Application of blocking CYLD shearing at D215 site in preparation of sepsis treatment medicine

Abstract

The invention relates to the technical field of biological medicines, and provides an application of blocking CYLD (Cytochrome-Yield-Diode) shearing at a D215 site in preparation of a sepsis treatment medicine, and a substance for blocking CYLD shearing at the D215 site is selected from a combination of any one or more of the following conditions: (1) a Caspase8 or FADD inhibitor, (2) a CYLD inhibitor, (3) a CYLD inhibitor, (4) a CYLD inhibitor, and (5) a CYLD inhibitor; and (2) a system for editing the 215 site of the CYLD. The invention further provides a recombinant expression vector and a sepsis treatment pharmaceutical composition, based on the discovery of the invention, the pathophysiology of sepsis inflammatory diseases can be better known, and a new opportunity is provided for diagnosis and treatment of sepsis, so that the clinical service of patients with sepsis inflammatory diseases is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to the application of the cleavage product of CYLD at position D215 as a target in the treatment of sepsis, specifically to the application of blocking the cleavage of CYLD at position D215 in the preparation of drugs for treating sepsis. Background Art

[0002] Sepsis is a common complication after severe burns, traumas, and surgical operations. It has now become one of the main causes of death in critically ill patients and is a type of infectious disease with very high morbidity and mortality worldwide, seriously endangering public health. Sepsis is a systemic inflammatory response caused by host infection. Clinically, more than 95% of sepsis cases are caused by bacterial infections, including Gram-positive bacteria and Gram-negative bacteria.

[0003] The cleavage of CYLD exists in various cells, such as L929, BMDM, U937, and MEF. The cleavage of CYLD can be detected in L929 and U937 cells under LPS stimulation, and the cleavage of CYLD can also be detected in BMDM and MEF cells under TNFα stimulation. Moreover, with the prolongation of the stimulation time, the CYLD protein will decrease, while the protein band of the cleavage product P25 at its 215th site will gradually increase; similar experimental results are also shown in animal model experiments induced by LPS and DSS. Summary of the Invention

[0004] The purpose of the present invention is to provide a treatment target for sepsis, and the purpose of the present invention is also to provide the application of blocking the cleavage of CYLD at position D215 in the preparation of drugs for treating sepsis.

[0005] The inventors have conducted in-depth research and found that: Mechanistic analysis shows that the cleavage of the 215th site of CYLD to produce the P25 protein is mediated by Caspase8 and FADD and depends on Trif to complete, does not depend on the secretion and exosome pathways mediated by TMED10, but depends on Caspase8; in terms of localization, it can be secreted by cells to the extracellular space and cannot enter the nucleus. Thus, the inventors have discovered a new use of the P25 protein as a treatment target for sepsis. Inhibiting the activity of the P25 protein or the activity of key sites during its generation is helpful to achieve the treatment of sepsis, and thus the present invention is completed on this basis.

[0006] In the first aspect of the present invention, there is provided the application of a substance that blocks the cleavage of CYLD at position D215 in the preparation of drugs for treating sepsis.

[0007] Since the cleavage of the 215th site of CYLD produces the P25 protein, which is mediated by Caspase8 and FADD and depends on Trif to complete.

[0008] The amino acid sequence of P25 protein is shown as follows:

[0009] MSSGLWSQEKVTSPYWEERIFYLLLQECSVTDKQTQKLLKVPKGSIGQYIQDRSVGHSRIPSAKGKKNQIGLKILEQPHAVLFVDEKDVVEINEKFTELLLAITNCEERFSLFKNRNRLSKGLQIDVGCPVKVQLRSGEEKFPGVVRFRGPLLAERTVSGIFFGVELLEEGRGQGFTDGVYQGKQLFQCDEDCGVFVALDKLELIEDDDTALESD(SEQ ID NO.1);

[0010] The amino acid sequence of human CYLD is shown as follows. Caspase8 cleavage occurs after LESD (bold).

[0011] MSSGLWSQEKVTSPYWEERIFYLLLQECSVTDKQTQKLLKVPKGSIGQYIQDRSVGHSRIPSAKGKKNQIGLKILEQPHAVLFVDEKDVVEINEKFTELLLAITNCEERFSLFKNRNRLSKGLQIDVGCPVKVQLRSGEEKFPGVVRFRGPLLAERTVSGIFFGVELLEEGRGQGFTDGVYQGKQLFQCDEDCGVFVALDKLELIEDDDTALESDYAGPGDTMQVELPPLEINSRVSLKVGETIESGTVIFCDVLPGKESLGYFVGVDMDNPIGNWDGRFDGVQLCSFACVESTILLHINDIIPALSESVTQERRPPKLAFMSRGVGDKGSSSHNKPKATGSTSDPGNRNRSELFYTLNGSSVDSQPQSKSKNTWYIDEVAEDPAKSLTEISTDFDRSSPPLQPPPVNSLTTENRFHSLPFSLTKMPNTNGSIGHSPLSLSAQSVMEELNTAPVQESPPLAMPPGNSHGLEVGSLAEVKENPPFYGVIRWIGQPPGLNEVLAGLELEDECAGCTDGTFRGTRYFTCALKKALFVKLKSCRPDSRFASLQPVSNQIERCNSLAFGGYLSEVVEENTPPKMEKEGLEIMIGKKKGIQGHYNSCYLDSTLFCLFAFSSVLDTVLLRPKEKNDVEYYSETQELLRTEIVNPLRIYGYVCATKIMKLRKILEKVEAASGFTSEEKDPEEFLNILFHHILRVEPLLKIRSAGQKVQDCYFYQIFMEKNEKVGVPTIQQLLEWSFINSNLKFAEAPSCLIIQMPRFGKDFKLFKKIFPSLELNITDLLEDTPRQCRICGGLAMYECRECYDDPDISAGKIKQFCKTCNTQVHLHPKRLNHKYNPVSLPKDLPDWDWRHGCIPCQNMELFAVLCIETSHYVAFVKYGKDDSAWLFFDSMADRDGGQNGFNIPQVTPCPEVGEYLKMSLEDLHSLDSRRIQGCARRLLCDAYMCMYQSPTMSLYK(SEQ ID NO.2).

[0012] Therefore, the substance that blocks the cleavage of CYLD at position D215 is selected from any one or a combination of the following cases:

[0013] (1) Caspase8 or FADD inhibitor;

[0014] (2) A substance that edits the 215th site of CYLD.

[0015] Among them, the cleavage protein of CYLD at position D215 is the P25 protein with a molecular weight of 25KD, and the amino acid sequence is as shown in SEQ ID NO.1.

[0016] Preferably, the Caspase8 inhibitor is selected from siRNA or shRNA targeting Caspase8; the FADD inhibitor is selected from siRNA or shRNA targeting FADD; the system for editing the 215th site of CYLD is the Caspase-8 CRISPR-Cas9 system or the FADD CRISPR-Cas9 system.

[0017] More preferably, (a) the siRNA targeting Caspase8 includes siRNA-1, siRNA-2, siRNA-3, and the specific sequences are as follows:

[0018] siRNA-1:

[0019] Sense strand: 5'-CCUCUAAUGAUUAACUCAAAC-3' (SEQ ID NO.3),

[0020] Antisense strand: 5'-UUGAGUUAAUCAUUAGAGGUG-3' (SEQ ID NO.4);

[0021] siRNA-2:

[0022] Sense strand: 5'-CAGAGUUGUCUUUAUGCUAUU-3' (SEQ ID NO.5),

[0023] Antisense strand: 5'-UAGCAUAAAGACAACUCUGGA-3' (SEQ ID NO.6);

[0024] siRNA-3:

[0025] Sense strand: 5'-GGUGGGACCUGCUGGUCAACU-3' (SEQ ID NO.7),

[0026] Antisense strand: 5'-UUGACCAGCAGGUCCCACCGA-3' (SEQ ID NO.8).

[0027] (b) The target sequence of the shRNA targeting Caspase8 is shown in SEQ ID NO.9, and the sense and antisense strand sequences of the shRNA are shown in SEQ ID NO.10 and 11 respectively, as follows:

[0028] Target sequence: 5'-CCUCUAAUGAUUAACUCAAAC-3' (SEQ ID NO.9);

[0029] The shRNA sequence is as follows:

[0030] Sense strand: 5'-GATCCCCUCUAAUGAUUAACUCAAACTTCAAGAGAUCCUGAGGUACUGCAUGCTTTTTTG-3' (SEQ ID NO.10),

[0031] Antisense strand: 5'-AATTCAAAAAACCUCUAAUGAUUAACUCAAACCUCUUGAAGAUCCUGAGGUACUGCAUGCG-3' (SEQ ID NO.11).

[0032] (c) The siRNAs targeting FADD include siRNA-4, siRNA-5, siRNA-6, and the specific sequences are as follows:

[0033] siRNA-4:

[0034] Sense strand: 5'-GGAAGAAUGCUGAGAAGAAGA-3' (SEQ ID NO.12),

[0035] Antisense strand: 5'-UUCUUCUCAGCAUUCUUCCAG-3' (SEQ ID NO.13);

[0036] siRNA-5:

[0037] Sense strand: 5'-CGGUGCUGCUGGAGCAGAACG-3' (SEQ ID NO.14),

[0038] Antisense strand: 5'-UUCUGCUCCAGCAGCACCGUG-3' (SEQ ID NO.15);

[0039] siRNA-6:

[0040] Sense strand: 5'-GCUGCGCCGACACGAUCUACU-3' (SEQ ID NO.16)

[0041] Antisense strand: 5'-UAGAUCGUGUCGGCGCAGCGA-3' (SEQ ID NO.17);

[0042] (d) The target sequence of the shRNA targeting FADD is shown in SEQ ID NO.18, and the sense and antisense strand sequences of the shRNA are shown in SEQ ID NO.19 and 20 respectively, as follows:

[0043] Target sequence: 5'-GGAAGAAUGCUGAGAAGAAGA-3' (SEQ ID NO.18);

[0044] The shRNA sequence is as follows:

[0045] Sense strand: 5'-GATCCCGGAAGAAUGCUGAGAAGAAGATTCAAGAGAUCCUGAGGUACUGCAUGCTTTTTTG-3' (SEQ ID NO.19),

[0046] Antisense strand: 5'-AATTCAAAAAAGGAAGAAUGCUGAGAAGAAGACUCUUGAAGAUCCUGAGGUACUGCAUGCG-3' (SEQ ID NO.20).

[0047] (e) In the Caspase-8 CRISPR-Cas9 system, the target sequence is shown as any one of the following,

[0048] Target sequence 1: 5'-TAGCTTCTGGGCATCCTCGA-3' (PAM: TGG) (SEQ ID NO.21),

[0049] Target sequence 2: 5'-CTTCCTAGACTGCAACCGAG-3' (PAM: AGG) (SEQ ID NO.22),

[0050] Target sequence 3: 5'-GCAGGTCCCACCGACTGATG-3' (PAM: AGG) (SEQ ID NO.23);

[0051] The CRISPR-Cas9 sgRNA sequence is the 20bp upstream of the target sequence + PAM;

[0052] (f) In the FADD CRISPR-Cas9 system, the target sequence is shown as any one of the following:

[0053] Target sequence 1: 5'-TTCGTTTGCTCACGCGCTCG-3' (PAM: TGG) (SEQ ID NO.24),

[0054] Target sequence 2: 5'-GAGCAGAACGACCTGGAGCG-3' (PAM: AGG) (SEQ ID NO.25),

[0055] Target sequence 3: 5'-GCGCGTGAGCAAACGAAAGC-3' (PAM: TGG) (SEQ ID NO.26);

[0056] The FADD Cas9 sgRNA sequence is 20 bp upstream of the target sequence + PAM.

[0057] The subjects targeted by the drugs of the present invention are selected from the following groups: patients who have been diagnosed with sepsis; sepsis patients who are ineffective or have poor effects or are expected to have poor effects after treatment with existing drugs.

[0058] Generally, the full-length sequences or fragments of the sgRNA and CRISPR-Cas9 mRNA herein can be obtained by PCR amplification, recombination or artificial synthesis methods. For the PCR amplification method, primers can be designed according to the nucleotide sequences disclosed in the present invention to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified each time together in the correct order.

[0059] It should be understood that the P25 protein molecule herein is preferably obtained from humans, and other P25 protein molecules obtained from other animals that are highly homologous to human P25 protein (such as having a sequence identity of more than 70%, more than 75%, more than 80%, more preferably more than 85% such as 85%, 90%, 95%, 98% or even 99% or more) are also within the equivalent scope preferably considered in the present invention. Methods and tools for aligning sequence identity are also well known in the art, such as BLAST.

[0060] In terms of source, the inhibitors of the present invention are selected from: natural purified substances, modified natural purified substances, semi-synthetic substances, chemically synthesized substances; further, the inhibitors are derived from mammals, such as humans, non-human primates (such as orangutans, apes), rodents (such as rats, mice, guinea pigs), pets (such as cats, dogs), livestock (such as horses, cows, sheep, pigs, rabbits).

[0061] In a second aspect of the present invention, a recombinant expression vector is provided, which comprises an expression vector and an sgRNA or siRNA of Caspase8 or FADD inserted and disposed on the expression vector, or a Caspase-8 CRISPR-Cas9 system or an FADD CRISPR-Cas9 system, and the specific sequences are as shown above.

[0062] In a third aspect of the present invention, the application of the above recombinant expression vector in the preparation of a therapeutic drug for sepsis is provided.

[0063] In a fourth aspect of the present invention, a therapeutic drug composition for sepsis is provided, which comprises an active ingredient and a pharmaceutically or immunologically acceptable excipient, carrier or diluent. Among them, the active ingredient is the above-mentioned substance that blocks the cleavage of CYLD at position D215 or the recombinant expression vector.

[0064] The term "pharmaceutically / immunologically acceptable" ingredient is a substance that is applicable to humans and / or animals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), that is, a substance with a reasonable benefit / risk ratio. As used herein, the term "effective amount" refers to an amount that can produce a function or activity in humans and / or animals and is acceptable to humans and / or animals.

[0065] The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent, including various excipients and diluents. This term refers to such pharmaceutical carriers: they are not necessarily the active ingredients themselves and have no excessive toxicity after administration. Suitable carriers are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington’s Pharmaceutical Sciences, Mack Pub. Co., N.J. 1991.

[0066] In the composition, the pharmaceutically acceptable carrier may contain liquids, such as water, saline, glycerol and ethanol. In addition, auxiliary substances may also be present in these carriers, such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting agents or emulsifiers, flavoring agents, pH buffering substances, etc. Usually, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably, the pH is about 6-8.

[0067] The active substance in the composition herein accounts for 0.001-99.9 wt% of the total weight of the composition; preferably 1-95 wt% of the total weight of the composition, more preferably 5-90 wt%, and even more preferably 10-80 wt%, and the balance is pharmaceutically acceptable carriers and other additives and the like.

[0068] The composition of the present invention can be in solid state (such as granules, tablets, lyophilized powder, suppositories, capsules, sublingual tablets), liquid state (such as oral liquid) or other suitable forms. The administration routes can be: (1) direct naked DNA / RNA injection method; (2) connecting the relevant sgRNA and CRISPR-Cas9 mRNA with transferrin / poly-L-lysine complex to enhance its biological effect; (3) forming a complex of sgRNA and CRISPR-Cas9 mRNA with positively charged lipids to overcome the difficulty of crossing cell membrane caused by the negative charge of the phosphate backbone; (4) mediating the entry of sgRNA and CRISPR-Cas9 mRNA into cells after encapsulating them with liposomes, which is beneficial for the smooth entry of macromolecules and protects them from hydrolysis by various extracellular enzymes; (5) binding sgRNA and CRISPR-Cas9 mRNA with cholesterol to increase the cytoplasmic retention time by 10 times; (6) transporting sgRNA and CRISPR-Cas9 mRNA with immunoliposomes can specifically transport them to target tissues and target cells; (7) in vitro transfection of sgRNA and CRISPR-Cas9 mRNA into carrier cells (such as fibroblasts) can also load the relevant drugs into target cells well; (8) electroporation, that is, introducing sgRNA and CRISPR-Cas9 mRNA into target cells by means of electric current.

[0069] As used in the present invention, the term "unit dosage form" refers to a dosage form prepared from the composition of the present invention for convenient administration, which is the dosage form required for a single administration, including but not limited to various solid forms (such as tablets), liquid forms, capsule forms, sustained-release forms.

[0070] In some embodiments of the present invention, the composition is in unit dosage form or multiple dosage forms. "Unit dosage form" refers to a dosage form prepared from the product for convenient administration, which is the dosage form required for a single administration, including but not limited to various solid forms (such as tablets), liquid forms, capsule forms, sustained-release forms. In some embodiments herein, the composition herein is administered at an appropriate frequency, for example, daily, every other day, weekly, every other week or two weeks, monthly, every other month or two months, for example, 1 to 6 doses are administered.

[0071] It should be understood that the effective dose of the active substance used may vary with the severity of the subject to be administered or treated. The specific situation is determined according to the individual situation of the subject (such as the subject's body weight, age, physical condition, the effect to be achieved), which is within the scope that can be judged by a skilled physician.

[0072] In the fifth aspect of the present invention, a method for treating sepsis is provided, which includes administering a therapeutically effective amount of the related substance that blocks the cleavage of CYLD at position D215 to a subject in need. To improve the therapeutic effect, the pharmaceutical composition is used in combination with other drugs for treating sepsis.

[0073] The administration methods of the present invention are diverse, including oral administration, injection (such as direct naked DNA or protein injection method, liposome-encapsulated DNA, RNA or protein injection method), gold-coated gene gun bombardment method, plasmid DNA carried by replication-defective bacteria method, target DNA carried by replication-defective adenovirus method or protein encoded by the target gene, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, transdermal administration.

[0074] The beneficial guarantees and effects of the present invention are as follows:

[0075] The present invention reveals a new use of the P25 protein molecule in the early diagnosis of sepsis, and also provides new ideas and approaches for the research and development and utilization of key sites or substances in its generation process as therapeutic targets. The present invention for the first time discloses that the P25 protein molecule can be effectively used as a therapeutic target for sepsis, thereby providing a novel biomarker in this field, which has clinical application prospects. Description of the Drawings

[0076] Figure 1 Cleavage of CYLD can be detected in a variety of cells. A is L929, B is BMDM, C is U937, and D is MEF. As shown in the figure, cleavage of CYLD can be detected in L929 and U937 cells stimulated by LPS; cleavage of CYLD can also be detected in BMDM and MEF cells stimulated by TNFα. As the stimulation time of these cells prolongs, the CYLD protein will decrease, while the P25 protein band will gradually increase.

[0077] Figure 2 Caspase8 and FADD mediate the cleavage of CYLD. Under TNFα stimulation, in cells with Caspase8 - / - RIP3 - / - and FADD - / - RIP3 - / - the P25 protein band cannot be detected, while in cells with RIP3 - / - the results consistent with those after stimulation of WT cells are shown, indicating that Caspase8 and FADD can mediate the cleavage of CYLD.

[0078] Figure 3 It shows that the cleavage of CYLD at position 215 to generate P25 is Trif-dependent, and the basal cleavage of CYLD in cells comes from the stimulation of autocrine TNF in cells.

[0079] Figure 4 showed the intracellular localization of P25 protein. The P25 protein fragment (CYLD D215A / D215A in cells was the full-length CYLD protein) could be secreted by cells into the extracellular space ( Figure 4 A and 4B), and the P25 protein fragment could not enter the nucleus ( Figure 4 C).

[0080] Figure 5 showed the secretion mechanism of the P25 protein fragment. The secretion of the P25 protein fragment was not dependent on TMED10-mediated secretion ( Figure 5 A) and the exosome pathway ( Figure 5 B), but was dependent on Caspase8 ( Figure 5 C).

[0081] Figure 6 showed that the P25 protein fragment increased in mice with the prolongation of LPS injection time, while the CYLD protein decreased with the prolongation of LPS injection time. A, B, C, and D were the spleen, liver, lung, and kidney tissues of mice, respectively.

[0082] Figure 7 showed that P25 could be detected in the DSS-induced colitis model. Among them, A was the representative result graph of the colon of the control group (C) and the DSS-treated group (T), B was the statistical graph of the colon length of the two groups of mice, C was the change in body weight of the DSS-treated group of mice, D was the change in the clinical score of the DSS-treated group of mice, and E was the situation of CYLD and P25 proteins in the control group (C) and the DSS-treated group (T) of mice. Detailed implementation manners

[0083] Now, in combination with the embodiments and the drawings, the present invention will be described in detail, but the implementation of the present invention is not limited thereto.

[0084] The reagents and raw materials used in the present invention are all commercially available or can be prepared according to the literature methods. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions such as those described in Sambrook et al., "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory Press, 1989), or under conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by volume.

[0085] All numerical ranges provided herein are intended to clearly include all numerical values falling between the range endpoints and the numerical ranges therebetween. The features mentioned in the present invention or the features mentioned in the embodiments can be combined. All features disclosed in this specification can be used in any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent or similar features.

[0086] As used herein, "comprising", "having" or "including" include "containing", "consisting essentially of", "consisting substantially of", and "consisting of"; "consisting essentially of", "consisting substantially of" and "consisting of" are subordinate concepts of "comprising", "having" or "including".

[0087] The numerical ranges herein include their endpoints as well as each specific numerical point and sub-range within the numerical range. For example, 1 to 3 includes the endpoints 1 and 3, the specific integer numerical points 2 therebetween, and non-integer numerical points (such as but not limited to: 1.2, 1.5, 1.8, 2.1, 2.3, 2.4, 2.8, etc.), as well as its sub-ranges (such as but not limited to: 1 to 2, 2 to 3, 1 to 1.2, 1.5 to 1.8, etc.).

[0088] 1. Cell experiments

[0089] Construct CYLD D215A / D215A genotype mice, and use in vitro and in vivo models to identify CYLD D215A / D215A whether cells and mice regulate apoptosis and necroptosis. The specific contents include:

[0090] Isolate mouse embryo fibroblasts (MEF); use drugs such as TNFα, BV6, and zVAD to induce apoptosis and necroptosis; measure cell viability through the Cell Titer-Glo Luminescent Cell Viability Assay Kit; detect related molecules of apoptosis and necrosis using Western Blot experiments; detect the effect of the cleavage at position 215 of CYLD on necrosis in vivo by injecting TNFα cell necrosis in vivo model into the mouse tail vein.

[0091] 2. Exploration of the cell types, stimulation conditions, cleavage rules and characteristics of CYLD cleavage

[0092] Detect whether CYLD cleavage occurs in cells such as 293T, L929, BMDM, U937, and MEF under TNFα or LPS stimulation; which molecules does the cleavage at position 215 depend on; which molecules are involved in the cleavage at position 215; and under which TLR stimulations will cleavage occur.

[0093] Figure 1 It shows that CYLD cleavage can be detected in multiple cells. As shown in the figure, CYLD cleavage can be detected in L929 and U937 cells under LPS stimulation; CYLD cleavage can also be detected in BMDM and MEF cells under TNFα stimulation. As the stimulation time prolongs in these cells, the CYLD protein will decrease, while the P25 protein band will gradually increase.

[0094] Figure 2 It shows that Caspase8 and FADD mediate the cleavage of CYLD. Under TNFα stimulation, in cells lacking Caspase8 - / - RIP3 - / - and FADD - / - RIP3 - / - the P25 protein band cannot be detected, while cells lacking RIP3 - / - show results consistent with those of WT cells after stimulation, indicating that Caspase8 and FADD can mediate the cleavage of CYLD.

[0095] Figure 3 It shows that the generation of P25 by cleavage at position 215 of CYLD is Trif-dependent, and the basal CYLD cleavage in cells comes from the stimulation of autocrine TNF in the cells, as follows:

[0096] As Figure 3 shown in A, in WT cells, after TLR3 (ligand is Poly(I:C)) and TLR4 (ligand is LPS) are activated, they can stimulate the cleavage of CYLD to generate P25, while after TLR7 (ligand is R837), TLR1 / 2 (ligand is Pam3csk4), and TLR9 (ligand is CpG) are activated, they cannot induce the cleavage of CYLD; in CYLD D215A / D215A knockout cells, various stimulants cannot stimulate CYLD cleavage to generate P25.

[0097] Figure 3 Figure B shows that TNFα and FasL can also cause CYLD cleavage to generate P25. Figure 3 Figure C shows that in Trif knockout cells, the activation of TLR3 and TLR4 cannot cause CYLD cleavage, and Trif is exactly the adaptor protein of TLR3 and TLR4, indicating that the CYLD cleavage triggered by Poly(I:C) and LPS stimulation depends on Trif.

[0098] Figure 3 D It is shown that the deletion of Trif does not affect the cleavage of CYLD induced by TNFα and FasL stimulation, which is consistent with the information that Trif is not involved in the above two signaling pathways.

[0099] Figure 4 It is shown that the P25 protein fragment (CYLD D215A / D215A in cells is the full-length CYLD protein) can be secreted by cells into the extracellular space ( Figure 4 A and 4B), and the P25 protein fragment cannot enter the nucleus ( Figure 4 C).

[0100] Figure 5 It is shown that the secretion of the P25 protein fragment is not dependent on TMED10-mediated secretion ( Figure 5 A) and the exosome pathway ( Figure 5 B), but is dependent on Caspase8 ( Figure 5 C).

[0101] 3. Animal model experiments

[0102] For the secretion of the P25 protein fragment and its correlation with the progression of the LPS model disease, further study the pathway by which it is secreted, and explore whether the P25 protein fragment will appear in other mouse models and whether the appearance of the P25 protein fragment can be used as a biomarker for a class of diseases. The specific contents include:

[0103] 3.1 After injecting LPS into CYLD D215A / D215A mice, different tissues were collected, lysed with RIPA, and Western Blot protein samples were prepared. The experimental results were analyzed by Western Blot.

[0104] The results are as Figure 6 shown. The P25 protein fragment increases in mice with the prolongation of LPS injection time, and the CYLD protein decreases with the prolongation of LPS injection time. Figure 6 A, 6B, 6C, and 6D are the spleen, liver, lung, and kidney tissues of mice respectively, and the results among different tissues are similar.

[0105] 3.2 WT mice were fed 2% dextran sulfate sodium (DSS) to induce an acute colitis model in mice. After successful modeling, the colon tissues were collected, and whether P25 also appears in the inflamed colon tissues was detected by Western Blot.

[0106] Figure 7 It is shown that P25 can be detected in the DSS-induced colitis model: Figure 7Figure A shows representative results of the colon in the control group (C) and the DSS-treated group (T). Figure 7 Figure B is a statistical graph of the colon lengths of the two groups of mice. The colon length of the DSS group was significantly shorter than that of the control group. Figure 7 Figure C shows the change in body weight of the mice in the DSS-treated group. As time went by, the body weight of the mice gradually decreased. Figure 7 Figure D shows the change in the clinical score of the mice in the DSS-treated group. Figure 7 Figure E shows the comparison of the expression of CYLD and P25 in the mice of the control group (C) and the DSS-treated group (T). The P25 protein was significantly increased in the DSS-treated group.

[0107] The above results indicate that: the P25 protein was significantly increased in the inflammation models induced by LPS and DSS. This result suggests a close correlation between the P25 protein and the occurrence and development of sepsis, and thus it can be used as a biomarker for tumor diagnosis, tumor treatment plan selection, and tumor prognosis evaluation.

[0108] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art can make various equivalent variations or substitutions without departing from the spirit of the present invention, and these equivalent variations or substitutions are all included within the scope defined by the claims of this application.

Claims

1. Application of substances that block CYLD shearing at position D215 in the preparation of drugs for the treatment of sepsis.

2. The use according to claim 1, characterized in that: in, The substance that blocks CYLD shearing at position D215 is selected from any of the following situations: (1) Caspase8 or FADD inhibitors; (2) A system for editing the 215 site of CYLD.

3. The use according to claim 2, characterized in that: in, The cleavage protein of CYLD at position D215 is the P25 protein with a molecular weight of 25KD, and the amino acid sequence is shown in SEQ ID NO.1; The Caspase8 inhibitor is selected from siRNA or shRNA targeting Caspase8; The FADD inhibitor is selected from siRNA or shRNA targeting FADD; The system used to edit the 215 site of CYLD is the Caspase-8 CRISPR-Cas9 system or the FADD CRISPR-Cas9 system.

4. The use according to claim 3, characterized in that: in, The siRNA targeting Caspase8 includes siRNA-1, siRNA-2, and siRNA-3; the sense strand and antisense strand sequences of siRNA-1 are shown in SEQ ID NOs. 3 and 4, respectively; the sense strand and antisense strand sequences of siRNA-2 are shown in SEQ ID NOs. 5 and 6, respectively; the sense strand and antisense strand sequences of siRNA-3 are shown in SEQ ID NOs. 7 and 8, respectively. The target sequence of the shRNA targeting Caspase8 is shown in SEQ ID NO.9, and the sense strand and antisense strand sequences of the shRNA are shown in SEQ ID NO.10 and 11, respectively. siRNAs targeting FADD include siRNA-4, siRNA-5, and siRNA-6; the sense strand and antisense strand sequences of siRNA-4 are shown in SEQ ID NOs. 12 and 13, respectively; the sense strand and antisense strand sequences of siRNA-5 are shown in SEQ ID NOs. 14 and 15, respectively; the sense strand and antisense strand sequences of siRNA-6 are shown in SEQ ID NOs. 16 and 17, respectively. The target sequence of the shRNA targeting FADD is shown in SEQ ID NO. 18, and the sense strand and antisense strand sequences of the shRNA are shown in SEQ ID NOs. 19 and 20, respectively.

5. The use according to claim 3, characterized in that: in, In the Caspase-8CRISPR-Cas9 system, the target sequence is shown in any one of SEQ ID NOs. 21 to 23, and the CRISPR-Cas9 sgRNA sequence is the target sequence + 20 bp upstream of the PAM; In the FADD CRISPR-Cas9 system, the target sequence is shown in any one of SEQ ID NOs. 24 to 26, and the FADD Cas9sgRNA sequence is the target sequence + 20 bp upstream of PAM.

6. The use according to any one of claims 1 to 5, characterized in that: Used in combination with other sepsis treatment drugs.

7. A recombinant expression vector, characterized in that: It includes an expression vector and an sgRNA or siRNA of Caspase8 or FADD inserted into the expression vector, or a Caspase-8 CRISPR-Cas9 system or a FADD CRISPR-Cas9 system, The sgRNA or siRNA of Caspase8 or FADD is as shown in claim 4, and the Caspase-8 CRISPR-Cas9 system or FADD CRISPR-Cas9 system is as shown in claim 5.

8. Use of the recombinant expression vector according to claim 7 in preparing a drug for treating sepsis.

9. A pharmaceutical composition for treating sepsis, characterized in that: It comprises an active ingredient and a pharmaceutically or immunologically acceptable excipient, carrier or diluent, Wherein, the active component is the substance that blocks the shearing of CYLD at position D215 as described in any one of claims 1 to 5 or the recombinant expression vector as described in claim 7.

10. The pharmaceutical composition for treating sepsis according to claim 9, characterized in that: The pharmaceutical composition is used in combination with other sepsis therapeutic drugs.

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