial culturing.Suitably host's representative example includes but not limited to: bacterial cell, as escherichia coli, streptomycete and salmonella typhimurium cell; The fungal cell is as yeast cells (as saccharomyces cerevisiae or pichia pastoris phaff); Insect cell is as fruit bat S2 and noctuid SF9 cell; Zooblast, as CHO, COS, NSO, 293 and the Bowes melanoma cells; And plant cell.The appropriate culture medium of above-mentioned host cell and condition of culture are known in the art.
For separation and purification effectively or secretion target protein, usually also can utilize label protein or the label polypeptide (Tag) of being convenient to separation and purification.Commonly used have glutathione-S-transferase (glutathione S-transferase, GST), six polyhistidyl peptides (His.Tag), a-protein (protein A) and cellulose binding site (cellulose binding domain) etc.By the form of particularity albumen or polypeptide and target protein formation fusion rotein, utilize the special nature of described label protein or label polypeptide to separate and purification after the expression to target protein.Combine with Ni-Chelating Sepharose post specificity as His.Tag.Described label protein or label polypeptide can be removed fusion sequence with the locus specificity protease digestion behind purification, as available thrombin, enterokinase and Xa factor etc., to obtain target protein.
The present invention also comprises the host cell that contains nucleotide sequence of the present invention, and described nucleotide sequence can be operated with one or more allos control zone (as promoter and/or enhancer) through technology known in the art and link to each other.Can select to regulate the expression of the gene order of inserting, or can be according to the required particular form modification and the host strain of processed gene product.In the presence of some inducer, the expression that some promoter starts can raise; Therefore, can control polypeptide expression through genetic modification.In addition, different host cells have distinctive and special translation, translation post-treatment and the proteinic mechanism of modification (as phosphorylation, cracking).Can select suitable cell line to guarantee that the exogenous proteins of expressing is carried out desirable modification and processing.
By the transfection of calcium phosphate transfection, the mediation of DEAE-glucosan, transfection, electroporation, transduction, infection or other method of cation lipid mediation, nucleic acid of the present invention and nucleic acid recombinant vector can be imported host cell.Described method is described in the laboratory manual of a plurality of standards, as Davis et al., and Basic Methods InMolecular Biology (1986).
The described proteic polynucleotide of the present invention of encoding can be connected to breed in the host with the carrier that contains selected marker.In general, can be at precipitate, import plasmid vector in the complex as calcium phosphate precipitation thing or itself and charged lipids.If carrier is a virus, can use suitable incasing cells to tie up to and external it be packed, transduce to host cell again.
Can identify by successful cell transformed by well-known technology, promptly contain the cell of DNA recombinant vector of the present invention.For example, can cultivate the cell of importing express recombinant carrier gained to produce required polypeptide.Collect and cell lysis, use as Southem (1975) J.Mol.Biol.95,503 or Berent etal (1985) Biotech.3,208 described methods detect the existence of DNA in its DNA content.Perhaps, use protein in the antibody test supernatant.
By well-known method from the reconstitution cell culture, reclaim and purification described albumen of the present invention comparatively favourable, described method comprise sulphuric acid by or ethanol precipitation, acid extraction, anion or cation-exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatograph, hydroxyapatite chromatography, dewatering electric charge effect chromatography and agglutinin chromatography.In some embodiments, can use high performance liquid chroma-tography (HPLC) to carry out purification.
In some embodiments, can use above-mentioned one or more chromatography method purification described albumen of the present invention.In other embodiments, can use following one or more chromatographic column purification described fusion rotein of the present invention, described chromatographic column has: Q sepharose FF post, SP sepharose FF post, Q sepharoseHigh Performance post, Blue sepharose FF post, Blue post, Phenyl Sepharose FF post, DEAE Sepharose FF, Ni-Chelating Sepharose FF post or Methyl post etc.
In addition, can use the method purification albumen of describing among the international publication number WO00/44772 (listing this paper in as a reference in full) of the present invention.Those skilled in the art can easily change wherein said method to be used for purification albumen of the present invention.Can be from comprising that for example the protokaryon or the eucaryon host of antibacterial, yeast, higher plant, insecticide and mammalian cell reclaim albumen of the present invention through the product that recombinant technique produces.
Pharmaceutical composition
Be used for the present invention or contain proteic group of medicine compound of the present invention.Usually, when pharmaceutical composition of the present invention is used for such use, described albumen can be made the pharmaceutical dosage form of different way of administration with one or more pharmaceutically acceptable carriers or mixed with excipients, as tablet, capsule, powder, granule, syrup, solution, oral liquid, spirit, tincture, aerosol, powder spray, injection, injectable sterile powder, suppository etc.
" pharmaceutically acceptable " composition is to be applicable to people and/or animal and not have the material that excessive bad side reaction (as toxicity, stimulation and allergy) promptly has rational benefit/risk ratio." pharmaceutically acceptable carrier " is acceptable solvent, suspending agent or the excipient pharmaceutically or on the food that is used for fusant albumen of the present invention is sent to the animal or human.Carrier can be a liquid or solid.
Albumen of the present invention can be through oral, intravenous, intramuscular or subcutaneous route administration.
But the dosage form of oral administration administration is in the above-mentioned dosage form: tablet, capsule, powder, granule, syrup, solution, spirit.Solid-state carrier comprises: starch, lactose, calcium hydrogen phosphate, microcrystalline Cellulose, sucrose, kaolin, micropowder silica gel, Pulvis Talci, low-substituted hydroxypropyl cellulose, carboxymethyl starch sodium, polyvinylpyrrolidone.And liquid carrier comprises: sterilized water, ethanol, Polyethylene Glycol, nonionic surfactant and edible oil (as Semen Maydis oil, Oleum Arachidis hypogaeae semen and Oleum sesami).Normally used adjuvant comprises in the process of pharmaceutical compositions: flavoring agent, coloring agent, antiseptic (as oxybenzene alkyl butyl ester, sodium benzoate, sorbic acid) and antioxidant (as vitamin E, vitamin C, sodium pyrosulfite and dibenzylatiooluene).
The dosage form that can be used for injection administration in the above-mentioned dosage form comprises: injection, injectable sterile powder, they are that medicine and one or more pharmaceutically acceptable mixed with excipients are made form for drug administration by injection.Solvent comprises: sterilized water, ethanol, glycerol, propylene glycol, Polyethylene Glycol.In addition, also need add antibacterial (as benzyl alcohol, butyl hydroxybenzoate, thimerosal), isoosmotic adjusting agent (as sodium chloride, glucose), suspending agent (as sodium carboxymethyl cellulose, methylcellulose), solubilizing agent (tween 80, lecithin), antioxidant (as vitamin E, vitamin C, sodium pyrosulfite) and filler (as lactose, mannitol).
From being easy to prepare the position with administration, preferred pharmaceutical composition is a solid-state composition, especially lyophilized injectable powder.
Pharmaceutical composition of the present invention can prepare according to the method that the pharmaceutical manufacturing of knowing and generally acknowledge requires.Pharmaceutical composition is suitable to comprise protein of the present invention and pharmaceutically acceptable carrier, and is suitably unit dosage form.Pharmaceutical composition of the present invention can comprise the protein of the present invention of prodrug forms, and this prodrug can change into the activity form of thing of the present invention at receiver's host intracellular metabolite.
Pharmaceutical composition of the present invention also can with the other therapies use in conjunction, as simultaneously, sequential or use respectively.Pharmaceutical composition of the present invention can include other active function thing.
Purposes
Disclose described albumen and can prevent or treat the method for acute hepatic failure or acute liver damage, subacute liver failure, slow extra urgaent dispatch (subacute) liver failure or chronic liver failure as active component, this method comprises above-mentioned (a) that give patient's effective dose or (b) described albumen.
In some embodiments, when described hepatic injury is acute hepatic failure or acute liver damage, subacute liver failure, slow extra urgaent dispatch (subacute) liver failure or chronic liver failure, described method can be by mode before the drug administration, behind the drug administration, before the drug administration and after the chemotherapeutics administration, gives above-mentioned (a) of patient's effective dose or (b) described albumen.
" effective dose " or " therapeutic dose " all is meant the amount that is enough to produce curative effect.Effective dose can divide one or multiple dosing.Usually, effective dose is enough to relax, improve, stablize, slow down or postpone further developing of disease.
The effective dose of used active component can change with the order of severity of mode of administration and disease to be treated.For most of large mammal, the accumulated dose that imposes effective ingredient every day is about 0.01-1000mg.Usually, the scope of adult's clinical administration amount is 0.01-200mg/ day, is preferably 0.05-100mg/ day.
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is usually according to the normal condition or the condition of advising according to manufacturer.Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
1.1rhIL-1Ra influence to the inductive ALF mice of the APAP of 650mg/kg survival rate
Experimental technique:
The C57BL/6 mice in 8 to 10 ages in week is divided into rhIL-1Ra group and normal saline (NS) group, and all mices carry out modeling according to body weight 650mg/kg disposable celiac injection APAP solution.After the modeling medication 1 hour, rhIL-1Ra group mice is treated according to the rhIL-1Ra subcutaneous injection that body weight gives 1mg/kg, and the NS group gives isopyknic normal saline subcutaneous injection and treats in contrast, and treatment is 12 hours once.Treatment is 7 days continuously.Observe continuously after the medication and write down and respectively organize dead mouse quantity, observed altogether 7 days.
Experimental result:
As shown in Figure 1, the survival rate (80%) for the treatment of rhIL-1Ra (human interleukin-11 receptor antagonist) group (n=10) after 48 hours will be significantly higher than the survival rate (33.33%) of NS group (n=18), has significant difference (Kaplan-Meier method statistic, P＜0.05).Two groups are not all had dead mouse after 48 hours.
1.2rhIL-1Ra influence to the inductive ALF mice of the APAP of 550mg/kg survival rate
Experimental technique:
The C57BL/6 mice in 8 to 10 ages in week is divided into rhIL-1Ra group and NS group, and all mices carry out modeling according to body weight 550mg/kg disposable celiac injection APAP solution.After the modeling medication 1 hour, rhIL-1Ra group mice is treated according to the rhIL-1Ra subcutaneous injection that body weight gives 1mg/kg, and the NS group gives isopyknic normal saline subcutaneous injection and treats in contrast, and treatment is 12 hours once.Treatment is 7 days continuously.Observe continuously after the medication and write down and respectively organize dead mouse quantity, observed altogether 7 days.
Experimental result:
As shown in Figure 2, the survival rate (100%) for the treatment of rhIL-1Ra group (n=14) after 48 hours will be significantly higher than the survival rate (35.71%) of NS group (n=14), has significant difference (Kaplan-Meier method statistic, P＜0.01).Two groups are not all had dead mouse after 48 hours.
1.3rhIL-1Ra to 2.6mL/kg CCl
4The influence of inductive acute liver damage model mice survival rate.
Experimental technique:
The male C57BL/6 mice in 8 ages in week of 20 health is as experimental group, every mouse peritoneal injection CCl
4LD50 dosage (Semen Maydis oil dilution in 1: 1), 1mg/kg body weight subcutaneous injection rhIL-1Ra is pressed in modeling after 1 hour, and 12h injects albumen once more at interval, injects continuously 5 days; The male C57BL/6 mice in healthy 8 ages in week is organized in contrast, every mouse peritoneal injection CCl
4LD50 dosage (1: 1 Semen Maydis oil dilution), and contrast as therapeutic injecting isopyknic PBS according to the rhIL-1Ra administration time thereafter.Observe continuously after the medication and write down and respectively organize dead mouse quantity, observed altogether 5 days.
Experimental result:
As shown in Figure 3, the survival rate (60%) for the treatment of rhIL-1Ra group (n=6) after 60 hours will be significantly higher than the survival rate (10%) of NS group (n=1), has significant difference (Kaplan-Meier method statistic, P＜0.01).Two groups are not all had dead mouse after 60 hours.
Pathology and the biochemistry detection of embodiment 2, rhIL-1Ra treatment ALF mice:
2.1 experimental technique:
2.1.1 animal packet design: at first, the C57BL/6 mice in 120 8 to 10 ages in week is divided into treatment group (T group, it observes terminal point is 3 hours, 6 hours, 24 hours, 48 hours, 96 hours and 168 hours, be designated as T3, T6, T24, T48, T96, T168, totally 6 groups, there are 10 mices in every group, the hourage of terminal point is observed in numerical value representative after the T) and matched group (C group, it observes terminal point is 3 hours, 6 hours, 24 hours, 48 hours, 96 hours and 168 hours, be designated as C3, C6, C24, C48, C96, C168, totally 6 groups, there are 10 mices in every group, the hourage of terminal point is observed in the numerical value representative after the C) two groups greatly.
2.1.2 animal modeling, processing and pattern detection: all mices are according to body weight 550mg/kg disposable celiac injection APAP solution.After the modeling medication 1 hour, treatment group mice all gives rhIL-1Ra subcutaneous injection according to body weight 1mg/kg, and matched group gives isopyknic NS subcutaneous injection and treats in contrast, and treatment is 12 hours once, and treatment is to observing terminal point continuously.Put to death mice when every group of mice observed terminal point, get liver immediately, a part is with carrying out pathological observation after the formaldehyde fixed, and another part is frozen to be used for biochemistry detection in-70 ℃.Liver samples is all taken from the lobus sinister position identical with right middle lobe.
2.2 experimental result:
Because the protection of survival rate before experiment has shown that dead mouse all occurred within 48 hours, and major part is to occur in 6-12 hour; Meanwhile, a spot of dead mouse is only arranged, before 6 hours and the death generation of only a few only arranged after 24 hours at 12-24 hour.In order to contrast the mechanism that statistics and explaination rhIL-1Ra can effectively improve the inductive acute hepatic failure mice of APAP survival rate better, this experiment emphasis has been set forth the contrast situation of three aspects:
I) the death time period occurred frequently (being 6-12 hour) before, promptly 3 hours and 6 hours, treatment group and matched group hepatic necrosis, apoptosis and regenerated contrast situation.
II) the mouse liver cell necrosis of surviving in each time point group after 24 hours, apoptosis and regenerated contrast situation in modeling.
III) after confirming that hepatocellular apoptosis is causing the important function of acute hepatic failure dead mouse final result in taking place, we have further studied the expression of signal of interest molecule in 3 hours and 6 hours treatment groups and matched group hepatocellular apoptosis signal transduction pathway and the contrast of vigor.
2.2.1rhIL-1Ra significantly reduced the ALT and the AST level of acute hepatic failure mice serum
After APAP inducing mouse acute hepatic failure 3 hours and 6 hours, the serum glutamic pyruvic transminase (ALT) of treatment group (T group) and matched group (C group) mice, glutamic oxaloacetic transaminase, GOT (AST) level all had remarkable rising than normal mice.But by contrast, treatment group mice serum ALT and AST level are all than matched group obviously low (Fig. 4); At 3 hours and 6 hours time points, treatment group mice ALT level reduced by 67.78% and 82.28% respectively than control group mice, and the AST level has then reduced by 62.85% and 78.99% respectively.
Legend: rhIL-1Ra has significantly reduced the ALT and the AST level of acute hepatic failure mice serum.Acute hepatic failure mice serum ALT and AST activity level have significantly been suppressed at 3 hours and 6 hours rhIL-1Ra.The comparison of (Fig. 4 A) Serum ALT activity level (n=10, * * p＜0.01).The comparison (* * p＜0.01) of (Fig. 4 B) serum AST activity level.
2.2.2rhIL-1Ra significantly reduced the necrosis area of acute hepatic failure mouse liver
After APAP inducing mouse acute hepatic failure 3 hours and 6 hours, significant change took place in the outward appearance of liver, and color is bronzing, profile enlargement, quality more tough (Fig. 5 A); From the histology, tangible amalgamation necrosis has appearred in hepatocyte, and necrosis is from lobule central authorities basically, to around spread, the hepatocyte of portal area is main pathological change with degeneration then; In hepatocellular necrotic zone, very a spot of inflammatory cell (Fig. 5 B) is only arranged.The treatment group is compared with matched group, the necrosis area of liver obviously little (Fig. 5 C), and prompting rhIL-1Ra has significantly reduced the hepatic necrosis area of acute hepatic failure mice.
Legend: rhIL-1Ra has significantly reduced the necrosis area of acute hepatic failure mouse liver.In 3 hours and 6 hours rhIL-1Ra influence to the inductive acute hepatic failure mouse liver of the APAP of 550mg/kg necrosis area.Fig. 5 A: treatment group and matched group liver are schemed substantially.Fig. 5 B: each organizes representative hepatic pathology slice map (HE dyeing, * 100).Fig. 5 C: each organizes the comparison (n=10, * * p＜0.01) of hepatic necrosis area (100 times of mirror downward view area statistics).
2.2.3rhIL-1Ra significantly reduced the hepatocellular apoptosis quantity of acute hepatic failure mice
At 3 hours and 6 hours time points, we found that the quantity of hepatocellular apoptosis of treatment group and matched group are compared and have reduced (p＜0.01) significantly; Particularly in the time of 6 hours, the hepatocellular apoptosis quantity highly significant of control group mice, the hepatocyte of apoptosis is except the periphery of necrotic zone, and other zones also have more widely and distribute; The apoptosis hepatocyte quantity of matched group then obviously will be lacked, the hepatocyte of apoptosis then mainly be the periphery that appears at necrotic zone (Fig. 6 A, B).This prompting rhIL-1Ra has significantly reduced the hepatocellular apoptosis quantity of acute hepatic failure mice.
Legend: rhIL-1Ra has significantly reduced the hepatocellular apoptosis quantity of acute hepatic failure mice.In 3 hours and 6 hours rhIL-1Ra influence to acute hepatic failure mouse liver apoptosis quantity.Fig. 6 A: each is organized the liver cell apoptosis and detects representative picture (TUNEL, * 200).Fig. 6 B: each organizes the comparison (n=10, * * p＜0.01) of apoptosis quantity (200 times of mirror downward view statistics).
2.2.4rhIL-1Ra significantly promoted acute hepatic failure mouse liver cell regeneration speed
At 3 hours and 6 hours, treatment group and matched group all had only very a spot of PCNA positive cell can be detected (Fig. 7 A); This explanation did not change even hepatocellular regeneration is not hepatic pathology in this stage; This also points out, and in the pathogenic process of acute hepatic failure mice, early stage liver regeneration may not be the deciding factor whether mice survives.But, at 3 hours and 6 hours time points, compare with matched group, the quantity of the PCNA positive cell of treatment group mouse liver has significantly increased (P＜0.01) (Fig. 7 B); RhIL-1Ra is in the process of acute hepatic failure progression of disease in this explanation, can promote liver regeneration significantly.
Legend: rhIL-1Ra has significantly promoted acute hepatic failure mouse liver cell regeneration speed.At 3 hours and 6 hours time point rhIL-1Ra to the regenerated influence of acute hepatic failure mouse liver cell.Fig. 7 A: each is organized liver PCNA positive cell and detects representative picture (SABC, * 100).Fig. 7 B:, compare treatment group positive cell quantity (100 times of mirror downward view statistics) more (n=10, * * p＜0.01) at 3 hours and 6 hours time points with matched group.
2.2.5rhIL-1Ra significantly reduced the necrosis area of acute hepatic failure mouse liver after the modeling
From substantially, after APAP inducing mouse generation acute hepatic failure 24 hours, the liver of two groups of mices all was dark red brown, and the profile enlargement is obvious, and quality is more tough; At 48 hours, the bronzing of two groups of livers was all significantly thin out, but still enlargement is obvious, and quality is still more tough; At 96 hours, liver presented the pink of comparison near normal liver, and swelling also obviously alleviates, and quality is soft; And at 168 hours, liver on color, profile, quality all very near normal liver.
Comparatively speaking, at each time point, it is light that the hepatic lesions of treatment group mice is obviously wanted, and the speed that returns to normal liver (Fig. 8 A) more rapidly.
From the Histological change shown in the HE dyeing liver section, from 24 hours until 48 hours, the hepatic necrosis situation is still continuing aggravation; But, at these two time points, remaining hepatocyte and necrotic zone boundary are very obvious, also do not have situations such as hydropic degeneration, acidophilia's degeneration especially significantly, and the inflammation that liver is described is by localization gradually; And can observe these remaining hepatocyte and relatively to enliven mitotic performance.And can not find the performance of liver necrosis recently at 96 hours liver sections, the general structure of liver is relatively near normal; At 168 hours, the liver of part control group mice still can be observed a large amount of inflammatory cells (mainly being neutrophilic granulocyte etc.) and engulf the performance of removing remaining dead hepatic tissue; But generally speaking, the structure of 168 hours livers very near the structure of normal liver (liver plate marshalling, the not swollen degeneration of water breakthrough, acidophilia's degeneration etc.) (Fig. 8 B).Two groups are comparatively speaking, at 24 hours and 48 hours, and treatment group hepatic necrosis area obviously little (P＜0.05) (Fig. 8 C); This is presented at the recovery stage of acute hepatic failure, and injection rhIL-1Ra can significantly reduce the hepatic necrosis area of acute hepatic failure mice.
Legend: rhIL-1Ra has significantly reduced the necrosis area of acute hepatic failure mouse liver after the modeling.The contrast of matched group and treatment group hepatic necrosis situation after the modeling.Fig. 8 A: treatment group and matched group liver are schemed substantially.Fig. 8 B: each organizes representative hepatic pathology slice map (HE dyeing, * 100).On picture C168, can also observe a large amount of inflammatory cells and engulf the performance of removing remaining dead hepatic tissue.The comparison (* p＜0.05) of Fig. 8 C:24 hour and 48 hours treatment groups and matched group hepatic necrosis area (100 times of mirror downward views statistics); When 96 hours and 168 hours, treatment group and matched group liver section are not all observed the performance of hepatic necrosis recently.
2.2.6rhIL-1Ra can significantly alleviate mouse liver apoptosis after the acute hepatic failure modeling
After modeling, at 24 hours, the quantity of matched group and treatment group hepatocellular apoptosis did not have significant difference (p＞0.05); But, there is significant difference (p＜0.05) (Fig. 9 A, B) between two groups at 48 hours.Treatment group apoptotic cell quantity obviously is less than contrast.At time point thereafter, two groups are not all had apoptotic cell, illustrate that the apoptosis of liver has obtained repairing.This experimental result shows that rhIL-1Ra can significantly alleviate the liver cell apoptosis.
Legend: rhIL-1Ra can significantly alleviate mouse liver apoptosis after the acute hepatic failure modeling.The contrast of modeling matched group and treatment group survival mouse liver cell apoptosis quantity after 24 hours.Fig. 9 A: each is organized the liver cell apoptosis and detects representative picture (TUNEL, * 200).Comparing of Fig. 9 B:48 hour treatment group and matched group hepatocellular apoptosis quantity (200 times of mirror downward views statistics) has significant difference (* p＜0.05).And all can not detect hepatocellular apoptosis in 96 hours and 168 hours time point treatment groups and matched group.
2.2.7rhIL-1Ra can significantly accelerate the acute hepatic failure mouse liver cell regeneration speed of surviving after the modeling
At 24 hours, all there is more positive hepatocyte to be detected in treatment group and matched group; And along with the prolongation of time, the quantity of positive cell increases sharply; Treatment group and matched group reach the peak of hepatocyte growth respectively when 48 hours and 96 hours; The quantity of positive cell all begins to descend afterwards, and hepatocyte growth then draws to an end during to 168 hours, have only the positive cell of minute quantity to be detected (Figure 10 A, B).This shows that rhIL-1Ra also can promote liver regeneration in the liver recovery stage of acute hepatic failure mice.
Legend: rhIL-1Ra can significantly accelerate the acute hepatic failure mouse liver cell regeneration speed of surviving after the modeling.The contrast of treatment group and matched group survival mouse liver PCNA positive cell quantity after 24 hours.Figure 10 A: each matched group and each treatment group mice PCNA detect representative picture.Figure 10 B: compare with matched group, the peak value of treatment group PCNA positive cell quantity (100 times of mirror downward view statistics) has obviously been shifted to an earlier date (48 hours vs.96 hour).
2.2.8rhIL-1Ra can significantly suppress the activity of acute hepatic failure murine liver tissue apoptosis signal transduction pathway
After having confirmed that rhIL-1Ra can significantly reduce the apoptosis of acute hepatic failure mouse liver cell, we have further studied the active influence of rhIL-1Ra to acute hepatic failure murine liver tissue apoptosis signal transduction pathway, suppress apoptotic mechanism of action to explore it.We have selected to occupy signaling molecule cytochrome C, Bax, caspase-3, caspase-8 and the caspase-9 of critical role as object of study in the signal transduction pathway of hepatocellular apoptosis.By discovering, at 3 hours and 6 hours, to compare with matched group, the cytochrome C in the liver cell mitochondria of treatment group leaks into cytoplasmic level significantly have been reduced; The expression of hepatic tissue Bax is significantly suppressed; And the caspase-3 of hepatic tissue, caspase-8 and caspase-9 activity have also significantly been reduced (p＜0.01) (Figure 11).This shows that rhIL-1Ra has suppressed the hepatocellular apoptosis of the inductive acute hepatic failure mice of APAP by the apoptosis signal transduction pathway that suppresses cytochrome C, Bax, caspase-3, caspase-8 and caspase-9 mediation.
Legend: rhIL-1Ra can significantly suppress the activity of acute hepatic failure murine liver tissue apoptosis signal transduction pathway.Figure 11 A: at 3 hours and 6 hours, compare with matched group, treatment group hepatocyte inner cell pigment C leaks into cytoplasmic amount from mitochondrion and obviously will lack, and the expression of hepatic tissue Bax is also obviously low.β-the actin horizontal detection in contrast.This picture has one of experiment of analog result from three times.Figure 11 B be ca spase-3 specific activity, Figure 11 C be the caspase-8 specific activity and Figure 11 D be the caspase-8 specific activity: at 3 hours and 6 hours, compare with matched group, the activity of treatment group hepatocyte caspase-3, caspase-8 and caspase-9 all significantly reduces (n=10, * * p＜0.01).Each organize multiple value for the ratio of normal mouse detected value.
3.1 experimental technique:
The male C57BL/6 mice in 8 ages in week of 60 health is divided into two groups at random:
Group 1: every mouse peritoneal injection CCl
4, dosage is 1mL/kg body weight (Semen Maydis oil dilution in 1: 3), presses 1mg/kg body weight subcutaneous injection rhIL-1Ra behind the 1h, 12h injects continuous 5 days once more at interval;
Group 2: processing method on the same group 1, every mouse peritoneal injection CCl
4, dosage is 1mL/kg body weight (Semen Maydis oil dilution in 1: 3), injects isopyknic PBS in contrast;
3.2 experimental result:
In the acute liver damage model mice, rhIL-1Ra (human interleukin-11 receptor antagonist) treatment group mice glutamic oxaloacetic transaminase, GOT (AST) level all significantly is lower than matched group (p＜0.01) when 3 days and 5 days, see Figure 12, and 3 days serum glutamic pyruvic transminase (ALT) significantly be lower than matched group (p＜0.05), see Figure 13.As Figure 14, the rhIL-1Ra treatment effectively alleviates hepatic injury, and tissue injury's area is significantly less than matched group (p＜0.01).
Scope of the present invention is not subjected to the restriction of described specific embodiments, and described embodiment also comprises the method and the component of functional equivalent only as the single example of illustrating various aspects of the present invention in the scope of the invention.In fact, except content as herein described, those skilled in the art can easily grasp multiple improvement of the present invention with reference to above description and accompanying drawing.Described improvement also falls within the scope of appended claims.Every piece of list of references mentioned above is listed this paper in as a reference all in full.
Sequence table
＜110〉Shanghai Communications University
＜120〉purposes of interleukin-1 receptor antagonist
＜130〉description sequence table
<160>2
<170>PatentIn?version?3.3
<210>1
<211>152
<212>PRT
＜213〉people source (Homo sapiens)
<400>1
Arg?Pro?Ser?Gly?Arg?Lys?Ser?Ser?Lys?Met?Gln?Ala?Phe?Arg?Ile?Trp
1 5 10 15
Asp?Val?Asn?Gln?Lys?Thr?Phe?Tyr?Leu?Arg?Asn?Asn?Gln?Leu?Val?Ala
20 25 30
Gly?Tyr?Leu?Gln?Gly?Pro?Asn?Val?Asn?Leu?Glu?Glu?Lys?Ile?Asp?Val
35 40 45
Val?Pro?Ile?Glu?Pro?His?Ala?Leu?Phe?Leu?Gly?Ile?His?Gly?Gly?Lys
50 55 60
Met?Cys?Leu?Ser?Cys?Val?Lys?Ser?Gly?Asp?Glu?Thr?Arg?Leu?Gln?Leu
65 70 75 80
Glu?Ala?Val?Asn?Ile?Thr?Asp?Leu?Ser?Glu?Asn?Arg?Lys?Gln?Asp?Lys
85 90 95
Arg?Phe?Ala?Phe?Ile?Arg?Ser?Asp?Ser?Gly?Pro?Thr?Thr?Ser?Phe?Glu
100 105 110
Ser?Ala?Ala?Cys?Pro?Gly?Trp?Phe?Leu?Cys?Thr?Ala?Met?Glu?Ala?Asp
115 120 125
Gln?Pro?Val?Ser?Leu?Thr?Asn?Met?Pro?Asp?Glu?Gly?Val?Met?Val?Thr
130 135 140
Lys?Phe?Tyr?Phe?Gln?Glu?Asp?Glu
145 150
<210>2
<211>152
<212>PRT
＜213〉mice (Mus musculus)
<400>2
Arg?Pro?Ser?Gly?Lys?Arg?Pro?Cys?Lys?Met?Gln?Ala?Phe?Arg?Ile?Trp
1 5 10 15
Asp?Thr?Asn?Gln?Lys?Thr?Phe?Tyr?Leu?Arg?Asn?Asn?Gln?Leu?Ile?Ala
20 25 30
Gly?Tyr?Leu?Gln?Gly?Pro?Asn?Ile?Lys?Leu?Glu?Glu?Lys?Ile?Asp?Met
35 40 45
Val?Pro?Ile?Asp?Leu?His?Ser?Val?Phe?Leu?Gly?Ile?His?Gly?Gly?Lys
50 55 60
Leu?Cys?Leu?Ser?Cys?Ala?Lys?Ser?Gly?Asp?Asp?Ile?Lys?Leu?Gln?Leu
65 70 75 80
Glu?Glu?Val?Asn?Ile?Thr?Asp?Leu?Ser?Lys?Asn?Lys?Glu?Glu?Asp?Lys
85 90 95
Arg?Phe?Thr?Phe?Ile?Arg?Ser?Glu?Lys?Gly?Pro?Thr?Thr?Ser?Phe?Glu
100 105 110
Ser?Ala?Ala?Cys?Pro?Gly?Trp?Phe?Leu?Cys?Thr?Thr?Leu?Glu?Ala?Asp
115 120 125
Arg?Pro?Val?Ser?Leu?Thr?Asn?Thr?Pro?Glu?Glu?Pro?Leu?Ile?Val?Thr
130 135 140
Lys?Phe?Tyr?Phe?Gln?Glu?Asp?Gln
145 150
Claims (9)
1. one kind following (a) or protein (b) are used for the treatment of the purposes of the medicine of hepatic injury in preparation:
(a) albumen of its aminoacid sequence shown in SEQ ID NO:1;
(b) at least with (a) in aminoacid sequence 70% homology is arranged and has the albumen of liver protection agent of activity.
2. purposes according to claim 1 is characterized in that acute hepatic failure or acute liver damage, subacute liver failure, slow extra urgaent dispatch liver failure, chronic liver failure or chronic hepatic injury that described hepatic injury causes for the toxicity nosetiology.
3. purposes according to claim 2 is characterized in that the hepatic injury of described hepatic injury for being caused by toxicant.
4. purposes according to claim 3 is characterized in that described toxicant is interior extracellular toxin, flavacin, nitrosamine, ethanol, medicine or virus.
5. purposes according to claim 4 is characterized in that described medicine is antitubercular agent, antibiotic, Chinese patent medicine, Chinese herbal medicine, antipyretic analgesic or hypolipidemic.
6. purposes according to claim 1 is characterized in that the aminoacid sequence shown in described proteinic aminoacid sequence and the SEQID NO:1 has at least 77% homology and has liver protection agent of activity.
7. purposes according to claim 6 is characterized in that, described proteinic aminoacid sequence is shown in SEQID NO:2.
8. a pharmaceutical composition is characterized in that it is protein and pharmaceutically acceptable carrier or the excipient of aminoacid sequence shown in SEQID NO:1 that said composition contains active component.
9. pharmaceutical composition according to claim 8 is characterized in that, the preparation of described pharmaceutical composition is an intestinal external administration preparation.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110772637A (en) * | 2019-11-22 | 2020-02-11 | 上海市第六人民医院 | Application of rhIL-1Ra in preparation of drug for treating acute liver failure |
| CN111202840A (en) * | 2019-11-22 | 2020-05-29 | 上海市第六人民医院 | Kupffer cell activity inhibitor and application thereof in preparation of drug for treating acute liver failure |
| CN114177295A (en) * | 2021-12-16 | 2022-03-15 | 上海市第五人民医院 | Use of interleukin 1receptor antagonist for treating non-alcoholic fatty liver disease |
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| CN101690801A (en) * | 2009-10-26 | 2010-04-07 | 上海交通大学 | Application of interleukin-1 receptor antagonist and medicinal composition thereof |
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| CN101690801A (en) * | 2009-10-26 | 2010-04-07 | 上海交通大学 | Application of interleukin-1 receptor antagonist and medicinal composition thereof |
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| 《TOXICOLOGIC PATHOLOGY》 19961231 MARK E. B1 LAZKA等 Histopathology of Acetaminophen-Induced Liver Changes: Role of Interleukin 1alpha; and Tumor Necrosis Factor alpha 第181－189页 1－7 第24卷, 第2期 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110772637A (en) * | 2019-11-22 | 2020-02-11 | 上海市第六人民医院 | Application of rhIL-1Ra in preparation of drug for treating acute liver failure |
| CN111202840A (en) * | 2019-11-22 | 2020-05-29 | 上海市第六人民医院 | Kupffer cell activity inhibitor and application thereof in preparation of drug for treating acute liver failure |
| CN114177295A (en) * | 2021-12-16 | 2022-03-15 | 上海市第五人民医院 | Use of interleukin 1receptor antagonist for treating non-alcoholic fatty liver disease |
| CN114177295B (en) * | 2021-12-16 | 2023-09-19 | 上海市第五人民医院 | Use of interleukin 1receptor antagonist for treating non-alcoholic fatty liver disease |
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