A polypeptide drug for treating sepsis encephalopathy

Abstract

This invention discloses a polypeptide drug for treating septic encephalopathy. The invention's research discovered a direct interaction between ATF2 and c-JUN protein in SAE mice, forming an ATF2-c-JUN complex. Based on this discovery, a cell-penetrating peptide that competitively inhibits this direct interaction was designed and synthesized. Experiments have demonstrated that this cell-penetrating peptide can effectively improve brain damage in SAE mice.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a polypeptide drug for treating septic encephalopathy. Background Technology

[0002] Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection, and is considered one of the leading causes of morbidity and mortality in intensive care units (ICUs) worldwide. As a severe systemic inflammatory response, sepsis is also closely linked to the global disease burden. When microorganisms invade the body, they trigger a systemic immune response to fight off the invading microbes, leading to an inflammatory response. This instinctive protective response can promptly identify, eliminate, and control the infection locally. However, the overactivation of the immune response during sepsis leads to sequential damage to host cells and tissues, causing dysfunction and life-threatening multi-organ failure. The pathophysiology of sepsis is defined as an early, sustained, high-intensity inflammatory state followed by a prolonged state of immunosuppression. Both phases can result in high mortality rates, with the early high mortality attributed to a massive inflammatory response (also known as an inflammatory cytokine storm). The excessive release of inflammatory mediators during a cytokine storm leads to significant endothelial cell damage, resulting in the disruption of the protective barrier, vasodilation, activation of coagulation pathways, platelet aggregation and adhesion, and mitochondrial dysfunction, among other things. Excessive inflammatory and immune dysregulation, and its consequences, ultimately lead to microvascular thrombosis, hypotension, impaired cellular function, insufficient local perfusion, tissue hypoxia, and progressive tissue damage, eventually resulting in refractory shock and multiple organ failure. Cardiovascular dysfunction, acute lung injury and acute respiratory distress syndrome, acute kidney injury, liver dysfunction, central nervous system dysfunction, and related encephalopathy are well-known complications of sepsis, and their underlying mechanisms have attracted considerable attention. These abnormal changes and dysfunctions in tissues and organs collectively contribute to the high mortality rate of sepsis.

[0003] Sepsis-associated encephalopathy (SAE), a major complication of sepsis, is characterized by a range of brain dysfunctions, from mild confusion to coma. A large-scale retrospective analysis of a multicenter database showed that 53% (1341 / 2351) of sepsis patients presented with delusions and coma upon ICU admission. This study also indicated that older patients with a history of chronic alcoholism, neurological disorders, prior cognitive impairment, and long-term use of psychoactive drugs may be more prone to SAE. Furthermore, complications including acute renal failure, metabolic disorders, glycemic instability, hypercapnia, and hypernatremia may be risk factors for SAE incidence. Overall, sepsis patients with brain dysfunction appear to have a heavier systemic disease burden and are associated with higher mortality rates. However, whether these systemic diseases and disturbances are considered confounding factors or diagnostic indicators of SAE is debatable. Researchers generally agree that patients with septic shock are more likely to develop brain dysfunction. Hypoperfusion, hypoxia, microthrombosis, and internal environment disturbances are considered major causes of multiple organ dysfunction, including brain dysfunction. Guidelines such as the "Surviving Sepsis Campaign" recommend early goal-directed therapy and organ replacement therapy to reverse shock and protect organs. However, given the fragility and irreplaceability of the brain (compared to the kidneys and liver), specific neuroprotective interventions are urgently needed. Although the understanding and research on sepsis-associated encephalopathy (SAE) continues, there is still no specific, evidence-based treatment for treating SAE in patients. Considering that SAE is secondary to sepsis and does not involve a direct central nervous system infection, the focus of treatment remains on preventing SAE by treating sepsis and suppressing systemic inflammatory response syndrome. Statistics from the United States in 1979-2000 show that bacterial infections accounted for 90% of all sepsis cases, with Gram-positive bacteria accounting for 52%, Gram-negative bacteria for 38%, and multibacterial and fungal infections accounting for 4.7% and 4.6% of all cases, respectively. Furthermore, by definition, viruses can also cause sepsis; COVID-19 has been found to cause critical conditions such as respiratory failure, septic shock, and multiple organ dysfunction in approximately 5% of patients. Therefore, both broad-spectrum and narrow-spectrum antibiotics are essential. Other treatments for sepsis and septic shock, such as fluid resuscitation, administration of vasoactive drugs, glycemic control, and nutritional support, are also recommended.

[0004] In sepsis management, bioactive peptides are considered to possess both therapeutic and protective properties against sepsis, thus becoming a novel option among currently effective treatment methods. Among different classes of bioactive peptides, antimicrobial peptides (AMPs) are naturally occurring peptides capable of resisting microbial infections and their associated complications, such as sepsis. Due to their unique antimicrobial mechanism of action, potent antimicrobial efficacy, minimal drug residues, and simplicity of production and modification, AMPs have shown great potential as a promising alternative to antibiotics for decades. Furthermore, AMPs exhibit significantly lower levels of antimicrobial resistance due to their multiple mechanisms not addressed by traditional antibiotics. In addition, anti-inflammatory peptides (AIPs) have shown beneficial effects in bacterial infections and sepsis. The use of these peptides has been shown to reduce inflammation by targeting various nodes in the sepsis inflammatory cascade, thereby reducing the extent of sepsis-related tissue and organ damage and achieving effective therapeutic effects. Therefore, the unique characteristics and potential of AMPs and AIPs in bacterial infections and sepsis make them a promising area of ​​research for developing new therapies. However, due to the problems with the bioavailability and tolerability of bioactive peptides, improving their efficiency and safety is currently a hot research focus. Summary of the Invention

[0005] This invention discovered that ATF2 and c-JUN protein directly interact in SAE mice, and the formation of the ATF2-c-JUN complex is related to the pathophysiological mechanism of SAE in mice. Based on this discovery, a cell-penetrating peptide that competitively inhibits this direct interaction was designed and synthesized. Experiments have shown that this cell-penetrating peptide can effectively improve brain damage in SAE mice. Based on the above research results, this invention provides the following protection: The present invention provides a polypeptide or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO.1.

[0006] Furthermore, the N-terminus of the polypeptide is acetylated and / or the C-terminus is amidated.

[0007] In this invention, the modified polypeptide sequences also fall within the scope of protection. The term "modification" refers to any chemical modification of an amino acid sequence, such as substitution, deletion, insertion, and / or addition of amino acids. The term "substitution" refers to replacing one or more amino acids with different amino acids. "Deletion" refers to the reduction of one or more amino acids in the amino acid sequence. "Insertion" or "addition" refers to a change in the amino acid sequence resulting in an increase of one or more amino acids compared to the naturally occurring molecule.

[0008] The present invention also provides a fusion cell-penetrating peptide, wherein the fusion cell-penetrating peptide comprises the aforementioned peptide segments, as well as the cell-penetrating peptide or its transduction domain.

[0009] Preferably, the cell-penetrating peptide or its transduction domain is connected to the C-terminus of the peptide segment.

[0010] The term “cell penetrating peptide (CPP)”, also known as “cell-penetrating peptide,” “protein translocation domain (PTD),” “Trojan horse peptides,” or “transduction peptide,” refers to a polypeptide that promotes the cellular uptake of various molecules (e.g., various macromolecules including proteins or nucleic acids). Such polypeptides are well known in the art and described, for example, Stewart KM, et al. Org Biomol Chem. 2008 Jul 7; 6(13):2242-55 and Chinese patent application CN101490081A (all of which are incorporated herein by reference); or can be obtained by methods known in the art, such as those described in detail in U.S. patent application US2008 / 0234183, all of which are incorporated herein by reference.

[0011] CPPs that can be used in this invention include, but are not limited to: cationic: Penetratin, HIV-TAT-47-57, HIV-1 Rev 34-50, FHV coat-35-49, oligoarginines (R9-R12), CCMV Gag-7-25, S413-PV, VP22, BP16, DPV3, DPV6, FAH shell protein, and protamine 1. 1) Human cJun, Engrailed-2, Islet-1, HoxA-13, TP10, etc.; Amphiphilic: Transportan, Transportan10, Pep-1, MPGα, MPGβ, CATY, Pepfect6, Pepfect14, Pepfect15, NickFect, Hel, sC18, pVEC, ARF (1-22), YTA2, PAR1 (Palmitoyl-SFLLRN), F2Pal10 (Palmitoyl-SFLLRN), BPrPp (1-30), hLF peptide (19-40), Buforin 2, Crotamine, Azurin p18, hCT peptide (18-32), S413-PVrev, etc.; Hydrophobic: Kaposi's sarcoma fibroblast growth factor, derived from Caiman Crocodylus contains the Ig light chain signal peptide, integrin β3 fragment, Grb2-SH2 domain, HIV-1gp41 (1-23), HBV translocation motif, sperm-egg fusion protein (89-111), human calcitonin (9-32), Pep-7, C105Y, K-FGF, etc.

[0012] Furthermore, the CPP used in this invention can also be selected from polypeptide sequences that have approximately 60, 70, 80, 90, 95, 99% or 100% sequence identity with any polypeptide sequence as described above, as long as the polypeptide sequence still retains its biological activity, i.e., promotes cellular uptake of the molecule.

[0013] In some embodiments, the cell-penetrating peptide is selected from penetratin, Tat or its derivative peptides, Rev(34-50), VP22, transport peptide, Pep-1, Pep-7, and any combination thereof.

[0014] In some embodiments, the cell-penetrating peptide is Tat or a derivative thereof.

[0015] In some embodiments, the amino acid sequence of the cell-penetrating peptide is shown in SEQ ID NO.3.

[0016] Preferably, the amino acid sequence of the fusion peptide is shown in SEQ ID NO.2.

[0017] The present invention also provides a modified peptide segment comprising the peptide segment described above or the fusion peptide segment described above.

[0018] Preferably, the modifications that may be made on the modified peptides include a series of predictable chemical modification methods such as detectable marker conjugation modification or imaging agent conjugation modification, cyclization modification, acetylation modification, PAS modification, PEG modification, fatty acid modification, albumin modification, albumin affinity peptide modification, NOA modification, radionuclide modification, tumor homing peptide conjugation, and nanocarrier conjugation.

[0019] Preferably, the detectable marker includes one or more of the following: fluorescent dyes, fluorescent molecules, chemiluminescent markers, dye molecules, phosphorescent molecules, biotin, radioactive isotopes, molecules that absorb in the UV spectrum, molecules that absorb in near-infrared radiation, or molecules that absorb in far-infrared radiation.

[0020] Preferably, the fluorescent dyes include, but are not limited to, rhodamine, p-methaminophenol, fluorescein, thiofluorescein, aminofluorescein, carboxyfluorescein, chlorofluorescein, methylfluorescein, sulfonylfluorescein, amino-p-methaminophenol, carboxy-p-methaminophenol, chloro-p-methaminophenol, methyl-p-methaminophenol, sulfonyl-p-methaminophenol, aminorhodamine, carboxyrhodamine, chlororhodamine, methylrhodamine, sulfonylrhodamine, as well as thiorhodamine, cyanine, indolecarbazocyanine, oxacarbazocyanine, thiacarbazocyanine, cyanine dyes (e.g., cyanine 2, cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine 7), oxadiazole derivatives, pyridyloxazole, and nitro... Benzodiazole, benzonitrobenzene, pyrene derivatives, waterfall blue, oxazine derivatives, Nile red, Nile blue, cresol purple, oxazine 170, acrylonitrile derivatives, proflavin, acridine orange, acridine yellow, arylmethyl benzoate derivatives, auramine, thioxanthate dyes, sulfonated thioxanthate dyes, Alexa Fluor (e.g., Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 700), crystal violet, malachite green, tetrapyrrole derivatives, porphyrin, phthalocyanine, bilirubin, Cy5.5, indocyanine green (ICG), DyLight 750 or IRdye 800.

[0021] Preferably, the fluorescent molecules include, but are not limited to, FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, TexasRed, or LCRED460.

[0022] Preferably, the chemiluminescent label includes, but is not limited to, peroxidase, alkaline phosphatase, luciferase, jellyfish luminescent protein, functionalized iron-porphyrin derivatives, lumina, luminol, isoluminol, acridine ester, sulfonamide, etc.

[0023] Preferably, the luciferase includes, but is not limited to, Gaussia luciferase, Renilla luciferase, Diplodocus luciferase, firefly luciferase, fungal luciferase, bacterial luciferase, and vargula luciferase.

[0024] In a specific embodiment of the present invention, the detectable marker is CY5.

[0025] The present invention also provides a biomaterial, the biomaterial comprising: 1) A nucleic acid molecule encoding the polypeptide or the fusion cell membrane-penetrating peptide described above in this invention; 2) A vector comprising the nucleic acid molecule described in 1); 3) A host cell containing the nucleic acid molecule described in 1) or the vector described in 2).

[0026] In this invention, the terms "polynucleotide," "nucleic acid molecule," and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, namely deoxyribonucleotides or ribonucleotides or their analogues. Polynucleotides may be further modified after polymerization, for example by conjugation with labeled components. The term also refers to double-stranded and single-stranded molecules.

[0027] In this invention, the term "vector" refers to a nonchromosomal nucleic acid containing a complete replicon, such that the vector can be replicated when placed within a permitted cell, for example, through a transformation process. A vector can replicate in one cell type (e.g., bacteria) but has limited ability to replicate in another cell type (e.g., mammalian cells). Vectors can be viral or non-viral. Exemplary non-viral vectors for delivering nucleic acids include naked DNA; and DNA, alone or in combination with a cationic polymer, in a cationic lipid complex; anionic and cationic liposomes; DNA-protein complexes; and particles containing DNA condensed with cationic polymers (such as heteropolymers of polylysine, fixed-length oligopeptides, and polyethyleneimine), and in some cases, also contained in liposomes.

[0028] In some embodiments, the vectors described in this invention include plasmids (expression plasmids, cloning vectors, small loops, microvectors, double microchromosomes), lentiviral vectors, adenoviral vectors, or retroviral vectors.

[0029] Preferably, the lentiviral vector includes a recombinant lentiviral vector from primates, namely a recombinant human immunodeficiency virus (HIV) vector or a recombinant simian immunodeficiency virus (SIV) vector.

[0030] Preferably, the lentiviral vector includes a non-primate recombinant lentiviral vector, namely recombinant equine infectious anemia virus (EIAV), recombinant feline immunodeficiency virus (FIV), or recombinant caprine arthritis-encephalitis virus (CAEV).

[0031] Preferably, the carrier further includes one or more control elements.

[0032] Preferably, the regulatory element comprises a promoter, an enhancer, a ribosome binding site for translation initiation, a terminator, a polyadenylate sequence, and a selection marker gene.

[0033] Preferably, the promoter is an inducible promoter, a constitutive promoter, a tissue-specific promoter, a suicide promoter, or any combination thereof.

[0034] In some embodiments, the host cells described in this invention include one or more of Escherichia coli, Streptomyces, Agrobacterium, yeast cells, plant cells, animal cells, or viruses.

[0035] The present invention also provides a detection product, which includes the aforementioned polypeptide, the aforementioned fusion cell membrane-penetrating peptide, and the aforementioned modified polypeptide.

[0036] The present invention also provides a medicament for treating sepsis or sepsis-associated encephalopathy, the medicament comprising the aforementioned polypeptide, the aforementioned fusion cell-penetrating peptide, the aforementioned modified polypeptide, the aforementioned nucleic acid molecule, a carrier, or a host cell.

[0037] Preferably, the drug further includes a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are described in detail in Remington's Pharmaceutical Sciences.

[0038] Preferably, the drug can be tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, oral tablets, etc.), pills, powders, granules, capsules (including soft capsules, microcapsules), lozenges, syrups, liquids, emulsions, suspensions, controlled-release formulations (e.g., instantaneous-release formulations, sustained-release formulations, sustained-release microcapsules), aerosols, films (e.g., orally disintegrating films, oral mucosa-adhesive films), injections (e.g., subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), intravenous infusions, transdermal absorption formulations, ointments, lotions, adhesive formulations, suppositories (e.g., rectal suppositories, vaginal suppositories), small pills, nasal preparations, pulmonary preparations (inhalers), eye drops, etc., oral or parenteral preparations (e.g., intravenous, intramuscular, subcutaneous, intra-organ, intranasal, intradermal, infusion, intracerebral, rectal, etc., administered near the lesion and directly to the lesion).

[0039] The present invention also provides the use of reagents that inhibit the formation of complexes between ATF2 protein and c-JUN in the preparation of medicaments for treating sepsis or sepsis-associated encephalopathy.

[0040] Preferably, the reagent is a substance that competitively binds to c-JUN with the ATF2 protein.

[0041] Preferably, the substance includes the aforementioned polypeptide, the aforementioned fusion cell-penetrating peptide, and the aforementioned modified polypeptide.

[0042] In the description of this invention, the term "amino acid" refers to the basic unit that constitutes a protein, giving the protein a specific molecular structure and morphology, and endowing its molecules with biochemical activity. For example, the "amino acid" used in this invention includes the following 20 natural amino acids: alanine (Ala or A), glycine (Gly or G), isoleucine (Ile or I), asparagine (Asn or N), arginine (Arg or R), lysine (Lys or K), cysteine ​​(Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), glutamine (Gln or Q), histidine (His or H), leucine (Leu or L), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), valine (Val or V), and tyrosine (Tyr or Y). Attached Figure Description

[0043] Figure 1The results of successfully establishing a mouse model of sepsis-associated encephalopathy are shown in the figure. (A) Survival rate of mice 72 h after CLP modeling (n=20); (B) Sepsis score (MSS) of mice (n=10); (C) Postoperative rectal temperature measurement of mice (n=6); (D) Detection of mRNA levels of inflammatory factors in the hippocampus of mice 24 h after surgery (n=6); (E) Immunofluorescence staining of early apoptosis factor Caspase-3 in the hippocampus region of mouse brain tissue sections 24 h after surgery (n=3). All data are mean ± standard deviation. Compared with the Sham group, *P < 0.05 and ****P < 0.0001. Figure 2 The results of the interaction between ATF2 and c-JUN protein are shown in the figure. (A) Coomassie brilliant blue staining of mouse hippocampal tissue co-IP protein sample after gel running; (B) Specific peptide sequence of c-JUN protein interacting with ATF2 detected by LC-MS / MS; (C) Co-IP experimental results showing that ATF2 and c-JUN interact. Figure 3 The results of molecular docking prediction and SPR verification are shown. (A) Schematic diagram of predicted molecular docking, which shows the molecular peptide and its docking schematic diagram, hydrogen bond docking schematic diagram, and cartoon illustration of the complex docking process from left to right; (B) Schematic diagram of plasma resonance SPR technology experiment; (C) Analysis of SPR experimental results and affinity constant of intermolecular binding; (DG) Crosslinking mass spectrometry analysis shows that the FLKNCE peptide of ATF2 is bound to multiple amino acid sequence peptides of c-JUN. Figure 4 The results of the self-designed and synthesized functional short peptide FLKNCE-Tat are shown in the figure. (A) is a schematic diagram of the design and synthesis of FLKNCE-Tat short peptide; (B) the micro-thermophoretic MST experiment shows that FLKNCE-Tat can effectively bind to c-JUN. Figure 5The results of the toxicity test of FLKNCE-Tat short peptide in mice are shown in the figure. (A) Record of body weight change of mice in each group after 7 consecutive days of administration (n=6); (BI) Blood biochemical indicators of liver and kidney function in mice (n=6); ALT: Alanine Aminotransferase; AST: Aspartate Aminotransferase; ALP: Alkaline Phosphatase; TBIL: Total Bilirubin; TP: Total Protein; ALB: Albumin; CRE: Creatinine; BUN: Blood Urea Nitrogen; (J) Comparison of histopathological H&E staining of heart, liver, lung and kidney tissues of normal mice and the FLKNCE-Tat treatment group treated with 50 mg / kg dose for 7 consecutive days of intravenous injection (n=6). Figure 6 The results show that FLKNCE-Tat alleviated CLP-induced brain injury in CLP-induced septic mice. (A) Survival rate of the five groups of mice at 72 h (n=20); (B) Comparison of sepsis (MSS) scores of the five groups of mice at 24 h post-surgery (n=10); (CE) Detection of serum levels of pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 in the five groups of mice at 24 h post-surgery (n=6); (F) and (G) H&E staining and statistical analysis of hippocampal tissue sections (CA1 region) of the five groups of mice at 24 h post-surgery (n=6); Scale bar: 200µm; 50µm. (H) and (I) Nissl staining and statistical analysis of hippocampal tissue sections (CA1 region) of the five groups of mice at 24 h post-surgery (n=6); Scale bar: 200µm; 50µm. All data are expressed as mean ± standard deviation; *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; Figure 7 The results show that FLKNCE-Tat can effectively penetrate the blood-brain barrier. (A) Mouse in vivo imaging experiment after injection of Cy5-labeled FLKNCE-Tat (n=3); (B) Mean plasma concentration-time curve after intravenous injection of 10 mg / kg FLKNCE-Tat (n=6). Figure 8The figure shows the results of FLKNCE-Tat effectively reducing neuroinflammatory response and improving cognitive dysfunction in SAE mice. (A) Rectal body temperature of mice in the three groups 24 hours post-surgery after different treatments (n=6); (B) ELISA assay of inflammatory factors (IL-1β, IL-6, and TNF-α) in the hippocampus of the three groups of mice 24 hours post-surgery. Expression levels of α) (n=6); Detection of S100a8 and S100a9 levels, potential early diagnostic markers of sepsis, in the serum of mice in groups (C) and (D) 24 h post-surgery (n=6); Monitoring of in vivo electrophysiology of mice in groups (EH) 24 h post-surgery, recording and analyzing the firing frequency of neurons in the CA1 region of the hippocampus (n=6); Statistical analysis of Y-maze test and time for mice to explore new arms in groups (I) and (L) 3 days post-surgery (n=6); Statistical analysis of new object recognition test and time for mice to explore new objects in groups (J) and (M) 3 days post-surgery (n=6); Statistical analysis of open field test and total distance traveled in groups (K) and (N) 3 days post-surgery (n=6); All data are mean ± standard deviation. *P <0.05, **P <0.01, ***P <0.001 and ****P <0.0001; Figure 9 The results of FLKNCE-Tat in alleviating oxidative stress damage and regulating the inflammatory microenvironment of the brain in SAE mice are shown in the following figures: (A) and (B) Superoxide DHE fluorescence staining and statistical analysis of brain tissue sections from different treatment groups 24 h after treatment (n=6); scale bar: 50µm; (CE) Expression levels of oxidative stress damage-related indicators (MDA, SOD, and GSH) in brain tissue homogenates from different treatment groups 24 h after treatment (n=6); (F) and (I) Immunofluorescence staining and fluorescence intensity statistical analysis of CD31 (red) in microvascular endothelial cells of the hippocampus (CA1 region) of mice (n=6); scale bar: 20µm; 50µm; (G) and (J) Expression levels and fluorescence intensity statistical analysis of GFAP (red) in astrocyte activation indicators of the hippocampus (CA1 region) of mice 24 h after surgery (n=6); scale bar: 50µm. Statistical analysis of expression level and fluorescence intensity of Iba-1 (green), a microglia activation marker, in hippocampal tissue sections (CA1 region) of (H) and (K) mice 24 h post-surgery (n=6); scale bar: 50µm; all data are mean ± standard deviation. *P <0.05, **P <0.01, ***P <0.001 and ****P <0.0001. Detailed Implementation

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0045] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0046] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0047] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0048] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0049] Unless otherwise specified, the "%" in this invention refers to a percentage by mass.

[0050] Example 1: Establishment of a mouse model of sepsis-associated encephalopathy I. Experimental Materials and Methods 1. Laboratory animals All research protocols described in this article were approved by the Laboratory Animal Ethics Committee of Tianjin Medical University General Hospital, and mice were handled in strict accordance with ethical requirements. The experimental animals used in this study were healthy adult male C57BL / 6J mice (6-8 weeks old, 20-25 g), carefully selected from the Laboratory Animal Center of the Academy of Military Medical Sciences in Beijing. The mice were housed in an environment meeting the following conditions: room temperature of 20-25°C, relative humidity of 55-65%; light-dark cycle twice daily; allowing the mice to acclimatize to their new environment for one week; and free access to food and water.

[0051] 2. Establishment of a mouse model of sepsis-associated encephalopathy A mouse model of sepsis-associated encephalopathy was established using the cecum ligation and puncture (CLP) method. After mice had fully acclimatized to the laboratory environment for one week, they were anesthetized with isoflurane and placed in a prone position. The skin was disinfected, and a 1 cm incision was made in the abdomen to expose the cecum, which was then ligated at 35%. The cecum was then punctured twice with a 21-gauge needle, and approximately 0.3 mL of cecum contents were squeezed out using sterile forceps. The cecum and squeezed contents were then returned to the abdominal cavity, and the abdominal muscles and tissues were sutured using specialized surgical suture needles and sutures. The sham-operated group underwent the same surgical procedure, but without cecum ligation and puncture; only exploratory laparotomy was performed. After model establishment, the experimental animals were subcutaneously injected with 1 mL of room-temperature saline, and lidocaine cream (Cat# H20063466, Ziguang, Beijing) was applied to the suture wound to relieve postoperative pain. The temperature of the postoperative mouse housing environment was controlled at 20-25°C, and a warming blanket was used to keep the mice warm to prevent hypothermia.

[0052] 3. Experimental Design Experimental mice were randomly divided into a sham-operated group (Sham) and a sepsis-associated encephalopathy (CLP) group. Animals in the CLP group underwent cecal ligation and perforation, while mice in the Sham group only underwent exploratory laparotomy. Twenty mice were randomly selected from each group, and survival rates were recorded and analyzed at 12h, 24h, 48h, and 72h post-surgery. Twenty-four hours post-surgery, ten mice from each group were randomly selected for sepsis scoring (MSS score), and six mice from each group were selected to measure rectal temperature before surgery, 12h post-surgery, and 24h post-surgery. Mice anesthetized with an overdose of isoflurane underwent cervical dislocation and were euthanized 24 hours after CLP or sham surgery. Brain tissue was collected, hippocampal homogenate was obtained, and the levels of cellular inflammatory factors, including interleukin (IL)-1β, IL-6, and TNF-α, were measured by qPCR.

[0053] 4. Mouse survival rate and mouse MSS score The survival rates of the two groups of mice were recorded and analyzed at different time points (12h, 24h, 48h, and 72h) within 72 hours after surgery. Under the same conditions as before sepsis modeling, the two groups of mice were housed in four different cages, and the survival status of the mice in both groups was observed and recorded (if a mouse died, its carcass was removed and disposed of). The mice were continuously observed for 72 hours, and their survival status was analyzed and statistically analyzed (n=20).

[0054] MSS score for septic mice: The Murine sepsis score (MSS) was used to assess the morbidity of mice after CLP modeling. Mice were grouped based on their physical characteristics, level of consciousness, activity level, stimulus response, ocular manifestations, respiratory rate, and respiratory quality (each indicator scored from 0 to 4 points) at 24 hours post-modeling. Mice that died within 6 hours of CLP modeling were excluded from subsequent experiments.

[0055] 5. Real-time quantitative PCR (1) Extraction of RNA from hippocampal tissue Weigh 10-20 mg of hippocampal tissue and add 500 µl of Buffer RL1. Homogenize the tissue using an electric homogenizer. Transfer the homogenized tissue solution to a DNA purification column, reserving the supernatant in the collection tube. Then, take 500 µl of the reserved supernatant and add 1.6 times the volume of Buffer RL2, gently mixing. Transfer the mixture to an RNA purification column and centrifuge at 12000×g for 1 min, discarding the waste liquid. Next, add 500 µl of Buffer RW1 to the purification column and centrifuge at 12000×g for 1 min, discarding the waste liquid again. Then, add 700 µl of Buffer RW2 to the purification column and repeat the centrifugation and waste liquid discarding steps. Place the purification column back into the collection tube and centrifuge at 12000×g for 2 minutes to remove any residual Buffer RW2 from the column. Finally, the purification column was transferred to a new centrifuge tube, and 50 µl of RNase-free ddH2O preheated at 65 °C was added to the purification column. After incubation at room temperature for 2 min, the column was centrifuged for 1 min, and the RNA solution was finally collected.

[0056] (2) Reverse transcription of hippocampal RNA Prepare a 10 µl mixture according to the instructions, containing 2 µl of 5×Evo M-MLVRT Master Mix, and then add 8 µl of a mixture of RNA and RNase-Free water. Place it in a PCR reverse transcription apparatus and set the reaction conditions as follows: 37°C for 15 min, then 85°C for 5 sec, and finally lower the temperature to 4°C to stop the reaction, obtaining cDNA for subsequent experiments. Note the following points: ① When preparing the RT reaction solution, be sure to mix it gently.

[0057] ② The amount of RNA can be added as needed. In a 10 μl reverse transcription system, when using the SYBR Green qPCR method, a maximum of 500 ng Total RNA can be used; when using the probe method, a maximum of 1 μg Total RNA can be used.

[0058] ③ If the reaction product is to be used immediately in subsequent qPCR reactions, it can be temporarily stored at 4℃ or on ice; for short-term storage, it is recommended to store at -20℃; for long-term storage, it is recommended to store at -80℃.

[0059] (3) qPCR reaction of hippocampal tissue The qPCR reaction was performed in a 10 µl volume containing 0.5 µl of the first primer, 0.5 µl of the second primer, 4 µl of 2×SYBR Green, and 5 µl of cDNA. The PCR instrument was set as follows: first, 95°C for 30 s, one cycle; second, 95°C for 5 s; and finally, 60°C for 30 s, for a total of 40 cycles. The primer sequences used are shown in Table 1.

[0060] Table 1 Primer Sequences

[0061] 6. Experimental Data Analysis All data were analyzed and visualized using GraphPad Prism (Version 8.0). Normality of the data was verified using the Shapiro-Wilk test. For normally distributed data, independent samples t-tests were used for comparisons between groups, and one-way ANOVA was used for comparisons among multiple groups. Results are expressed as mean ± standard deviation (Mean ± SD). For non-normally distributed data, the Mann-Whitney U test was used for comparisons between groups. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (i.e., P < 0.05) were considered statistically significant, and ns indicated no statistically significant difference.

[0062] II. Experimental Results A mouse model of SAE was successfully established using the CLP method. A mouse model of sepsis-associated encephalopathy (SAE) was established using the CLP method, the gold standard for animal models of sepsis. Compared with the sham-operated group, the survival rate of mice in the CLP group was significantly lower within 73 hours. Figure 1 A, P < 0.05), and CLP group mice showed typical deterioration of sepsis physiological indicators, including an increase in sepsis score (MSS score). Figure 1 B, P < 0.05), body temperature decreased 24 hours postoperatively ( Figure 1 C, P < 0.05. Meanwhile, the release of inflammatory factors in the hippocampus of CLP group mice was significantly higher than that in Sham group (C, P < 0.05). Figure 1 D, P < 0.05. Furthermore, fluorescence staining of early apoptosis factor Caspase-3 in mouse brain tissue sections showed that, compared with the Sham group, the CLP group mice exhibited a large number of apoptotic events in hippocampal neurons (D, P < 0.05). Figure 1 E, P < 0.05). The above experimental results show that a mouse model of sepsis-associated encephalopathy has been successfully established.

[0063] Example 2: Studying key molecules in neuronal damage caused by sepsis-associated encephalopathy 1. Constructing a mouse model of sepsis-associated encephalopathy Same as Example 1.

[0064] 2. Experimental Design Experimental mice were randomly divided into a sham-operated group (Sham) and a sepsis-associated encephalopathy (CLP) group. Animals in the CLP group underwent cecal ligation and perforation, while mice in the Sham group only underwent exploratory laparotomy. Twenty-four hours post-operation, six mice from each group were randomly selected and euthanized by cervical dislocation after inhaling an overdose of isoflurane. Brain tissue was collected, and hippocampal homogenate was obtained. Total protein was extracted from the samples, and immunoprecipitation-Coomassie brilliant blue staining was performed. High-performance liquid chromatography-mass spectrometry (LC-MS / MS) was used to screen for interacting proteins that interact with ATF2. Western blotting was used to detect the expression levels of apoptosis-related markers (Bax and Bcl-2) and the screened interacting proteins in the mouse hippocampus. Interferometric analysis (IF) was used to detect the expression of the screened interacting proteins in the mouse brain tissue.

[0065] 3. Western blot analysis Perform according to standard procedures.

[0066] 4. Co-immunoprecipitation (co-IP) assay to detect protein-protein interactions. This experiment was conducted in accordance with the experimental protocol recommended by the immunoprecipitation magnetic beads and kit (BeaverBeads™ Protein A (or A / G) Immunoprecipitation Kit) provided by Suzhou Beaver Biomedical Co., Ltd.

[0067] Clean the glass grinder and allow it to air dry. Weigh the mouse hippocampus tissue and place it in the grinder on ice. Add 100 μl of non-denaturing tissue lysis buffer RIPA to 1 μl of PMSF to prepare a 1 mM PMSF mixture. Add 1000 μl of the prepared mixture to each 0.1 g sample and grind thoroughly. After allowing it to stand completely, transfer the sample to a 1.5 ml centrifuge tube using a pipette. Centrifuge at 4°C for 14000 g × 10 min, and collect the supernatant as the total protein from the hippocampus tissue. Take an appropriate amount of sample for immunoprecipitation. Add 1 / 4 of the total sample volume of loading buffer (4×) to the remaining sample and mix thoroughly. Boil in a 95°C metal bath for 10 min, then allow it to cool naturally at room temperature. After cooling to room temperature, store it in a -20°C freezer to verify the input portion of the immunoblotting SDS-PAGE electrophoresis experiment.

[0068] 5. High-performance liquid chromatography-mass spectrometry (LC-MS / MS) Liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis was performed using a Vanquish Neo UHPLC system coupled with an OrbitrapFusion Lumos mass spectrometer (Thermo Fisher Scientific). Chromatographic separation was performed using a reversed-phase capillary column (50 μm inner diameter × 170 mm, Reprosil-Pur 120 C18-AQ, 1.9 μm; constant temperature 60 °C). The mobile phase consisted of (A) an aqueous solution containing 0.1% formic acid and (B) 80% acetonitrile containing 0.1% formic acid. A gradient elution program was performed at a flow rate of 0.6 μL / min: 0–2 min B phase 4–8%, 2–35 min 8–28%, 35–55 min 28–40%, 55–56 min 40–95%, 56–66 min 95% B phase. Mass spectrometry data acquisition was performed in data-dependent mode, with the entire scan range (m / z 300-1800) recorded in profile mode at a resolution of 120,000, automatic gain control (AGC) target set to "standard," and a maximum injection time of 20 ms. For MS / MS analysis of the most potent precursor, a resolution of 15,000, AGC target set to "standard," and a maximum injection time of 22 ms were used. Fragmentation was performed using a step-normalized collision energy (NCE) of 30. Data analysis was performed using pLink software (version 2.3.11).

[0069] II. Experimental Results The ATF2-c-JUN complex was clearly identified in the hippocampus of SAE mice. Immunoprecipitation was performed using two groups of fresh mouse hippocampal tissue homogenates, followed by Western blotting and Coomassie brilliant blue staining to confirm the success of the immunoprecipitation experiment. Figure 2 A). Further LC-MS / MS mass spectrometry experiments on the protein samples obtained by co-immunoprecipitation revealed an interaction between c-JUN and ATF2 protein, with the interacting peptide sequence being NSDLLTSPDVGLLK ( Figure 2 B). Further co-IP experiments indicated that c-JUN protein could be successfully detected in the protein sample precipitated by the anti-ATF2 antibody. Figure 2 C). This indicates that the formation of the ATF2-c-JUN complex is closely related to the pathogenesis of SAE.

[0070] Example 3: Study on the therapeutic effect of the designed and synthesized peptide on sepsis-induced brain injury. I. Experimental Materials and Methods 1. Constructing a mouse model of sepsis-associated encephalopathy Same as Example 1.

[0071] 2. Experimental Design Experimental mice were randomly divided into 5 groups: sham surgery group, sepsis-associated encephalopathy (CLP) group, and CLP + different doses of peptide treatment groups (CLP + 5 mg / kg group, CLP + 10 mg / kg group, CLP + 20 mg / kg group). Mice in the CLP + different doses of peptide treatment groups received a short peptide injection via tail vein at 2 h and 6 h post-modeling, while the other groups received an equal volume of saline. Animals in the CLP group underwent cecal ligation and perforation, while mice in the Sham group only underwent exploratory laparotomy. Twenty mice were randomly selected from each group, and the survival rate of each group was recorded and analyzed at 12 h, 24 h, 48 h, and 72 h post-surgery (method as in Example 1). Twenty mice were randomly selected from each group 24 hours post-surgery for sepsis scoring (MSS score) (method as in Example 1). Mice were euthanized by cervical dislocation 24 hours after CLP or sham surgery following anesthesia with an inhaled overdose of isoflurane. Serum and brain tissue samples were collected, and serum levels of cytotoxic cytokines IL-1β, IL-6, and TNF-α were measured using ELISA. Brain tissue was collected from six mice in each group for sectioning, H&E staining, and Nissl staining to observe the degree of neuronal damage. The polypeptide sequence is FLKNCEYGRKKRRQRRR (SEQ ID NO.2).

[0072] Mouse drug toxicity assay: The toxicological safety characteristics of the self-designed and synthesized short peptide were assessed in experimental mice. The experiment consisted of a normal control group and a drug-treated group. Mice in the drug-treated group received daily tail vein injections of the self-designed and synthesized short peptide at doses of 20 mg / kg or 50 mg / kg for 7 consecutive days, while mice in the normal control group received an equal volume of physiological saline. During the drug administration period, changes in body weight and the presence of toxic reactions or abnormal behavioral signs were observed in each group. Seven days after continuous drug administration, the mice were euthanized, and blood biochemical tests were performed on the heart, liver, and kidney function. Simultaneously, H&E staining was performed on the heart, liver, lung, and kidney tissues of both the normal control group mice and the mice that received the 50 mg / kg short peptide for 7 consecutive days to observe for histopathological damage in each organ and tissue.

[0073] 3. Western blot Perform according to standard procedures.

[0074] 4. Brain tissue slices Mice were taken 24 hours post-surgery, and after complete anesthesia (using isoflurane), they were euthanized by cervical dislocation. The mice were fixed on the operating table, the thoracic cavity was opened, and the heart was exposed. The heart was perfused with pre-cooled PBS, followed by perfusion with 4% paraformaldehyde until the mice's generalized muscle spasms disappeared, their limbs and trunk became rigid, and their posture was fixed. Immediately after this, the mice's brain tissue was harvested by decapitation. The brain tissue was cleaned with PBS and then soaked in paraformaldehyde solution for 24 hours for fixation. The brain tissue was then removed and successively soaked in 20% and 30% sucrose solutions for dehydration until it sank to the bottom of the solution bottle. Excess water was then absorbed, and the tissue was embedded in OCT for frozen sectioning.

[0075] 5. Hematoxylin-eosin (H&E) staining Perform according to standard procedures.

[0076] 6. Nissl staining Perform according to standard procedures.

[0077] 7. Detection of inflammatory factors using enzyme-linked immunosorbent assay (ELISA). Five groups of mice were included: the Sham group, the CLP group, and the CLP+ cell-penetrating peptide treatment group at different doses (5 mg / kg, 10 mg / kg, 20 mg / kg). After different treatments, the mice were euthanized 24 hours later, and serum samples were collected from the five groups of mice. The levels of systemic inflammatory factors (TNF-α, IL-1β, IL-6) in the serum were detected according to the instructions of the corresponding ELISA kits.

[0078] 8. Establish a molecular docking prediction model (1) Molecular docking preparation: First, the three-dimensional structural information of human ATF2 and c-JUN proteins, which have been experimentally verified, was searched and obtained from the UniProt (Universal Protein Resource) database to ensure the accuracy and reliability of the docking experiment. At the same time, the specific peptide sequence of c-JUN protein screened by co-IP combined with mass spectrometry was referenced.

[0079] (2) Molecular docking prediction: The peptide sequences obtained above were used to construct a model using I-TASSER, and protein-protein molecular docking was performed using HDOCKServer to obtain the complex structure.

[0080] (3) Molecular docking interaction mode analysis and visualization: The interaction mode analysis of the obtained complex structure was performed using LIGPlus software to evaluate the stability of protein-protein interactions. PyMOL was used for molecular visualization analysis to further analyze the spatial conformation of key amino acid residues, interaction interfaces, and molecular binding modes, so as to provide theoretical support for subsequent functional studies and experimental verification.

[0081] 9. Surface Plasmon Resonance (SPR) SPR (Surface Plasma Resonance) affinity detection is a powerful technique for studying interactions between biomolecules. As a real-time, label-free detection method, SPR plays a crucial role in biomolecular interaction research. SPR is a physical optical phenomenon where a beam of polarized light incident on the surface of a metal thin film within a certain angular range excites surface plasmon resonance (SPR). When biomolecules bind to the metal film surface, the resonance conditions of the SPR change, and by detecting this change, interactions between biomolecules can be monitored in real time. The specific operational procedures follow standard procedures.

[0082] 10. Protein cross-linking identification and analysis based on LC-MS / MS Performed using conventional techniques.

[0083] 11. Microfluidic Streaming Flow Technology (MST) Microfluidic thermal manipulation (MST) is a thermal effect technique based on microfluidic principles that uses thermal energy to manipulate and analyze tiny fluids. The core of MST is the use of temperature gradients to generate thermal surges within microchannels, thereby achieving fluid manipulation. A typical MST device includes a heating element and a cooling element, located on opposite sides of a microfluidic channel. When the heating element is activated, it heats one side of the channel, causing the fluid temperature to rise, while the cooling element on the other side maintains a lower temperature. This temperature difference induces thermal surges in the fluid, creating periodic flow patterns within the channel.

[0084] 12. Statistical Analysis Performed according to Example 2.

[0085] II. Experimental Results 1. Molecular docking prediction and verification Molecular docking prediction model results show that the amino acid sequence 73-82 of the ATF2 protein can directly bind to the peptide segment of the c-JUN protein sequence 33-107, and there is a strong binding, with a binding energy of -144.88 kcal / mol. This complex is stabilized through multiple interactions between key residues at the active site. Figure 3 A). Validation experiments following the predictive model: SPR experiments confirmed that purified recombinant human protein ATF2 directly binds to purified recombinant protein c-JUN, with an affinity constant of 1.68 nM ( Figure 3 B and 3C). Crosslinking mass spectrometry analysis identified the FLKNCE (SEQ ID NO.1) peptide of ATF2 as being bound to a peptide segment containing multiple amino acid sequences of c-JUN. Figure 3 (D-3G). This demonstrates that the FLKNCE peptide sequence of ATF2 is a key sequence peptide for the formation of the ATF2-c-JUN complex.

[0086] 2. Independent design and synthesis of functional short peptides Based on the above experimental results, our research group independently designed and synthesized a functional short peptide FLKNCEYGRKKRRQRRR (FLKNCE-Tat, SEQ ID NO.2). Its design aims to competitively bind c-JUN and disrupt the formation of the ATF2-c-JUN complex. First, the ATF2 cleavage peptide FLKNCE was selected. To enhance cellular uptake efficiency, a cell-penetrating peptide sequence with increased cell membrane penetration function, the human immunodeficiency virus type 1 (HIV-1) transduction domain Tat, was linked to its C-terminus. Its amino acid sequence is YGRKKRRQRRR (SEQ ID NO.3). Figure 4A). MST experimental results showed that the self-designed and synthesized short peptide FLKNCE-Tat effectively bound to the purified recombinant protein c-JUN, with a measured affinity constant of 9.11 μM (A). Figure 4 B).

[0087] 3. FLKNCE-Tat short peptide was well tolerated in mice with no toxic reactions. First, we evaluated the toxicological safety profile of the self-designed and synthesized short peptide in experimental mice. Mice were administered FLKNCE-Tat short peptide via tail vein injection at doses of 20 mg / kg or 50 mg / kg daily for 7 consecutive days. During the administration period, no toxic reactions or signs of abnormal behavior were observed in mice of any group, and there was no significant difference in body weight. Figure 5 A, P>0.05). Blood biochemical analysis of liver and kidney function in mice showed no significant differences between the different doses of short peptide treatment groups and the control group (A, P>0.05). Figure 5 B-5I, P>0.05). Furthermore, histopathological H&E staining of the heart, liver, lung, and kidney tissues showed no significant pathological changes compared to the control group treated with FLKNCE-Tat at a dose of 50 mg / kg for 7 consecutive days. Figure 5 J, P>0.05). Therefore, these results indicate that the FLKNCE-Tat short peptide was well tolerated in mice, and no toxic reactions were detected.

[0088] 4. FLKNCE-Tat alleviated CLP-induced brain injury in septic mice. To evaluate the therapeutic potential of FLKNCE-Ta in mice with septic brain injury, we administered different doses (5, 10, and 20 mg / kg) of FLKNCE-Tat short peptide at 2 and 6 hours post-CLP surgery. FLKNCE-Tat treatment improved CLP-induced septic brain injury in mice in a dose-dependent manner. Compared with CLP mice, the survival rate of mice treated with CLP + 10 mg / kg - FLKNCE-Tat (CLP + 10 mg) and CLP + 20 mg / kg - FLKNCE-Tat (CLP + 20 mg) was significantly increased. Figure 6 A, P<0.05 (CLP group: 30%; CLP+10 mg group: 70%; CLP+20 mg group: 75%), while the survival rate of mice treated with CLP+5 mg / kg-FLKNCE-Tat (CLP+5 mg) did not change significantly. Figure 6 A, P>0.05. Compared with the CLP group, mice treated with FLKNCE-Tat peptide also showed a dose-dependent decrease in MSS score at 24 h post-surgery. Figure 6B, P<0.05. Furthermore, compared to the CLP group, the serum levels of pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 were significantly reduced in the FLKNCE-Tat treatment group (B < 0.05). Figure 6 C-6E, P<0.05. H&E staining results showed that, compared with the CLP group, the FLKNCE-Tat short peptide treatment groups (CLP+5mg, CLP+10mg and CLP+20mg groups) showed a significant reduction in damaged pyramidal neurons in the hippocampus of mice, and a significant increase in morphologically regular neurons. Figure 6 F and 6G, P<0.05). Nissl staining results also showed that, compared with the CLP group, mice in the CLP+5mg, CLP+10mg and CLP+20mg groups had increased Nissl bodies and decreased dissolution (F and 6G, P<0.05). Figure 6 H and 6I (P<0.05). This indicates that the self-designed and synthesized short peptide FLKNCE-Tat can effectively alleviate CLP-induced brain damage in CLP-induced septic mice.

[0089] Example 4: Cell-penetrating peptide FLKNCE-Tat synergistically improves brain injury in SAE mice through multiple mechanisms. I. Experimental Materials and Methods 1. Constructing a mouse model of sepsis-associated encephalopathy Same as Example 1.

[0090] 2. Experimental Design To further evaluate the potential of the short peptide FLKNCE-Tat in treating sepsis-associated encephalopathy, we investigated its pharmacokinetic properties and blood-brain barrier (BBB) ​​penetration ability. Two hours post-surgery, mice in the Sham and CLP groups were intravenously injected with a 10 mg / kg dose of the Cy5-tagged FLKNCE-Tat short peptide via tail vein. In vivo imaging was performed to observe the fluorescence signal detected in brain tissue and to determine whether the drug crossed the BBB. Furthermore, the plasma concentration of FLKNCE-Tat in normal mice was quantitatively analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS), and mean plasma concentration-time curves were plotted.

[0091] Experimental mice were randomly divided into three groups: a sham-operated group (Sham), a sepsis-associated encephalopathy (CLP) group, and a CLP+FLKNCE-Tat (CLP+FT) treatment group. Animals in the CLP group underwent cecal ligation and perforation, while mice in the Sham group only underwent exploratory laparotomy. Mice in the CLP+FT group were treated with intravenous injection of 10 mg / kg FLKNCE-Tat short peptide via the tail vein 2 and 6 hours after CLP modeling, while the other two groups received an equal volume of saline. Six mice from each group were randomly selected to measure rectal temperature before surgery, 12 hours post-surgery, and 24 hours post-surgery. Mice anesthetized with an overdose of isoflurane after CLP or sham surgery were euthanized by cervical dislocation 24 hours post-surgery. Brain tissue was collected, and hippocampal tissue homogenate was obtained. The levels of pro-inflammatory cytokines, namely IL-1β, IL-6, and TNF-α, as well as the levels of S100a8 and S100a9, potential early diagnostic biomarkers for sepsis neuroinflammation, were measured using ELISA. Four mice were randomly selected from each group 24 hours post-surgery for in vivo electrophysiological experiments, and the firing activity of local neurons in the mouse brain was observed and recorded under resting conditions. Thirty mice from each group were randomly selected for behavioral experiments 3 days post-surgery, including the Y-maze, novel object recognition test, and open field test. Brain tissue was collected from each group 24 hours post-surgery, homogenized, and oxidative stress-related indicators (MDA, SOD, GSH) were detected. Brain tissue sections were also collected for immunofluorescence staining.

[0092] 3. Small animal live imaging technology Two hours after modeling, Cy5-labeled FLKNCE-Tat probes were injected via the tail vein, while the control group received an equal dose of saline. Imaging was performed at 0.5 and 4 hours post-administration, focusing on signal distribution in the central nervous system (head). Mice were anesthetized with isoflurane (inhaled concentration 1.5-2.0%, oxygen flow rate 500 ml / min) to maintain a body temperature of 36.5-37.5℃ (using a constant-temperature heating pad). The mouse head was fixed in the center of the imaging field to prevent signal deviation due to movement. In vivo imaging of the mice was performed using the IVIS SPECTRUM small animal in vivo imaging system, following the procedures outlined in the instrument's user manual. Three images were acquired for each mouse under the same parameters, and the average value was used. Simultaneously, images of the blank control group (untreated mice) were acquired for background subtraction.

[0093] 4. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS / MS) Technology Performed using conventional techniques.

[0094] 5. Enzyme-linked immunosorbent assay (ELISA) 24 hours after surgery, mice in each group were euthanized and brain tissue samples were collected. After homogenization, the tissue supernatant was collected, and the expression levels of TNF-α, IL-1β, IL-6, S100a8, and S100a9 in the tissue samples were detected according to the instructions of the corresponding ELISA kits.

[0095] 6. In vivo electrophysiology of mice Mice were anesthetized with isoflurane and placed within a stereotaxic frame. After creating a small cranial window, tungsten microelectrodes (16-channel microfilaments) were implanted into the CA1 region of the hippocampus. Following implantation, the mice were housed alone for two weeks. After the experimental procedure was completed, local neuronal firing activity was recorded in a resting state. The signals were amplified using a differential amplifier, and data were collected using a digital acquisition system.

[0096] 7. Behavioral experiments 7.1 Y Maze To assess the short-term spatial recognition and memory ability of mice. The maze was placed in a quiet, temperature-controlled, and uniformly lit room. Animals were allowed 72 hours of acclimatization before the experiment. The Y-maze consisted of three interconnected arms: the initial arm, the novel arm, and the other arms. The experiment consisted of two phases: a training phase and a testing phase. In the training phase, mice were placed on the initial arm and given 10 minutes to explore the familiar arm independently. In the testing phase, mice were placed on the initial arm and allowed to choose an arm they had not previously explored for 5 minutes. Spatial memory ability was assessed by counting the number of times mice entered the novel arm and the time spent exploring the novel arm during the testing phase.

[0097] 7.2 Novel Object Recognition (NOR) Test Novel Object Recognition Test: Cognitive memory assessment employed the Novel Object Recognition (NOR) paradigm, conducted in an open area (50×50×50 cm). After a 10-minute acclimatization period 24 hours prior to the test, mice were exposed to two identical familiar objects for 10 minutes. Two hours later, one object was replaced with a novel object, and exploration behavior was monitored for 5 minutes using SuperMaze software (Shanghai Xinran). The discrimination index was calculated as the percentage of time spent exploring the novel object relative to the total time spent exploring both objects.

[0098] 7.3 Open field test (OFT) Motor activity, exploratory behavior, and anxiety-like behavior were assessed in a 60×60×50 cm open field experimental area (divided into 9 equally divided grids). Mice completed a 5-minute adaptation period in the lower left quadrant, and their behavior was automatically tracked. The frequency of crossing horizontal lines (reflecting motor activity) and the frequency of entering the central area (reflecting low anxiety levels) were used as the main observation indicators.

[0099] 8. Statistical Analysis Same as Example 1.

[0100] II. Experimental Results 1. FLKNCE-Tat can effectively penetrate the blood-brain barrier. To evaluate the blood-brain barrier penetration ability of FLKNCE-Tat, we performed in vivo imaging experiments in mice after injecting Cy5-labeled FLKNCE-Tat into them. Figure 7 As shown in Figure A, after intravenous injection of 10 mg / kg Cy5-labeled FLKNCE-Tat into sham-operated mice, a significant fluorescent signal was detected in their brain tissue, indicating that the peptide successfully crossed the blood-brain barrier. Notably, compared to the sham group, the CLP group mice showed stronger fluorescence intensity in their brain tissue 2 hours after administration. A persistent fluorescent signal was still observed at 6 hours. Furthermore, we used liquid chromatography-tandem mass spectrometry (LC-MS / MS) to quantify the plasma concentration of FLKNCE-Tat. Figure 7 B shows the mean plasma concentration-time curve after intravenous injection of 10 mg / kg FLKNCE-Tat. These results combined suggest that FLKNCE-Tat can effectively cross the blood-brain barrier and may play a functional role in brain tissue.

[0101] 2. FLKNCE-Tat effectively reduces neuroinflammation and cognitive impairment in SAE mice. To explore the therapeutic potential of FLKNCE-Tat in severe acute brain injury (SAE), we evaluated neuroinflammation and cognitive function in a CLP-induced mouse model. Compared with the sham-operated group, CLP mice exhibited significant neuroinflammation, brain tissue damage, and cognitive deficits. Figure 8 AK, P<0.05. Compared with the CLP group, the FLKNCE-Tat treatment group (CLP+FT) showed a significantly reduced decrease in rectal temperature 24 hours after surgery. Figure 8 A, P<0.05), the levels of key inflammatory cytokines (IL-1β, IL-6, and TNF-α) in brain tissue were significantly reduced ( Figure 8 B, P<0.05. S100a8 and S100a9, potential early diagnostic markers for sepsis, were significantly elevated in the blood of CLP mice compared to the Sham group (B, P<0.05). Figure 8 CD, P<0.05; compared with the CLP group, the levels of S100a8 and S100a9 in the blood of mice in the CLP+FT group were significantly reduced (CD, P<0.05); Figure 8CD, P<0.05, indicating that FLKNCE-Tat treatment can reduce the severity of systemic inflammation. Furthermore, in vivo electrophysiological recordings showed that, compared with Sham, CLP-induced septic mice exhibited a significantly reduced neuronal firing frequency in the CA1 region of the hippocampus (CLP). Figure 8 EH, P<0.05. However, FLKNCE-Tat treatment reversed this downward trend; compared with the CLP group, the CLP+FT group showed a significantly reduced decrease in the firing frequency of neurons in the CA1 region of the hippocampus. Figure 8 EH, P<0.05). The results of the cognitive behavioral experiments on the third day after surgery showed that, compared with the Sham group, the CLP group mice exhibited significant cognitive behavioral abnormalities, namely, significantly reduced exploration time in the Y maze and new object recognition (NOR) tests, and significantly reduced total movement distance in the open field (OFT) test. Figure 8 IN, P<0.05). Mice treated with FLKNCE-Tat (CLP+FT) showed significant improvement in cognitive impairment and abnormal behavior, as evidenced by significantly increased exploration time in the Y maze and NOR tests, and a significantly greater total distance traveled in the OFT than untreated CLP mice. Figure 8 IN, P<0.05). In conclusion, FLKNCE-Tat can effectively reduce neuroinflammation, improve brain damage, reverse abnormal neuronal firing frequency, and improve sepsis-related cognitive impairment.

[0102] 3. FLKNCE-Tat reduces oxidative stress damage and regulates the inflammatory microenvironment in the brain of SAE mice. Oxidative stress is one of the core mechanisms in the development of SAE. It disrupts the oxidative-antioxidant balance in brain cells, triggering a series of pathological damages such as blood-brain barrier disruption, neuronal damage, glial cell activation, and amplified inflammation, ultimately leading to brain dysfunction. Figure 9 AE showed that, compared with the Sham group mice, CLP mice exhibited abnormal oxidative stress-related damage markers in their brain tissue, namely, a significant increase in superoxide dismutase (DHE) and malondialdehyde (MDA) levels in brain homogenate, while superoxide dismutase (SOD) activity and the antioxidant glutathione (GSH) levels were significantly decreased (P<0.05). In contrast, compared with the CLP group, the CLP+FT group mice showed a significant decrease in superoxide levels in their brain tissue (manifested as weakened DHE fluorescence intensity). Figure 9 AB, P<0.05), while the damaged tissue area was reduced by about half. Furthermore, MDA levels in the brain homogenate of mice in the CLP+FT group were significantly inhibited, and SOD activity was significantly enhanced, indicating that lipid peroxidation was effectively inhibited and antioxidant capacity was improved. Figure 9CD, P<0.05. GSH levels also showed a similar upward trend, further confirming the enhanced antioxidant activity after treatment. Figure 9 E, P<0.05).

[0103] Damaged neurons are known to release endogenous damage-associated molecular patterns (DAMPs), which can induce excessive activation of microglia and astrocytes, leading to excessive secretion of pro-inflammatory cytokines. This response creates a pro-inflammatory microenvironment in the brain, further triggering endothelial damage, homeostasis dysregulation, and exacerbating neuronal injury. As shown in the figure, compared with the Sham group, the CLP group showed significant glial cell overactivation and endothelial cell damage, specifically a significant decrease in CD31 (an indicator of microvascular endothelial cells) fluorescence signal, while a significant increase in GFAP (an indicator of astrocyte activation) and Iba-1 (an indicator of microglia activation) fluorescence signals. Figure 9 FK, P<0.05. However, compared with the CLP group, the CLP+FT group increased the abundance of CD31-labeled microvascular endothelial cells in the mouse brain, suggesting that it has a protective effect against vascular endothelial injury. Figure 9 F, I, P<0.05), and the GFAP fluorescence signal intensity in activated astrocytes in the hippocampus of mice treated with CLP+FT was significantly reduced ( Figure 9 G, J, P<0.05, the number of Iba1-positive microglia in the brain was significantly reduced ( Figure 9 H, K, P<0.05). Therefore, these findings indicate that FLKNCE-Tat alleviates neuroinflammation by inhibiting the migration and activation of astrocytes and microglia, thereby blocking the inflammatory cascade, promoting the repair of brain microvascular endothelial cells, remodeling the neuroinflammatory microenvironment, and promoting brain tissue recovery.

[0104] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. A fusion cell-penetrating peptide, characterized in that, The fusion cell-penetrating peptide comprises the polypeptide of claim 1, and the cell-penetrating peptide or its transduction domain thereof, preferably, the cell-penetrating peptide or its transduction domain is connected to the C-terminus of the polypeptide; Preferably, the amino acid sequence of the transmembrane peptide's conduction domain is shown in SEQ ID NO.3; Preferably, the amino acid sequence of the fusion cell-penetrating peptide is shown in SEQ ID NO.

2.

3. A nucleic acid molecule or a carrier containing the same, characterized in that, The nucleic acid molecule encodes the polypeptide of claim 1 or the fusion cell-penetrating peptide of claim 2.

4. A host cell, characterized in that, The host cell contains the nucleic acid molecule of claim 3 or a vector containing it.

5. A modified polypeptide, characterized in that, The modified polypeptide comprises the polypeptide of claim 1 or the fusion cell-penetrating peptide of claim 2; preferably, the modified polypeptide further comprises a detectable marker; preferably, the detectable marker is CY5.

6. A detection product comprising the polypeptide of claim 1, the fusion cell-penetrating peptide of claim 2, and the modified polypeptide of claim 5.

7. A drug for treating sepsis or sepsis-related encephalopathy, characterized in that, The drug comprises the polypeptide of claim 1, the fusion cell-penetrating peptide of claim 2, the modified polypeptide of claim 5, the nucleic acid molecule of claim 3 or a carrier containing the thereof, or the host cell of claim 4; preferably, the drug further comprises a pharmaceutically acceptable carrier.

8. Application of reagents that inhibit the formation of complexes between ATF2 protein and c-JUN in the preparation of drugs for treating sepsis or sepsis-associated encephalopathy.

9. The application according to claim 8, characterized in that, The reagent is a substance that competitively binds to c-JUN with the ATF2 protein.

10. The application according to claim 9, characterized in that, The substance includes the drug comprising the polypeptide of claim 1, the fusion cell-penetrating peptide of claim 2, and the modified polypeptide of claim 5.

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