CN115671140B - Application of propionibacterium acnes in preparation of medicines for treating nasal polyp - Google Patents
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Abstract
The invention discloses an application of propionibacterium acnes in preparing a medicine for treating nasal polyps, wherein a medicinal preparation for treating nasal polyps consists of an effective amount of propionibacterium acnes and pharmaceutically acceptable auxiliary materials. The content of the propionibacterium acnes in the pharmaceutical preparation is 10 6 ‑10 8 CFU/mL. The propionibacterium acnes is symbiotic bacteria, and is relatively safe and easy to obtain, so that the treatment cost of a nasal polyp patient can be greatly reduced, and a new treatment choice is provided for the treatment of the nasal polyp; the medicinal preparation for treating nasal polyp is nasal spray/nasal drops/flushing agent, has simple and convenient administration route, is easy to operate and is convenient for patients to use.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to application of propionibacterium acnes in preparing a medicine for treating nasal polyp.
Background
Chronic sinusitis with nasal polyps (chronic rhinosinusitis with nasal polyps, CRSwNP) is a chronic inflammatory disease that occurs mainly in the mucosa of the sinuses and is accompanied by the formation of polyps. The prevalence rate is high in the world and is as high as 2% -4% in China; CRSwNP not only significantly affects the quality of life of patients, consumes a large amount of medical and health resources, but also affects the occurrence and prognosis of chronic diseases such as asthma, chronic obstructive pulmonary disease and the like, and causes great burden to the nation and the patients. However, CRSwNP has heretofore remained a severely underestimated, undertreated and poorly treated disease. One of the important reasons is that CRSwNP endoprosthesis has a high degree of heterogeneity and CRSwNP of different ethnicities, countries or even regions have different immunopathological characteristics. However, both domestic and foreign studies show that nasal polyp eosinophilic inflammation is a key risk factor for poor prognosis in CRSwNP patients. For type 2 cytokines and eosinophilic inflammation in nasal polyps, a number of phase II or phase III clinical studies have shown in recent years that blocking type 2 cytokines, including anti-IL-5R, anti-IL-4rα and anti-IgE therapies, has encouraging clinical efficacy in the treatment of poorly responsive, type 2 inflammatory dominant eosinophilic CRSwNP with glucocorticoids; these targeted biologies can significantly reduce polyp volume, improve clinical symptoms, and reduce the chance of re-surgery. However, it is notable that even with these type 2 cytokine-targeted biologics, 40% -60% of patients still have nasal polyp volumes or nasal congestion symptoms that cannot be significantly improved. This suggests that there is a need to further develop an action target or drug against the formation of eosinophilic inflammation of nasal polyps.
Bacteria play a key role in host innate and adaptive immune system training and development; imbalance in bacterial composition is involved in the development of a variety of immune-mediated diseases, such as: inflammatory bowel disease, rheumatoid arthritis, malignant tumor, and the like. The nasal mucosa is the first line of defense of the respiratory tract against the outside world, and the surface colonises with a considerable amount of bacteria. However, previous studies based on conventional bacterial culture techniques have not led to a clear and uniform conclusion on the role of bacteria in the pathogenesis of CRSwNP.
At present, no report of a medicine for treating nasal polyp by utilizing bacteria exists.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides application of propionibacterium acnes in preparing medicines for treating nasal polyps.
In order to achieve the purpose, the invention designs an application of the acne propionibacterium in preparing a medicine for treating nasal polyp.
The invention also provides a pharmaceutical preparation for treating nasal polyp, which consists of effective amount of acnes propionici and pharmaceutically acceptable auxiliary materials.
Further, the auxiliary material is any one of normal saline, glucose, vitamin C and amino acid.
Still further, the pharmaceutical formulation is a nasal drop or spray formulation or a rinse.
Still further, the spray formulation is any one of an atomizer, a spray and a suspension.
Still further, the content of Propionibacterium acnes in the pharmaceutical preparation was 10 6 -10 8 CFU/mL。
Still further, the content of Propionibacterium acnes in the pharmaceutical preparation was 10 7 CFU/mL。
The principle of the invention is as follows:
1. the invention adopts a high-throughput sequencing technology in the early stage to compare the difference of the bacterial groups on the mucous membrane of the nasal meatus/nasal polyp surface in eosinophilic CRSwNP patients, non-eosinophilic CRSwNP patients and normal control groups. We found that the abundance of propionibacterium acnes in actinomycota was significantly reduced in eosinophilic CRSwNP relative to normal control, but not significantly altered in non-eosinophilic CRSwNP. Acne propionibacteria are gram-positive anaerobic cocci that often colonize the skin, mouth, gastrointestinal tract, and genitourinary tract. Previous researches show that the propionibacterium acnes is an important symbiotic bacterium for skin and is also a conditional pathogenic bacterium, and is closely related to the pathogenesis of acne. The propionibacterium acnes can be detected in 90% of nasal specimens, and the relative abundance of the propionibacterium acnes in nearly 40% of nasal specimens is greater than 1%, which suggests that the propionibacterium acnes are also an important component of nasal bacterial groups, and further the relative abundance of the propionibacterium acnes is found to have correlation with eosinophilic inflammation in nasal polyps, and in vitro cell experiments and in vivo animal experiments prove that the propionibacterium acnes can inhibit eosinophilic inflammation, so that the method has great guiding significance for further researching the propionibacterium acnes to treat nasal polyps.
2. The invention provides the potential treatment effect of the acnes propionics and the main active components thereof on the CRSwNP eosinophil inflammation on the basis of the combination of the early histopathology, high throughput sequencing, cell biology, molecular biology, chemistry and spectrum identification technology with in vitro cell culture and in vivo animal experiments, so that the acnes propionics can be proved to be capable of preparing medicines for treating nasal polyps.
The invention has the beneficial effects that:
1. the propionibacterium acnes is symbiotic bacteria, and is relatively safe and easy to obtain, so that the treatment cost of a nasal polyp patient can be greatly reduced, and a new treatment choice is provided for the treatment of the nasal polyp;
2. the medicinal preparation for treating nasal polyp is nasal spray/nasal drops/flushing agent, has simple and convenient administration route, is easy to operate and is convenient for patients to use.
Drawings
FIG. 1 is a graph of the population composition of bacteria on the middle nasal meatus mucosa/nasal polyp surface and the abundance level of Propionibacterium acnes in normal control and nasal polyp patients;
in the figure, a:16sRNA sequencing data; b: q-PCR data validated by another independent queue;
FIG. 2 is a graph showing the negative correlation of the abundance of Propionibacterium acnes in patients with CRSwNP and the IL-5 and CCL11 (eotaxin-1) levels in nasal polyp tissues
FIG. 3 is an expression pattern of nasal mucosa epithelial cells CCL11 and CCL24 in both the basal and IL-13 stimulated states of live Propionibacterium acnes (panels A, B) and inactivated Propionibacterium acnes (panels C, D)
FIG. 4 is a graph showing that the culture supernatant of Propionibacterium acnes failed to inhibit the production of nasal mucosal epithelial cells CCL11 and CCL 24.
FIG. 5 is a graph showing that inactivated Staphylococcus aureus is unable to regulate the expression of CCL11 and CCL24 mRNA in nasal mucosal epithelial cells;
FIG. 6 is a diagram of a BALB/c mouse modeling protocol for treatment with acne propionicum or Staphylococcus aureus and IL-13 nasal drops;
FIG. 7 is a graph showing that A.propionicum can reduce IL-13 mediated eosinophilic inflammation in animal models
In the figure, a: a-PAS staining showed a graph of reduction in goblet cell number in p.acnes treated mice;
b: MBP staining showed a graph of eosinophil number reduction in IL-13 treated mice after P.acnes;
c: RT-PCR was performed on nasal mucosa total RNA to detect mRNA patterns of TH2 cytokines (IL-4, IL-5 and IL-13) and eosinophil chemokines (CCL 11 and CCL 24);
d: ELISA method for measuring concentration patterns of IL-4, IL-5, IL-13, CCL11 and CCL24 in nasal lavage fluid.
Detailed Description
The present invention is described in further detail below in conjunction with specific embodiments for understanding by those skilled in the art.
The following examples study subject grouping and specimen collection and Source description
1. The study subjects were divided into:
(1) the normal control group is a patient with simple nasal septum deflection or simple nasal sinus cyst, nasal tumor and traumatic optic nerve injury performing decompression operation, and the middle nasal airway epithelial cells and mucus are scraped in the operation and the resected uncrushed or/and nasal mucosa (without obvious inflammation) tissues are collected;
(2) eosinophilic CRSwNP group, eosinophil count in nasal polyp tissue greater than 10% of total inflammatory cells, scraping nasal polyp surface epithelial cells and mucus during surgery and collecting resected nasal polyp tissue;
(3) a group of non-eosinophilic CRSwNP, having eosinophil count of less than 10% of total inflammatory cells in nasal polyp tissue, intraoperatively scraping nasal polyp surface epithelial cells and mucus and collecting resected nasal polyp tissue; systemic or local glucocorticoids, as well as leukotriene receptor antagonists, were not used for one month prior to surgery in all subjects; all subjects did not receive immunotherapy; patients with post-culling nasal polyps, mycotic sinusitis, cystic fibrosis, primary ciliated dyskinesia, immunodeficiency, systemic vasculitis, igG 4-related diseases, acute upper respiratory tract infections or asthma attacks within 4 weeks, and other nasal diseases. Diagnosis of combined allergic rhinitis is based on the diagnostic criteria of ARIA (Brozek JL, et al journal of Allergy and Clinical Immunology 2017; 140:950-8); diagnosis of asthma is based on the criteria of GINA (https:// ginasthma. Org /).
EXAMPLE 1 nasal mucosa histopathology and inflammatory mediator level detection
Detection of inflammatory mediator protein levels in nasal mucosa and nasal polyp tissues, obtaining tissue homogenate supernatants, and detecting expression levels of type 2 cytokines (IL-4, IL-5 and IL-13), type 1 cytokines (IL-12 and IFN-gamma), type 3 cytokines (IL-17A), monocyte chemokines (MIP-1α and MIP-1β), epithelial-derived cytokines (IL-25, IL-33, TSLP and IL-36 gamma), eosinophil chemokines (CCL 11, CCL24 and CCL 26), and neutrophil chemokines (IL-8) by Bio-Plex or ELISA techniques (FIG. 2).
As shown in the figure: expression levels of various cytokines and chemokines in nasal polyp tissue.
Example 2 nasal cavity bacterial group detection
1) Nasal cavity bacterial group high throughput sequencing study:
scraping epithelial cells and mucus from the nasal mucosa of a normal control group and the nasal polyp surface of a CRSwNP patient during operation, extracting total DNA of a microflora according to the instructions of Qiagen DNA Mini kit (Qiagen, CA, USA), and determining the concentration and purity of the DNA by using a Nanodrop 2000; PCR amplification of the V3-V5 region of the 16S rRNA Gene Using the 338F (5 '-CCGTCAATTCMTTTGAGTTT-3') and 806R (5'-ACTCCTACGGGAGGCAGCAG-3') primer pairs, recovery of PCR products by 2% agarose gel, purification of the recovered products by AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, union City, calif., USA), and purification with Quantus TM The recovered product was detected and quantified by a Fluorometer (Promega, madison, wis., USA); by means ofNEXTFLEX Rapid DNA-Seq Kit (bio Scientific inc., austin, TX, USA) library building, sequencing by using a micqpe 300 platform of Illumina company, quality control of the original sequencing sequence is performed by using Fastp software, and splicing is performed by using Flash software; using Upparse software, carrying out OTU clustering on the sequences according to a 97% similarity threshold value, removing chimeras, carrying out species taxonomy annotation on the OTU representing sequences by RDP classifiers, and setting the confidence threshold value to be 0.7 to obtain species taxonomy annotation results; analysis compares the differences between normal control, eosinophilic and non-eosinophilic CRSwNP (fig. 1, a).
The results show that: the expression of propionibacterium acnes in eosinophilic nasal polyps was significantly down-regulated compared to non-eosinophilic nasal polyps and the expression of control (fig. 1, a).
2) PCR detection of Propionibacterium acnes:
the experimental method of reference 1) extracts total DNA, and uses PCR method, large samples verify the abundance of bacteria such as acnes propionici in normal control, eosinophilic and non-eosinophilic CRSwNP patients. The primer sequences of the p.acnes16s rRNA are:
(F)5’-TTTTGTGGGGTGCTCGAG-3’，
(R)5’-CCAACCGCCGAAACTTTC-3’；
the total bacterial group 16s rRNA primer sequences were:
(F)5’-AGAGTTTGATCCTGGCTCAG-3’，
(R) 5'-CTGCTGCCTYCCGTA-3' (FIG. 1B).
3) The CRSwNP patient was analyzed for the correlation of propionibacterium acnes with the following indicators:
(1) important inflammatory mediator expression levels in nasal polyps (CCL 11, CCL24, CCL26, etc.);
(2) inflammatory cell counts (eosinophils, neutrophils, T cells, etc.) in nasal polyps (fig. 2).
The results show that: the expression of propionibacterium nasal polyp had a negative correlation with tissue IL-5, eosinophil chemokines and eosinophil counts (fig. 1, b and fig. 2).
EXAMPLE 3 modulation and mechanism study of propionibacterium acnes on nasal mucosal epithelial cells FOXM1, CCL11 and CCL24
1) Culturing of propionibacterium acnes and staphylococcus aureus:
(1) acne propionibacterium ATCC6919 is purchased from China medical Collection center, culture methods: acnes propionicum was inoculated from glycerol stock to 10mL of clostridium medium and cultured under anaerobic conditions at 37 ℃; after the bacterial growth reaches the logarithmic growth phase, centrifugally collecting bacteria, and transferring the bacteria into 50mL of clostridium culture medium to be cultured for 40 hours under anaerobic conditions at 37 ℃; after centrifugation at 4000g for 10 minutes, the propionibacterium bacterial cells and the culture supernatant were collected, respectively. Filtering culture supernatant with 0.2 μm filter screen; bacterial inactivation: incubating the acnes propionici at 60 ℃ for 30 minutes;
(2) culturing staphylococcus aureus: the clinical isolates of staphylococcus aureus were provided by the clinical laboratory of the affiliated homotaxial hospital of the university of Huazhong science and technology. The culture method comprises the following steps: staphylococcus aureus was inoculated onto tryptic soy agar plates and cultured under aerobic conditions at 37 ℃ for 24 hours; then, single colonies are selected and cultured for 18 hours under aerobic conditions of the pancreatin soybean culture medium at 37 ℃; after the bacterial growth reaches the logarithmic growth phase, centrifugally collecting the bacteria, and transferring the bacteria into 50mL of pancreatin soybean culture medium to be cultured for 40 hours under the aerobic condition at 37 ℃; after centrifugation at 4000g for 10 minutes, staphylococcus aureus bacterial cells were collected. The inactivation method comprises the following steps: staphylococcus aureus was incubated at 120 ℃ for 30 minutes.
2) Gas-liquid interaction surface culture of primary epithelial cells of nasal mucosa: scraping nasal mucosa epithelial cells in a normal control group in an operation, and immersing and culturing the nasal mucosa epithelial cells in a bronchial primary epithelial cell culture medium; after the cells grow fully, transferring the cells into a Transwell small plate with the size of 0.4 mu m, and continuously culturing in a bronchial epithelium growth medium; after the cells grow up the Transwell plate, the medium on the upper layer of the Transwell plate is removed, and the cells are continuously cultured in the gas-liquid interaction plane medium for about 21 days to finish the differentiation.
3) By the method of the above 2), the nasal mucosa epithelial cells were cultured on the gas-liquid interface, and then the viable propionibacterium acnes (1×10) was used 6 CFU), inactivated Propionibacterium acnes (1×10) 6 CFU), inactivated staphylococcus aureus (1×)10 6 CFU), propionibacterium acnes culture supernatant (10% of total volume of cell culture supernatant), after 24 hours of stimulation, collecting epithelial cells, extracting RNA, and detecting gene levels of CCL11, CCL24, by RT-PCR; the cell culture supernatants were extracted and assayed for protein levels of CCL11, CCL24 using ELISA.
The results are shown in the following: it was found that acnes propionici, but not the culture supernatant thereof, inhibited the production of the epithelial cells CCL11 and CCL24 (fig. 3, 4 and 5).
EXAMPLE 4 animal model study of Propionibacterium acnes against nasal mucosal eosinophilic inflammation
1) Establishing an IL-13 nasal mucosa stimulation mouse model:
(1) referring to the previous study (Sun LF, et al Sci Signal 2017; 10), a mouse IL-13 nasal mucosa stimulation model was constructed as follows: 20. Mu.L of recombinant mouse IL-13 (0.5. Mu.g) was instilled into each nostril on days 1, 3, and 4, and mice were sacrificed on day 6;
(2) bacterial treatment: at the time of model modeling of mice, 20. Mu.L of inactivated Propionibacterium acnes (1X 107 CFU), propionibacterium acnes bacterial wall active ingredient or inactivated Staphylococcus aureus (1X 107 CFU) was instilled every day from day 0 to day 5, and mice were sacrificed on day 6 (FIG. 6).
2) Sampling and detecting: after deep anesthesia of the mice, nasal cavity lavage fluid is obtained and stored at-70 ℃; after the sacrifice, separating the head, removing the skin and soft tissues of the head, making a coronal incision 1mm behind the eyes, separating nasal cavity-paranasal sinus parts, decalcification, fixation, paraffin embedding, and continuously running 4-mu m thick serial slices of the coronal position; or dissecting nasal cavity and nasal sinus under microscope to obtain nasal-nasal sinus mucosa and storing at-70deg.C; the following study was then performed:
(1) histomorphometric observations, AB-PAS and MBP staining to observe inflammatory cell infiltration, goblet cell proliferation and metaplasia, tissue fibrosis, etc. (fig. 7A and B);
(2) ELISA detects lavage fluid cytokine and chemokine levels, such as: IL-4, IL-13, IFN-gamma, CCL11, CCL24, etc. (FIG. 7C);
(3) RT-PCR was performed using the following primer pairs to detect the expression of IL-4, IL-13, IFN- γ, CCL11, CCL24, FOXM1 (FIG. 7D); the primer pair sequences were as follows:
human GUSB-F：GTCTGCGGCATTTTGTCGG，
human GUSB-R：CACACGATGGCATAGGAATGG；
humanβ-actin-F：CATGTACGTTGCTATCCAGGC，
humanβ-actin-R：CTCCTTAATGTCACGCACGAT；
human CCL26-F：AACTCCGAAACAATTGTACTCAGCTG，
human CCL26-R：GTAACTCTGGGAGGAAACACCCTCTCC；
human CCL11-F：CCCCTTCAGCGACTAGAGAG，
human CCL11-R：TCTTGGGGTCGGCACAGAT；
human CCL24-F：GGAGTGGGTCCAGAGGTACAT，
human CCL24-R：CAGGTGGTTTGGTTGCCAG；
house mouse CCL11-F：GAATCACCAACAACAGATGCAC，
house mouse CCL11-R：ATCCTGGACCCACTTCTTCTT；
house mouse CCL24-F：ATTCTGTGACCATCCCCTCAT，
house mouse CCL24-R：TGTATGTGCCTCTGAACCCAC；
house mouse IL-5-F：GCAATGAGACGATGAGGCTTC，
house mouse IL-5-R：GCCCCTGAAAGATTTCTCCAATG；
house mouse IL-13-F：TGAGCAACATCACACAAGACC，
house mouse IL-13-R：GGCCTTGCGGTTACAGAGG。
the results show that: the A.propionicum was able to reduce the expression levels of eosinophils, tissue IL-5, IL-13, eosinophil chemokine CCL11/CCL24 in the mouse model (FIGS. 6-7).
Example 4
A nasal drop for treating nasal polyp comprises effective amount of Propionibacterium acnes and pharmaceutically acceptable physiological saline; the content of Propionibacterium acnes in the nasal drops is 10 7 CFU/mL。
Other parts not described in detail are prior art. Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (6)
1. Use of acnes propionicum ATCC6919 in the manufacture of a medicament for the treatment of eosinophilic nasal polyps.
2. A pharmaceutical formulation for treating eosinophilic nasal polyp, characterized in that: consists of effective dose of acnes propionicum ATCC6919 and pharmaceutically acceptable auxiliary materials; and the content of the propionibacterium acnes ATCC6919 in the pharmaceutical preparation is 10 6 -10 8 CFU/mL。
3. A pharmaceutical formulation for the treatment of eosinophilic nasal polyp according to claim 2, wherein: the auxiliary materials are any one of physiological saline, glucose, vitamin C and amino acid.
4. A pharmaceutical formulation for the treatment of eosinophilic nasal polyp according to claim 2, wherein: the medicinal preparation is nasal drops, spray preparation or flushing agent.
5. The pharmaceutical formulation for treating eosinophilic nasal polyp of claim 4 wherein: the spray preparation is any one of an atomizing agent, a spray and a suspension.
6. A pharmaceutical formulation for the treatment of eosinophilic nasal polyp according to any of claims 2 to 5, characterized in that: the content of the propionibacterium acnes in the pharmaceutical preparation is 10 7 CFU/mL。
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