Application of kaempferol in preparation of medicine for promoting cilia growth

Abstract

The invention relates to the field of biological medicine, in particular to application of kaempferol in preparation of a medicine for promoting cilia growth, and provides application of kaempferol or pharmaceutically acceptable salt, stereoisomer, solvate, prodrug or derivative thereof in preparation of the medicine for promoting cilia growth. And a pharmaceutical composition or combination comprising a therapeutically effective amount of kaempferol or a pharmaceutically acceptable salt, stereoisomer, solvate, prodrug or derivative thereof.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to application of kaempferol in preparation of a drug for promoting growth of cilia. BACKGROUND

[0002] Cilia are microtubule-based antennae-like sensory organelles that project from the apical surface of most mammalian cells and are used to sense extracellular signals and stimuli. Cilia are mainly composed of basal body, axoneme, ciliary membrane and ciliary matrix. According to the structure of axoneme, cilia can be divided into motile cilia (9+2 structure) and primary cilia (9+0 structure). The basal body required for cilia formation is derived from the mother centriole, and there is a transition zone (TZ) between the basal body and the axoneme, which regulates the entry and exit of proteins and membrane components into the cilia. After the proteins required for cilia assembly enter the cilia, directional transport mainly depends on the Intraflagellar transport (IFT) family, including anterograde transport (through kinesin 2 and IFT-B complex) and retrograde transport (through dynein 2 and IFT-A complex), thereby promoting rapid extension of cilia. Once formed, cilia will be maintained at a specific length.

[0003] Cilia in each cell type will have a normal length range, and some congenital genetic factors (IFT, transition zone protein and signal pathway related gene mutations) and acquired environmental factors (viral infection, environmental pollution and drug toxicity, etc.) will cause cilia loss and dysfunction, which will trigger a series of diseases, collectively known as ciliopathies. Primary cilia are located on the surface of most eukaryotic cells and mainly participate in signal transduction, and their dysfunction will cause polycystic kidney disease (PKD), Leber congenital amaurosis (LCA), Bardet-Biedl syndrome, Joubert syndrome and Meckel-Gruber syndrome, etc.; motile cilia mainly exist in respiratory tract, ventricles and reproductive system, etc., and generate movement by coordinating swing, such as respiratory tract cilia removing mucus and sperm flagella driving sperm movement, and their dysfunction will cause respiratory diseases, infertility and hydrocephalus, etc.

[0004] Currently, the treatment of cilia disease in Western medicine mainly focuses on symptomatic support, which cannot repair or regenerate cilia, and the treatment mainly aims to relieve symptoms. For example: for respiratory tract infection, antibiotics are often used to treat infection, supplemented by expectorants, but they cannot promote cilia regeneration and restore cilia clearance function, and may also cause drug resistance or side effects; for infertility, assisted reproductive technology (such as in vitro fertilization) is used, but it cannot repair sperm flagellum function; for hydrocephalus, surgical treatment (such as ventricular shunt) is used, but it cannot restore ventricular cilia function; and the application of gene editing (such as CRISPR) in the treatment of cilia disease is still in the experimental stage, and there are problems such as off-target effects, low editing efficiency, and poor tissue specificity.

[0005] Traditional Chinese medicine has shown significant potential advantages in treating and restoring cilia function, especially in anti-inflammatory and antioxidant and restoring cilia dynamics. In terms of anti-inflammatory: such as quercetin, on the one hand, it can inhibit the release of cytokine release syndrome driven by nuclear factor kappa-B (NF-κB), inflammasome and interleukin-6 (IL-6), and on the other hand, it can activate the nuclear factor erythroid 2-related factor 2 (NRF2) pathway to reduce cytokine release syndrome and alleviate acute lung injury. Astragalus polysaccharide can promote high levels of hemagglutination inhibition titer and specific antibody immunoglobulin G (IgG) induction, achieving bidirectional immune regulation of cellular and humoral immunity. In terms of restoring cilia dynamics: such as gypsium in Maxingshigan decoction can accelerate cilia beat frequency by activating transient receptor potential vanilloid 1 (TRPV1) receptors. Naringin can inhibit intracellular Ca 2+ level to relax tracheal smooth muscle and increase cilia oscillation frequency, thereby accelerating viral clearance. Hesperidin can increase trans-epithelial Cl − secretion, improve airway cilia movement capacity, and enhance the function of the mucociliary clearance (MCC) system.

[0006] In terms of combination therapy, traditional Chinese medicine monomers combined with Western medicine also show good synergy and complementary characteristics. On the one hand, traditional Chinese medicine monomers can enhance the efficacy of antibiotics and expectorants, such as baicalein combined with azithromycin, which can enhance the antibacterial spectrum, reduce drug resistance, and reduce inflammation damage to cilia; on the other hand, traditional Chinese medicine monomers can reduce the adverse reactions of Western medicine, such as relieving the irritation of expectorants to the gastrointestinal tract or the bacterial flora imbalance caused by long-term use of antibiotics.

[0007] At present, although western medicine, traditional Chinese medicine and combined application of traditional Chinese and western medicine can improve cilia function through symptomatic auxiliary treatment or promoting cilia movement, and then promote disease recovery, there is still lack of drugs directly promoting cilia growth. Therefore, the research and development of drugs for promoting cilia stem cell differentiation and promoting cilia growth to promote cilia "structural reconstruction" and "functional repair" are expected to provide new pharmacological basis and practical strategy for the treatment of cilia disease. SUMMARY

[0008] In one aspect, the present disclosure relates to the use of kaempferol or a pharmaceutically acceptable salt, stereoisomer, solvate, prodrug or derivative thereof in the preparation of a medicament for promoting cilia growth.

[0009] In some embodiments, the use of the foregoing uses has at least one of the following applications: (1) promoting the increase of cilia length; (2) promoting cilia generation; (3) promoting the recovery of cilia function; (4) rescuing and / or repairing cilia function, cilia generation and / or cilia length defects in damaged cilia cells.

[0010] In some preferred embodiments, the foregoing damaged cilia cells are selected from respiratory cilia epithelial cells, reproductive system cilia cells, sensory system cilia cells, embryonic development related cilia cells, and kidney and liver cilia cells.

[0011] In some embodiments, the foregoing damaged cilia cells are bacterial or viral cilia damage cells, IFT system damaged cilia cells and / or CEP290 gene expression abnormal cilia cells.

[0012] In another aspect, the present disclosure relates to a pharmaceutical composition or combination comprising a therapeutically effective amount of kaempferol or a pharmaceutically acceptable salt, stereoisomer, solvate, prodrug or derivative thereof; optionally, comprising a pharmaceutically acceptable carrier.

[0013] In some embodiments, the foregoing pharmaceutical composition or combination comprises: a) a therapeutically effective amount of kaempferol or a pharmaceutically acceptable salt, stereoisomer, solvate, prodrug or derivative thereof; b) at least one therapeutically effective amount of a drug for increasing cilia movement or a pharmaceutically acceptable salt, stereoisomer, solvate, prodrug or derivative thereof; optionally, comprising a pharmaceutically acceptable carrier; The drug for increasing ciliary movement comprises a mucolytic agent, a physical / osmotic mucous clearance agent, a bronchodilator, a ciliary movement and function direct promoter, an immunomodulator, an antioxidant, an ion channel modulator, a complex preparation, and / or an auxiliary therapeutic agent.

[0014] In some embodiments, the aforementioned auxiliary therapeutic agent comprises an antibiotic and / or a drug for treating systemic complications.

[0015] In some embodiments, the aforementioned drug for increasing ciliary movement comprises ambroxol (mucolytic agent, ciliary movement enhancer, stimulates pulmonary surfactant synthesis and secretion, immunomodulation, antioxidant, etc.), acetylcysteine (mucolytic agent, antioxidant), erdosteine (mucolytic agent), hypertonic saline, mannitol, β2-receptor agonists (salbutamol, formoterol), anticholinergic drugs (ipratropium bromide, tiotropium bromide), P2Y2 receptor agonists (diquafos, myrtle oil), inhaled glucocorticoids (budesonide, fluticasone), macrolide antibiotics (azithromycin, erythromycin), antioxidant N-acetylcysteine, ion channel modulator ivacaftor.

[0016] In some preferred embodiments, the aforementioned drug for increasing ciliary movement is ambroxol.

[0017] In some embodiments, the molar ratio of kaempferol to the aforementioned drug for increasing ciliary movement in the aforementioned pharmaceutical composition or drug combination is 0.001-10.

[0018] In some embodiments, the molar ratio of kaempferol to the aforementioned drug for increasing ciliary movement in the aforementioned pharmaceutical composition or drug combination is 0.02-0.04, 0.04-0.06, 0.06-0.08, 0.08-0.10, 0.10-0.12, 0.12-0.14, 0.14-0.16, 0.16-0.18, or 0.18-0.20.

[0019] In some embodiments, the molar ratio of kaempferol to the aforementioned drug for increasing ciliary movement in the aforementioned pharmaceutical composition or drug combination is (1-5):50.

[0020] In some preferred embodiments, the molar ratio of kaempferol to ambroxol in the aforementioned pharmaceutical composition or drug combination is 0.001-10.

[0021] In some preferred embodiments, the molar ratio of kaempferol and ambroxol in the aforementioned pharmaceutical composition or pharmaceutical combination is 0.02-0.04, 0.04-0.06, 0.06-0.08, 0.08-0.10, 0.10-0.12, 0.12-0.14, 0.14-0.16, 0.16-0.18, or 0.18-0.20.

[0022] In some preferred embodiments, the molar ratio of kaempferol and ambroxol in the aforementioned pharmaceutical composition or pharmaceutical combination is (1-5):50. In some more preferred embodiments, the molar ratio of kaempferol and ambroxol in the aforementioned pharmaceutical composition or pharmaceutical combination is 1:50.

[0023] In some more preferred embodiments, the molar ratio of kaempferol and ambroxol in the aforementioned pharmaceutical composition or pharmaceutical combination is 3:50.

[0024] In some more preferred embodiments, the molar ratio of kaempferol and ambroxol in the aforementioned pharmaceutical composition or pharmaceutical combination is 5:50.

[0025] In some embodiments, the concentration of kaempferol in the aforementioned pharmaceutical composition or pharmaceutical combination is 0.1-100 μΜ.

[0026] In some preferred embodiments, the concentration of kaempferol in the aforementioned pharmaceutical composition or pharmaceutical combination is 1-3 μΜ.

[0027] In some preferred embodiments, the concentration of kaempferol in the aforementioned pharmaceutical composition or pharmaceutical combination is 3-5 μΜ.

[0028] In some preferred embodiments, the concentration of kaempferol in the aforementioned pharmaceutical composition or pharmaceutical combination is 1 μΜ.

[0029] In some preferred embodiments, the concentration of kaempferol in the aforementioned pharmaceutical composition or pharmaceutical combination is 3 μΜ.

[0030] In some preferred embodiments, the concentration of kaempferol in the aforementioned pharmaceutical composition or pharmaceutical combination is 5 μΜ.

[0031] In some more preferred embodiments, the concentration of kaempferol in the aforementioned pharmaceutical composition or pharmaceutical combination is 1 μΜ, and the molar ratio of kaempferol and ambroxol is 1:50.

[0032] In some more preferred embodiments, the concentration of kaempferol in the aforementioned pharmaceutical composition or pharmaceutical combination is 3 μΜ, and the molar ratio of kaempferol and ambroxol is 3:50.

[0033] In some more preferred embodiments, the concentration of kaempferol in the aforementioned pharmaceutical composition or pharmaceutical combination is 5 μM, and the molar ratio of kaempferol to ambroxol is 5:50.

[0034] In some embodiments, the aforementioned drug, pharmaceutical composition or pharmaceutical combination is used for treating a cilia dysfunction-related disease or disorder.

[0035] In some preferred embodiments, the aforementioned cilia dysfunction-related disease or disorder is selected from the group consisting of diseases or disorders caused by abnormal cilia structure, defective motility function or impaired signal transduction function, including primary ciliary dyskinesia, secondary cilia dysfunction, ciliopathies.

[0036] In some more preferred embodiments, the aforementioned primary ciliary dyskinesia includes Kartagener syndrome; the aforementioned secondary cilia dysfunction-related disease includes mucus clearance disorder and chronic airway inflammatory disease caused by chronic obstructive pulmonary disease, asthma, cystic fibrosis and respiratory tract infection and bronchiectasis; the aforementioned ciliopathy includes polycystic kidney disease, Bardet-Biedl syndrome, Joubert syndrome, Meckel-Gruber syndrome, Usher syndrome and Leber congenital amaurosis; and the aforementioned clinical manifestation includes infertility caused by abnormal sperm flagellum or oviduct ciliary motility, and hydrocephalus caused by impaired ependymal ciliary flow function.

[0037] In some more preferred embodiments, the aforementioned cilia dysfunction-related disease or disorder is cilia damage caused by acute or chronic bacterial infection.

[0038] In some more preferred embodiments, the aforementioned cilia dysfunction-related disease or disorder is respiratory tract cilia loss caused by bacterial infection.

[0039] In some specific embodiments, the aforementioned bacteria is gram-negative bacteria.

[0040] In some embodiments, the aforementioned drug, pharmaceutical composition or pharmaceutical combination is a topical administration preparation, an inhalation administration preparation or an oral administration preparation.

[0041] In some preferred embodiments, the aforementioned drug, pharmaceutical composition or pharmaceutical combination is a tablet, a lozenge, a capsule, a pill, a solution, a suspension, a syrup, an injection, a suppository, an inhalation solution, an inhalation powder mist, an aerosol or a spray.

[0042] The beneficial effects of the present application at least include: In the present application, kaempferol monotherapy has a significant effect on restoring cilia, and its effect on promoting cilia growth is concentration-dependent, and can widely restore cilia defects caused by various reasons (such as viral injury, IFT system damage, and CEP290 deletion).

[0043] Ambroxol monotherapy has limited effect on cilia shortening caused by IFT system damage or CEP290 deletion. However, when kaempferol is used in combination with ambroxol, a synergistic effect is exhibited: the combination therapy not only can rescue viral cilia damage, but also has a better effect on restoring cilia damage caused by IFT system damage or CEP290 deletion than kaempferol monotherapy, and the synergistic effect is more significant at a higher concentration. BRIEF DESCRIPTION OF DRAWINGS

[0044] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present specification and serve to explain the principles of the present specification.

[0045] Figure 1 The effect of kaempferol on cilia formation and cilia length is shown.

[0046] Figure 2 The recovery effect of kaempferol on cilia loss caused by viral infection is shown.

[0047] Figure 3 The recovery effect of kaempferol on cilia loss in IFT system damaged cells is shown.

[0048] Figure 4 The recovery effect of kaempferol on various genetic cilia loss caused by CEP290 reduction (siCEP290) is shown.

[0049] Figure 5 The effect of kaempferol combined with ambroxol on cilia number and length is shown.

[0050] Figure 6 The recovery effect of kaempferol combined with ambroxol on cilia loss caused by viral infection is shown.

[0051] Figure 7 The recovery effect of kaempferol combined with ambroxol on cilia loss in IFT system damaged cells is shown.

[0052] Figure 8 The recovery effect of kaempferol combined with ambroxol on various genetic cilia loss caused by CEP290 reduction (siCEP290) is shown.

[0053] Figure 9 The recovery effect of kaempferol on respiratory cilia damage in a bacterial infection model (lipopolysaccharide induction) is shown. DETAILED DESCRIPTION

[0054] I. Definitions In the present disclosure, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the terms and phrases related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and immunology are used in accordance with their broadest generally accepted and customary meanings in the art, and the chemical formulas written herein are intended to represent the broadest form of the compounds. Furthermore, for better understanding of the present disclosure, the definitions and explanations of the related terms are provided below.

[0055] As used herein and unless otherwise indicated, the terms "comprising," "including," "having," "containing," including grammatical variants thereof, are open-ended and non-limiting, for example, in that they do not exclude additional unrecited elements or steps.

[0056] All ranges disclosed herein are to be interpreted as having the endpoints and all points within the range. For example, a disclosure of a range of 1-1000 is to be interpreted to include the endpoints 1 and 1000, as well as all points within the range, such as 200, 300, 400, 500, 600, 700, 800, 900, etc., as well as the points 200, 300, 400, 500, 600, 700, 800, 900, and 1000.

[0057] The compounds can exist in the pharmaceutical composition as pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present application that are found to have particular substituents, which are prepared from the compounds of the present application by reaction with a relatively nontoxic acid or base. When the compounds of the present application contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the base in either a neat solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salts or similar salts. When the compounds of the present application contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the acid in either a neat solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, bicarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, hydrogensulfuric, hydriodic, phosphorous, and the like; and organic acids such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, ethanesulfonic, and the like; also salts of amino acids such as arginate, gluconate, and the like; and salts of organic acids like glucuronic, and the like. Certain specific compounds of the present application contain both basic and acidic functionalities and as such can be converted into either base or acid addition salts.

[0058] The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, the salt will be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of both.

[0059] In addition to salt forms, the compounds provided herein can be in a prodrug form. Prodrugs of the compounds described herein are readily converted by chemical or physiological action into the compounds of the present application. Additionally, prodrugs can be converted by chemical or physiological action to a compound of the present application in an environment of the body. For example, a prodrug can be slowly converted into a compound of the present application when placed in a transdermal patch reservoir containing the appropriate enzyme or chemical reagent.

[0060] Certain compounds of the present application can exist in unsolvated as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are included in the scope of the present application. Certain compounds of the present application can exist in different polymorphic or amorphous forms. In general, all physical forms are equivalent and are intended to be within the scope of the present application.

[0061] As used herein, the term "kaempferol derivative" refers to a natural flavonoid compound, kaempferol (chemical formula C 15 H 10O6) structural analogs formed by chemical modification (e.g., glycosylation, methylation, acetylation, prenylation, etc.) or bioconversion. Exemplary examples of kaempferol derivatives include, but are not limited to, kaempferol-3-glucoside, kaempferol-3-glucorhamnoside, 6-hydroxykaempferol-3-glucoside, 6-hydroxykaempferol-3,6-diglucoside, 6-hydroxykaempferol-3,6,7-triglucoside, kaempferol-3,7-dirhamnoside, 7-rhamnosiokaempferol-3-glucoside, 7-glucosiokaempferol-3-rhamnoside, kaempferol-4'-glucoside, 3-acetyIkaempferol-7-glucoside, kaempferol-7-glucoside, 4'-acetyIkaempferol-7-glucoside, 3-hydroxyethylkaempferol-7-rhamnoside, kaempferol-7,4'-dirhamnoside glucoside, kaempferol-5-methylether-7-glucoside, kaempferol-3-rhamnoside, kaempferol-3-arabinoside, kaempferol-3-galactoside, kaempferol-3-xyloside, kaempferol-3-glucuronide, kaempferol-3-coumaroylarabinoside, kaempferol-3-feruloylarabinoside, 7,4'-dihydroxyethyl-kaempferol-3-rutinoside, 4'-methylkaempferol-7-glucoside, kaempferol-5,4'-diglucoside, kaempferol-3-(3,4,5-trihydroxy)benzoylglucoside, and the like.

[0062] As used herein, the use of the term "promote" or "inhibit" is relative to a control. One of skill in the art will readily determine the appropriate control for each experiment. For example, an elevated or reduced response in a subject or cell treated with a compound is compared to the response in a subject or cell not treated with the compound.

[0063] As used herein, the term "in combination" or "in combination with" is a mode of administration that includes all cases of simultaneous, concurrent, sequential, continuous, alternating, or separate administration of the two agents. As used herein, "simultaneous" includes administration within the same dosing cycle, e.g., within 2 days, or within 1 day, of each other. As used herein, "sequential," "continuous," "alternating," or "separate" administration includes administration within different dosing cycles. The mode of administration "in combination" includes administration simultaneously, separately formulated and co-administered, or separately formulated and administered sequentially. Each of the above modes of administration is encompassed by the present application.

[0064] As used herein, the term "effective amount" or "therapeutically effective amount" includes an amount that is sufficient to ameliorate or prevent the symptoms or conditions of a medical disorder. An effective amount also means an amount that is sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health status of the patient, the method route and dose of administration, and the severity of side effects. The effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxic effects.

[0065] The term "pharmaceutically acceptable" with respect to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0066] As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation, formulation aid or carrier, which is commonly used in the art of pharmaceutical formulation, together with the therapeutic agent, which together make up a "pharmaceutical composition" for administration to an individual. The pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed, and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is suitable for the formulation employed. Suitable carriers are well known to those skilled in the art, such as carbohydrates, waxes, water soluble and / or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, liposomes, polymeric micelles, or inorganic nanocarriers, and the like.

[0067] As used herein, the term "pharmaceutical composition" refers to a mixture of one or more components. The purpose of the pharmaceutical composition is to facilitate administration of the compounds of the disclosure to an organism. The pharmaceutical composition of the disclosure can include one or more pharmaceutically acceptable salts, pharmaceutically acceptable carriers and / or adjuvants, such as antioxidants, preservatives, wetting agents, emulsifying agents and dispersing agents, and the like.

[0068] The "pharmaceutical composition" of the disclosure can also be administered to a patient or subject in need of such treatment in any suitable administration manner, such as oral, intravenous, parenteral, rectal, pulmonary, or topical administration, and the like. When used for oral administration, the pharmaceutical composition can be formulated into oral preparations, such as oral solid preparations, such as tablets, capsules, pills, granules, and the like; or oral liquid preparations, such as oral solutions, oral suspensions, syrups, and the like. When formulated into oral preparations, the pharmaceutical preparation can further include suitable fillers, binders, disintegrants, lubricants, and the like.

[0069] The pharmaceutical composition of the disclosure can be formulated into any pharmaceutically acceptable dosage form for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration, for example, can be formulated into tablets, troches, capsules, pills, solutions, suspensions, syrups, injections, suppositories, inhalation solutions, inhalation powders, aerosols, or sprays.

[0070] As used herein, the term injured ciliated cell or "ciliopathy cell" refers to a cell with cilia structural damage (e.g., abnormal arrangement of axonemal microtubules, basal body misplacement) or dysfunction (e.g., reduced beating frequency, impaired signal transduction) due to genetic mutation, infection (e.g., bacteria, virus), environmental stimulus (e.g., pollutants, chemical toxins), or disease state (e.g., chronic inflammation), with the core features of shortened cilia length, reduced cilia number, or impaired cilia function, which in turn affects cell motility, signal sensing, and tissue homeostasis.

[0071] As used herein, the term "ciliopathy-associated disease or disorder" refers to a class of diseases or disorders caused by abnormal cilia structure or function, mainly manifested as impaired cilia clearance, motility, or signaling ability, which in turn leads to multi-system dysfunction.

[0072] As used herein, the term "bacterial infection-induced cilia injury" refers to direct or indirect structural damage (e.g., shortened cilia, cilia shedding, or disordered cilia arrangement) and functional impairment (e.g., reduced beating frequency, abnormal rhythm) of respiratory mucosa epithelial cell cilia caused by pathogenic bacterial toxins (e.g., lipopolysaccharide LPS, hemolysin, etc.) or their metabolites, and the excessive immune inflammatory response of the host, which in turn weakens the defense function of the mucociliary clearance system and further exacerbates the vicious cycle of pathogenic colonization and persistent inflammation. Bacterial infection can be used to construct an experimental model of cilia injury. For example, in some embodiments of the present disclosure, a secondary cilia dysfunction model is established by administering lipopolysaccharide (LPS) to simulate gram-negative bacterial infection, which is classified in the category of respiratory diseases and mucus clearance disorders, and is used to verify the restorative effect of kaempferol on such dysfunction.

[0073] II. DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS For the purpose of clarity and brevity, features are described herein as part of the same or separate embodiments, however, it will be understood that the scope of the disclosure can include embodiments having combinations of all or some of the described features. The following examples are merely illustrative of the present application and should not be considered limiting thereof. Where specific techniques or conditions are not described in the following examples, techniques or conditions described in the literature or according to the product specification are used. Where the manufacturer of a reagent or instrument is not indicated, conventional products available from commercial vendors are used.

[0074] EXAMPLES Example 1: Kaempferol regulates cilia length in a concentration-dependent manner Human retinal pigment epithelial (ARPE19) cells (purchased from Wuhan Ponsay Biotech Co., Ltd. and subjected to STR identification) were used to verify whether kaempferol regulates cilia genesis and growth, which undergo ciliogenesis when starved (serum removal).

[0075] Cells were routinely cultured in DMEM / F12 medium containing 10% fetal bovine serum at 37 °C, 5% CO2. To induce cilia formation, when cells grew to about 90% confluence, the medium was replaced with serum-free medium and the cells were starved for another 24 hours. Then, different final concentrations of kaempferol (1 µM, 3 µM and 5 µM) were added to the starved cells, respectively, and the drug treatment lasted for 24 hours. Then, the cells were fixed, stained with DAPI for nuclei and ARL13B for cilia, and imaged and analyzed by immunofluorescence microscopy.

[0076] The staining process first fixed the cells with 4% paraformaldehyde for 15 minutes, and then permeabilized with 0.2% Triton X-100 solution for 20 minutes after two PBS washes. Then, 10% goat serum was used for blocking. Then, the antibody incubation was performed: the cells were first incubated with rabbit anti-ARL13B primary antibody and mouse anti- ACTUB primary antibody at 4 °C overnight; the next day, the corresponding Alexa Fluor 488 and Alexa Fluor 568 labeled secondary antibodies were incubated at room temperature for 2 hours in the dark. Finally, the anti-quenching mounting medium containing DAPI was used for mounting. The stained cells were observed and imaged using a confocal microscope, and the cilia formation and growth were imaged and counted by analyzing the fluorescence signals of ARL13B and DAPI. The primary antibodies included rabbit anti-ARL13B (1:1000; 17711-1-AP, Proteintech) and mouse anti- ACTUB (1:2000; T6793, Sigma). The secondary antibodies included goat anti-rabbit IgG-Alexa Fluor 488 conjugated antibody (1:1000; A11036, Invitrogen) and goat anti-mouse IgG-Alexa Fluor 568 conjugated antibody (1:1000; A11004, Invitrogen).

[0077] Results are shown in Figure 1As shown, Figure A is a fluorescence image of ciliated cells after treatment with kaempferol. Cilia are marked in red with ARL13B, and cell nuclei are marked in blue; the scale bars in the figures are all 10 µm. Figure B shows the ciliary length analysis; Figure C shows the frequency analysis of different ciliary lengths; Figure D shows the quantitative analysis of the percentage of ciliated cells. After treating starved cells with kaempferol for 24 hours, the number of cilia did not change significantly, but the average length of the cilia increased significantly, and the average length of the cilia increased significantly with increasing kaempferol concentration (blank control = 4.42 ± 0.07 µm, dimethyl sulfoxide solvent control = 4.22 ± 0.07 µm, 1 µM kaempferol = 4.93 ± 0.09 µm, 3 µM kaempferol = 4.97 ± 0.07 µm, 5 µM kaempferol = 5.88 ± 0.09 µm; **** p <0.001 Compared with the dimethyl sulfoxide solvent control group, ns: no statistical significance, all values ​​in the figure are mean ± standard error, n ≥ 3).

[0078] In addition, the expression of ciliary-associated proteins (ACTUB and ARL13B) in starved ARPE19 cells after treatment with 5 µM kaempferol for 24 hours was detected by Western blotting, and semi-quantitative analysis was performed. The antibodies used included: primary antibody rabbit anti-ARL13B (1:1000; 17711-1-AP, Proteintech), mouse anti-ACTUB (1:2000; T6793, Sigma), and mouse anti-GAPDH (1:5000; 60004-1-Ig, Proteintech), and HRP-labeled secondary antibody (1:4000; SA00001, Proteintech).

[0079] The results are as follows Figure 1 As shown in Figures E, F, and G, the effects of kaempferol on the expression of ciliary-related proteins (ACTUB and ARL13B) are illustrated. E represents the representative Western Blot results, while F and G show the quantitative analyses of ACTUB and ARL13B, respectively. Compared to the blank control group, the expression of ciliary-related proteins (ACTUB and ARL13B) in the kaempferol-treated group was significantly upregulated, with statistically significant differences. p <0.05 and ** p Values ​​<0.01 are compared with the blank control group. All values ​​in the figure are mean ± standard error, n ≥ 3.

[0080] These results indicate that kaempferol promotes an increase in ciliary length in a concentration-dependent manner.

[0081] Example 2: Kaempferol restores the number and length of cilia in cells damaged by viral cilia.

[0082] To establish a viral cilia loss model, transfection of SARS-CoV-2 structural protein NSP13 in ARPE19 cells led to a large number of cilia loss. Starvio transfection reagent (T21003, Shanghai Starbio Biotechnology Co., Ltd.) was used to transfect cells after reaching 80% density, and the transfection system is shown in Table 1. Then, after removing the serum, the cilia-deficient ARPE19 cells were treated with 1 µM, 3 µM and 5 µM kaempferol for 24 hours, respectively, and the cilia were imaged and analyzed by immunofluorescence microscopy, and the immunofluorescence staining method was the same as in Example 1; the primary antibody used further included mouse anti-FLAG (1:1000; 66008-3-lg, MBL).

[0083] Table 1. Starvio transfection reagent transfection system formula Results are shown in Figure 2 Figure A is a fluorescence image of viral cilia loss recovered by kaempferol drugs, cilia are labeled with ARL13B as red and virus-infected cells are labeled with FLAG as green, and cell nuclei are labeled as blue; the scale bars in the figure are 10 µm. Figure B is an analysis of cilia length; Figure C is an analysis of the frequency of different cilia lengths; Figure D is a quantitative analysis of the percentage of cilia cells. The average length of the cilia of the cells transfected with SARS-CoV-2 structural protein NSP13 and the percentage of cilia cells were significantly reduced. However, after treating the cells transfected with NSP13 with 1 µM, 3 µM and 5 µM kaempferol, respectively, it was observed that the defects in cilia length and the reduction in the number of cilia were rescued, and the cilia length was higher than the control level p <0.01 compared with the blank control group, p< 0.05, p< 0.01 and p <0.001 compared with the NSP13 virus infection model group, all the figures are mean ± standard error, n ≥ 3; see Table 2 for data).

[0084] Table 2 In addition, the expression of cilia-related proteins (ACTUB and ARL13B) after transfection of NSP13 and treatment of starved ARPE19 cells with 5 µM kaempferol for 24 hours was detected by immunoblotting, and semi-quantitative analysis was performed. The results are shown in Figure 2Figure E, F, and G show the effect of kaempferol on the expression of cilia-related proteins (ACTUB and ARL13B) in viral infection cilia loss. E shows the representative Western Blot results, and F and G show the quantitative analysis of ACTUB and ARL13B, respectively. Compared with the blank control group, the expression of cilia protein markers ACTUB and ARL13B was significantly reduced after transfection of the SARS-CoV-2 NSP13 plasmid. However, compared with the NSP13 transfection group, the expression of cilia-related proteins (ACTUB and ARL13B) in the kaempferol treatment group was significantly up-regulated, with a statistically significant difference. p <0.05 and **** p <0.001 compared with the blank control group, ## p< 0.01 and ### p <0.001 compared with the NSP13 viral infection model group, and all the figures show mean ± standard error, n ≥ 3.

[0085] These results show that kaempferol rescues ciliogenesis and cilia length defects in viral cilia damage cells.

[0086] Example 3: Kaempferol restores cilia length in IFT system impaired cells.

[0087] A small molecule cilia inhibitor, Ciliobrevin D, which can inhibit the function of the important component of the IFT system, dynein-2 complex, was used to construct a cilia reduction and shortening model to verify whether kaempferol can restore cilia shortening caused by IFT system impairment. ARPE19 cells were treated with 50 µM Ciliobrevin D, 50 µM Ciliobrevin D + 1 µM kaempferol, 50 µM Ciliobrevin D + 3 µM kaempferol, and 50 µM Ciliobrevin D + 5 µM kaempferol for 24 hours under serum-free conditions. Cilia were immunofluorescently stained with cilia marker proteins (ACTUB and ARL13B), and cilia were imaged and analyzed by immunofluorescence microscopy.

[0088] The results are shown in Figure H, I, and J. Figure 3Figure A shows the fluorescence images of kaempferol rescuing cilia of IFT system impaired cells. Cilia were labeled with ARL13B in red and ACTUB in green, and nuclei were labeled in blue. Scale bars, 20 µm. Figure B shows the cilia length analysis. Figure C shows the frequency analysis of different cilia length. Figure D shows the quantitative analysis of ciliated cell percentage. Compared with the control group, the number and length of cilia were significantly reduced after 50 µM Ciliobrevin D intervention. The length of cilia was recovered after treatment with different concentrations of kaempferol, but the number of cilia was not improved. p <0.05 and **** p <0.001 compared with dimethyl sulfoxide group, p <0.001 compared with Ciliobrevin D group, ns: not statistically significant, all mean ± standard error, n ≥ 3; see Table 3.

[0089] Table 3 In addition, the expression of cilia-related proteins (ACTUB and ARL13B) was detected by immunoblotting after 50 µM cilia inhibitor Ciliobrevin D treatment, and the starved ARPE19 cells were treated with 5 µM kaempferol for 24 hours, and semi-quantitative analysis was performed. The results are shown in Figure 3 Figure E, F, and G show the effect of kaempferol on the expression of related proteins (ACTUB and ARL13B) in Ciliobrevin D-treated IFT system impaired cilia loss. E is a representative Western Blot result, F and G are quantitative analysis of ACTUB and ARL13B, respectively. Compared with the blank control group, the expression of cilia protein markers ACTUB and ARL13B was significantly reduced after Ciliobrevin D treatment. However, compared with the Ciliobrevin D group, the expression of cilia-related proteins (ACTUB and ARL13B) in the kaempferol treatment group was significantly up-regulated, with statistical difference. p <0.005 compared with blank control group, p <0.05 compared with Ciliobrevin D group, all mean ± standard error, n ≥ 3.

[0090] These results show that kaempferol can rescue cilia shortening caused by IFT system impairment.

[0091] Example 4: Kaempferol restores cilia length in siCEP290 cells.

[0092] CEP290 is a cilia transition zone protein that mainly maintains the normal structure of cilia. To determine the recovery of kaempferol on siCEP290 cilia inhibition, after knocking down CEP290 in ARPE19 cells, 1 µM, 3 µM and 5 µM kaempferol were added respectively under serum-free conditions for 24 hours of intervention, and cilia were immunofluorescently stained with cilia marker protein ACTUB and CEP290 antibody, and imaged and analyzed by immunofluorescence microscopy, the immunofluorescent staining method was the same as in Example 1; the primary antibody used further included rabbit anti-CEP290 (1:1000; 22490-1-AP, Proteintech).

[0093] Results are shown in Figure 4 Figure A is a fluorescence picture of kaempferol drug recovery of siCEP290 cilia, cilia are labeled with ACTUB as green, CEP290 as red, and cell nucleus as blue; the scale bars in the figure are all 20 µm. Figure B is the analysis of cilia length; Figure C is the frequency analysis of different cilia lengths; Figure D is the quantitative analysis of cilia cell percentage. Compared with the blank control group, the number and length of cilia in the cells after knocking down CEP290 were significantly down-regulated, and the cilia length was recovered after adding different concentrations of kaempferol, but the number of cilia was not improved. p <0.05 and **** p <0.001 compared with the blank control group, p <0.001 and ns compared with the siCEP290 group, ns: not statistically significant, all in the figure are mean ± standard error, n ≥ 3; see Table 4 for data.

[0094] Table 4 In addition, the expression of cilia-related proteins (ACTUB and ARL13B) was detected by immunoblotting after siCEP290 treatment and 5 µM kaempferol treatment of starved ARPE19 cells for 24 hours, and semi-quantitative analysis was performed. Results are shown in Figure 4 Figure E, F, and G are the effects of kaempferol on the expression of related proteins (ACTUB and ARL13B) in ARPE19 cells after siCEP290 treatment. E is a representative Western Blot result, F and G are quantitative analysis of ACTUB and ARL13B, respectively. Compared with the blank control group, the expression of cilia protein markers ACTUB and ARL13B was significantly reduced after siCEP290 treatment. However, compared with the siCEP290 group, the expression of cilia-related proteins (ACTUB and ARL13B) in the kaempferol treatment group was significantly up-regulated, with statistical difference. p <0.05 compared with the blank control group,p <0.05 compared with siCEP290 group, all mean ± standard error, n ≥ 3.

[0095] These results show that kaempferol can rescue the cilia shortening caused by CEP290 deletion.

[0096] Example 5: Kaempferol and common expectorant ambroxol combined medication to regulate cilia length Ambroxol is a new type of agonist that can dilute sputum and enhance airway cilia beating. To determine the effect of kaempferol and ambroxol combined medication on cilia number and length, we added different concentrations of ambroxol (5 µM, 10 µM and 50 µM) and 50 µM ambroxol + 1 µM kaempferol, 50 µM ambroxol + 3 µM kaempferol and 50 µM ambroxol + 5 µM kaempferol to ARPE19 cells under serum-free conditions, and after 24 hours of drug intervention, the cells were fixed, the nuclei were stained with DAPI, the cilia were stained with cilia-related proteins (ACTUB and ARL13B), and the cilia were imaged and analyzed by immunofluorescence microscopy.

[0097] The results are shown in Figure 5 Figure A is a fluorescence image of cilia cells after treatment with kaempferol and ambroxol combined medication, cilia are labeled with ARL13B as red and ACTUB as green, and nuclei are labeled as blue; the scale bars in the figure are all 20 µm. Figure B is the analysis of cilia length; Figure C is the frequency analysis of different cilia lengths; Figure D is the quantitative analysis of cilia cell percentage. Compared with the blank control group, the cilia length of cells treated with 5 µM and 10 µM ambroxol did not change significantly, the cilia length of cells treated with 50 µM ambroxol increased significantly, and the cilia length of cells treated with different concentrations of kaempferol increased significantly in a concentration-dependent manner. Compared with the 50 µM ambroxol group, the combination of ambroxol and different concentrations of kaempferol promoted the growth of cilia length, and the cilia length was longer than that after ambroxol medication alone; and the cilia length after the combination of 50 µM ambroxol and 1 µM, 3 µM and 5 µM kaempferol was also longer than that after 1 µM, 3 µM or 5 µM kaempferol medication alone. p <0.01 and p <0.001 compared with the blank control group, p <0.05 and p <0.001 compared with the 50 µM ambroxol group, p <0.05 compared with the 1 µM kaempferol group, p <0.001 compared with the 3 µM kaempferol group,p <0.05 and 5 µM baicalein group, ns: not statistically significant, all in the figure are mean ± standard error, n ≥ 3; see Table 5 for data).

[0098] Table 5 These results show that the combination of ambroxol and baicalein can effectively synergistically promote the elongation of cilium length.

[0099] Example 6: Baicalein and commonly used expectorant ambroxol combination therapy restores cilium number and cilium length in viral cilium injury cells.

[0100] The same model as in Example 2 was used to verify whether the combination of baicalein and ambroxol could restore cilium injury caused by viruses. Then after removing serum, 1 µM baicalein, 3 µM baicalein, 5 µM baicalein, 50 µM ambroxol, 50 µM ambroxol + 1 µM baicalein, 50 µM ambroxol + 3 µM baicalein and 50 µM ambroxol + 5 µM baicalein were used to treat cilium-depleted ARPE19 cells for 24 hours, and cilium imaging and analysis were performed by immunofluorescence microscopy.

[0101] The results are shown in Figure 6 Figure A is a fluorescence image of cilium cells after treatment with the combination of baicalein and ambroxol, with cilium labeled red by ARL13B and virus-infected positive cells labeled green by FLAG, and cell nuclei labeled blue; the scale bar in the figure is 10 µm. Figure B is a quantitative analysis of the percentage of cilium cells; Figure C is an analysis of cilium length; and Figure D is an analysis of the frequency of different cilium lengths. Compared with the blank control group, the average length of the cilia of the cells transfected with the new coronavirus structural protein NSP13 and the percentage of cilium cells were significantly reduced. However, when the cells transfected with NSP13 were treated with 50 µM ambroxol, no recovery of cilium length and number was observed, but the cilium length of the cells treated with different concentrations of baicalein increased significantly in a concentration-dependent manner, and the cilium number was also restored. Compared with the 50 µM ambroxol alone group, the combination of ambroxol and different concentrations of baicalein in treating cells transfected with NSP13 showed more obvious rescue of cilium length defects and cilium number reduction; and the combination of high-concentration (5 µM) baicalein and ambroxol was superior to the recovery effect of baicalein alone. p <0.05 and 5 µM baicalein group, ns: not statistically significant, all in the figure are mean ± standard error, n ≥ 3; see Table 5 for data). p <0.01 compared with the blank control group, p <0.05, ## p <0.01 and p <0.001 compared with the NSP13 group, p <0.05, && p<0.01, &&& p <0.005 and &&& p <0.001 compared with 50 µM ambroxol,† p <0.05 compared with 5 µM kaempferol, ns: not statistically significant, all in the figure are mean ± standard error, n≥3; see Table 6).

[0102] Table 6 These results show that the combination of kaempferol and ambroxol can rescue the defects of ciliogenesis and cilium length in virus-induced cilia-damaged cells.

[0103] Example 7: Kaempferol and commonly used expectorant ambroxol combination therapy restores cilium length in IFT system-damaged cells.

[0104] The same model as in Example 3 was used to verify whether the combination of kaempferol and ambroxol can restore cilium shortening caused by IFT system damage; ARPE19 cells were treated with 50 µM Ciliobrevin D, 50 µM Ciliobrevin D + 50 µM ambroxol, 50 µM Ciliobrevin D + 1 µM kaempferol, 50 µM Ciliobrevin D + 3 µM kaempferol, 50 µM Ciliobrevin D + 5 µM kaempferol, 50 µM Ciliobrevin D + 50 µM ambroxol + 1 µM kaempferol, 50 µM Ciliobrevin D + 50 µM ambroxol + 3 µM kaempferol and 50 µM Ciliobrevin D + 50 µM ambroxol + 5 µM kaempferol for 24 hours, and the cilia were immunofluorescently stained with cilia marker proteins (ACTUB and ARL13B), and imaged and analyzed by immunofluorescence microscopy.

[0105] Results are shown in Table 6 and Figure 6. Figure 7Figure A shows the fluorescence images of ciliated cells treated with kaempferol and ambroxol combination, cilia are labeled with ARL13B in red and ACTUB in green, and nuclei are labeled in blue; the scale bar in the figure is 20 μm. Figure B shows the quantitative analysis of the percentage of ciliated cells; Figure C shows the analysis of cilia length; Figure D shows the frequency analysis of different cilia length. Compared with the blank control group, the number and length of cilia in cells treated with 50 μM Ciliobrevin D were significantly reduced, and the number and length of cilia were not significantly restored after the addition of 50 μM ambroxol. However, after the addition of 1 μM kaempferol, 3 μM kaempferol, 5 μM kaempferol, and 50 μM ambroxol combined with different concentrations of 1 μM, 3 μM and 5 μM kaempferol, respectively, the number of cilia was not restored, but the length of cilia was found to be restored, and the combination of higher concentrations (3 μM and 5 μM) of kaempferol and ambroxol was also superior to the restoration effect of ambroxol or kaempferol alone. p <0.005 and p <0.001 compared with the blank control group, p <0.01 and p <0.001 compared with the 50 μM Ciliobrevin D group, p <0.05 and p <0.01 compared with the 50 μM ambroxol group, p <0.01 compared with the 3 μM kaempferol group, p <0.05 compared with the 5 μM kaempferol group, ns: not statistically significant, all in the figure are mean ± standard error, n ≥ 3; see Table 7).

[0106] Table 7 These results suggest that ambroxol alone may not be able to restore the shortened cilia in cells with impaired IFT system, while higher concentrations of ambroxol combined with kaempferol can restore the length of cilia in cells with impaired IFT system, and are superior to the restoration effect of ambroxol or kaempferol alone.

[0107] Example 8: Kaempferol and commonly used expectorant ambroxol combination to restore the length of cilia in siCEP290 cells.

[0108] Using the same model as in Example 4, we verified whether the combined use of kaempferol and ambroxol could restore ciliary shortening caused by CEP290 deficiency. After knocking down CEP290 in ARPE19 cells, we treated the cells for 24 hours with 1 µM kaempferol, 3 µM kaempferol, 5 µM kaempferol, 50 µM ambroxol, 50 µM ambroxol + 1 µM kaempferol, 50 µM ambroxol + 3 µM kaempferol, and 50 µM ambroxol + 5 µM kaempferol, respectively, after removing serum. We then used ciliary marker proteins (ACTUB and ARL13B) to perform immunofluorescence staining on the cilia and imaged and analyzed the cilia using immunofluorescence microscopy.

[0109] The results are as follows Figure 8 As shown, Figure A is a fluorescence image of ciliated cells after treatment with a combination of kaempferol and ambroxol. Cilia are labeled green with ACTUB, red with CEP290, and cell nuclei are labeled blue. The scale bars in the magnified images are all 10 µm. Figure B shows the quantitative analysis of the percentage of ciliated cells; Figure C shows the analysis of ciliary length; and Figure D shows the frequency analysis of different ciliary lengths. Compared with the blank control group, the number and length of cilia in cells knocked down CEP290 were significantly reduced. The addition of 50 µM ambroxol did not significantly restore cilia length or number. However, treatment with 1 µM, 3 µM, and 5 µM kaempferol, and with different concentrations of 1 µM, 3 µM, and 5 µM kaempferol respectively, significantly restored cilia length. Furthermore, the combined use of different concentrations of kaempferol and ambroxol was superior to the recovery effect of 50 µM ambroxol alone. The combination of high concentration (5 µM) kaempferol and ambroxol was also superior to the recovery effect of kaempferol alone. (*) p <0.05 and **** p All values ​​<0.001 were compared with the blank control group. p <0.001 compared with the siCEP290 group, &&&& p <0.001 compared to the 50 µM ambroxol group,† p Compared with the 5 µM kaempferol group, the difference in ns was not statistically significant. All values ​​in the figure are mean ± standard error, n ≥ 3; data are shown in Table 8.

[0110] Table 8 These results suggest that ambroxol alone may not have a restorative effect on ciliary shortening caused by siCEP290, while kaempferol alone and in combination with ambroxol can restore ciliary shortening. Moreover, the combination of kaempferol and ambroxol has a better restorative effect than ambroxol alone, and the combination of high concentrations of kaempferol and ambroxol also has a better restorative effect than kaempferol alone.

[0111] Example 9: Kaempferol restores respiratory cilia loss induced by bacterial infection.

[0112] Respiratory cilia damage or dysfunction is widespread in various acute and chronic respiratory diseases. In order to determine whether kaempferol can restore respiratory cilia damage induced by acute and chronic bacterial infection, a mouse model of bacterial infection (lipopolysaccharide induction) induced respiratory cilia damage was constructed.

[0113] (1) Construction of animal model 6-week-old SPF C57 mice were anesthetized by intraperitoneal injection of tribromoethanol, and then airway surgery was performed: The chronic stimulation group was intratracheally injected with 0.4 μg / μL of LPS (50 μL); the acute stimulation group was intratracheally injected with 1 μg / μL of LPS (50 μL); the control group was intratracheally injected with the same volume of normal saline; after surgery, the wound was sutured, and the mice were returned to the cage after they woke up.

[0114] (2) Drug treatment A 10 mg / mL kaempferol suspension was prepared with 0.5% carboxymethylcellulose sodium (CMC-Na). The mice were divided into three groups: blank group, modeling group (0.5% CMC-Na, 10 mL / kg / d), and kaempferol gavage group (25 mg / kg, 50 mg / kg, and 100 mg / kg), with three repeated mice in each group. The dosing regimen was as follows: Chronic model: Kaempferol gavage treatment started 24 hours after LPS injection, once every 12 hours, for a total of 24 hours.

[0115] Acute model: Kaempferol gavage treatment started 1 hour after LPS injection, once every 12 hours, for a total of 24 hours.

[0116] One hour after the last kaempferol gavage, the tracheal tissue of the mice was taken; it was fixed with 4% paraformaldehyde solution overnight. The airway cilia was observed by immunofluorescence staining (using mouse anti- ACTUB primary antibody and Alexa Fluor 568 labeled secondary antibody) and scanning electron microscopy, respectively.

[0117] The results are as follows: Figure 9As shown, Figure A is a fluorescence picture of airway cilia after treatment with different concentrations of kaempferol after acute and chronic bacterial infection, cilia are labeled green with ACTUB, and nuclei are labeled blue; the scale bars in the figure are all 10 pm. Figure B is a scanning electron microscope picture of airway cilia after treatment with different concentrations of kaempferol after acute and chronic bacterial infection, the scale bars in the figure are all 5 pm. Compared with the blank control group, after airway injection of lipopolysaccharide 0.4 pg / pL for 24 hours and 1 pg / pL for 1 hour, the airway cilia of mice showed a large number of loss, the cilia arrangement was sparse, and part of the epithelial surface was exposed; in the chronic stimulation model group injected with lipopolysaccharide 0.4 pg / pL for 24 hours, it was found that after treatment with different concentrations of kaempferol, the airway cilia were restored, the number of cilia increased significantly, the arrangement was dense, and the epithelial surface was covered with cilia again, among which the recovery effect of cilia after treatment with kaempferol at a concentration of 50 mg / kg was significantly better than that after treatment with kaempferol at a concentration of 100 mg / kg. In the acute model group injected with lipopolysaccharide 1 pg / pL for 1 hour, it was observed that with the increase of the concentration of kaempferol, the number and density of cilia increased significantly, and after treatment with kaempferol at a concentration of 100 mg / kg, the recovery effect of cilia was the best, the number of cilia increased significantly, and the arrangement was more dense. In summary, kaempferol may be more advantageous in restoring cilia loss caused by chronic bacterial infection.

[0118] These results suggest that certain concentrations of kaempferol drugs can reverse airway cilia damage caused by acute or chronic bacterial infection to some extent.

Claims

1. Use of kaempferol or a pharmaceutically acceptable salt, stereoisomer, solvate, prodrug or derivative thereof in the manufacture of a medicament for promoting cilia growth.

2. The use according to claim 1, wherein the kaempferol or a pharmaceutically acceptable salt, stereoisomer, solvate, prodrug or derivative thereof has at least one of the following uses: (1) promoting increase in cilia length; (2) promoting ciliogenesis; (3) promoting recovery of cilia function; (4) rescuing and / or repairing defects in cilia function, ciliogenesis and / or cilia length in damaged ciliated cells; Preferably, the damaged ciliated cells are selected from respiratory ciliated epithelial cells, reproductive system ciliated cells, sensory system ciliated cells, embryonic development related ciliated cells, and kidney and liver ciliated cells.

3. The use according to claim 1 or 2, wherein the damaged ciliated cells are bacterial or viral cilia-damaged cells, IFT system-impaired ciliated cells, and / or CEP290 gene expression abnormal ciliated cells.

4. A pharmaceutical composition or combination comprising a therapeutically effective amount of kaempferol or a pharmaceutically acceptable salt, stereoisomer, solvate, prodrug or derivative thereof; optionally, a pharmaceutically acceptable carrier.

5. The pharmaceutical composition or combination according to claim 4, comprising: a) a therapeutically effective amount of kaempferol or a pharmaceutically acceptable salt, stereoisomer, solvate, prodrug or derivative thereof; and b) at least one therapeutically effective amount of a drug for increasing cilia movement or a pharmaceutically acceptable salt, stereoisomer, solvate, prodrug or derivative thereof; optionally, a pharmaceutically acceptable carrier; wherein the drug for increasing cilia movement comprises mucolytic agents, physical / osmotic mucous clearance agents, bronchodilators, cilia movement and function direct promoters, immunomodulators, antioxidants, ion channel modulators, complex preparations and / or adjuvant therapeutic agents; the adjuvant therapeutic agents comprise antibiotics and / or drugs for treating systemic complications; preferably, the drug for increasing cilia movement comprises ambroxol, acetylcysteine, erdosteine, hypertonic saline, mannitol, β2-receptor agonists, anticholinergic drugs, P2Y2 receptor agonists, inhaled glucocorticoids, macrolide antibiotics, N-acetylcysteine, edarovate.

6. The pharmaceutical composition or combination according to claim 5, wherein the drug for increasing cilia movement is ambroxol.

7. The pharmaceutical composition or combination according to any one of claims 4-6, wherein the molar ratio of kaempferol to the drug for increasing cilia movement in the pharmaceutical composition or combination is 0.001-10.

8. The pharmaceutical composition or combination according to any one of claims 4-7, wherein the concentration of kaempferol in the pharmaceutical composition or combination is 0.1-100 μM, preferably 1-3 μM or 3-5 μM.

9. The use according to any one of claims 1-3 or the pharmaceutical composition or the pharmaceutical combination according to any one of claims 4-8, wherein the drug, the pharmaceutical composition or the pharmaceutical combination is for treating a cilia dysfunction-related disease or disorder; Preferably, the cilia dysfunction-related disease or disorder is selected from the group consisting of diseases or disorders caused by abnormal cilia structure, defective motor function or impaired signal transduction function, including primary ciliary dyskinesia, secondary cilia dysfunction, ciliopathies and their clinical manifestations. More preferably, the primary ciliary dyskinesia comprises Kartagener syndrome; the secondary cilia dysfunction-related disease comprises mucus clearance disorder and chronic airway inflammatory disease caused by chronic obstructive pulmonary disease, asthma, cystic fibrosis and respiratory tract infection; the ciliopathy comprises polycystic kidney disease, Bardet-Biedl syndrome, Joubert syndrome, Meckel-Gruber syndrome, Usher syndrome and Leber congenital amaurosis; and the clinical manifestation comprises infertility caused by abnormal sperm flagellum or oviduct ciliary movement, and hydrocephalus caused by defective ependymal ciliary flow function.

10. The use, the pharmaceutical composition or the pharmaceutical combination according to claim 9, wherein the drug, the pharmaceutical composition or the pharmaceutical combination is a topical administration preparation, an inhalation administration preparation or an oral administration preparation. Preferably, the drug, the pharmaceutical composition or the pharmaceutical combination is a tablet, a lozenge, a capsule, a pill, a solution, a suspension, a syrup, an injection, a suppository, an inhalation solution, an inhalation powder mist, an aerosol or a spray.

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