Application of EIF2AK3 inhibitor in preparation of medicine for treating Kawasaki disease

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of an EIF2AK3 inhibitor in preparation of a medicine for treating Kawasaki disease. The invention discovers that EIF2AK3 can up-regulate the level of proinflammatory cytokines, so that vasculitis in KD is promoted through an MAPK-ERK1 / 2 pathway; in addition, EndoMT can be inhibited by knocking down the EIF2AK3, so that coronary injury is reduced. The new mechanism of KD-related vasculitis is clarified, and a theoretical basis is provided for researching the genetic background of Kawasaki disease vasculitis. The EIF2AK3 can provide a new direction and target spot for treating kawasaki disease vasculitis, and the EIF2AK3 inhibitor has great potential value in preparation of the medicine for treating kawasaki disease.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceutical technology, and specifically relates to the application of an EIF2AK3 inhibitor in the preparation of a drug for treating Kawasaki disease. Background Art

[0002] Kawasaki disease (KD) is an acute vasculitis disease that mainly affects infants and young children under 5 years old and is the most common cause of acquired heart disease in children in developed countries. 1,2 The main pathological features of KD are vascular inflammatory infiltration and coronary artery injury (CAL). Currently, the cause of KD is still unclear, but a widely accepted hypothesis is that KD is triggered by infectious pathogens that evoke abnormal immune responses in patients with susceptible genes, while mobilizing both the innate immune system and the adaptive immune system. 3,4 There is evidence that during the acute phase of KD, pro-inflammatory cytokines (such as IL-6, IL-8, IL-17A, IL-1β, and TNFα) are significantly increased. 5-8 In addition, reducing IL-1β and TNFα can significantly relieve KD 9-11 associated inflammatory responses. These studies indicate that pro-inflammatory cytokines play a crucial role in the progression of KD vasculitis, suggesting that treatment targeting these cytokines may be a useful clinical treatment strategy.

[0003] Currently, intravenous immunoglobulin (IVIG) is the main treatment method for Kawasaki disease (KD), aiming to relieve inflammation. However, 10 - 20% of patients do not respond to IVIG treatment. 12 The inflammation in these IVIG non-responders persists and they face a higher risk of developing coronary artery aneurysms 13 which can be life-threatening. Therefore, there is an urgent need to find new treatment strategies for anti-inflammatory treatment of KD.

[0004] Eukaryotic translation initiation factor 2α kinase 3 (EIF2AK3), also known as PERK, encodes an endoplasmic reticulum (ER)-resident type I transmembrane protein and is one of the eIF2α protein kinases. 14 Currently, the mechanism of action of the gene EIF2AK3 in inflammatory diseases and cardiovascular diseases is not clear, and there is almost no report on the involvement of EIF2AK3 in KD, and it is also unclear whether it is involved in KD coronary artery injury. Therefore, the present invention explores the regulatory mechanism of EIF2AK3 in the occurrence and development of Kawasaki disease and its potential in the treatment of KD.

[0005] References:

[0006] 1. McCrindle BW, Rowley AH, Newburger JW, et al. Diagnosis, Treatment, and Long - Term Management of Kawasaki Disease: A Scientific Statement for Health Professionals From the American Heart Association. Circulation. 2017;135(17):e927 - e999.

[0007] 2. Kawasaki T. [Acute febrile mucocutaneous syndrome with lymphoid involvement with specific desquamation of the fingers and toes in children]. Arerugi. 1967;16(3):178 - 222.

[0008] 3. Rife E, Gedalia A. Kawasaki Disease: an Update. Curr Rheumatol Rep. 2020;22(10):75.

[0009] 4. Hara T, Nakashima Y, Sakai Y, Nishio H, Motomura Y, Yamasaki S. Kawasaki disease: a matter of innate immunity. Clinical and experimental immunology. 2016;186(2):134 - 143.

[0010] 5. Huang J, Wu S, Cao S, Zhu X, Zhang S. Neutrophil - Derived Semaphorin 4D Induces Inflammatory Cytokine Production of Endothelial Cells via Different Plexin Receptors in Kawasaki Disease. BioMed research international. 2020;2020:6663291.

[0011] 6. Si F, Wu Y, Gao F, Feng S, Liu R, Yi Q. Relationship between IL-27 and coronary arterial lesions in children with Kawasaki disease. Clin Exp Med. 2017;17(4):451 - 457.

[0012] 7. Guo MM, Tseng WN, Ko CH, Pan HM, Hsieh KS, Kuo HC. Th17- and Treg-related cytokine and mRNA expression are associated with acute and resolving Kawasaki disease. Allergy. 2015;70(3):310 - 318.

[0013] 8. Hoang LT, Shimizu C, Ling L, et al. Global gene expression profiling identifies new therapeutic targets in acute Kawasaki disease. Genome Med. 2014;6(11):541.

[0014] 9. Chen J, Liao J, Xiang L, Zhang S, Yan Y. Current knowledge of TNF-alpha monoclonal antibody infliximab in treating Kawasaki disease: a comprehensive review. Front Immunol. 2023;14:1237670.

[0015] 10. Yamaji N, da Silva Lopes K, Shoda T, et al. TNF-alpha blockers for the treatment of Kawasaki disease in children. Cochrane Database Syst Rev. 2019;8(8):CD012448.

[0016] 11. Lee Y, Schulte DJ, Shimada K, et al. Interleukin-1beta is crucial for the induction of coronary artery inflammation in a mouse model of Kawasaki disease. Circulation. 2012;125(12):1542-1550.

[0017] 12. Tremoulet AH, Best BM, Song S, et al. Resistance to intravenous immunoglobulin in children with Kawasaki disease. J Pediatr. 2008;153(1):117-121.

[0018] 13. Friedman KG, Jone PN. Update on the Management of Kawasaki Disease. Pediatr Clin North Am. 2020;67(5):811-819.

[0019] 14. Bettigole SE, Glimcher LH. Endoplasmic reticulum stress in immunity. Annual review of immunology. 2015;33:107-138. Summary of the Invention

[0020] To solve the above technical problems, the present invention first proposes a solution for treating Kawasaki disease by knocking down or knocking out EIF2AK3, which specifically includes the following aspects.

[0021] The purpose of the first aspect of the present invention is to provide the use of an EIF2AK3 inhibitor in the preparation of a drug for treating Kawasaki disease.

[0022] The purpose of the second aspect of the present invention is to provide a nucleic acid molecule that inhibits the expression of EIF2AK3.

[0023] The purpose of the third aspect of the present invention is to provide a recombinant vector that inhibits the expression of EIF2AK3.

[0024] The purpose of the fourth aspect of the present invention is to provide a virus that inhibits the expression of EIF2AK3.

[0025] The object of the fifth aspect of the present invention is to provide a drug.

[0026] To achieve the above object of the present invention, the technical solution adopted by the present invention is:

[0027] The first aspect of the present invention provides an application of an EIF2AK3 inhibitor in the preparation of a drug for treating Kawasaki disease.

[0028] The EIF2AK3 inhibitor includes at least one of 1) and 2):

[0029] 1) A substance that inhibits the EIF2AK3 gene at the gene level;

[0030] 2) A substance that inhibits the EIF2AK3 protein at the protein level.

[0031] In some embodiments of the present invention, specifically, the EIF2AK3 inhibitor includes at least one of a substance that reduces the content or expression level of the EIF2AK3 gene or protein, a substance that reduces the activity of the EIF2AK3 gene or protein, and a substance that promotes the degradation of the EIF2AK3 gene or protein.

[0032] In some embodiments of the present invention, the amino acid sequence of the EIF2AK3 protein is as shown in a1) to a4):

[0033] a1) A protein having an amino acid sequence as shown in SEQ ID NO: 1 (Protein_ID = "NP_001300844.1");

[0034] SEQ ID NO: 1 is as follows: miipsldgal fqwdqdresm etvpftvesl lessykfgdd vvlvggkslttyglsaysgk vryicsalgc rqwdsdemeq eedilllqrt qktvravgpr sgnekwnfsv ghfelryipdmetragfies tfkpnenteeskiisdveeq eaaimdivik vsvadwkvma fskkgghlew eyqfctpiasawllkdgkvi pislfddtsy tsnddvledeediveaarga tensvylgmy rgqlylqssv risekfpsspkalesvtnen aiiplptikw kplihspsrt pvlvgsdefdkclsndkfsh eeysngalsi lqypydngyylpyykrernk rstqitvrfl dnphynknir kkdpvlllhw wkeivatilf ciiattfivrrlfhphphrqrkesetqcqt enkydsvsge andsswndik nsgyisrylt dfepiqclgr ggfgvvfeaknkvddcnyaikrirlpnrel arekvmrevk alaklehpgi vryfnawlea ppekwqekmd eiwlkdestdwplsspspmd apsvkirrmdpfatkehieiiapspqrsrs fsvgiscdqt sssesqfspl efsgmdhedisesvdaaynl qdscltdcdv edgtmdgndeghsfelcpse aspyvrsrer tsssivfeds gcdnasskeepktnrlhign hcankltafk ptssksssea tlsispprpt tlsldltkntteklqpsspk vylyiqmqlcrkenlkdwmn grctieerer svclhiflqi aeaveflhsk glmhrdlkps nifftmddvvkvgdfglvtamdqdeeeqtv ltpmpayarh tgqvgtklym speqihgnsy shkvdifslg lilfellypfstqmervrtltdvrnlkfpp lftqkypcey vmvqdmlspspmerpeainiienavfedld fpgktvlrqrsrslsssgtk hsrqsnnshs plpsn, whose sequence has a Protein_ID of NP_001300844.1 on NCBI and has a full-length of 965 AA sequences.

[0035] a2) A protein with the same function obtained by substitution and / or deletion and / or addition of one or several amino acid residues to the amino acid sequence shown in SEQ ID NO: 1;

[0036] a3) An amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO: 1; a4) A derivative related to any one of the proteins described in a1), a2), a3).

[0037] Preferably, the proteins with the same function in a2) are generally regarded as conservative substitutions, such as the substitution of each other among the aliphatic amino acids Ala, Val, Leu, and Ile, the exchange of the hydroxyl residues Ser and Thr, the exchange of the acidic residues Asp and Glu, the substitution between the amide residues Asn and Gln, the exchange of the basic residues Lys and Arg, and the substitution between the aromatic residues Phe and Tyr.

[0038] Preferably, a3) means that a given amino acid sequence shares at least 85% identity with a reference sequence. For example, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. Alternatively, it means that a given amino acid sequence differs from the reference sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids. For polypeptides, this difference is preferably an amino acid substitution or deletion. The difference in identity here can come from conservative substitutions of amino acids, or the introduction of a tag protein, or the introduction of a nuclear localization sequence, or the introduction or modification of a signal peptide sequence. For example, adding tags such as His, GFP, HA, etc. to the C-terminus of SEQ ID NO: 1, thereby resulting in a change in identity, or adding a signal peptide to the N-terminus of SEQ ID NO: 1, or adding a conventional nuclear localization sequence to the N-terminus, thereby resulting in a change in identity.

[0039] Preferably, the derivatives related to the protein in a4) include an EIF2AK3 fusion protein linked to a tag protein, a pharmaceutically acceptable salt of the EIF2AK3 protein, and a pharmaceutically acceptable chemical modification of the EIF2AK3 protein.

[0040] Preferably, the tag protein includes but is not limited to at least one of His, Flag, Myc, GST, GFP, HA.

[0041] In some embodiments of the present invention, the nucleic acid sequence encoding the EIF2AK3 protein comprises:

[0042] 1) the transcript nucleotide sequence as shown in SEQ ID NO: 2 (NCBI GeneID: 9451);

[0043]

[0044] 2) A nucleotide sequence that has been substituted, deleted, and / or added with one or several nucleotides to the nucleotide sequence shown in SEQ ID NO: 2 and encodes the same active protein.

[0045] In some embodiments of the present invention, the substances that reduce the content or expression level of the EIF2AK3 gene or protein include small molecule inhibitors targeting the EIF2AK3 protein, or nucleotides that reduce the content or expression level of the EIF2AK3 protein.

[0046] In some embodiments of the present invention, the nucleotides that reduce the content or expression level of the EIF2AK3 protein include at least one of small guide RNA (sgRNA) that inhibits the expression of EIF2AK3, short hairpin RNA (shRNA) that inhibits the expression of EIF2AK3, Knock Out system for gene knockout, dsRNA that inhibits the expression of EIF2AK3, dsRNA microRNA that inhibits the expression of EIF2AK3, and dsRNAsiRNA that inhibits the expression of EIF2AK3.

[0047] In some embodiments of the present invention, the sequence of the shRNA that inhibits the expression of EIF2AK3 is as shown in SEQ ID NO: 3 (GCATCTGCCTGGTTACTTAAG).

[0048] In some embodiments of the present invention, the sequence of the siRNA that inhibits the expression of EIF2AK3 is as shown in SEQ ID NO: 4 (GCATCTGCCTGGTTACTTAAG). The sgRNA can be applied to the conventional CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene editing technology in the art to achieve the inhibition of EIF2AK3. The shRNA can be applied to the conventional shRNA-mediated gene editing technology in the art to achieve the inhibition of EIF2AK3.

[0049] Preferably, the Knock Out system includes at least one of complete gene knockout and conditional gene knockout.

[0050] Preferably, the Knock Out system includes at least one of the Cre / LoxP system, Gin / Gix system, FLP / FRT system, R / RS system, and CRISPR-Cas9 system.

[0051] Editing the EIF2AK3 gene using the above-mentioned nucleotides (including but not limited to sgRNA, shRNA, siRNA, microRNA, dsRNA, Knock Out system) that reduce the content or expression level of EIF2AK3 can be carried out in vitro or in vivo. The above-mentioned nucleotides that reduce the content or expression level of EIF2AK3 and the supporting related components can be delivered in vivo using delivery systems known in the prior art to achieve the editing of the EIF2AK3 gene in target cells in vivo, thereby inhibiting the expression of EIF2AK3. The related delivery systems include but are not limited to liposome delivery systems, adenovirus delivery systems, adeno-associated virus delivery systems, lentivirus delivery systems, and nanoemulsion delivery systems.

[0052] The second aspect of the present invention provides a nucleic acid molecule for inhibiting the expression of EIF2AK3, and the nucleic acid molecule includes shRNA and siRNA.

[0053] In some embodiments of the present invention, the nucleotide sequence of the shRNA for inhibiting the expression of EIF2AK3 is as shown in SEQ ID NO: 3.

[0054] In some embodiments of the present invention, the nucleotide sequence of the siRNA for inhibiting the expression of EIF2AK3 is as shown in SEQ ID NO: 4. The third aspect of the present invention provides a recombinant vector for inhibiting the expression of EIF2AK3, and the recombinant vector includes the shRNA described in the second aspect of the present invention.

[0055] In some embodiments of the present invention, the recombinant vector is a lentiviral vector.

[0056] The present invention takes the plasmid with the vector structure of pLKO.1-U6-Puro as an example for experimental testing, which does not mean that only the recombinant plasmid with this structure can achieve the expected technical effects. Those skilled in the art should know that other conventional lentiviral vector systems can all be paired with the shRNA described in the second aspect of the present invention and achieve the expected technical effects.

[0057] The fourth aspect of the present invention provides a virus for inhibiting the expression of EIF2AK3, and the virus includes the recombinant vector described in the third aspect of the present invention.

[0058] In some embodiments of the present invention, the virus is a lentivirus, and other common virus types in the art can also achieve the technical effects of the present invention.

[0059] The fifth aspect of the present invention provides a drug, including the shRNA described in the second aspect of the present invention, the recombinant vector described in the third aspect of the present invention, or the virus described in the fourth aspect of the present invention.

[0060] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient. Such excipients are generally recognized for this purpose and serve as inactive ingredients of the medicament. Compilations of pharmaceutically acceptable excipients can be found in "Handbook of Pharmaceutical Excipients" (2nd edition, edited by A. Wade and P. J. Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994); "Pharmacopoeia of the People's Republic of China - List of Medicinal Excipients" and other reference books.

[0061] Preferably, the drug is administered by one or more of the following methods: oral administration, injection, gold-coated gene gun bombardment, plasmid DNA carried by replication-defective bacteria, target DNA carried by replication-defective adenovirus, or the protein encoded by the target gene, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, transdermal administration.

[0062] Preferably, the dosage form of the drug comprises at least one of capsules, tablets, microcapsule preparations, freeze-dried powder injection preparations, injections, suppositories, sprays, powders, soft capsules, sustained-release or controlled-release preparations.

[0063] The beneficial effects of the present invention are as follows:

[0064] The present invention discovers that EIF2AK3 can up-regulate the levels of pro-inflammatory cytokines, thereby promoting vasculitis in KD through the MAPK-ERK1 / 2 pathway; in addition, knocking down EIF2AK3 can inhibit EndoMT, thereby reducing coronary artery injury. This study clarifies a new mechanism of KD-related vasculitis and provides a theoretical basis for studying the genetic background of Kawasaki disease vasculitis. EIF2AK3 can provide a new direction and target for the treatment of Kawasaki disease vasculitis, and EIF2AK3 inhibitors have great potential value in the preparation of drugs for treating Kawasaki disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] The present invention will be further described below with reference to the drawings and embodiments, wherein:

[0066] Figure 1Results of Example 1 of the present invention. Under KD inflammatory conditions, the expression level of EIF2AK3 was significantly upregulated. Among them, (A - B) Protein expression of EIF2AK3 in PBMCs was determined by Western blotting (A); quantification of relative protein expression levels is shown in (B); (C - D) Expression levels of EIF2AK3 were determined by qRT - PCR (C) and Western blotting (D); (E - F) Representative images (E) and quantification (F) of EIF2AK3 immunofluorescence staining in the thoracic aorta of mice injected with PBS or LCWE; scale bar = 250 μm or 50 μm; (G - H) Expression levels of EIF2AK3 were detected by qRT - PCR (G) and Western blotting (H). THP - 1 cells were stimulated with different concentrations of LCWE. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0067] Figure 2 Results of Example 2 of the present invention. EIF2AK3 increased the expression of pro - inflammatory cytokines in KD. Among them, (A - B) qRT - PCR (A) and Western blotting (B) were used to detect the expression levels of pro - inflammatory cytokines (IL - 1β, IL - 6, IL - 8 and TNFα) in 293T cells transfected with negative control siRNA or EIF2AK3 siRNA; (C - D) qRT - PCR (C) and Western blotting (D) were used to detect the expression levels of these pro - inflammatory cytokines in EIF2AK3 - knockout THP - 1 stable cells and control cells after 24 - hour LCWE treatment; (E - F) Expression levels of these pro - inflammatory cytokines were determined by qRT - PCR (E) and Western blotting (F); THP - 1 cells were treated with the EIF2AK3 inhibitor (GSK2606414) for 24 hours; (G - H) In 293T cells overexpressing EIF2AK3, expression levels of these pro - inflammatory cytokines were detected by qRT - PCR (G) and Western blotting (H); (I) Representative protein immunoblotting of the expression of these pro - inflammatory cytokines after 24 - hour treatment with the EIF2AK3 agonist (CCT020312) (same as before). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0068] Figure 3This is the result of Example 3 of the present invention. EIF2AK3 promotes Kawasaki disease vasculitis through the MAPK-ERK1 / 2 pathway. Among them, (A) Volcano plot of the transcriptome showing upregulated genes (red) and downregulated genes (blue) after EIF2AK3 knockout in THP-1 cells; (B) KEGG pathways enriched according to the number of genes; KEGG: Kyoto Encyclopedia of Genes and Genomes; (C) Heat map of GO enrichment terms (regulation of ERK1 and ERK2 cascades); GO, Gene Ontology; (D) Key protein levels of the MAPK signaling pathway in EIF2AK3-knockout THP-1 stable cells and control cells after 24 hours of LCWE treatment; (E-F) 293T cells overexpressing EIF2AK3 were treated with the ERK1 / 2 specific inhibitor (SCH772984) for 24 hours; The expression levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNFα) were measured by qRT-PCR (E) and Western blot (F); * p < 0.05.

[0069] Figure 4 This is the result of Example 4 of the present invention. Knockdown of EIF2AK3 can inhibit EndoMT. Among them, (A-B) HUVECs were treated with TNFα and IL-1β for 6 days; The expression level of EIF2AK3 was detected by qRT-PCR (A) and Western blot (B); β-actin was used as an internal control; Data are expressed as mean ± SD (n = 3); (C-D) The expression levels of EndoMT markers were detected by qRT-PCR (C) and Western blot (D); Under LCWE treatment, HUVECs were transfected with negative control siRNA or EIF2AK3 siRNA; (E-F) EIF2AK3-knockdown HUVECs were pretreated with LCWE; Representative images of migrating HUVECs stained with crystal violet (E); (Scale bar = 100 μm); The number of migrating cells was counted as shown in (F); Data are expressed as mean ± SD (n = 7). p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Detailed implementation manners

[0070] The concept of the present invention and the technical effects generated will be clearly and completely described below in conjunction with the embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present invention.

[0071] The materials and methods of the present invention are as follows:

[0072] 1) LCWE-induced KD mouse model

[0073] This study was approved by the Animal Ethics Committee of Guangzhou Medical University (approval number: 2024082711252097) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals. All mice used in the experiment were of the C57BL / 6 strain and were purchased from Guangdong Yaokang Biotechnology Co., Ltd. The mice were housed in a specific pathogen-free (SPF) environment and fed a standard diet. The KD model was established by intraperitoneal injection of Lactobacillus casei cell wall extract (LCWE) (500 μg per mouse) once into 5-week-old mice, and the PBS (500 μl per mouse) group was used as an age- and sex-matched control group (reference: Noval Rivas M, Arditi M. Kawasaki disease: pathophysiology and insights from mouse models. Nat Rev Rheumatol. 2020;16(7):391-405.). The preparation method of LCWE was as described in the literature (Liang X, Wu S, Geng Z, et al. LARP7 Suppresses Endothelial-to-Mesenchymal Transition by Coupling With TRIM28. Circ Res. 2021;129(9):843-856), and it was prepared in advance using Lactobacillus casei (#11578, ATCC). LCWE was quantified using a total sugar content detection kit (Solarbio, #BC2710, Beijing, China) and then intraperitoneally injected into the experimental mice. The mice were euthanized on the 7th day, and the thoracic aorta was collected and embedded in paraffin for further histological examination.

[0074] 2) Cell culture and treatment

[0075] THP-1 cells and 293T cells were from the American Type Culture Collection (ATCC). Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cord samples of healthy newborns and were approved by the legal guardians of the infants and the Ethics Review Committee of Guangzhou Women and Children's Medical Center (number: 2021290B01). THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin; 293T cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin; HUVEC cells were cultured in ECM medium supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% vascular endothelial growth factor. The cell number was 1×10^6 The THP-1 cells were treated with different reagents, such as GSK2606414 (MCE, #HY-18072, USA) at concentrations of 0, 0.25, and 1 μM, CCT020312 (Selleck, #SE1030, USA) at concentrations of 0, 1, and 4 μM, and SCH772984 (Selleck, #S7101, USA) at concentrations of 0 and 2.0 μM for 24 hours before collection. Unless otherwise stated, all cell experiments were carried out under LCWE stimulation.

[0076] 3) Lentivirus packaging and infection

[0077] The lentiviral vector and two helper plasmids (psPAX2, pMD2.G) were co-transfected into 293T cells using Lipofectamine 3000 transfection reagent (Invitrogen, #L3000015, USA). The cell culture supernatant was collected 48 hours later and centrifuged at 4000 × g for 30 minutes. A stable EIF2AK3 knockout cell line was created using lentivirus-based short hairpin RNA (shRNA) under puromycin (Sigma, #540222, Germany) selection. A stable cell line producing shRNA was finally established. shRNA sequence: GCATCTGCCTGGTTACTTAAG (SEQ ID NO: 3).

[0078] 4) siRNA transfection

[0079] Before transfection, exponentially growing untreated 293T cells and HUVECs were plated in six-well plates for 24 hours. When the cells reached 60 - 70% confluence, 25 pmol of specific small interfering RNA (siRNA) targeting EIF2AK3 mRNA or scrambled siRNA (si-NC) was added, and siRNA transfection was carried out with Lipofectamine RNAiMAX (Invitrogen, #13778150, USA) for 48 hours. The interference efficiency was confirmed by qRT-PCR and Western blot, and then the cells were harvested for further detection. si-RNA sequence: GGAAACAGCTATTCTCATA (SEQ ID NO: 4).

[0080] 5) Cell migration assay

[0081] The cell migration ability was evaluated using the Transwell assay (Corning, USA). 10% FBS medium was placed in the lower chamber, and then 2 × 10 ^4A suspension of cells. After incubation for the specified time, the cells on the upper surface of the membrane were removed. The migrated cells were fixed with methanol and stained with crystal violet. Then, the migrated cells were photographed under a microscope and quantified using ImageJ (version 1.54, National Institutes of Health, USA).

[0082] 6) HE staining

[0083] The thoracic aorta of mice was collected and fixed with 4 wt% paraformaldehyde. After fixation, the tissues were dehydrated and embedded in paraffin to form sections with a thickness of 5 μm. After dewaxing, the sections were stained with hematoxylin and eosin, and then permeabilized in xylene. After drying, the sections were sealed with neutral resin. Finally, the tissue morphology was observed under an optical microscope.

[0084] 7) Immunofluorescence

[0085] The paraffin-embedded sections of the thoracic aorta of mice were deparaffinized, hydrated, and antigen repaired. Then, the sections were incubated at room temperature in a blocking solution containing 0.1% Triton X-100 and 5% BSA. The primary antibody (EIF2AK3 antibody, 1:100, ZenBioScience, #340751, China) was incubated overnight at 4°C, and then incubated with the fluorescent secondary antibody (1:1000, prepared with 1 wt% BSA) at room temperature. Finally, the slides were treated with an anti-quencher containing DAPI (Beyotime, #P0131, Shanghai, China), and images were acquired using a Leica SP8 confocal microscope (Leica, USA).

[0086] 8) Real-time quantitative PCR (qRT-PCR)

[0087] Total cellular RNA was extracted using RNAiso Plus reagent (Takara, #9109, Osaka, Japan). The extracted RNA was reverse transcribed into cDNA using Takara's PrimeScript RT reagent kit (Takara, #RR036A, Osaka, Japan). The quantitative expression levels of mRNA were determined using a real-time fluorescence quantitative PCR instrument (ABI Q6 system, Applied Biosystems, USA). The primer sequences are shown in Table 1.

[0088] Table 1

[0089]

[0090]

[0091] 9) Western blot analysis

[0092] Total cellular proteins were extracted from cells on ice using cell lysis buffer (Beyotime, #P0013B, Shanghai, China), and protease and phosphatase inhibitors were added. Protein concentration was measured by the BCA method. Samples were separated by SDS-PAGE (EpiZyme, #PG113, Shanghai, China) and then transferred to a polyvinylidene difluoride membrane (PVDF) (Merck Millipore, #ISEQ00010, Darmstadt, Germany). The membrane was incubated overnight with diluted primary antibody at 4 °C. Then, the membrane was incubated with the secondary antibody for 1 h at room temperature. After enhanced chemiluminescence exposure, protein expression was visualized using a ChemiDoc Touch imaging system (Amersham Imager 600UV, California, USA).

[0093] 10) RNA sequencing

[0094] mRNA sequencing was performed on the stable THP-1 cell line with EIF2AK3 knockout (sh-EIF2AK3) and the control group (sh-NC) using the Illumina Novaseq 6000 / MGISEQ-T7 instrument. The data generated from the Illumina / BGI platform were used for bioinformatics analysis. All analyses were performed using an in-house pipeline developed by Shanghai Applied Protein Technology Co., Ltd. Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the clusterProfiler R package.

[0095] 11) Statistics

[0096] GraphPad Prism 9 (GraphPad Software Prism 8.0, La Jolla, CA, USA) was used for statistical analysis and graph production. Unless otherwise specified, all statistical analyses were performed using two-tailed tests. A p-value of 0.05 or less was considered statistically significant.

[0097] Example 1 Upregulation of EIF2AK3 expression level under KD inflammatory conditions

[0098] To preliminarily evaluate the potential involvement of EIF2AK3 in the pathophysiology of KD, this example compared the expression of EIF2AK3 protein in peripheral blood mononuclear cells (PBMCs) of KD patients and healthy controls (HCs) (samples were from patients and healthy children recruited at Guangzhou Women and Children's Medical Center between January 2014 and December 2019, and their guardians had signed informed consent forms). The results showed that the expression of EIF2AK3 was significantly increased in KD patients ( Figure 1 , A - B). In vitro studies showed that both the mRNA and protein levels of EIF2AK3 were increased in the KD serum co-culture group (Figure 1 , C-D).

[0099] Furthermore, in this example, the expression of EIF2AK3 was further verified in a KD mouse model induced by LCWE, which replicated the histopathological, functional, and immunological characteristics of human KD vasculitis. Immunofluorescence (IF) analysis showed that the expression of EIF2AK3 was also upregulated in the thoracic aorta of LCWE-induced mice ( Figure 1 , E-F).

[0100] To simulate the inflammatory environment of KD, in this example, subsequent experiments were conducted using THP-1 cells with 1×10^6 cells and 293T cells with 5×10^5 cells treated with complete medium containing 10% KD serum or LCWE (concentration 2 μg / ml) in six-well plates. Notably, after LCWE treatment, the expression of EIF2AK3 increased in a dose-dependent manner ( Figure 1 , G-H). In summary, these research results indicate that under KD inflammatory conditions, the expression of EIF2AK3 is significantly elevated, suggesting that it may play a key role in the inflammatory pathology of KD.

[0101] Example 2 EIF2AK3 increases the expression of pro-inflammatory cytokines in KD

[0102] To clarify the role of EIF2AK3 in vasculitis, in this example, the expression of pro-inflammatory cytokines was first detected after knocking out EIF2AK3. The results showed that knocking out EIF2AK3 significantly reduced the expression levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNFα) at the mRNA and protein levels ( Figure 2 , A-D). In contrast, overexpression of EIF2AK3 significantly increased the levels of these pro-inflammatory cytokines ( Figure 2 , G-H).

[0103] To confirm that the expression of pro-inflammatory cytokines depends on EIF2AK3, THP-1 cells were treated with different concentrations of the EIF2AK3 inhibitor (GSK2606414). The results showed that as the concentration of GSK2606414 increased, the mRNA and protein levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNFα) gradually decreased ( Figure 2 , E-F). In contrast, treatment with the EIF2AK3 activator (CCT020312) led to a dose-dependent increase in the protein levels of these pro-inflammatory cytokines ( Figure 2 , I). In summary, these data indicate that inhibiting EIF2AK3 by gene or drug can reduce the expression levels of pro-inflammatory cytokines, suggesting that EIF2AK3 plays a key role in regulating the expression of pro-inflammatory cytokines in KD.

[0104] Example 3: EIF2AK3 promotes the expression of pro-inflammatory cytokines through the MAPK-ERK1 / 2 pathway

[0105] To investigate the molecular mechanism by which EIF2AK3 affects the levels of pro-inflammatory cytokines, transcriptome sequencing was performed in this example. In the EIF2AK3 knockout group, 14 genes were up-regulated and 27 genes were down-regulated ( Figure 3 in A). In addition, KEGG pathway analysis showed that the MAPK signaling pathway was enriched according to the number of genes ( Figure 3 in B), and this pathway plays an important role in various inflammatory diseases.

[0106] Therefore, in this example, EIF2AK3 in THP-1 cells was knocked out, and the expression levels of key molecules in the MAPK pathway were evaluated. The results showed that the protein levels of p-MEK and p-ERK1 / 2 were significantly decreased, while the levels of p-JNK and p-p38 did not change significantly ( Figure 3 in D). The heat map of GO enrichment analysis (regulation of ERK1 and ERK2 cascades) further confirmed these results ( Figure 3 in C).

[0107] To further confirm whether EIF2AK3 regulates KD vasculitis through the MAPK-ERK1 / 2 pathway, EIF2AK3 was overexpressed in 293T cells with a cell number of 5×10^ 5 , and treated with an ERK1 / 2 specific inhibitor (SCH772984, concentrations of 0 and 2.0 μM) for 24 hours. Then, in this example, the expression levels of key proteins in the MAPK-ERK1 / 2 pathway and the expression levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNFα) were measured. The results showed that the ERK1 / 2 inhibitor (SCH772984) could significantly inhibit the increase in cytokines induced by EIF2AK3 overexpression ( Figure 3 , E-F). These findings indicate that EIF2AK3 promotes the transcription and expression of pro-inflammatory cytokines in KD through the MAPK-ERK1 / 2 pathway.

[0108] Example 4: Knockdown of EIF2AK3 inhibits EndoMT.

[0109] Example 3 has demonstrated that EIF2AK3 can enhance the expression of TNFα and IL-1β, which are associated with the induction of endothelial-to-mesenchymal transition (EndoMT) in endothelial cells (Liang X, Wu S, Geng Z, et al. LARP7 SuppressesEndothelial-to-Mesenchymal Transition by Coupling With TRIM28. Circ Res. 2021;129(9):843-856.). Notably, EndoMT is considered a key process in the pathogenesis of KD coronary artery injury (He M, Chen Z, Martin M, et al. miR-483Targeting of CTGF Suppresses Endothelial-to-Mesenchymal Transition: Therapeutic Implications in Kawasaki Disease. CircRes. 2017;120(2):354-365.). In addition, pretreatment of 1×10 ^5 HUVECs with exogenous TNFα and IL-1β at a concentration of 10 ng / mL can significantly increase the expression of EIF2AK3( Figure 4 , A-B). In summary, these observations suggest that EIF2AK3 may be involved in EndoMT of endothelial cells.

[0110] Then, this example evaluated the key indicators of EndoMT in HUVECs after EIF2AK3 knockdown. The results showed that knockdown of EIF2AK3 reduced the mRNA and protein expression levels of mesenchymal markers such as N-cadherin and FSP1, while increasing the levels of endothelial markers including ZO-1 and VE-cadherin( Figure 4 , C-D). These findings indicate that the reduction of EIF2AK3 inhibits EndoMT in the context of KD. In addition, the transwell assay showed that the reduction of EIF2AK3 significantly inhibited the migration of HUVEC cells( Figure 4 , E-F). In summary, knockdown of EIF2AK3 may inhibit the migration of HUVEC cells in KD by suppressing EndoMT, thereby alleviating CAL.

Claims

1. Use of an EIF2AK3 inhibitor in the preparation of a medicament for treating Kawasaki disease.

2. The use according to claim 1, wherein: The EIF2AK3 inhibitor comprises at least one of 1) and 2): 1) A substance that inhibits the EIF2AK3 gene at the gene level; 2) A substance that inhibits the EIF2AK3 protein at the protein level; Preferably, the EIF2AK3 inhibitor comprises at least one of a substance that reduces the content or expression level of the EIF2AK3 gene or protein, a substance that reduces the activity of the EIF2AK3 gene or protein, and a substance that promotes the degradation of the EIF2AK3 gene or protein.

3. The use according to claim 2, wherein: The amino acid sequence of the EIF2AK3 is as shown in a1) to a4): a1) A protein with an amino acid sequence as shown in SEQ ID NO: 1; a2) A protein with the same function obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence as shown in SEQ ID NO: 1; a3) An amino acid sequence having at least 85% identity with the amino acid sequence as shown in SEQ ID NO: 1; a4) A derivative related to any one of the proteins of a1), a2), and a3).

4. The use according to claim 2, wherein: The substance that reduces the content or expression level of the EIF2AK3 gene or protein comprises a small molecule inhibitor targeting the EIF2AK3 gene or protein, or a nucleotide that reduces the content or expression level of the EIF2AK3 gene or protein; Preferably, the nucleotide that reduces the content or expression level of the EIF2AK3 protein comprises at least one of a small guide RNA that inhibits the expression of the EIF2AK3 protein, a short hairpin RNA that inhibits the expression of the EIF2AK3 protein, a Knock Out system for gene knockout, a dsRNA that inhibits the expression of the EIF2AK3, a dsRNA microRNA that inhibits the expression of the EIF2AK3, and a dsRNA siRNA that inhibits the expression of the EIF2AK3; Preferably, the interference sequence of the shRNA that inhibits the expression of the EIF2AK3 is as shown in SEQ ID NO:

3.

5. A nucleotide molecule that inhibits EIF2AK3, wherein: The nucleotide molecule comprises shRNA and siRNA; The interference sequence of the shRNA is as shown in SEQ ID NO: 3; The sequence of the siRNA shown is as shown in SEQ ID NO:

4.

6. A recombinant vector that inhibits the expression of EIF2AK3, wherein: The recombinant vector comprises the shRNA and / or siRNA described in claim 5.

7. A virus that inhibits the expression of EIF2AK3, wherein: The virus comprises the recombinant vector described in claim 6.

8. A medicament comprising the nucleic acid molecule described in claim 5, the recombinant vector described in claim 6, or the virus described in claim 7.

9. The medicament according to claim 8, wherein: The drug also includes pharmaceutically acceptable excipients.

10. A method for non-therapeutically inhibiting inflammation in vitro, comprising the following steps: Treating cells with the drug according to claim 8 to reduce the level of EIF2AK3 in the cells.

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