Application of PTN in preparation of product for treating cognitive impairment caused by serious infection

Abstract

The invention discloses application of PTN in preparation of a product for treating cognitive impairment caused by serious infection. The PTN inhibitor provided by the invention can inhibit chronic neuroinflammation so as to prevent and inhibit the formation and development of advanced cognitive impairment caused by serious infection, and meanwhile, the damage of infiltrating macrophages to microglial cells can be effectively blocked by inhibiting the expression or function of PTN, so that a new direction is provided for the treatment of related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of PTN in the preparation of products for treating cognitive impairment caused by severe infections. Background Technology

[0002] Sepsis is a life-threatening organ dysfunction caused by a dysregulation of the host response to infection. Millions of people worldwide are diagnosed with sepsis each year, and it is one of the leading causes of death in intensive care units (ICUs). More alarmingly, a large number of sepsis survivors experience long-term neurological sequelae, the most common manifestation of which is cognitive impairment, known as sepsis-associated encephalopathy (SAE) or post-infectious cognitive impairment. These cognitive deficits can last for months or even years, severely impairing patients' quality of life and imposing a heavy medical and economic burden on society.

[0003] Clinical and basic research indicates that severe infectious events in early life are a significant risk factor for cognitive decline and an increased risk of neurodegenerative diseases such as Alzheimer's disease in later life. However, the underlying cellular and molecular mechanisms of this pathological process, from acute peripheral infection to chronic central nervous system dysfunction, remain largely unknown. Currently, treatment options for post-infectious cognitive impairment are extremely limited, primarily focusing on supportive care during the acute infection phase; there are no specific therapies that can effectively prevent or reverse long-term cognitive impairment.

[0004] Neuroinflammation, or the immune response within the central nervous system (CNS), is considered a core pathological factor driving post-infectious cognitive impairment. Traditionally, it was believed that the CNS immune environment would return to homeostasis after the acute infection subsides and the blood-brain barrier (BBB) ​​integrity is restored. However, new evidence suggests that transient increases in BBB permeability during acute infection allow peripheral immune cells, particularly macrophages derived from monocytes, to enter the CNS. These cells may establish long-lived resident populations in the brain, persistently disrupting neuroimmune homeostasis, thereby leading to chronic neuroinflammation and cognitive dysfunction.

[0005] In previous studies, a batch of MHCII mice were found in a sepsis mouse model. low / Ly6C +Infiltrating macrophages enter key brain regions such as the hippocampus during the acute phase of infection and remain there for an extended period (at least 6 months). These long-lived infiltrating macrophages secrete signaling molecules that pathologically interact with the brain's inherent immune cells—microglia—driving persistent microglia activation and abnormal myelination, ultimately leading to neuronal dysfunction and cognitive impairment. However, the molecules mediating this crucial "cross-boundary dialogue" remain unknown, thus limiting the development of new therapies. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, the present invention adopts the following technical solution to provide a novel and specific treatment strategy for preventing or treating late-stage cognitive impairment caused by severe infection.

[0007] A first aspect of the present invention provides the use of PTN inhibitors in the preparation of medicaments for treating cognitive impairment caused by severe infections.

[0008] Furthermore, the cognitive impairment caused by the severe infection is sepsis-associated encephalopathy.

[0009] Furthermore, the serious infections include systemic infections, respiratory infections, CNS infections, viral infections, chronic infections, and parasitic infections.

[0010] Furthermore, the systemic infections include sepsis, pneumonia, bacterial septicemia, and septic shock; the respiratory system infections include; the CNS infections include bacterial meningitis, viral encephalitis, fungal meningitis, tuberculous meningitis, and brain abscess; the viral infections include herpes simplex virus (HSV) infection, human immunodeficiency virus (HIV) infection, influenza virus infection, coronavirus infection (including SARS-CoV-2), and Japanese encephalitis virus infection; the chronic infections include HIV infection, chronic tuberculosis infection, chronic viral hepatitis, and neurosyphilis; and the parasitic infections include Plasmodium infection, Toxoplasma gondii infection, schistosomiasis infection, and cysticercosis.

[0011] In a specific embodiment of the present invention, the severe infection is sepsis.

[0012] In this invention, the term "severe infection" refers to life-threatening organ dysfunction caused by infection; the term "cognitive impairment" refers to a decline in one or more cognitive functions such as memory, attention, executive function, language ability, learning ability, or information processing speed, including but not limited to: mild cognitive impairment, infection-related cognitive impairment, and cognitive deficits caused by sepsis-related encephalopathy.

[0013] Furthermore, the inhibitors include protein inhibitors and nucleic acid inhibitors.

[0014] In this invention, the protein inhibitor refers to an inhibitor that acts at the protein level and exerts its effect through mechanisms such as antigen binding, neutralization, or signal inhibition; the nucleic acid inhibitor refers to an inhibitor that can reduce the transcription or translation level of the PTN gene in cells by interfering with the mRNA production or protein synthesis of the PTN gene, thereby reducing the expression level of PTN.

[0015] Furthermore, the nucleic acid inhibitors include siRNA, shRNA, ribozymes, and antisense oligonucleotides.

[0016] In a specific embodiment of the present invention, the nucleic acid inhibitor is siRNA.

[0017] Furthermore, the siRNA sequence is an RNA molecule with the sense strand sequence SEQ ID NO:1 and the antisense strand sequence SEQ ID NO:2.

[0018] Furthermore, chemical modifications that enhance stability are introduced into the sequence of the siRNA.

[0019] Furthermore, the chemical modification includes introducing two thiophosphate bonds and / or making a 2'-O-methyl modification on the pyrimidine nucleotide.

[0020] A second aspect of the present invention provides a pharmaceutical composition for treating cognitive impairment caused by severe infection, the composition comprising the siRNA described in the first aspect of the present invention.

[0021] Furthermore, the pharmaceutical composition also includes LNP, SLN, and NLC.

[0022] In a specific embodiment of the present invention, the pharmaceutical composition further includes LNP.

[0023] In this invention, the term "LNP" refers to lipid nanoparticles, which are nanoscale delivery carrier systems formed by the self-assembly of lipid components, used to encapsulate, protect, and deliver nucleic acid molecules into target cells, thereby realizing the biological functions of the nucleic acid molecules.

[0024] Furthermore, the pharmaceutical composition further includes pharmaceutically acceptable excipients and / or carriers.

[0025] The pharmaceutically acceptable carriers and / or excipients described in this invention include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). These materials can be used to formulate various dosage forms, including but not limited to injections, tablets, capsules, oral liquids, granules, ointments, creams, creams, eye drops, suspensions, and other external or topical dosage forms. These can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems. To formulate unit-dose dosage forms into injectable formulations, such as solutions, emulsions, lyophilized powders for injection, and suspensions, all diluents commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxidized isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. In addition, to prepare isotonic injection solutions, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injectable formulation. Furthermore, conventional solubilizers, buffers, pH adjusters, etc., can also be added. Furthermore, if necessary, colorants, preservatives, flavorings, tasters, sweeteners, or other materials can be added to the pharmaceutical formulation. To formulate unit-dose dosage forms into tablets, a wide variety of carriers known in the art can be widely used. Examples of carriers include, for instance, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; humectants and binders such as water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.; and disintegrants. Examples of carriers include dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors include sucrose, tristearate, cocoa butter, and hydrogenated oil; absorption enhancers include quaternary ammonium salts and sodium dodecyl sulfate; and lubricants include talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into pills. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, gelucire, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc.The above-mentioned dosage forms can be administered via injection, including subcutaneous injection, intravenous injection, intramuscular injection, and intracavitary injection. Injection is the preferred route of administration. When patients experience local poisoning symptoms due to organophosphate contamination of the skin, mucous membranes, or eyes, topical or local application dosage forms can be used.

[0026] A third aspect of the present invention provides a method for screening candidate drugs for treating cognitive impairment caused by severe infection.

[0027] Further, the method includes: testing the effect of the candidate drug on PTN levels in samples obtained from the subject, wherein a decrease in PTN levels after use of the candidate drug indicates that the candidate drug has a therapeutic effect on cognitive impairment caused by severe infection.

[0028] The advantages and beneficial effects of this invention are as follows: 1. Novel targets and mechanisms: For the first time, PTN has been revealed as a key signaling molecule connecting peripheral immune infiltration and central chronic neuroinflammation, providing a novel drug target for the treatment of post-infectious cognitive impairment.

[0029] 2. Precision treatment: Targeting the PTN signaling pathway, rather than broadly suppressing the immune system, achieves precise intervention in the core pathological process, which is expected to improve efficacy and reduce side effects.

[0030] 3. Broad application prospects: The strategy of this invention is not only applicable to sepsis, but may also be applicable to other systemic infections caused by viruses (such as COVID-19), bacteria, etc., or cognitive impairment caused by aseptic inflammation (such as traumatic brain injury, stroke).

[0031] 4. Wider treatment window: Because it targets the maintenance mechanism of chronic inflammation, this therapy may be used not only for acute prevention but also for chronic treatment, bringing hope to a large number of patients already suffering from cognitive impairment. Attached Figure Description

[0032] Figure 1 This study demonstrates the construction of a mouse infection model using CLP (cecal ligation model) and the subsequent single-cell sequencing in the later stages of infection. Figure 2 The results of single-cell sequencing showed a group of MHCII cells that had infiltrated the central nervous system and remained there for a long time. low / Ly6C + Infiltrating macrophages; Figure 3 Single-cell interaction analysis was presented, showing that macrophages that infiltrate and reside in the central nervous system act on microglia through PTN molecules; Figure 4It was demonstrated that PTN acts on microglia, coordinating with inflammatory signals, leading to the activation of microglia and phagocytosis, which engulfs the myelin sheath of neurons, ultimately causing cognitive dysfunction. Figure 5 This is a schematic diagram of the PTN siRNA dosing regimen in a mouse model of sepsis. Figure 6 The results of the Y-maze spontaneous alternation experiment showed that PTN siRNA significantly improved working memory impairment in CLP mice; Figure 7 The results of the novel object recognition experiment showed that PTN siRNA significantly improved the recognition memory impairment in CLP mice; Figure 8 The results of the Barnes maze experiment showed that PTN siRNA significantly improved spatial learning and memory impairment in CLP mice; Figure 9 Immunofluorescence images of the hippocampus show that, compared with the control siRNA group, PTN siRNA treatment significantly reduced the amount of myelin (MBP+) phagocytosed by microglia (Iba1+) in the hippocampus of CLP mice. Detailed Implementation

[0033] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0034] Experimental Materials and Methods Animal model: Male C57BL / 6J mice aged 8-10 weeks were used. Sepsis was induced by cecal ligation and punchcture (CLP) surgery. In short, after anesthetizing the mice, a 1 cm incision was made along the midline of the abdomen to expose the cecum. The cecum was ligated at approximately 50% of its distal end using 4-0 suture, and then punctured twice distal to the ligation using a 21-gauge needle. A small amount of feces was expelled to induce infection, the cecum was repositioned, and the abdominal wall was sutured. In the sham surgery group (Sham mice), only the cecum was exposed without ligation and punchcture. All mice underwent immediate postoperative resuscitation with a subcutaneous injection of 1 mL of preheated sterile saline and were placed on a heating pad until awakening.

[0035] Single-cell RNA sequencing (scRNA-seq): Six months after CLP, mice were anesthetized and perfused with PBS via the heart. Hippocampal tissue was harvested and digested with papain and DNase I to prepare single-cell suspensions. Single-cell libraries were prepared and sequenced using a 10x GenomicsChromium platform.

[0036] Bioinformatics analysis: The Seurat software package was used for quality control, dimensionality reduction (UMAP), and cell clustering of scRNA-seq data. The CellChat software package was used to analyze ligand-receptor interactions among different cell subpopulations to identify key communication signaling pathways.

[0037] Design and preparation of siRNA: Three siRNA sequences targeting mouse Ptn gene mRNA (Ptn-siRNA-1, -2, -3, sequences shown in Table 1) and one negative control siRNA (control-siRNA) were designed. All siRNAs were synthesized by an external supplier (Sangon Biotech) and chemically modified to enhance stability: two phosphate thioester (PS) bonds were introduced at the 3' end of each double strand, and 2'-O-methyl (2'-OMe) modification was performed on all pyrimidine nucleotides (C and U).

[0038] Table 1. siRNA sequence design targeting the mouse Ptn gene

[0039] Animal treatment regimen: Starting from day 0 after CLP surgery, mice were injected intracerebroventricularly with siRNA at a dose of 3 mg / kg body weight once a week for 6 months. The CLP+Ptn-siRNA group received Ptn-siRNA, and the CLP+control-siRNA group received LNP preparation of control-siRNA. The Sham group received an equal volume of PBS.

[0040] Preparation of siRNA-LNP formulation: Ptn-targeting siRNA (Ptn-siRNA) or control siRNA was encapsulated in lipid nanoparticles (LNPs). Specifically, ionizable cationic lipid DLin-MC3-DMA, auxiliary lipid DSPC, cholesterol, and PEGylated lipid PEG-DMG 2000 were dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 to form a lipid organic phase. Simultaneously, siRNA was dissolved in sodium citrate buffer at pH 4.0 to form an aqueous phase. Using a microfluidic mixing system, the organic and aqueous phases were rapidly mixed at a flow rate of 3:1, allowing the liposomes to self-assemble and encapsulate the siRNA. The primary product was purified by ultrafiltration centrifugation, using PBS (pH 7.4) as the exchange medium to remove the organic solvent and replacing the external buffer with physiological saline. Finally, the formulation concentration was adjusted to the target concentration and sterilely filtered through a 0.22 μm filter to obtain the Ptn-siRNA-LNP formulation and the control-siRNA-LNP formulation, which were stored at 4 °C for later use.

[0041] Behavioral tests: These were initiated 6 months after the last dose of medication. The Y-maze test was used to assess working memory (spontaneous alternation rate was recorded); the new object recognition test was used to assess recognition memory (recognition index was recorded); and the Barnes maze test was used to assess spatial learning and memory (escape latency and time spent in the target quadrant were recorded).

[0042] Histological and Immunofluorescence Staining: After behavioral testing, hippocampal tissue was harvested from mice after perfusion. The tissue was dehydrated with sucrose and then frozen sectioned. The tissue was incubated with primary antibodies against Iba1 (a microglia marker) and MBP (a myelin basic protein marker), followed by labeling with fluorescent secondary antibodies. Nuclear counterstaining was performed using DAPI. Finally, images were acquired and analyzed using confocal microscopy.

[0043] Statistical analysis: All data are expressed as mean ± standard error (SEM). Statistical analysis was performed using GraphPad Prism software. T-tests were used for comparisons between two groups, and one-way or two-way ANOVA was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant. In the figures, This indicates that P < 0.05. This indicates that P < 0.01. This means P < 0.001.

[0044] Example 1: Infiltrating macrophages that reside in the hippocampus long after sepsis are key cells driving neuropathology. To identify key cell types leading to long-term cognitive impairment following severe infection, this invention first investigated CLP-induced septic mice at a long-term time point of 6 months post-surgery. Single-cell RNA sequencing (scRNA-seq) was performed on hippocampal tissue from CLP mice and sham-operated mice (experimental protocol detailed in...). Figure 1 ).

[0045] The results showed that, compared with the Sham group, a unique cell population appeared in the hippocampus of CLP mice, which was almost absent in the Sham group. This cell population was characterized by low MHCII expression levels and high Ly6C expression levels (MHCII...). low / Ly6C + ), identified as infiltrative macrophages of peripheral origin ( Figure 2 These cells remained stable for up to 6 months after infection, indicating that they had formed a long-lived resident population within the brain parenchyma.

[0046] Example 2: PTN is a key molecule connecting the pathological communication between infiltrating macrophages and microglia. Based on the above findings that infiltrating macrophages are key pathogenic cells, this invention further explores how they interact with the brain's inherent immune cells (microglia). Using the scRNA-seq data obtained in Example 1, we performed an analysis of intercellular communication networks.

[0047] The results showed that among numerous ligand-receptor pairs, pleiotrophin (PTN), derived from long-lived infiltrating macrophages, was significantly upregulated. Figure 3 This suggests that PTN may be a key signaling molecule mediating the pathological “dialogue” between these two cell types.

[0048] To verify this hypothesis, in vitro experiments were conducted. Treatment of the microglia cell line BV2 with recombinant PTN protein revealed that PTN synergistically enhanced the phagocytic activity of microglia by acting on inflammatory signals (such as LPS). These results collectively confirm that PTN is a key mediator driving pathological activation of microglia and leading to nerve damage. Figure 4 ).

[0049] Example 3: siRNA targeting PTN can effectively improve long-term cognitive impairment caused by sepsis. Based on the core role of PTN in the pathological mechanism, this invention proposes a strategy to treat post-infectious cognitive impairment by inhibiting PTN. We selected a highly efficient and specific gene silencing technology—small interfering RNA (siRNA).

[0050] We used the CLP sepsis model, starting on day 0 post-surgery, and administered siRNA preparations (CLP+Ptn-siRNA group) or negative control siRNA preparations (CLP+control-siRNA group) weekly via intracerebroventricular injection. A sham surgery group was established as a healthy control group (see experimental procedure). Figure 5 ).

[0051] At 6 months post-surgery, we performed a series of behavioral tests on all mice. The results showed: 1. Y-maze test (working memory): Compared with the Sham group, the spontaneous alternation rate in the CLP+control-siRNA group was significantly reduced, while the alternation rate in the Ptn-siRNA treatment group significantly increased, returning to near-normal levels. Figure 6 ).

[0052] 2. New object recognition test (recognition memory): The recognition index of the CLP+control-siRNA group was significantly lower than that of the Sham group, while the recognition index of the Ptn-siRNA treatment group recovered somewhat. Figure 7 ).

[0053] 3. Barnes Maze Test (Spatial Memory): The escape latency for finding the target hole was significantly longer in the CLP+control-siRNA group than in the Sham group, while the latency was significantly shorter in the Ptn-siRNA treatment group. Figure 8 ).

[0054] In summary, these results strongly demonstrate that administration of PTN-targeting siRNA during the chronic phase of sepsis can effectively improve long-term cognitive impairment involving multiple cognitive domains caused by severe infection.

[0055] Example 4: PTN siRNA protects neurons by inhibiting abnormal phagocytosis of myelin by microglia. To investigate the cellular mechanism by which PTN siRNA improves cognitive function, we performed immunofluorescence staining analysis on the hippocampus of mice after the behavioral tests were completed.

[0056] The results are as follows Figure 9 As shown, in the hippocampus of mice in the CLP+ control-siRNA group, a large number of activated microglia (Iba1+, red) were observed. These microglia were short and stout, with enlarged cell bodies, and contained a large number of phagocytosed myelin fragments (MBP+, gray). In contrast, in the hippocampus of mice treated with Ptn-siRNA, the activation level of microglia was significantly reduced, and the number of phagocytosed MBP-positive myelin fragments was also significantly decreased. Quantitative analysis showed that the average myelin sheath area phagocytosed by each microglia in the Ptn-siRNA treatment group was significantly lower than that in the control-siRNA group.

[0057] This result indicates that the PTN siRNA therapy provided by this invention works by inhibiting PTN signaling, blocking the pathological drive of infiltrating macrophages to microglia, thereby reducing the abnormal phagocytosis of myelin sheaths by microglia, protecting the structural and functional integrity of neurons, and ultimately improving cognitive function.

[0058] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Application of PTN inhibitors in the preparation of drugs for treating cognitive impairment caused by severe infections.

2. The application according to claim 1, characterized in that, The cognitive impairment caused by the severe infection is sepsis-associated encephalopathy.

3. The application according to claim 1, characterized in that, The inhibitor is a nucleic acid inhibitor.

4. The application according to claim 3, characterized in that, The nucleic acid inhibitors include siRNA, shRNA, ribozymes, and antisense oligonucleotides.

5. The application according to claim 4, characterized in that, The siRNA sequence is: an RNA molecule with the sense strand sequence SEQ ID NO:1 and the antisense strand sequence SEQ ID NO:

2.

6. The application according to claim 5, characterized in that, The siRNA is also modified with chemical additives to enhance its stability.

7. The application according to claim 6, characterized in that, The chemical modifications include introducing two thiophosphate bonds at the 3' end and / or performing 2'-O-methyl modification on the pyrimidine nucleotide.

8. A pharmaceutical composition for treating cognitive impairment caused by severe infection, characterized in that, The pharmaceutical composition comprises siRNA as described in any one of claims 5-7.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition also includes LNP.

10. A method for screening candidate drugs for treating cognitive impairment caused by severe infection, characterized in that, The method includes: testing the effect of the candidate drug on PTN levels in samples obtained from the subject, wherein a decrease in PTN levels after use of the candidate drug indicates that the candidate drug has a therapeutic effect on cognitive impairment caused by severe infection.

Images