A macrophage-targeting transmembrane peptide, its preparation and application

Abstract

This invention discloses a macrophage-targeting transmembrane peptide, its preparation, and its applications. The functional polypeptide is encoded by a transmembrane sequence and a leucine zipper sequence at the HBXIPC terminus. The polypeptide of this invention can specifically target macrophages, possessing immunomodulatory and anti-aging functions. It can reduce the levels of inflammatory factors in LPS-induced pneumonia and DSS-induced enteritis models, downregulate the expression of pulmonary fibrosis-related genes, and downregulate the expression of aging-related genes p16 and p21 in the lung tissue of aged mice. The CPP-AA proposed in this invention has a small molecular weight, easily enters cells to exert its effects, is easy to synthesize in large quantities, has good stability, and is cheaper than current immunomodulators and anti-aging agents. It has good application prospects in the preparation of immunomodulatory agents and anti-aging drugs for treating pneumonia, pulmonary fibrosis, and enteritis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a macrophage-targeting transmembrane peptide and its preparation and application. Background Technology

[0002] Individual aging is accompanied by the gradual decline of various biological functions of the body, one important characteristic of which is the accumulation of senescent cells in the body. Senescent cells accumulate with lifespan and are associated with many diseases. Senescent cells are present in many precancerous lesions, fibrotic diseases including neuropathology (such as cerebral aneurysms, Alzheimer's disease, and Parkinson's disease), pulmonary pathology (such as idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, and cystic fibrosis), ocular pathology (such as cataracts, glaucoma, and macular degeneration), musculoskeletal pathology (such as myopathy, intervertebral disc degeneration, and osteoarthritis), cardiovascular pathology (such as atherosclerosis, cardiac fibrosis, and aortic aneurysm), renal pathology (such as kidney disease and transplant complications), and other pathologies such as diabetes, mucositis, hypertension, and myelofibrosis (OMF).

[0003] Senescent cells undergo a period of arrested proliferation and restricted replication, while exhibiting various phenotypic changes, including chromatin reorganization, increased β-galactosidase activity, morphological and metabolic alterations, and secretion of various inflammatory factors and cytokines, collectively known as the aging-associated secretory phenotype (SASP). Immunity and aging are closely related; inflammatory factors produced during aging are significant contributors to organ dysfunction and accelerated aging processes. Dysregulation of the aging immune system, insufficient immune surveillance, and systemic humoral immune aging can lead to various age-related diseases and malignant tumors. Recent studies have shown that immune system aging accelerates the aging process of organs and tissue damage throughout the body, and reversing the pro-inflammatory senescent state of immune cells may offer promising therapeutic benefits for the prevention and treatment of some age-related diseases. Senescent macrophages are a key factor in inflammatory senescence. Sustained low-level secretion of inflammatory factors is believed to promote many age-related symptoms, such as cognitive decline, neurodegeneration, and atherosclerosis. Reducing macrophage inflammatory responses and enhancing macrophage metabolism can reverse age-related diseases such as cognitive decline in mice. Recent studies have shown that senescent cells and macrophages share many similarities in biological characteristics, including activation, metabolism, proliferation, and apoptosis. Based on these key pathways, developing drugs that target macrophages holds great potential for simultaneously regulating immunity, inflammation, and aging.

[0004] Cellular lifespan largely depends on lysosomal function, and mounting evidence shows that lysosomes are key hubs controlling cellular senescence. Lysosomes are not only the site of cellular waste processing but also important regulators of cellular homeostasis. They participate in regulating the effective composition of intracellular nutrients, such as stress responses, programmed cell death, plasma membrane repair, development, and cell differentiation. Lysosomal dysfunction in senescent and macrophage cells leads to impaired apoptotic cell clearance, protein body aggregation, and antigen-antibody complex formation, triggering inflammation and cellular senescence. Therefore, drug development and interventions targeting lysosomes and aiming at lysosomal repair are important areas and cutting-edge innovations in immunomodulation and anti-aging research. Currently discovered interventions such as calorie restriction, intermittent fasting, and some interventional drugs such as spermidine, metformin, and resveratrol are closely related to the regulation and repair of lysosomal function. This invention is a targeted small peptide designed based on previous research findings that HBXIP binds to the lysosomal v-ATPase subunit, regulates lysosomal pH, and promotes autophagy.

[0005] Peptide drugs typically refer to peptide chains of no more than 50 amino acids. In clinical applications, they are characterized by broad indications, high safety, and significant efficacy. Their production and preparation are highly controllable, highly pure, easily modified, and structurally stable, and they have been widely used in the prevention, diagnosis, and treatment of diseases such as cancer, cardiovascular and cerebrovascular diseases, hepatitis, diabetes, and AIDS. Cell-penetrating peptides (CPPs) are a class of positively charged polypeptide fragments, usually composed of no more than 30 amino acid residues, rich in basic amino acid residues such as arginine and lysine. They can carry active substances (such as small molecule drugs, peptides, proteins, siRNAs, and nanoparticles) into cells to exert biological activity, and have significant value in targeted formulations, transdermal drug delivery systems, and cosmetics. With the development and maturation of peptide synthesis technology, peptide drugs have become one of the hottest areas in drug development. Currently, more than 80 peptide drugs are marketed globally, covering a market of over $20 billion, with more than 150 undergoing clinical trials. However, the development of peptide drugs related to immune regulation and anti-aging is still in its early stages, with a relatively small overall scale, but significant development potential and application value. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a macrophage-targeting transmembrane peptide, its preparation, and its application in the preparation of immunomodulatory drugs or anti-aging drugs.

[0007] In a first aspect, the present invention provides a macrophage-targeted polypeptide CPP-AA with cell membrane crossing function, encoded by a membrane-penetrating sequence and an HBXIPC-terminal leucine zipper sequence, the amino acid sequence of which is KIKKVKKKGRKHAGVISVLAQQAAKLTSDPTDIP, as shown in SEQ NO. 1.

[0008] The transmembrane region sequence is KIKKVKKKGRK;

[0009] The zipper sequence in the HBXIP gene is a nucleotide (55-116), preferably a nucleotide (102-116) sequence or its degenerate sequence translated from it.

[0010] Secondly, the present invention provides an isolated polynucleotide encoding the aforementioned polypeptide CPP-AA.

[0011] Thirdly, the present invention provides an expression vector containing the polynucleotide encoding the polypeptide CPP-AA.

[0012] Fourthly, the present invention provides a recombinant cell containing the polynucleotide in an expression vector or in the genome of the polynucleotide.

[0013] Fifthly, the present invention provides the use of the above-mentioned polypeptide CPP-AA, or polynucleotide, or expression vector, or recombinant cell in the preparation of medicaments for immunomodulation or for treating age-related diseases or conditions.

[0014] Preferably, the immunomodulatory drug is a drug for the prevention, relief or treatment of acute pneumonia.

[0015] The immunomodulatory drugs mentioned are those that reduce pulmonary inflammatory response in acute lung injury, decrease pulmonary inflammatory infiltration, and repair lung tissue damage.

[0016] The immunomodulatory drug mentioned is a medication for treating ulcerative colitis.

[0017] The immunomodulatory drugs mentioned are those that reduce the inflammatory response of intestinal tissue in ulcerative colitis, repair intestinal tissue damage, and reduce weight loss caused by ulcerative colitis.

[0018] Preferably, the age-related disease or condition is one or more of the following: metabolic disease, inflammatory disease, lung disease, neurological disease, proliferative disease, kidney disease, eye disease, or dermatological disease.

[0019] Preferably, the anti-aging drug is a drug for treating age-related idiopathic pulmonary fibrosis and for anti-aging purposes.

[0020] Preferably, the anti-aging drug is one that reduces fibrosis-related genes, decreases collagen fibers, and improves the structural integrity of lung tissue in a pulmonary fibrosis model.

[0021] Preferably, the anti-aging drug is one that reduces the expression levels of aging genes p16 and p21 in aging lung tissue.

[0022] Preferably, the anti-aging drug is a drug that reduces β-galactosidase, a marker of cellular senescence, in aging lung tissue.

[0023] In a sixth aspect, the present invention provides a method for preparing the polypeptide CPP-AA, the method comprising: culturing the recombinant cells to recombinantly express the polypeptide CPP-AA; or, preparing the polypeptide CPP-AA by means of in vitro artificial synthesis.

[0024] In a seventh aspect, the present invention provides a pharmaceutical composition comprising: a polypeptide CPP-AA or a polynucleotide encoding thereon, or the expression vector or the recombinant cell; and a pharmaceutically or physiologically acceptable carrier.

[0025] Eighthly, the present invention provides a kit comprising: a polypeptide CPP-AA or a polynucleotide encoding thereas; or the expression vector; or the recombinant cells; or the pharmaceutical composition.

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] 1. The CPP-AA polypeptide provided by this invention can specifically target macrophages, inhibit their spontaneous differentiation into inflammatory cell phenotypes, and suppress the release of inflammatory factors and tissue damage, thereby treating inflammatory and age-related diseases. This polypeptide molecule has a transmembrane structure, specifically acts on lysosomes, has a well-defined target, and high efficacy.

[0028] 2. The CPP-AA polypeptide provided by this invention is prepared through biological and chemical synthesis. The technology is mature, easy to prepare in large quantities, and has good stability. It has potential application prospects for the development of anti-inflammatory and anti-aging drugs for which there are currently no effective treatments. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope.

[0030] Figure 1 The detection and analysis chromatograms after obtaining CPP-AA are shown below: A is the mass spectrometry chromatogram of CPP-AA membrane-penetrating peptide; B is the HPLC purity chromatogram of CPP-AA membrane-penetrating peptide.

[0031] Figure 2Effects of control group and CPP-AA treatment on LPS-induced pneumonia symptoms in mice: A represents the levels of inflammatory factors in lung tissue of pneumonia model mice treated with control group and CPP-AA treatment, where **P<0.01; B represents the H&E staining results of lung tissue of pneumonia model mice treated with control group and different doses of CPP-AA treatment, with a scale bar of 200 μm.

[0032] Figure 3 The effects of treatment in the control group and the CPP-AA treatment group on DSS-induced ulcerative colitis symptoms in mice were investigated. Specifically: AB represents the comparison of colon length and body weight in mice with enteritis in the control group and the CPP-AA treatment group (*P<0.05, **P<0.01, ***P<0.001); C represents the DAI score in mice with enteritis in the control group and the CPP-AA treatment group (***P<0.001); D represents the levels of inflammatory factors in colon tissue of mice with enteritis in the control group and the CPP-AA treatment group (**P<0.01, ***P<0.001, ****P<0.0001); E represents the H&E staining results of colon tissue in mice with enteritis in the control group and the CPP-AA treatment group (scale bar: 200 μm); and F represents the levels of tight junction genes in colon tissue of mice with enteritis in the control group and the CPP-AA treatment group (***P<0.001, ****P<0.0001).

[0033] Figure 4 The effects of the control group and CPP-AA treatment group on bleomycin-induced pulmonary fibrosis symptoms in mice were investigated. A represents the expression levels of pulmonary fibrosis genes in lung tissue of mice treated with the control group and CPP-AA treatment group (**P<0.01, ***P<0.001); B represents the H&E staining results of lung tissue in mice treated with the control group and CPP-AA treatment group (scale bar: 200 μm); C represents the Masson staining results of lung tissue in mice treated with the control group and CPP-AA treatment group (scale bar: 200 μm); D represents the expression levels of aging factors p16 and p21 in lung tissue of mice treated with the control group and CPP-AA treatment group (**P<0.01, ***P<0.001); E represents the β-Gal staining results of lung tissue in mice treated with the control group and CPP-AA treatment group (scale bar: 200 μm). Detailed Implementation

[0034] The present invention will be further described in detail below with reference to specific embodiments.

[0035] The experimental materials used in the following examples are as follows:

[0036] 1.1 Experimental drugs and reagents: LPS (Sigma); DSS (Shanghai Yisheng Biotechnology Co., Ltd.); Bleomycin (Selleck); CPP-AA (Nanjing Genscript Biotechnology Co., Ltd.); Reverse transcription kit (Shanghai Yisheng Biotechnology Co., Ltd.); SYBR Green dye (Shanghai Yisheng Biotechnology Co., Ltd.); β-galactosidase kit (Beyotime); Modified Gomori trichrome staining kit (Solepro).

[0037] 1.2 Experimental animals and their rearing: C57 / BL6 mice, 7-8 weeks old, were provided by Nanjing Annokang Biotechnology Co., Ltd.; all animals were housed in an SPF environment at a room temperature of 22±2℃ and a humidity of 40-60%, and were used in experiments after 1 week of acclimatization.

[0038] Example 1:

[0039] 2.1 Preparation of CPP-AA membrane-penetrating peptide storage solution.

[0040] 4 mg of CPP-AA membrane-penetrating peptide was dissolved in 50 μL of formic acid to prepare a stock solution with a concentration of 80 mg / mL. For subsequent animal experiments, the solution was diluted with PBS to prepare a working solution of the corresponding concentration. Figure 1 The detection and analysis chromatograms after obtaining CPP-AA are shown below: A is the mass spectrometry chromatogram of CPP-AA membrane-penetrating peptide; B is the HPLC purity chromatogram of CPP-AA membrane-penetrating peptide.

[0041] 2.2 Construction of a mouse LPS pneumonia model.

[0042] Mice were lightly anesthetized with ether and administered 2 mg / kg LPS intranasally (50 μL total volume). Two hours after LPS treatment, mice were divided into four groups: a control group, a low-dose treatment group (0.1 mg / kg), a medium-dose treatment group (1 mg / kg), and a high-dose treatment group (2 mg / kg), with four mice in each group. Mice in the treatment groups received 50 μL of the corresponding dose of CPP-AA intranasally, while mice in the control group received 50 μL of PBS intranasally. Lung tissue samples were collected from the mice 24 hours after model initiation.

[0043] 2.3 Construction of the mouse ulcerative colitis model DSS.

[0044] Mice were divided into a control group and a CPP-AA treatment group, with 4 mice in each group. Mice in the control group and the treatment group drank drinking water containing 3% DSS for 7 consecutive days, followed by drinking distilled water for 3 days. Mice in the CPP-AA treatment group were given 0.05 mg / kg CPP-AA by gavage on the day of DSS modeling, and gavage was performed every three days. Mice in the control group were given an equal volume of PBS by gavage. The weight, loose stools, and bloody stools of the mice were observed daily, and the weight and DAI score were recorded. After the modeling was completed, the colon tissue of the mice was collected for subsequent analysis.

[0045] 2.4 Construction of a mouse pulmonary fibrosis model.

[0046] After anesthetizing mice with ether, 50 uL of bleomycin (3 mg / kg) was instilled into the airway. After 24 hours, the mice were divided into a control group and a CPP-AA treatment group, with 4 mice in each group. The treatment group received 50 uL of 0.05 mg / kg CPP-AA via nasal instillation, while the control group received an equal volume of PBS via nasal instillation. The drugs were administered once every three days, and lung tissue samples were collected on the 7th day after modeling.

[0047] 2.5H&E staining.

[0048] Fresh lung tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 5 μm. The paraffin sections were dewaxed by placing them in xylene ① for 10 min, xylene ② for 10 min, 100% ethanol ① for 10 min, 100% ethanol ② for 10 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, and ddH2O for 1 min. The water on the slides was shaken off, and hematoxylin staining solution was added for 7 min. The staining solution was then rinsed with running water. The slides were then immersed in eosin staining solution for 20 s and rinsed with running water. After the slides were dried, they were mounted with neutral resin and images were acquired under a Leica inverted microscope.

[0049] 2.6 Masson staining.

[0050] Fresh lung tissue was fixed with 4% paraformaldehyde and frozen into sections with a thickness of 10 μm. After the sections were brought to room temperature, they were stained with prepared Weigert iron hematoxylin for 10 min, rinsed with running water for 2 min, rinsed with tap water for 10 min to regain blue color, and washed twice with distilled water. Gomori staining solution was added and stained for 40 min, followed by rinsing with running water. Gomori differentiation working solution was used for washing for 90 s. The sections were then rapidly dehydrated with 95% ethanol and dehydrated three times with anhydrous ethanol for 10 s each time. The sections were then soaked in xylene three times for 2 min each time, mounted with neutral resin, and images were acquired under a Leica inverted microscope.

[0051] 2.7β-galactosidase staining.

[0052] Fresh lung tissue was fixed with 4% paraformaldehyde and frozen into sections with a thickness of 10 μm. After thawing, the frozen sections were washed three times with PBS for 5 min each time. β-galactosidase staining fixative was added and the sections were fixed at room temperature for 15 min. The sections were washed three times with PBS for 5 min each time. An appropriate amount of staining working solution was added to cover the tissue, and the sections were incubated overnight in a humidified chamber at 37°C to prevent staining evaporation. The sections were washed with 70% ethanol to remove staining crystals from the tissue, and images were acquired under a Leica inverted microscope.

[0053] Experimental results

[0054] from Figure 2The study investigated the effect of CPP-AA treatment on pneumonia symptoms in mice. Compared with the control group, CPP-AA treatment significantly reduced the levels of inflammatory factors in lung tissue, significantly improved lung lesions, enhanced alveolar structure, and significantly reduced inflammatory cell infiltration in the alveolar cavity, indicating that CPP-AA can effectively improve inflammatory damage in the lungs.

[0055] from Figure 3 The study investigated the effects of CPP-AA treatment on the symptoms of ulcerative colitis in mice. Compared with the control group, the CPP-AA treatment group showed improvements in colon length, body weight, and DAI score, decreased levels of inflammatory factors in colonic tissue, upregulation of tight junction genes, more intact colonic epithelial tissue, and milder crypt and edema, indicating that CPP-AA can effectively alleviate ulcerative colitis in mice.

[0056] from Figure 4 The study investigated the effects of CPP-AA treatment on pulmonary fibrosis symptoms in mice. Compared to the control group, CPP-AA treatment significantly downregulated pulmonary fibrosis-related genes, markedly improved alveolar structure, reduced inflammatory cell infiltration and alveolar septal thickening, and decreased collagen fibers. Simultaneously, CPP-AA treatment significantly downregulated aging genes p16 and p21, and reduced β-galactosidase, indicating that CPP-AA can effectively inhibit pulmonary fibrosis in mice and downregulate aging indicators.

[0057] In summary, the therapeutic approach for inflammation and aging-related diseases presented in this invention has the following characteristics: 1. By targeting macrophages to regulate the activation of innate immune cells and reduce inflammatory responses, it has shown good efficacy for persistent low-level inflammation related to lung injury and aging for which there are currently no specific therapeutic drugs; 2. HBXIP improves lysosomal function related to aging by targeting lysosomal v-ATPase. This invention selects the core sequence of the functional region of HBXIP that specifically interacts with v-ATPase as the drug design target, which is a novel mechanism; 3. HBXIP has a dual effect of promoting macrophage lysosomal function and inhibiting mTORC1, and has a synergistic effect in promoting macrophage autophagy, anti-inflammation, and anti-aging pathways, possessing advantages that other single-function molecules do not have; 4. The expression level of HBXIP in autoimmune diseases, inflammatory diseases, and the elderly is lower than that in normal controls, indicating that quantifiable regulatory measures and drugs targeting this target have potential intervention and improvement effects; and selectively targeting macrophages can avoid the potential carcinogenic risk due to high expression of HBXIP in parenchymal cells;

[0058] In summary, the membrane-penetrating peptide targeting macrophages provided by this invention can effectively treat pneumonia, enteritis, pulmonary fibrosis, and reduce lung aging, thus it can be used to prepare immunomodulatory and anti-aging drugs. The above description is merely a preferred embodiment of this invention and does not impose any limitations on the embodiments. Simple improvements and modifications can be made based on the principles of this invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A macrophage-targeted polypeptide with cell membrane crossing function, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ NO.

1.

2. An isolated polynucleotide, characterized in that, It encodes the polypeptide described in claim 1.

3. An expression carrier, characterized in that, It contains the polynucleotide described in claim 2.

4. A recombinant cell, characterized in that, It contains the expression vector of claim 3 or its genome contains the polynucleotide of claim 2.

5. The use of the polypeptide of claim 1 in the preparation of a medicament for treating acute pneumonia, ulcerative colitis, and pulmonary fibrosis.

6. A method for preparing the polypeptide of claim 1, characterized in that, The method includes culturing the recombinant cells of claim 4 to achieve recombinant expression of a polypeptide with an amino acid sequence as shown in SEQ NO. 1; or, preparing a polypeptide with an amino acid sequence as shown in SEQ NO. 1 by in vitro artificial synthesis.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the polypeptide of claim 1, or the polynucleotide of claim 2, or the expression vector of claim 3, or the recombinant cell of claim 4; and a pharmaceutically or physiologically acceptable vector.

8. A kit for treating acute pneumonia, ulcerative colitis, and pulmonary fibrosis, characterized in that, The kit comprises the polypeptide of claim 1.

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