Application of IL-17a in treatment of age-related olfactory dysfunction

Abstract

The invention relates to the technical field of biological medicine, provides novel application of IL-17a, and particularly relates to application of an IL-17a inhibitor in preparation of a medicine for treating age-related olfactory dysfunction. The research result shows that the expression increase of the IL-17a can trigger an inflammation signal and damage the olfactory function. Administration of the IL-17a inhibitor Y-320 or a neutralizing antibody promotes sensory neuron regeneration in the olfactory epithelium (OE) and reverses age-related respiratory metaplasia. In addition, conditional knockout of IL-17a in T cells can promote olfactory epithelium regeneration by promoting recruitment and differentiation of horizontal basal cells (HBCs) into globular basal cells (GBCs). In general, the research of the invention determines the previously undetermined effect of the IL-17a in olfactory epithelium regeneration and age-related olfactory function defects, and provides evidence for the IL-17a as a potential therapeutic target for olfaction of the elderly.

Description

Application of IL-17a in the treatment of age-related olfactory dysfunction Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of IL-17a as a therapeutic target for age-related olfactory dysfunction. Background Technology

[0002] Smell is a vital sensory function in mammals, profoundly influencing human physiology, cognition, and emotion. Olfactory dysfunction (OD) refers to the deterioration or loss of olfactory function. It is an early indicator of neurodegenerative diseases, a hallmark symptom of viral infections, and a common manifestation of aging. Due to the accelerating aging of the population, the incidence of olfactory dysfunction is rising year by year, affecting up to 50% of the elderly. This condition not only seriously impacts quality of life but can also endanger the lives of the elderly due to the inability to detect dangerous odors such as gas leaks, smoke, or spoiled food in time.

[0003] Impaired olfactory epithelial (OE) regeneration is a key factor in the prevalence of age-related ophthalmopathy (OD). However, the pathogenesis and key regulatory mechanisms of age-related OE regeneration defects remain unclear. Inflammation is a complex physiological process involving the recruitment of immune cells and the secretion of pro-inflammatory cytokines. Inflammatory senescence refers to an age-related, low-grade, chronic, and progressive state of heightened pro-inflammatory inflammatory activity that occurs in the absence of infection. Studies have shown that inflammatory cytokines are involved in the progression of inflammatory senescence, which often occurs concurrently with various chronic age-related diseases. Therefore, older adults with chronic diseases may experience inflammatory senescence mediated by immune dysfunction.

[0004] The olfactory epithelium is a pseudostratified epithelial structure, primarily composed of horizontal basal cells (HBCs), bulbous basal cells (GBCs), supporting cells (SUS), and olfactory sensory neurons (OSNs). Olfactory function is mainly mediated by sensory neurons, which detect odors and transmit signals to the olfactory bulb and higher brain regions. Under homeostatic conditions, mitotically active GBCs continuously generate new neurons throughout life, while homeostatic HBCs attached to the basal layer do not participate in maintaining normal tissue renewal. When severely damaged, HBCs are activated and transform into GBCs, which then differentiate into sensory neurons and non-sensory supporting cells; HBCs can also directly generate supporting cells, thus completing epithelial regeneration. However, in inflammatory senescence, the accumulation of immune cells releases more inflammatory factors, leading to an imbalance in the inflammatory microenvironment. This imbalance hinders the activation and differentiation of basal cells, resulting in weakened neuronal renewal and consequently, olfactory dysfunction.

[0005] In the process of inflammatory senescence, T cells play a crucial role in tissue renewal, homeostasis, and repair. Multiple signaling pathways and inflammatory factors are involved in tissue aging, such as IL-6, TNFα, and IFNα. Among these, IL-17a has been reported to be elevated in aging tissues and organs, but its role in the development of age-related olfactory decline has not yet been reported. Summary of the Invention

[0006] Based on the above research, this invention aims to provide the application of interleukin-17a (IL-17a) as a target in alleviating age-related OE regeneration and olfactory function.

[0007] The results showed that inflammatory aging in older oocytes (OEs) promoted the recruitment and activation of immune cells, accompanied by interactions between HBCs and T cells. (Due to CD4+) + Th cells secrete significantly elevated levels of IL-17a, and elevated IL-17a expression triggers inflammatory signaling and impairs olfactory function. Administration of the IL-17a inhibitor Y-320 or a neutralizing antibody promotes sensory neuron regeneration in the oocyte endothelial layer (OE) and reverses age-related respiratory metaplasia. In summary, this study identifies a previously undefined role of IL-17a in OE regeneration and age-related olfactory dysfunction, providing evidence for IL-17a as a potential therapeutic target for age-related olfaction.

[0008] Specifically, the present invention provides the following technical solution: In a first aspect, the present invention provides the application of IL-17a as a therapeutic target for age-related olfactory dysfunction.

[0009] In a second aspect, the invention provides the use of IL-17a inhibitors in the preparation of medicaments for the treatment of age-related olfactory dysfunction.

[0010] Preferably, IL-17a is selected from any of the following substances: IL-17a gene, IL-17a mRNA or cDNA, IL-17a protein, or any of the aforementioned active or marker fragments.

[0011] Preferably, the IL-17a inhibitor is selected from any of the following substances: substances that reduce IL-17a expression levels, reduce IL-17a activity, or promote IL-17a metabolism.

[0012] Furthermore, the small molecule inhibitor of IL-17a was selected from Y-320.

[0013] In a third aspect, the present invention provides the application of the above-mentioned IL-17a inhibitor in the preparation of a therapeutic drug for age-related olfactory dysfunction.

[0014] In a fourth aspect, the present invention provides a pharmaceutical composition for treating age-related olfactory dysfunction, namely, the present invention provides a treatment product for age-related olfactory dysfunction comprising: (A) an inhibitor of IL-17a; and (B) an IL-17a neutralizing antibody. In some embodiments, the present invention also provides a treatment method for age-related olfactory dysfunction, the method comprising administering a therapeutically effective amount of an IL-17a inhibitor or a neutralizing antibody to a subject in need. Attached Figure Description

[0015] Figure 1 shows the characteristics of olfactory epithelial cell types and T cell interactions in young and aged mice: (a) UMAP visualization of scRNA-seq data from young and aged mice, showing the five major olfactory epithelial cell types and T cells; (b) UMAP plots by sample, comparing the differences in OE cell composition between young and aged mice; (c) Component analysis showing the abundance ratio of different cell types in the OE of young and aged mice; (d) Feature map showing the expression patterns of typical cell type markers, with color scales indicating expression levels from low (gray) to high (red / green); (e) Major cell type markers. (f) Representative confocal images of OE tissues stained with immunostains; (g) Quantification of positive cells for each cell type in young and aged mice; (h) Dot plot showing increased ligand-receptor mediated interactions between T cells and OE cell types in aged mice; (i) Immunofluorescence staining of MIF, ICAM1, CD74, and CD44 in OE tissues of young and aged mice; (j) Quantitative analysis of MIF+, CD74+, and CD44+ cells in OE tissues of young and aged mice; (d) Violin plot showing expression levels of Mif, Cd74, and Cd44 in different OE cell types. Cell nuclei in all images were stained with Hoechst. Scale bar, 50 μm, *P<0.05, ***P<0.001, ****P<0.0001.

[0016] Figure 2 shows the increased expression of IL-17a in aged mice: (a) UMAP visualization of scRNA-seq data, colored by annotated T cell subtypes; (b) Composite bar chart showing the abundance proportion of each T cell subtype; (c) Feature map showing the expression patterns of representative cell type marker genes; (d) Dot plot showing the expression levels of key marker genes for each T cell subtype. The size of the dots reflects the percentage of cells expressing 58 genes; color intensity represents the average expression level; (e) List of marker genes for CD4+Th cell subpopulations; (f) Violin plot showing representative marker genes for CD4+Th cell subpopulations; (g) Differentiation index score of CD4+Th cell subpopulations calculated based on the marker genes listed in (e); (h) Percentage of cells expressing IL-17a in each T cell subpopulation; (i) Concentrations of IL-17a and IL-23 in plasma and OE tissue of young and aged mice; (j) Western blot. (k) Western blot analysis of IL-17a protein expression levels in OE tissues of young and aged mice; (l) qPCR analysis of IL-17a mRNA expression levels in OE tissues of young and aged mice; (m) Violin plots based on scRNA-seq data showing IL-17a expression levels in OE tissues of young and aged mice; (n) Quantification of IL-17a+ cells in OE samples of young and aged mice based on scRNA-seq data; (o) Correlation analysis between IL-17a mRNA expression levels and time required to find a biscuit in young and aged mice; (p) Olfactory behavior test to measure the time required for young and aged mice to find a biscuit. All cell nuclei were stained with Hoechst. Scale bar, 50 μm, **P<0.01, ***P<0.001, ****P<0.0001.

[0017] Figure 3 shows that inhibition of IL-17a promotes olfactory epithelial regeneration in young and aged mice: (a) representative confocal images of OE tissue at specified time points (1, 2, 3, and 7 dpi) after methimazole-induced injury, immunostained for CD45, IL-17a-EGF, OMP, and Tuj1; (b) quantification of the number of positive cells labeled in (a); (c) experimental protocol for administering Y320 to young and aged mice; (d, e) olfactory behavior test, measuring the effects of Y320 on young and aged mice. (d) Time required to find cookies in methimazole-induced lesions before treatment (d) and after treatment (e); (f) Plasma concentration of IL-17a in aged mice treated with or without Y320; (g) Quantification of IL-17a+ / CD45+ double-positive cells in OE tissue; (h) Double immunohistochemical staining results of IL-17a and CD45 in OE of young and aged mice at 14 dpi, regardless of Y320 treatment; (i) Changes in OE thickness in young and aged mice with or without Y320 treatment; (j) Tuj1, OMP, and Gap43 staining in the dorsal OE region of young and aged mice at 7 dpi and 14 dpi; (km) Statistical analysis of Tuj1+ (k), Gap43+ (l), and OMP+ (m) cells in OE under specified conditions. All cell nuclei were stained with Hoechst. Scale bar, 50 μm, **P<0.01, ***P<0.001, ****P<0.0001.

[0018] Figure 4 shows the reversible methimazole-induced respiratory metaplasia of the olfactory epithelium induced by IL-17a inhibition: (a) Representative confocal images of the dorsal, medial, and plaque regions of the olfactory epithelium in young and aged mice treated with the IL-17a inhibitor (Y320) or DMSO at 14 dpi under control conditions or methimazole injury. Sections were examined for Krt19, Tuj1, and Icam1 by immunostaining; (b) Schematic diagram of OE tissue structure (left) and corresponding immunostaining results (right). Green indicates Krt19+ regions, red indicates Tuj1+ regions, and the boxed areas highlight metaplastic plaques in young and aged OEs; (c) Quantitative analysis of the length of Krt19+ metaplastic plaques in OE tissues; (d) Statistical analysis of the proportion of metaplastic and rehabilitated regions in young and aged OE tissues; (e) Flowchart of the experiment on treatment of aged mice with IL-17a neutralizing antibody (NAb); (f) Olfactory behavior test showing the change in the time required for aged mice to find cookies after Met-induced injury following treatment with two concentrations of IL-17aNAb (high and low); (g) Immunostaining of Krt19, Tuj1, and Icam1 in aged mouse OEs pretreated with low or high doses of IL-17aNAb before methimazole injury; (i) Quantification of the length of Krt19+ metaplastic plaques under different treatment conditions; (j) Quantification of Tuj1+ cells in aged mouse OEs under different treatment conditions. All immunostaining images show Hoechst staining of cell nuclei. The scale bar is 50 μm, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Detailed Implementation

[0019] The present invention will now be described in detail with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0020] All reagents and raw materials used in this invention are commercially available or can be prepared according to literature methods. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer.

[0021] 1. Materials and Methods 1.1 Experimental Animals C57BL / 6JCya-Il17a-[tm2(EGFP)] mice were purchased from Cyagen Biologics (Suzhou, China).

[0022] Wild-type C57BL / 6J mice aged 2 months (juvenile), 12 months (middle-aged), and 19-22 months (senior) were purchased from Shanghai Bikai Laboratory Animal Co., Ltd. (Shanghai, China). To avoid the effects of estrogen on aging, male mice were used for behavioral testing, and male and female mice were randomly assigned to other experimental groups. All mice were housed according to standard procedures, with free access to water and food, a 12:12 light-dark cycle, and a temperature maintained at 18–22°C. After all experimental procedures, mice were euthanized by slow inhalation of carbon dioxide (CO2) (30–70% volume / min), and death was confirmed by cervical dislocation. The animal handling procedures were approved by the Laboratory Animal Ethics Committee of Naval Medical University (License No.: SYXK2022-0011).

[0023] 1.2 Olfactory epithelial lesion induction: Young and aged mice were intraperitoneally injected with methimazole (100 μg / g, #60560, Sigma-Aldrich) and sacrificed on days 7 and 14 after methimazole treatment.

[0024] 1.3 Mice treated with Y-320 were given Y-320 (#HY-15898, MCE) by gavage one week prior to methimazole treatment at a dose of 3 mg / kg once daily. Y-320 was dissolved in corn oil containing 10% DMSO. Control mice were treated with the same dose of corn oil and DMSO.

[0025] 1.4 Neutralization Treatment in Aged Mice: Anti-mouse IL-17a neutralizing antibody (InVivoMAb, clone 17F3, BioXcell, #BE0173) was administered intravenously to each mouse at a dose of 105 µg or 150 µg. Control mice received an equivalent dose of IgG1 isotype control (InVivoMAb, clone MOPC-21, BioXcell, #BE0083) via the same route, at 150 µg per mouse. Prophylactic treatment began 7 days prior to thiamazole-induced OE lesions and was administered three times every other day for one week. After model establishment (designated as day 0), the same administration regimen (intravenous injection, three times a week) was maintained for another two weeks, for a total treatment duration of three weeks.

[0026] 1.5 Buried Food Test: The test was conducted according to the method described in the literature [Ma Z, et al. TMEM59 ablation leads to loss of olfactorysensory neurons and impairs olfactory functions via interaction withinflammation. Brain Behav Immun 111, 151-168 (2023)]. Two days before the test, mice were trained to find biscuits and were starved for 24 hours in advance, with water provided freely. Before the experiment, mice were placed in clean experimental cages (35 cm × 25 cm × 20 cm) with a 5 cm thick bedding and left for 10 minutes. Then, the mice were placed in the same experimental cages, and biscuits were buried 3 cm below the bedding. The time required to find the biscuits was recorded, with the cutoff time being 360 seconds (6 minutes). 1.6 Olfactory Habituation-De-habituation Test: Mice were exposed to two different odors to test olfactory habits and discrimination abilities. Each mouse was placed in an isolated test cage (45 cm × 30 cm × 20 cm) and acclimated to an odorless cotton tip for 30 minutes prior to testing. The odors used were geraniol and citrate, diluted 1:20 in mineral oil. After four trials with a cotton swab soaked in mineral oil, the mice were exposed to one odor four times, followed by four exposures to a different odor. The number of times the mice explored the odor within one minute after each exposure was recorded and compared to their response to mineral oil.

[0027] 1.7 Olfactory Threshold Testing Following induced olfactory epithelial injury, olfactory threshold testing was performed 7 or 14 days post-injury (dpi). Concentrated geraniol extract was diluted to various concentrations (1:160, 1:80, 1:40, 1:20, 1:10) using mineral oil. After an acclimatization period, mice were tested four times using cotton swabs soaked in mineral oil. Then, the odorant was dissolved in mineral oil, and a series of tests were performed using cotton swabs soaked in the odor diluent, arranged in order of concentration from weakest to strongest. Control cotton swabs soaked in mineral oil were also placed nearby. The lowest concentration of the odorant detected was determined when mice exhibited a significantly longer olfactory duration to the cotton swabs soaked in diluted odor compared to those soaked in mineral oil.

[0028] 1.8 RT-qPCR Analysis Total RNA was extracted from mouse tissues using an RNA isolation kit (#RC112-01, Vazyme). cDNA was synthesized via reverse transcription using RTSuperMix (#R333, Vazyme), followed by quantitative PCR (qPCR) on a real-time fluorescence PCR system (7900HT, ABI) using SYBR qPCR Master Mix (#Q711, Vazyme). The results were analyzed by 2... [-ΔΔCt] The cyclic threshold method was used to calculate the relative expression levels of the target genes and normalize the expression of β-actin. The primer sequences used in this study were synthesized by Shanghai Tsinghua Biotechnology Co., Ltd., and are as follows:

[0029]

[0030]

[0031]

[0032] 1.13 Flow Cytometry Assay To detect endogenous IL-17a, labeled cells were fixed after membrane labeling and then stained intracellularly with PE anti-mouse IL-17a (#506903, BioLegend) or BV421 anti-mouse IL-17a (#506925, BioLegend). Representative gating strategies can be found in the source data file.

[0033] 1.14 Enzyme-linked immunosorbent assay (ELISA) analysis: Mice were anesthetized and venous blood was drawn to collect serum. The blood sample was allowed to coagulate at room temperature for 30 minutes, and then centrifuged at 1200×g for 10 minutes at 4°C to obtain serum.

[0034] For tissue analysis, mice were euthanized and their nasal mucosa was dissected. The tissues were mechanically homogenized on ice at a ratio of 9 µL / mg tissue weight. The homogenate was centrifuged at 1000×g for 10 minutes, and the supernatant was collected for analysis. IL-17a and IL-23 levels were quantified using the following commercial ELISA kits: mouse IL-17a (#U96-3240E, YOBIBIO) and mouse IL-23 (#U96-3252E, YOBIBIO).

[0035] 1.15 Immunostaining For tissue immunostaining, mice were euthanized under deep anesthesia by intraperitoneal injection of ketamine-xulazine (200 and 15 mg / kg body weight). Hair and muscle tissue from the head were carefully removed under a microscope, the entire nasal cavity was excised, and the tissue was fixed with 4% PFA (#G1101, Servicebio) for 6 hours. The tissue was then dehydrated and decalcified using 15% and 30% sucrose (#A502792, Sangon) and EDTA decalcification solution (#G1105, Servicebio). Nasal tissue was embedded in OCT compound (#4583, Sakura) and cut into 10 μm thick coronal sections using a cryosectioning platform (CM1950, Leica). For immunostaining, tissue sections were infiltrated with PBS to remove the OCT compound and then repaired at 95°C using EDTA antigen retrieval solution (#P0086, Beyotime). Block the sections in 5% bovine serum albumin (BSA) in PBS containing 0.3% Triton X-100 (#20107ES20, Yeasen) for 1 hour, and then incubate with primary antibody overnight at 4°C.

[0036] 1.18 Statistical Analysis Statistical analysis was performed using IBM SPSS version 24.0 and Graphpad Prism version 8.0 (Graphpad Software, La Jolla, CA). For normally distributed data, we used unpaired Student t-tests (two-tailed) to compare differences between groups, and adjusted for multiple comparisons using ANOVA with Tukey post-hoc test or Sidak multiple comparison test (one-tailed). For non-normally distributed data, two-tailed Mann-Whitney U tests were used for comparisons between groups, and Kruskal-Wallis tests and Dunn post-hoc tests were used for multiple comparisons. Spearman's rank correlation was used to assess correlation. p-values ​​are shown in the figures. Each experiment was performed independently at least three times, and repetitions yielded similar results.

[0037] 2. Results 2.1 Loss of Olfactory Sensory Neurons in Aged Mice Accompanied by Changes in the Immune Microenvironment In previously published studies, single-cell resolution aging atlases of the OE were mapped. To further investigate the impact of T cells on OE aging, a detailed analysis of the major cell types in the OE was performed, including HBCs, GBCs, immature and mature neurons (iOSNs and mOSNs), SUSs, and T cells. Cluster maps of various OE cell types and T cells in young (3-month-old) and aged (24-month-old) mice were displayed using UMAP (Fig. 1a, b). The proportion of cell types is shown in Fig. 1c, highlighting a significant decrease in GBCs, mOSNs, and SUSs, while T and HBCs were significantly increased in aged mice. The feature map in Fig. 1d depicts the expression levels of marker genes specific to each cell type: Omp for mOSNs, Tubb3 (Tuj1) and Gap43 for iOSNs, Sox2 for SUS and GBCs, Krt5 for HBCs, and Cd3e for T cells. The number of Krt5[+] HBCs increased by 16.40 ± 6.98% in aged mice. Notably, we observed that the morphology of Krt5+ cells changed from the flattened shape of young mice to the irregular triangular shape of aged mice, indicating that HBCs transitioned from a dormant to an activated state. This is consistent with our previous findings that more activated HBCs are present in aged OEs.

[0038] In contrast, the number of immune cells in aged mice increased significantly. Most of these immune cells were located in the lamina propria of the oE, indicating that immune cells accumulate in aging tissues.

[0039] To investigate changes in intercellular interactions in aging oocytes (OEs), we performed cell-cell communication analysis (CellChat), focusing on T cells and various OE cell types. Data showed a significant increase in the macrophage migration inhibitory factor (MIF) signaling pathway in aged mice. This is a pleiotropic pro-inflammatory cytokine involved in multiple age-related functions, such as leukocyte recruitment, inflammation, immune responses, and tumorigenesis. As shown in Figure 1g, Mif-(Cd74+Cd44) is the most important ligand / receptor pair between T cells and HBCs in aging OEs. Immunostaining and subsequent statistical analysis revealed a significant increase in MIF[+], CD74[+], and CD44[+] cells within the OE with age. Meanwhile, the increased CD74[+] cells in aging tissues were primarily located near the basal cell layer (labeled by Icam1), while CD44[+] cells were distributed in both the basal cell layer and the lamina propria (Figure 1h, 1i). The violin plot further supports these observations, showing the accumulation of Mif in T cells and a significant increase in the expression of Cd74 and Cd44 in HBCs and T cells of aged OEs (Fig. 1j, p<0.0001). Overall, these findings suggest that the immune response patterns within the microenvironment of OE stem cells undergo age-dependent changes with aging.

[0040] 2.2 Increased IL-17a expression in aged OE is associated with olfactory function. To further investigate the effects of T cells on OE aging, we divided T cells into seven subsets based on the expression of known marker genes: Naïve T, CD4[+] Th, Treg T, γδT, CD8[+] T, NK T, and Cycling T

[23] , as shown in Figure 2a. Overall, there was a significant difference in the proportion of cell subsets between young and aged tissues: CD4[+] Th, Treg, and γδT were mainly present in the aged group (Figure 2b), and these cell types are important regulators in the immune response process. The feature map illustrates the expression of IL-17a and known marker genes CD4[+] Th (Ctla4), γδT (Trdc), and Treg (Foxp3) (Figure 2c). The characteristic marker genes of each cell subset are represented in the dot plot (Figure 2d). As the immune response progresses, CD4+Th cells are activated and subsequently differentiate into various subsets, including Th1, Th2, Th9, Th17, Th22, and Tfh. To gain a deeper understanding of the differentiation pathway of CD4+Th cells, we assessed differentiation index scores based on marker genes of different subsets (Fig. 2e). A violin plot illustrates the expression patterns of representative marker genes (Fig. 2f). Our results show that Th17 cells exhibited the highest score, indicating that CD4+Th cells are primarily polarized towards the Th17 lineage (Fig. 2g, p<0.0001). Subsequent analysis of scRNA-seq data was performed to characterize the cellular origin of IL-17a in senescent tissues. A pie chart shows that among all cells expressing IL-17a, CD4+Th cells constituted the majority (76.7%), followed by γδT cells (13.7%) and other immune populations (Fig. 2h).

[0041] We then validated IL-17a expression in OE tissues. Compared to young controls, ELISA results showed significantly increased concentrations of IL-17a and IL-23 in OE tissues and plasma from aged mice (Fig. 2i). Consistent with these results, Western blot and qPCR analyses further confirmed significantly increased levels of both IL-17a protein and mRNA expression in aged OE tissues (Fig. 2j, k). Violin plots also showed a significant upregulation of IL-17a transcript levels in aged mice (Fig. 2l). Furthermore, immunostaining of OE tissue sections revealed significantly increased expression levels of CD3[+], CD4[+], and IL-17a[+] cells in aged mice, while IL-17a signaling was almost undetectable in OE tissues from young mice (Fig. 2m). scRNA-seq data analysis showed that the IL-17a[+] cell count in aged OE tissues was approximately 32-fold higher than in young mice (Fig. 2n).

[0042] To assess olfactory function, we conducted a food-burying experiment on mice and observed that older mice took longer to find the biscuit than younger controls (Fig. 2p). Correlation analysis showed a negative correlation between Il-17a mRNA expression levels and olfactory function (Fig. 2o). In summary, these results suggest that elevated IL-17a expression in older OE mice triggers inflammatory signals, leading to a decline in olfactory function.

[0043] 2.3 IL-17 Inhibitor Y-320 Promotes OE Regeneration Given the potential association between elevated IL-17a in the OE and olfactory dysfunction, we sought to investigate whether IL-17a plays an inhibitory role in OE regeneration. An OE injury model was established by injecting methimazole (Met). Il17a-EGFP mice were used to directly observe the expression level of endogenous Il17a. As shown in Figure 3a, a large number of OMP[+] cells were present in the undamaged OE. On day 1 post-injection (1 dpi), only a monolayer of basal cells remained (demarcated by the white dashed line), and a large number of CD45[+] inflammatory cells entered the mucosal tissue. Simultaneously, the endogenous Il17a-EGFP expression image showed a significant increase in the number of IL-17a[+] cells after injury. A small number of newly formed immature Tuj1[+] olfactory neurons appeared at 2 dpi. Compared to 3 dpi, Tuj1[+] expression was more pronounced at 7 dpi, while the number of IL-17a[+] cells decreased (Fig. 3a, b).

[0044] Y-320 is a specific IL-17 inhibitor used to suppress the production of IL-17 secreted by CD4+ T cells. To investigate the role of IL-17a in post-injury OE regeneration, mice were administered Y-320 by gavage 7 days before Met administration, and tissues were collected at 7 or 14 dpi after injury for subsequent experiments (Fig. 3c). Mice treated with DMSO served as a simulated control group. To ensure that olfactory function was not affected by Y-320, we performed olfactory behavior tests on mice before Met injection. We found that older mice took significantly longer to find the biscuit compared to younger mice, indicating impaired olfactory function. However, Y-320 treatment did not alter olfactory behavior in mice compared to controls injected with DMSO in either younger or older animals (Fig. 3d), suggesting that inhibition of IL-17a does not affect olfactory function under normal conditions.

[0045] One day after Met administration (1 dpi), mice were unable to find the biscuit within the specified time, indicating a significant decline in olfactory function, with similar results in both young and aged mice. Olfactory assessments were subsequently repeated at 3, 7, and 14 dpi. In young mice treated with Y-320, olfactory function showed a significant improvement at 3 dpi compared to the DMSO group, and this improvement persisted until 14 dpi. Simultaneously, aged mice in the Y-320 group also showed improved olfactory function from 3 to 14 dpi compared to the DMSO group, as shown in Figure 3e. These findings suggest that inhibiting IL-17a production can accelerate the recovery of olfactory function in both young and aged mice.

[0046] At 7 dpi, we collected plasma samples from aged mice and detected IL-17a concentrations using ELISA. The results showed that Y-320 administration led to a decrease in peripheral plasma IL-17a levels (Figure 3f), indicating that Y-320 inhibits IL-17a production.

[0047] Furthermore, at 14 dpi, we performed immunostaining of IL-17a and CD45 in young and aged mice. IL-17a[+] and CD45[+] cells were located in the lamina propria of the mucosal tissue (Fig. 3h). Compared with young mice, the number of double-positive cells in the tissues of aged mice was significantly increased, while Y-320 administration reduced the percentage of IL-17a[+] cells in CD45[+] cells (Fig. 3g). These data indicate that Y-320 improves the level of inflammatory activation in the damaged OE.

[0048] Since OSNs are mainly concentrated in the dorsal and medial regions, we measured the OE thickness in these two regions in young and aged mice at 7 and 14 dpi. As shown in Figure 3i, the OE thickness in aged mice was significantly thinner compared to young mice treated with DMSO, and administration of Y-320 restored the OE thickness to some extent. Subsequently, we performed immunostaining using specific antibodies (Tuj1, Gap43) against mature (OMP) and immature OSNs. As shown in Figures 3j-3m, after 7 dpi of injury, we observed a certain degree of recovery in the number of mature and immature neurons in the dorsal OE. After using Y-320, the number of Gap43[+] and Tuj1[+] neonatal neurons in young mice increased significantly by 49.61±6.93% (p<0.0001) and 49.97±8.26% (p<0.0001), respectively, at 7 dpi of injury. At 14 dpi, Y-320 had a more significant effect on neuronal recovery in young mice, increasing the number of OMP[+] mature neurons by 81.67 ± 13.91% (p < 0.0001). In older mice, neuronal recovery was limited at both 7 and 14 dpi, with fewer Tuj1[+] and Gap43[+] cells in the OE.

[0049] 2.4 Inhibition of IL-17a can reverse age-related respiratory metaplasia in osteogenesis imperfecta (OE).

[0050] OE damage has been reported to lead to respiratory epithelial metaplasia and olfactory dysfunction [30, 31]. To determine whether Y-320 administration could reverse metaplastic changes in damaged OEs, we performed immunostaining of Krt19 (a specific marker of respiratory basal cells) in OE tissues from young and aged mice treated with DMSO or Y-320; untreated mice served as controls. As shown in Figures 4a and 4d, Krt19+ cells in untreated control mice were predominantly located in the RE region. In contrast, after Met-induced damage, DMSO-treated mice exhibited extensive Krt19[+] metaplasia in the dorsal, medial, and lateral regions of the OE, outlined as “patch areas” within the specified frame (green dashed lines, Figure 4b). In young mice, metaplasia remained relatively limited, with many areas retaining Tuj1[+]iOSN. However, in aged mice, the metaplastic areas covered almost the entire OE. Notably, Y-320 administration resulted in a significant reduction in the degree of metaplasia, an effect particularly pronounced in aged mice. The metaplastic growth area in the dorsal region was significantly shortened by 158.70 ± 9.83 μm, and in the medial region by 150.20 ± 12.18 μm (Fig. 4c). Quantitative analysis showed that the metaplastic area of ​​Krt19[+] in aged mice accounted for 77.97% of the total OE, significantly higher than that in the young group (43.5%). After Y-320 treatment, the metaplastic area in the aged group and the young group decreased by 44.99% and 21.65%, respectively (Fig. 4d). These results indicate that under the same injury conditions, OE in aged mice is more prone to metaplastic transformation and has significantly impaired regenerative capacity, which may be due to a decline in age-related intrinsic repair mechanisms.

[0051] To further validate the functional role of IL-17a in age-related olfactory decline, we injected aged mice with IL-17a neutralizing antibodies at two concentrations: 105 µg / mL (low) and 150 µg / mL (high). Olfactory function was assessed at 3 and 7 days after Met-induced injury (Fig. 4e). Mice receiving higher doses showed significantly shorter food-finding times, indicating accelerated functional recovery (Fig. 4f). Immunostaining and quantitative analysis showed that the neutralizing antibody reduced the area of ​​Krt19[+] respiratory metaplasia and significantly increased the density of Tuj1[+] neurons (Fig. 4g, i, j). The results indicate that IL-17a neutralization significantly improves olfactory recovery in a dose-dependent manner.

[0052] In summary, these findings suggest that neutralization of Y-320 and IL-17a can effectively counteract age-related pathologies in OE and promote tissue repair and functional recovery.

[0053] In summary, this invention demonstrates the important role of IL-17a in regulating inflammation levels and olfactory regeneration within the olfactory system, and provides a potential therapeutic target for treating age-related olfactory dysfunction.

[0054] The undescribed parts of this invention are the same as or implemented using existing technology. The applicant declares that this invention is illustrated through the above embodiments, but the invention is not limited to the above detailed methods, i.e., it does not mean that the invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. Application of IL-17a as a therapeutic target for age-related olfactory dysfunction.

2. Application of IL-17a inhibitors in the preparation of drugs for the treatment of age-related olfactory dysfunction.

3. The application according to claim 2, characterized in that: wherein, The IL-17a is selected from any of the following substances: IL-17a gene, IL-17a mRNA or cDNA, IL-17a protein or any of the above active or marker fragments, and the IL-17a inhibitor is selected from any of the following substances: substances that reduce IL-17a expression levels, reduce IL-17a activity or promote IL-17a metabolism.

4. The application according to claim 3, characterized in that: wherein, The IL-17a inhibitors include any one of IL-17a small interfering RNA molecules, short hairpin RNA, antisense nucleotides, or nanoparticles, viral vectors, PEG-modified proteins, protein microspheres, liposomes, or extracellular vesicles carrying any of the above substances, and also include small molecule drugs or neutralizing antibodies.

5. The application according to claim 4, characterized in that, The small molecule drug was selected from the IL-17a inhibitor Y-320.

6. A pharmaceutical composition for treating age-related olfactory dysfunction, characterized in that, It includes an active ingredient and pharmaceutically or immunologically acceptable excipients, carriers or diluents, wherein the active ingredient is an IL-17a inhibitor as described in any one of claims 2 to 5.

7. The pharmaceutical composition for treating age-related olfactory dysfunction according to claim 6, characterized in that, This pharmaceutical composition is used in combination with other treatments for age-related olfactory dysfunction.

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