CN114081940B

CN114081940B - Application of calcitonin gene-related peptide in treating meningitis

Info

Publication number: CN114081940B
Application number: CN202210063349.4A
Authority: CN
Inventors: 韩根成; 李宇翔; 郝莹; 李葛; 王智鼎; 高振芳; 侯春梅; 陈金婕; 莫荣亮
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2022-01-20
Filing date: 2022-01-20
Publication date: 2022-04-12
Anticipated expiration: 2042-01-20
Also published as: CN114081940A

Abstract

The invention relates to application of calcitonin gene-related peptide in treating meningitis. The invention provides application of calcitonin gene-related peptide in preparing a medicament for treating meningitis, and also provides application of calcitonin gene-related peptide in preparing a medicament for resisting bacterial infection.

Description

Application of calcitonin gene-related peptide in treating meningitis

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to application of calcitonin gene-related peptide in treating meningitis.

Background

Meningitis (meningitis) refers to a diffuse inflammatory change caused by invasion of pia mater by various biological pathogenic factors such as bacteria, viruses, fungi and the like, and is a clinical common disease and frequently encountered disease in neurology. Meningitis is one of ten fatal diseases, causing about 135000 deaths each year. The most common clinical manifestations of meningitis are headache, stiffness of neck, fever, vomiting, seizures and changes in consciousness, etc., for example, inflammation invades brain parenchyma, and nervous system signs such as coma, convulsion, abnormal mental behavior, etc. may appear. Meningitis has complex etiology and rapid disease progression, and can cause serious consequences when the meningitis is not treated in time, so that irreversible nervous system injury is brought to patients, and even the patients are threatened to life.

Bacterial Meningitis (BM) is characterized by inflammation of the meninges and subarachnoid space. Streptococcus pneumoniae is one of the most common pathogenic bacteria, accounting for 50% of community-acquired encephalitis under 5 years of age in developed countries. Although vaccines and antibiotics aiming at streptococcus pneumoniae infection are continuously applied and upgraded, bacterial meningitis still has high morbidity and mortality, and meanwhile, about 30-50% of survivors have serious brain injury and nervous system sequelae including sensory-motor disorder, hearing disorder, blindness, epilepsy, cognitive dysfunction and the like, so that the life safety of people is greatly threatened, and the social and family burdens are caused. It is therefore of great importance to find an effective treatment to reduce the mortality rate of bacterial meningitis and the incidence of neurological sequelae.

Disclosure of Invention

Aiming at the existing defects, the invention aims to provide a medicament for preventing or treating meningitis. In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a polypeptide for use in the treatment of bacterial meningitis, including the amino acid sequence of CGRP, or fragments, analogues, derivatives thereof. Wherein said fragment, analog, derivative exhibits activity for the treatment of bacterial meningitis.

The term "analog" as used herein includes any peptide having an amino acid sequence substantially identical to the amino acid sequence of the CGRP described herein, wherein at least one residue is conservatively substituted with a functionally similar residue. An "analog" has 60% or more (preferably 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99%) of its amino acid sequence homologous to the CGRP amino acid sequence and is a functional variant thereof. "analogs" include variants of the CGRP amino acid sequence which have a homologous three-dimensional conformation. "analog" further includes any pharmaceutically acceptable salt of the analog described herein. "variant" further includes any pharmaceutically acceptable salt of the variants described herein.

As used herein, a "derivative" refers to a peptide of the invention having one or more amino acids that are chemically derivatized by reaction of functional side chain groups. Representative derivatized molecules include, but are not limited to, salts or amides derivatized by the addition of acetyl, amine hydrochloride, benzyloxycarbonyl, chloroacetyl, formyl, p-toluenesulfonyl, or t-butoxycarbonyl groups, free amino groups in the peptide molecule. The free hydroxyl group can be derivatized to form an oxyacetyl or oxyalkyl derivative. Furthermore, free carboxyl groups can be derivatized to form salts, esters (e.g., methyl and ethyl esters) or hydrazides. Thus "derivative" further includes any pharmaceutically acceptable salt of the derivative described herein.

As a preferred embodiment, the polypeptide comprises the sequence shown in SEQ ID NO. 1.

In a second aspect, the invention provides a fusion protein comprising a polypeptide according to the first aspect of the invention. In a particular embodiment of the invention, the fusion protein further comprises an FC protein.

In a third aspect, the invention provides a nucleic acid molecule encoding a polypeptide according to the first aspect of the invention, or a fusion protein according to the second aspect of the invention.

The nucleic acid molecule of the invention may be DNA or RNA. They can be prepared by a variety of techniques known to those skilled in the art, including, but not limited to, automated oligonucleotide synthesis using a commercially available oligonucleotide synthesizer, such as an applied biosystems model392DNA/RNA synthesizer. In addition, the nucleic acid molecules of the invention may be labeled with one or more detectable labels or tags. Labeling of nucleic acid molecules can be accomplished by one of many methods known in the art, such as nick translation, end labeling, end-fill labeling, polynucleotide kinase exchange reactions, random primers, or SP6 polymerase (used to make RNA probes), as well as one of a variety of labels, for example, radioactive labels such as 35S, 32P, or 3H, or non-radioactive labels such as biotin, Fluorescein (FITC), acridine, cholesterol, or carboxy-X-Rhodamine (ROX).

In a fourth aspect, the present invention provides an expression vector comprising a nucleic acid molecule according to the third aspect of the invention.

As used herein, an "expression vector" is a DNA construct comprising a DNA sequence operably linked to a suitable control sequence capable of effecting the expression of the DNA in a suitable host. The vector may be, for example, a plasmid, a phage particle, or a gene insert. The term "operably linked" as further used herein describes a functional relationship between two DNA regions. Expression vectors suitable for use in the present invention include at least one expression control element (e.g., an operator, a promoter, a lac system, a leader sequence, a stop codon, and/or a polyadenylation signal) operably linked to a nucleic acid molecule encoding a peptide of the present invention. In one embodiment, the expression vector is a eukaryotic expression vector (e.g., a retroviral vector, a vaccinia viral vector, an adenoviral vector, a herpes viral vector, or a fowlpox viral vector) that functions in a eukaryotic cell.

Once operably linked to the nucleic acid molecule of the invention, the expression vector can be introduced into a recipient cell by any in vivo or in vitro method suitable for nucleic acid transfer, including but not limited to electroporation, DEAE dextran transfection, calcium phosphate transfection, lipofection, single cation liposome fusion, multi-cation liposome fusion, protoplast fusion, establishing an in vivo electric field, bombardment with DNA-encapsulated microparticles, injection of recombinant replication-defective viruses, homologous recombination, viral vectors, naked DNA transfer, or any combination thereof. Recombinant viral vectors suitable for nucleic acid transfer include, but are not limited to: vectors derived from viral genomes such as retrovirus, herpes simplex virus, adenovirus, adeno-associated virus, Semilikikifort virus, cytomegalovirus, vaccinia virus, and the like.

In a fifth aspect, the invention provides a cell comprising an expression vector according to the fourth aspect of the invention.

In the invention, the cell comprises a prokaryotic cell and a eukaryotic cell.

In a preferred embodiment, the prokaryotic cell comprises a bacterial cell.

As a preferred embodiment, the eukaryotic cell includes a protist cell, an animal cell, a plant cell, a fungal cell. As a more preferred embodiment, the animal cell includes mammalian cell, avian cell, insect cell.

In a sixth aspect, the invention provides a composition comprising a polypeptide according to the first aspect of the invention, or a fusion protein according to the second aspect of the invention.

As a preferred embodiment, the composition further comprises a pharmaceutically acceptable carrier, buffer or excipient.

By "pharmaceutically acceptable" is meant a non-toxic material that does not detract from the active ingredient. Such pharmaceutically acceptable buffers, carriers or Excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18 th edition, A.R Gennaro, eds., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3 rd edition, A.Kibbe eds., Pharmaceutical Press (2000)).

The term "buffer" is intended to mean an aqueous solution containing an acid-base mixture with the aim of stabilizing the pH. Examples of buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPSO, imidazole lactate, PIPES, SSPE, POPSO, TAPS, TABS, TAPSO and TES.

The carriers of the present invention include antimicrobial agents, isotonic agents, antioxidants, local anesthetics, suspending agents, dispersing agents, emulsifying agents, chelating agents, thickening agents, or solubilizing agents.

The excipient may be one or more of the following: carbohydrates, polymers, lipids, and minerals. Examples of carbohydrates include lactose, sucrose, mannitol, and cyclodextrins, which are added to the composition, for example, to facilitate lyophilization. Examples of polymers are starch, cellulose ethers, cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, ethyl cellulose, methyl cellulose, propyl cellulose, alginates (alginates), carrageenans (carageenans), hyaluronic acid and its derivatives, polyacrylic acid, polysulfonates (polysulfonates), polyethylene glycol/polyethylene oxide, polyethylene oxide/polypropylene oxide copolymers, polyvinyl alcohol/polyvinyl acetate, poly (lactic acid), poly (glycolic acid) or copolymers thereof with various compositions, and polyvinylpyrrolidone (all of different molecular weights) which are added to the composition, for example to control viscosity, to achieve bio-adhesion, or to protect the active ingredient from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, mono-, di-and triglycerides, ceramides, sphingolipids and glycolipids (all with different acyl chain lengths and saturations), lecithin (eg lecithin), soy lecithin, hydrogenated lecithin and soy lecithin, which are added to the composition for similar reasons as the polymers. Examples of minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain benefits such as reduced liquid accumulation or favorable pigment properties.

A seventh aspect of the invention provides the use of any one of:

(1) use of a polypeptide according to the first aspect of the invention in the manufacture of a medicament for the prevention or treatment of meningitis;

(2) the use of a fusion protein according to the second aspect of the invention in the manufacture of a medicament for the prevention or treatment of meningitis;

(3) use of a nucleic acid molecule according to the third aspect of the invention in the manufacture of a medicament for the prevention or treatment of meningitis;

(4) the use of an expression vector according to the fourth aspect of the invention in the manufacture of a medicament for the prevention or treatment of meningitis;

(5) use of a cell according to the fifth aspect of the invention in the manufacture of a medicament for the prevention or treatment of meningitis;

(6) use of a composition according to the sixth aspect of the invention in the manufacture of a medicament for the prevention or treatment of meningitis;

(7) the use of a polypeptide according to the first aspect of the invention in the manufacture of a medicament for the treatment of a bacterial infection or a disease associated with a bacterial infection;

(8) the use of a fusion protein according to the second aspect of the invention in the manufacture of a medicament for the treatment of a bacterial infection or a disease associated with a bacterial infection;

(9) use of a nucleic acid molecule according to the third aspect of the invention in the manufacture of a medicament for combating or relating to bacterial infection;

(10) the use of an expression vector according to the fourth aspect of the invention in the preparation of a medicament against bacterial infection or a disease associated with bacterial infection;

(11) use of a cell according to the fifth aspect of the invention in the manufacture of a medicament for the treatment of a bacterial infection or a disease associated with a bacterial infection;

(12) the use of a composition according to the sixth aspect of the invention in the manufacture of a medicament for the treatment of a bacterial infection or a disease associated with a bacterial infection;

(13) use of a polypeptide according to the first aspect of the invention in the manufacture of a medicament for the reduction of inflammatory factors;

(14) the use of a fusion protein according to the second aspect of the invention in the preparation of a medicament for reducing inflammatory factors;

(15) use of a nucleic acid molecule according to the third aspect of the invention in the manufacture of a medicament for reducing an inflammatory factor;

(16) the use of an expression vector according to the fourth aspect of the invention in the preparation of a medicament for the reduction of inflammatory factors;

(17) use of a cell according to the fifth aspect of the invention in the preparation of a medicament for reducing inflammatory factors;

(18) use of a composition according to the sixth aspect of the invention in the manufacture of a medicament for the reduction of inflammatory factors.

In the present invention, the meningitis includes bacterial meningitis, viral meningitis, tubercular meningitis, cryptococcal meningitis. In a preferred embodiment, the meningitis is bacterial meningitis.

As a preferred embodiment, the medicament may be administered orally, parenterally, transdermally, intranasally, topically, pulmonary, or by osmotic pumps.

The polypeptides of the invention, or nucleic acid sequences encoding the polypeptides, as disclosed herein, can be administered to a human or animal subject by known methods, including, but not limited to, oral administration, parenteral administration (e.g., suprafascial, intracapsular, intradermal, intramuscular injection, intraorbital, intraperitoneal, intraspinal, intrasternal, vascular, intravenous, parenchymal, or subcutaneous administration), transdermal absorption, nasal administration, pulmonary administration (e.g., intratracheal administration), and osmotic pump administration. In one embodiment, the method of administration is by intraperitoneal injection, parenteral administration.

For oral administration, the formulation of the peptide (or nucleic acid encoding the peptide) may be a capsule, tablet, powder, granule, or suspension or solution. The formulation may have conventional additives such as lactose, mannitol, corn starch or potato starch. The formulations may also contain binding agents, for example microcrystalline cellulose, cellulose derivatives, acacia, corn starch or gelatin. In addition, the formulations may contain disintegrating agents, such as corn starch, potato starch or sodium carboxymethyl cellulose. The preparation may further contain anhydrous calcium hydrogen phosphate or sodium carboxymethyl starch. Finally, the formulations may contain lubricating agents, such as talc or magnesium stearate.

For parenteral administration, the peptide (or nucleic acid encoding the peptide) may be combined with a sterile aqueous solution, preferably one that is isotonic with the blood of the subject. Such a formulation may be prepared by: the solid active ingredient is dissolved in a solution containing a physiologically compatible substance, such as sodium chloride, glycine and the like, and made into an aqueous solution in water having a buffered pH value compatible with physiological conditions, and then the solution is sterilized. The formulations may be presented in unit or multi-dose containers, for example sealed ampoules or vials. The formulations may also be administered by any injection means, including any of those described herein.

For transdermal administration, the peptide (or nucleic acid encoding the peptide) may be combined with a transdermal enhancer such as propylene glycol, polyethylene glycol, isopropyl alcohol, ethanol, oleic acid, N-methylpyrrolidone, and the like, which increases the skin permeability of the peptide or nucleic acid, allowing the peptide or nucleic acid to penetrate the skin into the blood. The composition of the promoter and the peptide or nucleic acid can be further combined with a polymer such as ethyl cellulose, hydroxypropyl cellulose, ethylene/vinyl acetate, polyvinylpyrrolidone, etc. to obtain a gel composition. Dissolving the gel composition in solvent such as dichloromethane, evaporating to desired viscosity, and coating on backing material to obtain patch. The peptide or nucleic acid may be administered transdermally at or near the site of infection in a subject. Alternatively, the peptide or nucleic acid may be administered transdermally at a site other than the affected area, thereby achieving systemic administration.

For nasal (e.g., nasal spray) and/or pulmonary (e.g., inhalation) administration, formulations of peptides or nucleic acids, including aerosols, can be prepared according to methods well known to those skilled in the art. The aerosol may comprise either solid particles or a solution (aqueous or non-aqueous). Nebulizers (e.g., jet nebulizers, ultrasonic nebulizers, etc.) and nebulizers may be used to produce aerosols from solutions (e.g., using solvents such as ethanol), metered dose inhalers, and dry powder inhalers may be used to produce aerosols of small particles. The desired aerosol particle size may be achieved by any of several methods known in the art, including but not limited to jet milling, spray drying and critical point condensation.

Pharmaceutical compositions for nasal administration may be in solid form (e.g. as a meal) and may contain excipients such as lactose. For solid dosage form administration, the container containing the powder is lifted to the nose and rapidly inhaled through the nasal passages. Compositions for nasal administration may also include aqueous or oily solutions of nasal sprays or nasal drops. For use with a nebulizer, the formulation of the peptide or nucleic acid may contain an aqueous solution and additives including, for example, an excipient, a buffer, an isotonic agent, a preservative, or a surfactant. Nasal sprays can be produced, for example, by passing a suspension or solution of the peptide or nucleic acid through a nozzle under pressure.

Formulations of the peptides or nucleic acids for pulmonary administration may be presented in a form suitable for administration by inhalation devices and may have particles of a size effective to reach the lower respiratory tract or sinuses of the lungs. For absorption through mucosal surfaces, including the pulmonary mucosa, the formulations of the present invention may include emulsions comprising, for example, bioactive peptides, a plurality of submicron particles, an adhesive polymer, and/or an aqueous continuous phase. Absorption via mucosal surfaces can be achieved by adhesion of emulsion particles.

Pharmaceutical compositions for use in metered dose inhalation devices may comprise fine powders containing peptides or nucleic acids, formulated as suspensions in non-aqueous media. For example, the peptide or nucleic acid may be suspended in the propellant with the aid of a surfactant such as sorbitan trioleate, soya lecithin or oleic acid. Metered dose inhalers typically use a propellant gas (e.g., a chlorofluorocarbon, hydrochlorofluorocarbon, hydrofluorocarbon, or hydrocarbon) stored in a container (e.g., a canister) as a mixture (e.g., as a liquefied compressed gas). The inhaler needs to be actuated during inhalation. For example, the metering valve actuation may release the mixture as an aerosol. Dry powder inhalers employ a breath-actuated mixing of powders.

The peptides or nucleic acids of the invention may also be released or administered from an osmotic micropump or other timed release device. The release rate of the primary osmotic pump may be adjusted by a microporous fast response gel disposed within the release orifice. The osmotic pressure micropump can be used for controlled drug release or targeted drug delivery of peptides or nucleic acids.

According to the methods described herein, the peptides of the invention can be administered to a subject by administering the peptide itself to the subject, or by administering to the subject a nucleic acid encoding the peptide in a manner that allows expression of the peptide. Thus, in one embodiment of the invention, the treatment or prevention of infection in a subject may be achieved by administering to the subject an amount of a peptide of the invention. In another embodiment of the invention, the treatment or prevention of infection in a subject may be by administering to the subject a nucleic acid sequence encoding a peptide of the invention in a manner that allows expression of the peptide in the subject.

The peptides of the invention may be administered or administered to a subject by known techniques for introducing peptides and other drugs, including, for example, injection and blood transfusion. Local infection of a particular part of a subject desirably involves introducing the therapeutic peptide directly into the area by injection or some other means, such as by introducing the peptide into the blood or other body fluid. The amount of peptide used is that amount which is effective to treat and/or prevent infection in a subject, as defined above, and which is readily determinable by the skilled artisan.

In the methods of the invention, the peptide may also be administered or introduced into a subject by introducing into a sufficient number of cells of the subject a nucleic acid encoding the peptide in a manner that allows expression of the peptide. The amount of nucleic acid encoding the therapeutic peptide is such that an effective amount of the peptide, as defined above, is produced to treat and/or prevent infection in the subject. The skilled artisan can readily determine this amount.

As used herein, a "subject" is an avian species (e.g., chicken, turkey, etc.) or a mammalian species (e.g., cow, dog, human, monkey, mouse, pig, rat, etc.). In one embodiment, the subject is a mouse. The subject may be infected, or at risk of infection. For example, the infection may be a microbial infection. Microbial infections that can be treated by the methods of the present invention include, but are not limited to, bacterial infections, fungal infections, and viral infections.

The medicament of the invention can be used for resisting bacterial infection. The type of the bacterium is not particularly limited. Examples of possible bacteria include, but are not limited to, the following: haemophilus influenzae, Moraxella catarrhalis, Chlamydia pneumoniae, Chlamydia trachomatis, Actinobacillus hemolyticus, Pasteurella koreana, Haemophilus ducreyi, Treponema pallidum, Diplococcus gonorrhoeae, helicobacter pylori, Leptospira rouxii, Borrelia burgdorferi, Campylobacter jejuni, Bacteroides, Bordetella pertussis, Staphylococcus aureus, coagulase-negative staphylococci, enterococcus faecalis, enterococcus faecium, enterococcus casseliflavus, enterococcus durans, Peptostreptococcus, Streptococcus pneumoniae, Streptococcus pyogenes, group A to G streptococci, Streptococcus agalactiae, Streptococcus viridis, Corynebacterium parvum, Mycobacterium microtuberculi, Mycoplasma urealyticum, Listeria, Mycoplasma pneumoniae, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium paratuberculosis, Mycobacterium tuberculosis, Mycobacterium kansasii, Mycobacterium terrapin, Cryptosporidium, Clostridium and/or Clostridium perfringens.

In a particular embodiment of the invention, the bacterium is streptococcus pneumoniae.

In the present invention, the diseases associated with bacterial infection include, but are not limited to, tuberculosis, bacillary dysentery, anematous fever, septicemia, brucellosis, bacterial food poisoning, epidemic cerebrospinal meningitis, bacterial infectious diarrhea, infectious endocarditis, rheumatoid arthritis, bronchiectasis, chronic bronchitis, superficial pyogenic infection of the skin, empyema, acute and chronic appendicitis, bacterial meningitis, anthrax, plague, brucellosis, cholera, epidemic cerebrospinal meningitis, scarlet fever, and pertussis.

Drawings

FIG. 1 is a graph of analysis of body weight, clinical score and survival rate after CGRP treatment of bacterial meningitis mice, wherein graph A is a graph of analysis of body weight, graph B is a graph of analysis of clinical score and graph C is a graph of analysis of survival rate;

FIG. 2 is a graph showing the analysis of bacterial load and inflammatory factor detection in CGRP-treated mice with bacterial meningitis, wherein A is a graph showing the analysis of bacterial load in brain tissue, B is a graph showing the analysis of IL-1. beta. in brain tissue, C is a graph showing the analysis of TNF-. alpha. in brain tissue, D is a graph showing the analysis of IL-6 in brain tissue, E is a graph showing the analysis of bacterial load in spleen tissue, F is a graph showing the analysis of IL-1. beta. in spleen tissue, G is a graph showing the analysis of TNF-. alpha. in spleen tissue, and H is a graph showing the analysis of IL-6 in spleen tissue;

FIG. 3 is a graph of pathological analysis of posterior meningococci in CGRP-treated bacterial meningitis mice, where Panel A is a graph of results obtained under 10-fold microscopy in the Sham group, Panel B is a graph of results obtained under 400-fold microscopy in the Sham group, Panel C is a graph of results obtained under 10-fold microscopy in the SP + Fc group, Panel D is a graph of results obtained under 400-fold microscopy in the SP + Fc group, Panel E is a graph of results obtained under 10-fold microscopy in the SP + CGRP-Fc group, and Panel F is a graph of results obtained under 400-fold microscopy in the SP + CGRP-Fc group;

fig. 4 is a graph of water maze experimental analysis after CGRP treatment of bacterial meningitis mice, wherein a is a statistical graph of escape distances of mice on day 7 of recovery, B is a statistical graph of escape distances of mice on day 14 of recovery, C is a statistical graph of escape distances of mice on day 28 of recovery, D is a statistical graph of escape latencies of mice on day 7 of recovery, E is a statistical graph of escape latencies of mice on day 14 of recovery, F is a statistical graph of escape latencies of mice on day 28 of recovery, G is a graph of Sham group escape routes, H is a graph of SP + Fc group escape routes, and I is a graph of SP + CGRP-Fc group escape routes.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. Those of ordinary skill in the art will understand that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.

The main instrument reagents and materials used in the invention are as follows:

1. instrument for measuring the position of a moving object

25 μL microsampler (Shanghai' an pavilion microsampler factory), 5 mL syringe, foam board, electronic weight scale (Beijing Kaiyouchun Biotech Co., Ltd.), constant temperature shaking table THZ-C (Suzhou Peiyin laboratory facilities Co., Ltd.), low speed centrifuge (Thermo Co.), clean bench (Qingdao Haier Co., Ltd.), biosafety cabinet (Qingdao Haier Co., Ltd.), CO₂Incubator (Thermo corporation), LightCycler480 II (Roche), Morris Water maze video analysis System (Tech scientific development, Inc., Tokyo Zhongdi).

2. Reagent

THB medium (Tech Co., Ltd., Beijing Sorboard technologies), sheep blood (Tech Co., Ltd., Beijing Kannuochun Biotech Co., Ltd.), sheep blood agar plate (Tech Co., Ltd., Beijing Kannuochun Biotech Co., Ltd.), normal saline for injection (national chemical reagents Co., Ltd.), TRIzol (Invitrogen), First Strand cDNA synthesis kit (Beijing King Kogyo Biotech Co., Ltd.); the CGRP-Fc protein and the control Fc protein are obtained by the laboratory through expression according to a conventional method, wherein the Fc protein is a full-length sequence, and the amino acid sequence of the CGRP is CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP (SEQ ID NO. 1).

3. Bacteria and laboratory mice

Streptococcus Pneumoniae (SP) was purchased from Baiohbowei Biotech Co., Ltd, Beijing, and 6-8 week male C57BL/6 mice were purchased from Sibefu Biotechnology Co., Ltd.

The experimental method used in the present invention is as follows:

1. preparation of bacterial suspensions

Streptococcus pneumoniae was inoculated in sheep blood agar medium at 37 deg.C and 5% CO₂Culturing overnight in a constant temperature incubator, taking a single colony to inoculate in 5 mL broth culture medium containing 5% sheep blood, shaking the bacteria for 16 h at 37 ℃ at 220 r/min, taking out 1mL bacterial liquid, adding 4 mL broth culture medium containing 5% sheep blood, shaking the bacteria for 16 h at 37 ℃ at 220 r/min. Centrifuging 1mL bacterial liquid (10000r/min), washing with physiological saline, centrifuging again, diluting bacterial liquid to required 6 × 10⁶ CFU/mL。

2. Bacterial meningitis model and construction

Self-made mouse fixing plate, fixing a 10 mL injector on the plate, making head and trunk of mouse at 120 deg, exposing large hole of occipital bone, injecting 10uL (6 × 10) through medulla oblongata pond⁶CFU/mL) Streptococcus pneumoniae method to construct a Streptococcus pneumoniae meningitis mouse model. Mice were evaluated for clinical score (according to evaluation criteria 1 of Diederik van de Beek), survival curve, and weight change.

3. Design of experiments

Experimental mice were divided into 3 groups: sham group as a Sham group (10 uL of saline was injected through the medullary canal to exclude the effects of brain damage caused by surgical procedures); the SP + Fc group was given control Fc protein treatment for a streptococcus pneumoniae meningitis mouse model; the SP + CGRP-Fc group was treated with the CGRP-Fc protein for a mouse model of Streptococcus pneumoniae meningitis. The SP + Fc group and SP + CGRP-Fc group were specifically performed by intraperitoneal injection of 7.5. mu.g/200. mu.L of Fc protein or 7.5. mu.g/200. mu.L of CGRP-Fc protein at 0.5h before modeling, 24 h and 48 h after modeling, respectively, and then the endpoints were observed.

4. Mouse survival curve, observation of body constitution

Body mass and time to death were recorded daily after cisterna bulbifera injection.

5. Mouse clinical score index (see Table 1)

The clinical course is divided into prophase (from inoculation time to clinical score of less than or equal to 10 points) and symptomatic phase (from clinical score of 10-15 points until death).

6. Mouse brain and spleen tissue bacterial load and inflammatory factor detection

On day 5 after modeling, mice were sacrificed, brain and spleen tissues were collected, cellular RNA was extracted, reverse transcription was performed, and the levels of bacteria and related inflammatory factor mRNA in mouse brain and spleen tissues were detected by RT-qPCR method.

7. Pathological analysis of mouse brain tissue

On day 5 after modeling, after cardiac perfusion was performed with anesthesia on the mice, brain tissue of the mice was carefully dissected out to make paraffin sections. The compound is subjected to xylene dewaxing, hydration, hematoxylin dyeing, differentiation, eosin dyeing, dehydration, transparent sealing, observation under an optical microscope and photographing and preservation.

8. Mouse Morris Water maze assay

The experimental training phase was continued for 4 days with 1 training session per day. During training, a mouse is placed into the water pool from four water inlet points facing the pool wall, the time from the water inlet of the mouse to the time of finding an underwater hidden platform and standing on the platform is recorded, the latency is expressed in seconds(s), and the mouse stands on the platform for 10 s after finding the platform. If the platform could not be found in the 60 s mice after entering the water, the mice were gently pulled from the water onto the platform and left for 10 s before the next training. Each mouse is respectively put into the water pool from four water inlet points for one training. And (5) carrying out water maze detection on the mice on the 5 th day, and removing the mice which can not find the platform. Mice were then modeled and water maze tests were performed on days 7, 14 and 28 after the end of the endpoint (12 days modeled) observation, respectively.

Example 1 Effect of CGRP on body weight, clinical score, survival Rate in mice with Streptococcus pneumoniae meningitis

Body mass, clinical scores and time to death were recorded daily for 3 groups of mice (Sham, SP + Fc, SP + CGRP-Fc). The results are shown in FIG. 1, and A-C in FIG. 1 show: compared with the Sham group of mice, meningitis onset is shown in the SP + Fc group and the SP + CGRP-Fc group of mice. CGRP-Fc treatment was able to significantly restore body weight in mice with streptococcus pneumoniae meningitis (a in fig. 1), improve clinical meningitis scores in mice (B in fig. 1), and increase survival rates in mice (C in fig. 1) compared to the Fc control protein treated group.

Example 2 Effect of CGRP on bacterial load and inflammatory factor expression in mice with Streptococcus pneumoniae meningitis

On day 5 after modeling, mouse brain and spleen tissues were collected and levels of bacteria and related inflammatory factor mRNA in mouse brain and spleen tissues were detected by RT-qPCR method. The results are shown in FIG. 2, and A-D in FIG. 2 show: compared with the Sham group, the SP + Fc group mice have obviously increased brain tissue bacteria and inflammation factor mRNA expression, and the CGRP-Fc protein treatment can reduce the brain tissue bacteria and inflammation factor mRNA expression of the streptococcus pneumoniae meningitis mice. Further comparing the difference of the expression of the bacteria and the mRNA of the inflammatory factor in the spleen tissue of the mice, the results are shown in E-H in FIG. 2, compared with the Sham group, the expression of the bacteria and the mRNA of the inflammatory factor in the spleen tissue of the mice with the SP + Fc group is obviously improved, and the expression of the bacteria and the mRNA of the inflammatory factor in the spleen tissue of the mice with the streptococcus pneumoniae meningitis can be reduced when the CGRP-Fc protein is treated.

Example 3 Effect of CGRP on meningeal injury and inflammatory cell infiltration in Streptococcus pneumoniae meningitis mice

On the 5 th day after modeling, all surviving mice were decapitated and brains were collected after abdominal anesthesia and cardiac perfusion, brain tissues were made into paraffin sections, hematoxylin-eosin staining was performed, and the severity of the streptococcus pneumoniae meningitis pathological tissues was observed under an optical microscope and photographed for storage. The results are shown in FIG. 3: compared with the Sham group, the brain tissue surface of the mice after the streptococcus pneumoniae infection is obviously congested and edematous, and the congestion and the oedema are scattered at a bleeding point and are accompanied with infiltration of red blood cells and inflammatory cells. And the treatment of CGRP-Fc can obviously reduce meningeal injury and inflammatory cell infiltration of a streptococcus pneumoniae meningitis mouse.

Example 4 Effect of CGRP on cognitive Performance in mice with Streptococcus pneumoniae meningitis

Mice were allowed to recover for one month after finishing the observation (12 days of modeling) and three groups of mice were subjected to Morris water maze test on days 7, 14 and 28 of recovery, respectively. The results are shown in FIG. 4: the mice with S.pneumoniae infection D had increased escape distance (A-C in FIG. 4), prolonged escape latency (D-F in FIG. 4), complex escape route (G-I in FIG. 4) at 7, 14 and 28 days of recovery as compared to Sham group mice, and the mice were all recovered to varying degrees after CGRP-Fc treatment.

The experiment proves that: the CGRP can obviously recover the weight of a meningitis mouse, improve the clinical score of the mouse, improve the survival rate of the mouse, reduce the inflammatory factor storm and meningeal injury caused by meningitis and improve the cognitive ability of the mouse. The results show that CGRP has definite antibacterial infection effect. The research result of the invention provides a new method and thought for clinically treating meningitis.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Sequence listing

<110> military medical research institute of military science institute of people's liberation force of China

<120> application of calcitonin gene related peptide in treating meningitis

<141> 2022-01-13

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 32

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Cys Gly Asn Leu Ser Thr Cys Met Leu Gly Thr Tyr Thr Gln Asp Phe

1 5 10 15

Asn Lys Phe His Thr Phe Pro Gln Thr Ala Ile Gly Val Gly Ala Pro

20 25 30

Claims

1. The application of the polypeptide in preparing the medicine for treating the bacterial meningitis is characterized in that the polypeptide is CGRP, and the sequence of the polypeptide is shown as SEQ ID No. 1.

2. Use of a fusion protein obtained by fusing the polypeptide of claim 1 to FC protein in the manufacture of a medicament for the treatment of bacterial meningitis.

3. Use of a nucleic acid molecule encoding a polypeptide according to claim 1 or a fusion protein according to claim 2 in the manufacture of a medicament for the treatment of bacterial meningitis.

4. Use of an expression vector in the manufacture of a medicament for the treatment of bacterial meningitis, wherein said expression vector comprises the nucleic acid molecule of claim 3.

5. Use of a cell comprising the expression vector of claim 4 in the manufacture of a medicament for the treatment of bacterial meningitis.

6. Use of a composition comprising the polypeptide of claim 1 or the fusion protein of claim 2 in the preparation of a medicament for treating meningitis, said composition further comprising a pharmaceutically acceptable carrier, buffer or excipient.