CN115671140A - Application of propionibacterium acnes in preparation of medicine 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 polyp. In the medicinal preparation, the content of the propionibacterium acnes is 10 6 ‑10 8 CFU/mL. The propionibacterium acnes is symbiotic bacteria which is relatively safe and easy to obtain, so that the treatment cost of nasal polyp patients can be greatly reduced, and a new treatment option is provided for the treatment of the nasal polyp; the medicinal preparation for treating nasal polyp is nasal spray, nasal drops or irrigant, and the administration routes of the medicinal preparation and the nasal drops are simple and convenient, easy to operate and convenient for patients to use.
Description
Technical Field
The invention relates to the field of biological medicines, and in particular relates to application of propionibacterium acnes in preparation of a medicine for treating nasal polyps.
Background
Chronic sinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease that occurs mainly in the mucosa of the sinuses with the formation of polyps. The prevalence rate is high in the world and is up to 2% -4% in China; CRSwNP not only obviously influences the life quality of patients and consumes a large amount of medical and health resources, but also influences the occurrence and prognosis of chronic diseases such as asthma, chronic obstructive pulmonary disease and the like, and causes great burden to countries and patients. However, CRSwNP is still a disease that has been severely underestimated, inadequately treated, and poorly treated to date. One important reason for this is that the CRSwNP intrinsic type is highly heterogeneous, and crswnps of different ethnicities, countries, or even regions have different immunopathological characteristics. However, domestic and foreign studies show that nasal polyp eosinophilic inflammation is a key risk factor for poor prognosis of patients with CRSwNP. In recent years, multiple phase II or III clinical studies have shown that blocking type 2 cytokines, including anti-IL-5R, anti-IL-4 Ra and anti-IgE treatment, have encouraging clinical efficacy against eosinophilic CRSwNP with predominant type 2 inflammation and poor glucocorticoid treatment response against nasal polyps, as well as eosinophilic inflammation; these targeted biologics can significantly reduce polyp volume, improve clinical symptoms, and reduce the chance of re-surgery. However, it is noteworthy that even with these biological agents targeting type 2 cytokines, nasal polyp volume or nasal congestion symptoms could not be significantly improved in 40% -60% of patients. This suggests that there is a need to further develop targets of action or drugs directed to the formation of eosinophilic inflammation of nasal polyps.
Bacteria play a key role in host innate and adaptive immune system training and development; imbalances in bacterial organization are involved in the development of a variety of immune-mediated diseases, such as: inflammatory bowel disease, rheumatoid arthritis, malignant tumor, etc. The nasal mucosa is the first line of defense for the respiratory tract to contact the outside world, and the surface is colonized with a considerable number of bacteria. The previous research based on the traditional bacterial culture technology has not been able to obtain a clear and uniform conclusion on the role of bacteria in the pathogenesis of CRSwNP.
At present, no medicine for treating nasal polyp by using bacteria is reported.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides application of propionibacterium acnes in preparing a medicine for treating nasal polyps.
In order to achieve the purpose, the invention designs an application of propionibacterium acnes in preparing a medicine for treating nasal polyp.
The invention also provides a medicinal preparation for treating nasal polyp, which consists of an effective amount of propionibacterium acnes 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 preparation is a nasal drop or a spray or a rinse.
Still further, the spray formulation is any one of an atomizing agent, a spray agent and a suspension agent.
Still further, the content of Propionibacterium acnes in the pharmaceutical preparation is 10 6 -10 8 CFU/mL。
Still further, the content of Propionibacterium acnes in the pharmaceutical preparation is 10 7 CFU/mL。
The principle of the invention is as follows:
1. the invention adopts high-throughput sequencing technology at the early stage to compare the difference of nasal mucosa/nasal polyp surface bacteria groups in eosinophilic CRSwNP patients, nonacidic CRSwNP patients and normal control groups. We found that the abundance of propionibacterium acnes in the phylum actinomycetales was significantly reduced in eosinophilic CRSwNP, but not significantly altered in non-eosinophilic CRSwNP, relative to the normal control group. Propionibacterium acnes is a gram-positive anaerobic coccus that often colonizes the skin, oral cavity, gastrointestinal tract, and urogenital tract. Previous researches show that the propionibacterium acnes is an important symbiotic bacterium of skin, is also a conditional pathogen and is closely related to the attack of acne. The propionibacterium acnes can be detected in 90% of nasal cavity samples, and the relative abundance of the propionibacterium acnes in nearly 40% of the nasal cavity samples is more than 1%, so that the propionibacterium acnes is also an important component of a nasal cavity bacterial group, the correlation between the relative abundance of the propionibacterium acnes and eosinophilic granulocytic inflammation in nasal polyps is further found, and in vitro cell experiments and in vivo animal experiments prove that the propionibacterium acnes can inhibit the eosinophilic granulocytic inflammation, so that the method plays an important guiding role in further researching the treatment of the propionibacterium acnes on the nasal polyps.
2. The invention provides the potential therapeutic action of the propionibacterium acnes and the main active ingredients thereof on the CRSwNP eosinophilic granulocyte inflammation on the basis of the combination of early histopathology, high-throughput sequencing, cytobiology, molecular biology, chemistry and spectral identification technologies and in-vitro cell culture and in-vivo animal experiments, so that the propionibacterium acnes can be proved to be capable of preparing the medicine 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 patient with nasal polyps can be greatly reduced, and a new treatment option is provided for the treatment of the nasal polyps;
2. the medicinal preparation for treating nasal polyp is nasal spray, nasal drops or irrigant, and the administration routes of the medicinal preparation and the nasal drops are simple and convenient, easy to operate and convenient for patients to use.
Drawings
FIG. 1 is a graph of the composition of the bacterial population on the middle nasal mucosa/surface of nasal polyps and the abundance level of Propionibacterium in normal controls and patients with nasal polyps;
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 between the abundance of Propionibacterium acnes and the levels of IL-5 and CCL11 (eotaxin-1) in nasal polyp tissues and eosinophil granulocytic count in CRSwNP patients
FIG. 3 is a graph showing that both live Propionibacterium acnes (panels A, B) and inactivated Propionibacterium acnes (panels C, D) were able to inhibit the expression of CCL11 and CCL24 in the basal state and IL-13 stimulated state of the primary nasal mucosal epithelial cells
FIG. 4 is a graph showing that the culture supernatant of Propionibacterium acnes cannot inhibit the production of CCL11 and CCL24 in nasal mucosal epithelial cells.
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 model for treatment with P.propionicum or S.aureus and nasal drops of IL-13;
FIG. 7 is a graph of the reduction of IL-13 mediated eosinophilic inflammation in animal models by P.acnes
In the figure, A: a-PAS staining shows a plot of the reduction in goblet cell number in p.acnes treated mice;
b: MBP staining shows a graph of eosinophil reduction in IL-13 treated mice after p.acnes;
c: RT-PCR of total RNA of nasal mucosa to detect the mRNA patterns of TH2 cytokines (IL-4, IL-5 and IL-13) and eotaxin (CCL 11 and CCL 24);
d: the ELISA method was used to determine the concentration profiles of IL-4, IL-5, IL-13, CCL11, and CCL24 in the nasal lavage fluid.
Detailed Description
The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.
The following examples study object Components and sample Collection and Source description
1. The study subjects were:
(1) the normal control group is a patient who is subjected to decompression operation by simple nasal septum deflection or simple sinus cyst, nasal tumor and traumatic optic nerve injury, middle nasal tract epithelial cells and mucus are scraped in the operation, and excised uncinate process or/and mucosa (without obvious inflammation) tissues of the sinus are collected;
(2) eosinophilic CRSwNP group, the eosinophilic count in the nasal polyp tissue is more than 10% of the total inflammatory cell count, nasal polyp surface epithelial cells and mucus are scraped in the operation and the resected nasal polyp tissue is collected;
(3) the non-eosinophilic granulocyte CRSwNP group, the eosinophilic granulocyte count in the nasal polyp tissue is less than 10% of the total inflammatory cell number, the epithelial cells and mucus on the surface of the nasal polyp are scraped in the operation and the resected nasal polyp tissue is collected; systemic or local glucocorticoids, as well as leukotriene receptor antagonists, were not used one month prior to surgery in all subjects; all subjects received no immunotherapy; patients with post-nasal polyps, fungal sinusitis, cystic fibrosis, primary ciliary dyskinesia, immunodeficiency, systemic vasculitis, igG 4-related disorders, acute upper respiratory tract infections or acute episodes of asthma within 4 weeks, and other nasal disorders. Diagnosis of incorporated allergic rhinitis was according to the diagnostic criteria of ARIA (Brozek JL, et al. Journal of Allergy and Clinical Immunology 2017; asthma was diagnosed according to the criteria for GINA (https:// ginasthma. Org /).
Example 1 nasal mucosal histopathology, inflammatory mediator level assay
The level of inflammatory mediator protein in sinus mucosa and nasal polyp tissue was measured by obtaining tissue homogenate supernatant and measuring the 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-1. Alpha. And MIP-1. Beta.), epithelium-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 technique (FIG. 2).
As shown in the figure: expression levels of various cytokines as well as chemokines in nasal polyp tissue.
Example 2 nasal bacterial group detection
1) Nasal bacterial group high throughput sequencing study:
epithelial cells and mucus from the nasal mucosa in the normal control group and the surface of nasal polyps of patients with CRSwNP were scraped in the operation, total DNA extraction of microbial community was performed according to Qiagen DNA Mini kit (Qiagen, CA, USA) instructions, and the concentration and purity of DNA were determined by NanoDrop 2000; PCR amplification of the 16S rRNA gene V3-V5 region was performed using 338F (5 '-CCGTCAATTCMTTTGAGTTT TT-3') and 806R (5 'ACTCCTACGGGAGGCAGCAG-3'), and the PCR product was recovered from a 2% agarose gelThe product was purified using AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, union City, calif., USA), and purified using Quantus TM A Fluorometer (Promega, madison, wis., USA) detects and quantifies the recovered product; establishing a library by using NEXTFLEX Rapid DNA-Seq Kit (Bio Scientific Inc., austin, TX, USA), sequencing by using a MiseqPE300 platform of Illumina company, performing quality control on an original sequencing sequence by Fastp software, and splicing by using Flash software; using Uprease software, carrying out OTU clustering on the sequence according to a 97% similarity threshold value and removing chimeras, carrying out species taxonomy annotation on the OTU representative sequence by using an RDP classifier, and setting a confidence threshold value to be 0.7 to obtain a species taxonomy annotation result; analysis compared 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 controls (fig. 1, a).
2) PCR detection of Propionibacterium acnes:
reference 1) total DNA was extracted and large samples were used to verify the abundance of bacteria such as propionibacterium acnes in normal control, eosinophilic and non-eosinophilic CRSwNP patients using PCR. The p.acnes16s rRNA primer sequence is:
(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 correlation of the propionibacterium acnes of the CRSwNP patient with the following indexes is analyzed:
(1) levels of expression of important inflammatory mediators in nasal polyps (CCL 11, CCL24, CCL26, etc.);
(2) inflammatory cell counts (eosinophils, neutrophils, and T cells, etc.) in nasal polyps (fig. 2).
The results show that: the expression of Propionibacterium rhinopolypus has a negative correlation with tissue IL-5, eotaxin, and eosinophil count (FIGS. 1, B and 2).
Example 3 research on the regulatory action and mechanism of Propionibacterium acnes on the nasal mucosal epithelial cells FOXM1, CCL11 and CCL24
1) Culturing of Propionibacterium acnes and Staphylococcus aureus:
(1) the propionibacterium acnes ATCC6919 is purchased from China medical bacteria collection and management center, and the culture method comprises the following steps: the propionibacterium acnes is inoculated to 10mL of clostridium culture medium from the glycerol storage solution and cultured under the anaerobic condition at 37 ℃; when the bacteria grow to reach the logarithmic growth phase, centrifugally collecting the bacteria, and transferring the bacteria into 50mL of clostridium culture medium to culture for 40 hours under the anaerobic condition at 37 ℃; the bacterial cells of the Propionibacterium acnes and the culture supernatant were collected by centrifugation at 4000g for 10 minutes. Filtering the culture supernatant by a filter screen with the diameter of 0.2 mu m; and (3) inactivating bacteria: incubating propionibacterium acnes at 60 ℃ for 30 minutes;
(2) culturing staphylococcus aureus: the clinical isolate of Staphylococcus aureus was provided by the clinical laboratory of the affiliated college Hospital, university of science and technology, huazhong. The culture method comprises the following steps: staphylococcus aureus inoculated to tryptic soy agar plate and at 37 degrees C aerobic conditions for 24 hours; then, selecting a single colony to be cultured for 18 hours in a tryptic soy culture medium under the aerobic condition of 37 ℃; when the bacteria grow to reach the logarithmic growth phase, centrifugally collecting the bacteria, and transferring the bacteria to 50mL of pancreatin soybean culture medium for culturing for 40 hours under the aerobic condition of 37 ℃; and centrifuging at 4000g for 10 minutes, and collecting staphylococcus aureus bacterial thalli. The inactivation method comprises the following steps: staphylococcus aureus was incubated at 120 ℃ for 30 minutes.
2) Culturing primary epithelial cells of nasal mucosa on a gas-liquid interaction surface: scraping nasal mucosa epithelial cells in a normal control group in the operation, and carrying out immersion culture on the nasal mucosa epithelial cells in a primary bronchial epithelial cell culture medium; after the cells are overgrown, the cells are transferred into Transwell platelets with the size of 0.4 mu m and are continuously cultured in a bronchial epithelium growth culture medium; after the cells were overgrown with the Transwell platelets, the upper medium layer of the Transwell platelets was removed, and the cells were cultured in a gas-liquid interaction planar medium, whereby cell differentiation was completed in about 21 days.
3) MiningCulturing nasal mucosa epithelial cells on gas-liquid interaction surface by the method of 2), and culturing live Propionibacterium acnes (1 × 10) 6 CFU), inactivated Propionibacterium acnes thallus (1 × 10) 6 CFU), inactivated Staphylococcus aureus (1X 10) 6 CFU), a propionibacterium acnes culture supernatant (accounting for 10% of the total volume of the cell culture supernatant) and stimulating for 24 hours, collecting epithelial cells, extracting RNA, and detecting the gene levels of CCL11 and CCL24 by RT-PCR; cell culture supernatants were extracted and assayed for CCL11, CCL24 protein levels by ELISA.
The results are shown as follows: it was found that propionibacterium acnes, but not the culture supernatant thereof, could inhibit the production of CCL11 and CCL24 in the epithelial cells (fig. 3, fig. 4, and fig. 5).
Example 4 animal model study of Propionibacterium acnes in modulating eosinophilic inflammation of nasal mucosa
1) Establishing an IL-13 nasal mucosa stimulation mouse model:
(1) with reference to the previous study (Sun LF, et al. Sci Signal 2017), a mouse IL-13 nasal mucosa stimulation model was constructed as follows: mu.L of recombinant mouse IL-13 (0.5. Mu.g) was instilled into each nostril on days 1, 3, and 4, and the mice were sacrificed on day 6;
(2) and (3) bacterial treatment: in the case of mouse model modeling, 20. Mu.L of inactivated Propionibacterium acnes (1X 107 CFU), propionibacterium mural active ingredient, or inactivated Staphylococcus aureus (1X 107 CFU) was dropped into each nostril of each side every day from day 0 to day 5, and the mice were sacrificed on day 6 (FIG. 6).
2) Material taking and detection: after deep anesthesia of the mice, obtaining nasal cavity lavage fluid and storing the nasal cavity lavage fluid at-70 ℃; after the death, separating the head, removing the skin and soft tissues of the head, making a coronal incision 1mm behind the eyes, separating the nasal cavity-sinus part, decalcifying, fixing, embedding in paraffin, and continuously performing serial sections with the thickness of 4 μm at the coronal position; or dissecting nasal and paranasal sinuses under microscope to obtain nasal-paranasal sinus mucosa and storing at-70 deg.C; the following studies were then performed:
(1) histomorphometry, AB-PAS and MBP staining for inflammatory cell infiltration, goblet cell proliferation and metaplasia, tissue fibrosis, etc. (fig. 7A and B);
(2) ELISA detects levels of lavage cytokines and chemokines, etc., as: IL-4, IL-13, IFN-. Gamma., CCL11, CCL24, and the like (FIG. 7C);
(3) RT-PCR was performed using the following primer pairs to detect the expression of IL-4, IL-13, IFN-. Gamma.CCL 11, CCL24, FOXM1 (FIG. 7D); the primer sequences are 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: propionibacterium acnes was able to reduce the expression levels of eosinophils, tissue IL-5, IL-13, and eotaxin CCL11/CCL24 in a mouse model (FIGS. 6-7).
Example 4
A nasal drop for treating nasal polyp is prepared from acne propionibacterium in effective dosage and pharmacologically acceptable carrierThe physiological saline solution of (4); the content of Propionibacterium acnes in the nasal drop is 10 7 CFU/mL。
Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (7)
1. Application of Propionibacterium acnes in preparation of medicine for treating nasal polyp is provided.
2. A pharmaceutical preparation for treating nasal polyps, characterized in that: consists of an effective amount of propionibacterium acnes and pharmaceutically acceptable auxiliary materials.
3. The pharmaceutical formulation for treating nasal polyps according to claim 2, wherein: the auxiliary material is any one of normal saline, glucose, vitamin C and amino acid.
4. The pharmaceutical formulation for treating nasal polyps according to claim 2, wherein: the medicinal preparation is a nasal drop or a spray preparation or a flushing agent.
5. The pharmaceutical formulation for treating nasal polyps according to claim 4, wherein: the spray preparation is any one of an atomizing agent, a spray agent and a suspension agent.
6. A pharmaceutical preparation for the treatment of nasal polyps according to any one of claims 2-5, characterized in that: in the medicinal preparation, the content of the propionibacterium acnes is 10 6 -10 8 CFU/mL。
7. The pharmaceutical preparation for treating nasal polyps according to any one of claim 6, wherein: in the medicinal preparation, the content of the propionibacterium acnes is 10 7 CFU/mL。
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