Application of Chil4 gene in screening of medicament for treating olfactory disorder
By constructing a mouse model of Chil4 knockout, the lack of rapid recovery of the sense of smell by the treatment of olfactory disorders was solved, and the screening of drugs that can significantly restore the sense of smell by short-term use was achieved, providing an effective drug screening model.
Patent Information
- Application Number
- CN202411723426.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-28
- Publication Date
- 2025-03-11
AI Technical Summary
The causes of olfactory disorders are complex, and existing treatments such as glucocorticoids and olfactory training have limitations, especially the lack of effective methods for rapid recovery of olfactory.
By constructing a mouse model of Chil4 knockout, this model is used to screen drugs for treating olfactory disorders, especially drugs that can significantly restore the sense of smell in short term use.
The Chil4 knockout mouse model can effectively simulate olfactory disorders, and the use of dexamethasone can significantly increase the number of immature and mature olfactory sensory neurons, thereby partially restoring olfactory function, providing an effective drug screening model.
Smart Images
Figure CN119592573A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to application of Chil4 gene in screening drugs for treating olfactory disorders. Background Art
[0002] Olfactory disorder refers to abnormal odor perception caused by functional and / or organic lesions in any link of the process of receiving, transmitting, integrating and processing odor information. The incidence of olfactory disorder in adults in my country is 4.5% to 21.6%, while in the elderly population aged 70 to 79 and 80 to 97, the incidence of olfactory disorder is as high as 29.2% and 62.5%. Abnormal olfactory perception seriously affects normal social interaction, nutritional intake and even threatens life safety. However, the etiology of olfactory disorder is complex and the pathogenesis is not yet fully understood. Clinically, olfactory disorder is divided into quantitative olfactory disorder (including olfactory hypersensitivity, hyposmia and anosmia) and qualitative olfactory disorder (including olfactory perversion and phantom olfaction). According to the location of the lesion, it can be divided into sensorineural, conductive, central and mixed olfactory disorders. Among them, the pathogenesis of conductive and sensorineural olfactory disorders is closely related to olfactory epithelial damage or lesions.
[0003] The olfactory epithelium (OE) is the starting point of olfaction. It contains multiple cell types such as horizontal basal cells (HBC), globular basal cells (GBC), immature olfactory sensory neurons (iOSN), mature olfactory sensory neurons (mOSN), supporting cells (SUS), etc., and has the characteristics of self-renewal. Abnormal homeostasis of the olfactory epithelium is an important cause of olfactory disorders. Factors such as injury, infection, inflammation, and aging can lead to changes in the structure and cell components of the olfactory epithelium, thereby causing olfactory disorders. Although the olfactory epithelium can also proliferate and differentiate from basal cells into all cell types in the olfactory epithelium after injury to complete the regeneration of the olfactory epithelium, its repair ability is also limited. Severe damage that causes the olfactory epithelium to be unable to completely repair will also cause olfactory disorders.
[0004] The diagnosis of olfactory disorders is divided into two aspects: subjective assessment and objective examination. Subjective assessment is mainly carried out through psychophysical test methods of olfactory function (olfactometer test, olfactory stick test, University of Pennsylvania olfactory identification test, etc.) and questionnaires or scales; objective examination is relatively more objective and stable, mainly judged by imaging (CT, MRI) and electrophysiological testing methods.
[0005] The treatment of olfactory disorders varies according to the cause and location of the disease. The treatment of olfactory disorders mainly includes etiological treatment, drug treatment, surgical treatment and olfactory training. At present, glucocorticoids are the most common drugs used to treat olfactory disorders. They are often used to treat inflammatory, traumatic, and post-superior olfactory disorders. Among them, the treatment effect on inflammatory olfactory disorders is the best. Taking prednisone as an example, short-term treatment with small to medium doses is usually recommended (within 1 month). Olfactory training requires patients to actively and repeatedly sniff various types of olfactory agents to improve their olfactory function. The course of treatment is longer and requires a high degree of self-discipline and cooperation from patients.
[0006] Compared with other sensory disorders, olfactory disorders rely more on subjective evaluation, and it is too risky to test new drugs directly on humans. In addition, for olfactory disorders without organic lesions, non-drug recovery through olfactory training may be recommended first in clinical practice. The cycle of olfactory training is long, often lasting for several months, and the prognosis is uncertain, so it is not useful for everyone. Therefore, finding the key genes that regulate the self-renewal of the olfactory epithelium is of great significance for understanding the pathogenesis of olfactory disorders, exploring potential therapeutic targets, and developing and screening drugs. In addition, it is necessary to develop a good and stable olfactory disorder model animal for screening drugs that can significantly restore the sense of smell with short-term use. Summary of the invention
[0007] The purpose of the present invention is to provide an application of Chil4 gene in screening drugs for treating olfactory disorders, and to provide a method for constructing an olfactory disorder model and its application.
[0008] The technical solution adopted by the present invention is:
[0009] Application of Chil4 gene in screening drugs for the treatment of olfactory disorders.
[0010] Furthermore, the application method is: using the Chil4 gene as a drug target, constructing an animal model of knocking out the Chil4 gene, and using it to screen drugs for treating olfactory disorders.
[0011] The present invention also provides a method for constructing an olfactory disorder animal model, the method comprising: knocking out the Chil4 gene of a target animal to construct the olfactory disorder animal model.
[0012] The target animal is a mammal; preferably, the mammal is selected from mice, rats and the like.
[0013] The present invention also provides an olfactory disorder animal model, which is a Chil4 gene knockout mouse.
[0014] The present invention also provides application of the olfactory disorder animal model in olfactory disorder disease research.
[0015] The present invention also provides application of the olfactory disorder animal model in screening drugs for treating olfactory disorders.
[0016] The present invention also provides the use of the olfactory disorder animal model in screening drugs that can significantly restore the sense of smell after short-term use.
[0017] Chil4 knockout mice are a good and stable model of olfactory dysfunction. The Chil4 knockout mice creatively used in the present invention have no significant differences in growth and development and olfactory epithelial thickness compared with wild-type mice, but the number of various types of cells in the olfactory epithelium is different from that of wild-type mice. + 、NeuroD1 + Neuronal progenitor cells were significantly reduced, GAP43 + 、Tuj1 + A significant reduction in immature olfactory sensory neurons, OMP + Significant reduction in mature olfactory sensory neurons, Iba1 + 、MPO + 、F4 / 80 + Inflammatory cells increased significantly, and olfactory behavior test showed decreased olfactory function. Therefore, Chil4 knockout mice can be used as a mouse model of olfactory dysfunction for drug screening. Intraperitoneal injection of Dex for 7 consecutive days can significantly increase GAP43 + 、Tuj1 + Immature olfactory sensory neurons and OMPs + The number of mature olfactory sensory neurons demonstrates that Chil4 knockout mice provide a good model for screening and dose testing of olfactory restoration drugs.
[0018] Compared with the prior art, the present invention has at least the following beneficial effects:
[0019] Experiments based on this animal model found that the Chil4 gene may play an important role in regulating olfactory disorders. Chil4 gene knockout mice have impaired olfactory function, reduced numbers of immature olfactory sensory neurons and supporting cells, and increased inflammatory cells. Intraperitoneal injection of dexamethasone (Dex) can significantly increase the number of immature olfactory sensory neurons and mature olfactory sensory neurons in Chil4 knockout mice. Therefore, the Chil4 gene can be used as a target for screening drugs for the treatment of olfactory disorders. Chil4 gene knockout mice can be used to construct an olfactory disorder model for the study of olfactory disorders, providing new research ideas for the clinical treatment of olfactory disorders and related diseases. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1Comparison of the counts of various types of cells in the olfactory epithelium of wild-type mice (WT) and Chil4 knockout mice (KO); the two groups were compared using an unpaired t-test, and P<0.05 indicated a statistically significant difference (ns, no significant difference, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).
[0021] Figure 2 The figure shows the comparison of latency of grabbing food pellets in behavioral tests of wild-type mice (WT) and Chil4 knockout mice (KO). The left figure shows the latency when the food pellets were buried under the litter, and the right figure shows the latency when the food pellets were placed on the litter. The two groups were compared using an unpaired t-test, and P<0.05 indicated a statistically significant difference (ns: no significant difference, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).
[0022] Figure 3 The figure shows the comparison of the counts of various types of olfactory sensory neurons in the olfactory epithelium of Chil4 knockout mice after intraperitoneal injection of dexamethasone (KO+Dex) or DMSO (KO+DMSO) for 7 consecutive days. The two groups were compared using the unpaired t-test, and P<0.05 indicated a statistically significant difference (ns: no significant difference, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). DETAILED DESCRIPTION
[0023] The technical solution of the present invention is described in detail below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto.
[0024] Example 1
[0025] Experimental animals: Specific pathogen free (SPF) grade C57BL / 6J wild-type mice and SPF grade Chil4 knockout mice (Strain NO. T027807) were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.
[0026] When Chil4 knockout mice were bred, it was found that their growth and development were no different from those of wild-type mice of the same age.
[0027] 1. Count the various types of cells in the olfactory epithelium of wild-type mice (WT) and Chil4 knockout mice (KO).
[0028] Wild-type mice and Chil4 knockout mice of the same age (2-4 months old) were perfused cardiacly, and the olfactory epithelium was sectioned and stained.
[0029] Specifically, after deeply anesthetizing mice, they were perfused with precooled PBS and 4% paraformaldehyde (PFA) to remove the olfactory epithelium, including part of the skull and palate, and fixed with 4% PFA in a 4°C refrigerator for 5-8 hours. They were then decalcified with 0.5MEDTA for 4-6 days. Then, they were dehydrated in 10%, 20%, and 30% sucrose in a gradient manner. Finally, the tissues were placed in OCT embedding medium and quickly frozen at low temperature to prepare coronal olfactory epithelial frozen sections with a thickness of 20 μm. The frozen tissue sections were washed with PBS, permeated with 0.3% Triton X-100, blocked at room temperature, and then an appropriate amount of primary antibody was added. They were incubated at 4°C overnight. The next day, the corresponding fluorescent secondary antibody was added and incubated at room temperature for 1 hour. The cell nuclei were counterstained with DAPI, and the expression of fluorescent labeled proteins was observed under a laser confocal microscope. The number of different types of cells in the olfactory epithelium was counted and compared between groups.
[0030] The olfactory epithelial cells of wild-type mice and Chil4 knockout mice were counted (n=4 / 4). Figure 1 As shown, Ki67 in the olfactory epithelium of Chil4 knockout mice + (t=5.242, P<0.0001****), NeuroD1 + (t=3.36, P=0.002**) Neuronal precursor cells decreased significantly, GAP43 + (t=4.887, P<0.0001****), Tuj1 + (t=9.157,P<0.0001****) The number of immature olfactory sensory neurons decreased significantly, and OMP + Olfactory sensory mature neurons (lateral) (t = 2.488, P = 0.0376*) were significantly reduced, Iba1 + Microglia (t=2.400, P=0.0249*), MPO + Neutrophils (t=4.062, P=0.0005***), F4 / 80 + The number of various inflammatory cells, such as macrophages (t=3.492, P=0.0017**), increased significantly, indicating that the olfactory epithelial structure of Chil4 knockout mice was damaged.
[0031] 2. Behavioral experiments were conducted on wild-type mice (WT) and Chil4 knockout mice (KO), and olfactory function was evaluated by the latency to grasp food pellets.
[0032] Behavioral experiments were performed on wild-type mice and Chil4 knockout mice aged 2 to 4 months to evaluate olfactory function. The experimental mice were fasted for 24 hours before the test and replaced with new bedding, and drinking water was not restricted. On the day of the test, several 45cm×24cm×20cm test cages were prepared and evenly covered with 3cm thick bedding. The mice were placed in clean test cages to adapt for 10 minutes, and then the mice were taken out. Food pellets (0.2g±0.02g) were buried 0.5cm under the bedding on one side of the test cage. The mice were placed in the test cage from the other side and the timing was started. The latency of the mouse's front paw or mouth and nose contacting the food pellet was recorded. If it exceeded 300s, it was recorded as 300s. As a control, food pellets were placed on the bedding 6 hours later, and the other conditions remained unchanged. The time when the mouse's front paw or mouth and nose contacted the food pellet was also recorded and compared between groups.
[0033] Behavioral tests were performed on wild-type mice and Chil4 knockout mice (n=12 / 11). Figure 2 As shown, it was found that when the food pellets were buried at the bottom of the litter, the time taken by Chil4 knockout mice to find food was significantly prolonged (t=2.35, P=0.028*), while when the food pellets were placed on the surface of the litter, there was no significant difference in the time the two groups of mice spent in contact with the food pellets (t=1.44, P=0.165), indicating that the olfactory function of Chil4 knockout mice was impaired.
[0034] 3. Dexamethasone (Dex, purchased from Sigma-Aldrich) was injected intraperitoneally into Chil4 knockout mice at a dose of 1 mg / kg for 7 consecutive days. The control group consisted of Chil4 knockout mice, which were injected with the same volume of DMSO. On the 8th day, the olfactory epithelium was obtained after cardiac perfusion, sectioned, stained, and cell counted to observe whether the number of each type of cells changed.
[0035] Comparison of the counts of various olfactory sensory neurons in the olfactory epithelium of Chil4 knockout mice after intraperitoneal injection of dexamethasone (KO+Dex) or DMSO (KO+DMSO) for 7 consecutive days. Figure 3 As shown (n=4 / 4).
[0036] The results showed that GAP43 was expressed in the olfactory epithelium of mice after dexamethasone injection (KO+Dex). + (t=3.301, P=0.0023**), Tuj1 + (t=2.851,P=0.0074**) The number of immature olfactory sensory neurons was significantly decreased, and OMP +The number of mature olfactory sensory neurons (t=3.697, P=0.0024**) increased significantly, indicating that dexamethasone can partially restore the olfactory epithelial structure of Chil4 knockout mice, further contributing to the recovery of olfactory function.
[0037] The above experiments show that:
[0038] (1) Compared with wild-type mice, Chil4 knockout mice have decreased olfactory function;
[0039] (2) Continuous intraperitoneal injection of dexamethasone for 7 days can partially reverse the olfactory impairment of Chil4 knockout mice.
[0040] Therefore, Chil4 knockout mice can be used to construct an olfactory impairment model and to screen drugs for the treatment of olfactory impairment.
[0041] The above are only some preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the technical solution of the present invention, and any changes and modifications made are within the protection scope of the present invention.
Claims
1. Application of Chil4 gene in screening drugs for the treatment of olfactory disorders.
2. The use according to claim 1, characterized in that The application method comprises: using the Chil4 gene as a drug target, constructing an animal model for knocking out the Chil4 gene, and screening drugs for treating olfactory disorders.
3. A method for constructing an animal model of olfactory disorder, characterized in that: The method comprises: knocking out the Chil4 gene of a target animal to construct an olfactory disorder animal model.
4. The method according to claim 3, characterized in that The target animal is a mammal.
5. The method according to claim 3, characterized in that The mammal is selected from a mouse or a rat.
6. The olfactory disorder animal model constructed according to any one of claims 3 to 5.
7. The animal model of olfactory impairment according to claim 6, wherein: The olfactory disorder animal model is Chil4 gene knockout mouse.
8. Use of the olfactory disorder animal model as claimed in claim 6 in the study of olfactory disorder diseases.
9. Use of the olfactory disorder animal model according to claim 6 in screening drugs for treating olfactory disorders.
10. Use of the olfactory disorder animal model according to claim 6 in screening drugs that can significantly restore the sense of smell after short-term use.