阅读说明：本技术 雷帕霉素在制备治疗中耳炎药物中的应用 (Application of rapamycin in preparing medicine for treating otitis media ) 是由 李博 郑庆印 赵彤 郑体花 张肖林 奎丽红 于 2021-03-01 设计创作，主要内容包括：本发明公开了雷帕霉素在制备治疗中耳炎药物中的应用,属于医药技术领域。雷帕霉素可减轻中耳炎炎症和听力损失,降低自噬体蛋白LC3-II和自噬底物蛋白p62的积累,治疗效果显著；并且其无肾毒性,在研发治疗中耳炎的药物方面具有广阔前景。(The invention discloses application of rapamycin in preparing a medicament for treating otitis media, and belongs to the technical field of medicines. Rapamycin can relieve inflammation and hearing loss of otitis media, reduce accumulation of autophagosome protein LC3-II and autophagy substrate protein p62, and has remarkable treatment effect; and the medicine has no nephrotoxicity, and has wide prospect in the aspect of researching and developing medicines for treating otitis media.)

1. Application of rapamycin in preparing medicine for treating tympanitis is provided.

2. A medicament for the treatment of otitis media comprising rapamycin.

3. The medicament for treating otitis media according to claim 2, further comprising pharmaceutically acceptable excipients.

Technical Field

The invention relates to the technical field of medicines, in particular to application of rapamycin in preparing a medicine for treating otitis media.

Background

Otitis Media (OM) is an inflammatory lesion that affects all or part of the structure of the middle ear (including eustachian tube, tympanic cavity, tympanic sinus and mastoid chamber), is one of the most common clinical childhood diseases, and can cause serious complications inside and outside the cranium. The pathogenesis of OM is related to multiple factors such as immune system dysfunction, genetic susceptibility, pathogen exposure, eustachian tube dysfunction and the like, and the exact pathogenesis is not clear at present.

Rapamycin (RPM) is a novel macrolide immunosuppressant, has no nephrotoxicity and small toxic and side effects, can resist fungi and inhibit tumor growth, and can also block signal transduction through different cytokine receptors and block the progress of T lymphocytes and other cells from the G1 stage to the S stage, thereby exerting immunosuppressive effect and being used for treating rejection reaction of organ transplantation.

There are no reports of the use of rapamycin for treating otitis media.

Disclosure of Invention

In view of the above, the invention provides the application of rapamycin in preparing the medicament for treating otitis media for the first time.

Toll-like receptor 2(Tlr2) is involved in the initiation and clearance of OM inflammation; if no Tlr2 is involved, the inflammatory reaction is aggravated and the disease course is prolonged. The invention inoculates streptococcal peptidoglycan polysaccharide (PGPS) to Tlr2tm1Kir (Tlr2) by tympanocentesis^-/-) The middle ear of a mouse (the Tlr2 gene is knocked out) is constructed into an OM mouse model; compared with Wild Type (WT) mice, the OM mice inoculated with PGPS can show severe and persistent inflammation and tissue damage, show relatively stable OM symptoms, and provide a longer time window for drug screening and research of prevention and treatment mechanisms.

The OM mice were administered RPM, which reduced OM inflammation symptoms and saved hearing loss via (auditory brainstem response) ABR and tympanometry and middle ear histological analysis.

Further study of its mechanism of action revealed that PGPS-induced OM mice develop autophagic degradation dysfunction (increased accumulation of LC3-II and p62 proteins), while RPM treatment reduced autophagic degradation dysfunction; thus, the therapeutic effect of RPM may be associated with modulation of mTORC1 and/or autophagy activity.

Furthermore, the medicine for treating otitis media is prepared by taking rapamycin as a medicinal effective component, and pharmaceutically acceptable auxiliary materials can be added.

Has the advantages that: rapamycin can relieve inflammation and hearing loss of otitis media, reduce accumulation of autophagosome protein LC3-II and autophagy substrate protein p62, and has remarkable treatment effect; and the medicine has no nephrotoxicity, and has wide prospect in the aspect of researching and developing medicines for treating otitis media.

Drawings

FIG. 1 shows various sets of ABR waveforms;

FIG. 2 shows various sets of ABR thresholds;

FIG. 3 shows the I-wave latencies for each group;

FIG. 4 shows H & E staining patterns for each group;

FIG. 5 shows the relative coverage of inflammatory cells in the Middle Ear (ME) of each group of mice;

FIG. 6 is a graph showing immunohistochemical staining of each group; EC in the figure is middle ear epithelium;

FIG. 7 shows sets of western blot strips;

FIG. 8 shows the accumulation amounts of each histone.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Examples

OM mouse model construction and RPM treatment

Tlr2tm1Kir(Tlr2^-/-) Mice (from Jackon Lab) were randomly divided into 4 groups of 5 mice each and the mice in each group were administered the following drugs:

NS + DMSO group: saline + 0.0032% DMSO;

PGPS group: saline +60 μ g pgps;

PGPS +0.35 μ M RPM set: saline +60 μ g pgps + 0.0032% DMSO +0.35 μ M RPM;

PGPS +0.7 μ M RPM set: saline +60 μ g pgps + 0.0032% DMSO +0.7 μ M RPM;

each group of drugs was injected into the middle ear of the right ear of a mouse by tympanocentesis, the injection dose was 10. mu.L, and the function and mechanism of the tissues of the middle ear and the inner ear of the mouse were studied on day 3 after injection.

2. Auditory Brainstem Response (ABR) and tympanometry

ABR and tympanometry measurements were evaluated for each group of experimental mice on day 3 post-injection.

ABR thresholds (referring to the lowest stimulus level that identifies a clear and repeatable waveform) were measured using a computer-assisted evoked potential system (IHS3.30 Intelligent Hearing Systems, Miami, FL, USA). Specifically, audio at click, 8kHz, 16kHz and 32kHz is inserted into the right ear through the headphones.

Tympanometry MT 10 tympanometer (interacoutics, Assens, Denmark) was used.

The experimental results are shown in fig. 1-3, compared with the NS + DMSO group, the ABR threshold of the OM mice in the PGPS group is significantly increased, and the I-wave latency is delayed; while OM mice had significantly reduced hearing loss after RPM treatment.

3. Middle ear histological analysis

Groups of experimental mice were sacrificed on day 3 post-injection, and right ear blisters (including the middle and inner ear) were dissected and pathologically examined.

Specifically, the auricular vesicle tissue was fixed with 4% paraformaldehyde at 4 ℃ for 24 hours, decalcified with 10% EDTA solution for 5 days, and embedded in paraffin; paraffin sections were stained with hematoxylin-eosin (H & E) and viewed under light.

The results are shown in fig. 4-5, where the inflammatory cell coverage area in the middle ear portion of OM mice in the PGPS group was significantly increased compared to the NS + DMSO group, while inflammation was significantly reduced after RPM treatment.

4. Immunohistochemical assay

Fixing right ear blisters of each group of mice by 4% paraformaldehyde, decalcifying and embedding paraffin; the auricular tissue sections were 5-7 μm, deparaffinized, rehydrated and antigen-restored, and immunohistochemical staining was performed with anti-p 62 antibody (Abcam, ab56416, Cambridge, England, UK).

The sample slides were viewed under an optical microscope and imaged using LAS X software (Leica DM 4500B, Leica Microsystems inc., Buffalo Grove, IL, USA).

The results are shown in fig. 6, compared with the NS + DMSO group, the OM mice in the PGPS group had thickened middle ear epithelium and accumulated p62 protein in a large amount, while the middle ear epithelium was reduced in thickness and the accumulation of p62 protein was significantly reduced after RPM treatment.

5.Western blot

Ear vacuole tissues of each group of mice were lysed with RIPA lysis buffer and extraction buffer (Thermo Fisher Scientific,89900, Rockford, IL, USA) to obtain protein extracts. 30-50. mu.g of protein extract was separated on polyacrylamide gel, then transferred onto PVDF (Merck Millipore, IPVH00010), transferred with semi-dry transfer system at 15V for 40 minutes, blocked with 5% nonfat dry milk, and then tested overnight at 4 ℃ with the next antibody: anti-LC 3b (Novus biolicals, NB100-2220), anti-p 62(Abcam, ab56416), anti-TNF-alpha (Proteintetech, 17590-1-AP) and anti-GAPDH (Proteintech, 10494-1-AP).

After washing with Tris buffered Saline-Tween 20(TBS-T), the membranes were incubated with species specific secondary antibody and horseradish peroxidase for 1 hour at room temperature. Western blot bands were detected using HRP chemiluminescent substrate kit (Merck Millipore, WBKLS0100) and visualized using a ChemiDoc XRS + system (Bio-Rad, Hercules, Calif., USA). The intensity of the grey value of the protein bands was measured and quantified using ImageJ software from the national institutes of health, USA, as described by Miller (http:// Lukemiller. org/journel/2007/08/quantification-western-blocks-with. html).

The results are shown in FIGS. 7 and 8, and the accumulation of TNF-alpha, LC3-I, LC3-2, and p62 protein is significantly reduced compared with that of the PGPS group after RPM treatment.

The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.