Application of kaempferol in the preparation of drugs for promoting ciliary growth

Abstract

This disclosure relates to the biopharmaceutical field, specifically to the use of kaempferol in the preparation of medicaments for promoting ciliary growth. It provides the use of kaempferol or its pharmaceutically acceptable salts, stereoisomers, solvates, prodrugs or derivatives thereof in the preparation of medicaments for promoting ciliary growth, as well as pharmaceutical compositions or combinations thereof comprising a therapeutically effective amount of kaempferol or its pharmaceutically acceptable salts, stereoisomers, solvates, prodrugs or derivatives thereof.

Description

[0001] This application is a divisional application. The original application was filed on November 7, 2025, with application number 2025116313108, and the invention title was "Use of Kaempferol in the Preparation of a Drug for Promoting Cilia Growth". Technical Field

[0002] This invention belongs to the field of biomedicine, specifically relating to the application of kaempferol in the preparation of drugs for promoting ciliary growth. Background Technology

[0003] Cilia are antenna-like sensory organelles based on microtubules, protruding from the apex of most mammalian cells, used to sense extracellular signals and stimuli. Cilia are mainly composed of the basal body, axonal filaments, ciliary membrane, and ciliary matrix. Based on the axonal filament structure, they can be divided into motor cilia (“9+2” structure) and primary cilia (“9+0” structure). The basal body required for cilia formation originates from the maternal centriole. Between the basal body and axonal filament lies a transition zone (TZ) that regulates the movement of proteins and membrane components into and out of the cilia. After the proteins required for cilia assembly enter the cilia, they rely primarily on directional transport by the intraflagellar transport (IFT) family, including anterograde transport (via the kinin 2 and IFT-B complex) and retrograde transport (via the dynein 2 and IFT-A complex), thereby promoting rapid cilia elongation. Once formed, cilia maintain a specific length.

[0004] Each cell type has a normal range of cilia length. However, some congenital genetic factors (mutations in IFT, transition region proteins, and signaling pathway-related genes) and acquired environmental factors (viral infections, environmental pollution, and drug toxicity, etc.) can lead to cilia loss and dysfunction, which can trigger a series of diseases collectively known as ciliopathies. Primary cilia are located on the surface of most eukaryotic cells and are mainly involved in signal transduction. Their dysfunction can lead to polycystic kidney disease (PKD), Leber congenital amaurosis (LCA), Bardet-Biedl syndrome, Joubert syndrome, and Meckel-Gruber syndrome. Motor cilia are mainly found in the respiratory tract, ventricles of the brain, and reproductive system. They generate movement through coordinated beating, such as respiratory cilia clearing mucus and sperm flagella propelling sperm. Their dysfunction can lead to respiratory diseases, infertility, and hydrocephalus.

[0005] Currently, Western medicine's treatment of ciliopathy mainly focuses on symptomatic relief, without repairing or regenerating cilia. Treatment primarily aims to alleviate symptoms. For example, respiratory infections are often treated with antibiotics and expectorants, but these cannot promote ciliary regeneration or restore ciliary clearance function, and may even lead to drug resistance or side effects. Infertility is treated with assisted reproductive technologies (such as in vitro fertilization), but these cannot repair sperm flagellar function. Hydrocephalus is treated surgically (such as ventriculoperitoneum), but this cannot restore ventricular ciliary function. Furthermore, the application of gene editing (such as CRISPR) in the treatment of ciliopathy is still in the experimental stage, with issues such as off-target effects, low editing efficiency, and poor tissue specificity.

[0006] Traditional Chinese medicine (TCM) demonstrates significant potential advantages in treating and restoring ciliary function, particularly in its unique pharmacological effects on anti-inflammation, anti-oxidation, and ciliary dynamics. Regarding anti-inflammation, quercetin, for example, can inhibit cytokine release syndrome driven by nuclear factor kappa-B (NF-κB), inflammasomes, and interleukin-6 (IL-6), and simultaneously activate the nuclear factor erythroid 2-related factor 2 (NRF2) pathway to alleviate cytokine release syndrome and reduce acute lung injury. Astragalus polysaccharides can promote high levels of hemagglutination inhibition titers and the induction of specific antibody immunoglobulin G (IgG), achieving bidirectional immunomodulation of cellular and humoral immunity. In terms of restoring ciliary dynamics, gypsum in Ma Xing Gan Shi Tang can accelerate ciliary pulsation frequency by activating transient receptor potential vanilloid 1 (TRPV1). Naringin can inhibit intracellular calcium 2+ The horizontal movement relaxes the smooth muscle of the trachea and increases the frequency of ciliary beating, thereby accelerating viral clearance. Hesperidin, on the other hand, can increase the transepithelial chorionic villus concentration (C4). - It secretes secretions, enhances the ability of airway cilia to move, and strengthens the function of the mucociliary clearance system (MCC).

[0007] In terms of combined drug therapy, the use of traditional Chinese medicine (TCM) monomers in combination with Western medicines also demonstrates good synergistic and complementary characteristics. On the one hand, TCM monomers can enhance the efficacy of antibiotics and expectorants. For example, the combination of baicalin and azithromycin can enhance the antibacterial spectrum, reduce drug resistance, and alleviate the damage of inflammation to cilia. On the other hand, TCM monomers can reduce the adverse reactions of Western medicines, such as alleviating the gastrointestinal irritation caused by expectorants or the dysbiosis caused by long-term use of antibiotics.

[0008] Currently, while the combined use of Western medicine, traditional Chinese medicine, and traditional Chinese and Western medicine can improve ciliary function and thus promote disease recovery through symptomatic adjuvant treatment or by promoting ciliary movement, there is still a lack of drugs that directly promote ciliary growth. Therefore, the development of drugs that promote ciliary stem cell differentiation and ciliary growth, in order to drive ciliary "structural reconstruction" and "functional repair," is expected to provide a new pharmacological basis and practical strategy for the treatment of cilia diseases. Summary of the Invention

[0009] On the one hand, this disclosure relates to the use of kaempferol or its pharmaceutically acceptable salts, stereoisomers, solvates, prodrugs or derivatives thereof in the preparation of medicaments that promote ciliary growth.

[0010] In some embodiments, the aforementioned applications of kaempferol or its pharmaceutically acceptable salts, stereoisomers, solvates, prodrugs or derivatives thereof have at least one of the following applications: (1) Promotes an increase in cilia length; (2) Promotes ciliary development; (3) Promotes the recovery of ciliary function; (4) To rescue and / or repair defects in ciliary function, ciliation and / or ciliary length in damaged ciliated cells.

[0011] In some preferred embodiments, the aforementioned damaged ciliated cells are selected from respiratory ciliated epithelial cells, reproductive ciliated cells, sensory ciliated cells, embryonic development-related ciliated cells, and kidney and liver ciliated cells.

[0012] In some implementations, the aforementioned damaged ciliated cells are bacterial or viral ciliated cells, ciliated cells with impaired IFT system, and / or ciliated cells with abnormal CEP290 gene expression.

[0013] On the other hand, this disclosure relates to a pharmaceutical composition or combination of pharmaceuticals comprising a therapeutically effective amount of kaempferol or a pharmaceutically acceptable salt, stereoisomer, solvate, prodrug or derivative thereof; optionally, comprising a pharmaceutically acceptable carrier.

[0014] In some embodiments, the aforementioned pharmaceutical composition or combination of pharmaceuticals comprises: a) A therapeutically effective amount of kaempferol or its pharmaceutically acceptable salt, stereoisomer, solvate, prodrug or derivative thereof; b) At least one therapeutically effective amount of a drug that increases ciliary motility or a pharmaceutically acceptable salt, stereoisomer, solvate, prodrug or derivative thereof; Optionally, it may include a pharmaceutically acceptable carrier; The drugs that increase ciliary movement include mucolytic agents, physical / osmotic mucus removers, bronchodilators, direct promoters of ciliary movement and function, immunomodulators, antioxidants, ion channel modulators, compound preparations, and / or adjuvant therapeutic agents.

[0015] In some implementations, the aforementioned adjunctive therapy includes antibiotics and / or drugs used to treat systemic complications.

[0016] In some implementation schemes, the aforementioned drugs that increase ciliary movement include ambroxol (a mucolytic agent, enhances ciliary movement, stimulates the synthesis and secretion of pulmonary surfactant, has immunomodulatory and antioxidant properties), acetylcysteine ​​(a mucolytic agent and antioxidant), erdosteine ​​(a mucomodulator), hypertonic saline, mannitol, β2-receptor agonists (salbutamol, formoterol), anticholinergic drugs (ipratropium bromide, tiotropium bromide), P2Y2 receptor agonists (diquafos, myrtle oil), inhaled corticosteroids (budesonide, fluticasone), macrolide antibiotics (azithromycin, erythromycin), antioxidant N-acetylcysteine, and ion channel modulator ivacastor.

[0017] In some preferred embodiments, the aforementioned drug that increases ciliary movement is ambroxol.

[0018] In some embodiments, the molar ratio of kaempferol to the ciliary motility-enhancing drug in the aforementioned pharmaceutical composition or combination is 0.001-10.

[0019] In some embodiments, the molar ratio of kaempferol to the ciliary motility-enhancing drug in the aforementioned pharmaceutical composition or 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.

[0020] In some embodiments, the molar ratio of kaempferol to the ciliary motility-enhancing drug in the aforementioned pharmaceutical composition or combination is (1-5):50.

[0021] In some preferred embodiments, the molar ratio of kaempferol to ambroxol in the aforementioned pharmaceutical composition or combination is 0.001-10.

[0022] In some preferred embodiments, the molar ratio of kaempferol to ambroxol in the aforementioned pharmaceutical composition or 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.

[0023] In some preferred embodiments, the molar ratio of kaempferol to ambroxol in the aforementioned pharmaceutical composition or combination is (1-5):50. In some more preferred embodiments, the molar ratio of kaempferol to ambroxol in the aforementioned pharmaceutical composition or combination is 1:50.

[0024] In some preferred embodiments, the molar ratio of kaempferol to ambroxol in the aforementioned pharmaceutical composition or combination is 3:50.

[0025] In some preferred embodiments, the molar ratio of kaempferol to ambroxol in the aforementioned pharmaceutical composition or combination is 5:50.

[0026] In some embodiments, the concentration of kaempferol in the aforementioned pharmaceutical composition or combination is 0.1-100 μM.

[0027] In some preferred embodiments, the concentration of kaempferol in the aforementioned pharmaceutical composition or combination is 1-3 μM.

[0028] In some preferred embodiments, the concentration of kaempferol in the aforementioned pharmaceutical composition or combination is 3-5 μM.

[0029] In some preferred embodiments, the concentration of kaempferol in the aforementioned pharmaceutical composition or combination is 1 μM.

[0030] In some preferred embodiments, the concentration of kaempferol in the aforementioned pharmaceutical composition or combination is 3 μM.

[0031] In some preferred embodiments, the concentration of kaempferol in the aforementioned pharmaceutical composition or combination is 5 μM.

[0032] In some more preferred embodiments, the concentration of kaempferol in the aforementioned pharmaceutical composition or combination is 1 μM, and the molar ratio of kaempferol to ambroxol is 1:50.

[0033] In some more preferred embodiments, the concentration of kaempferol in the aforementioned pharmaceutical composition or combination is 3 μM, and the molar ratio of kaempferol to ambroxol is 3:50.

[0034] In some preferred embodiments, the concentration of kaempferol in the aforementioned pharmaceutical composition or combination is 5 μM, and the molar ratio of kaempferol to ambroxol is 5:50.

[0035] In some implementations, the aforementioned drugs, pharmaceutical compositions, or combinations of drugs are used to treat diseases or disorders related to ciliary dysfunction.

[0036] In some preferred embodiments, the aforementioned ciliary dysfunction-related diseases or disorders are selected from diseases or disorders caused by abnormal ciliary structure, motor function defects, or impaired signal transduction function, including primary ciliary dyskinesia, secondary ciliary dysfunction, and ciliopathy.

[0037] In some preferred embodiments, the aforementioned primary ciliary dyskinesia includes Kartagener syndrome; the diseases associated with secondary ciliary dysfunction include chronic obstructive pulmonary disease, asthma, cystic fibrosis, and mucus clearance disorders and chronic airway inflammatory diseases caused by respiratory tract infections and bronchiectasis; the ciliary diseases include polycystic kidney disease, Bardet-Biedl syndrome, Joubert syndrome, Meckel-Gruber syndrome, Usher syndrome, and Leber congenital amaurosis; the clinical manifestations include infertility due to abnormal sperm flagella or fallopian tube ciliary dyskinesia, and hydrocephalus due to impaired ependymal ciliary flow.

[0038] In some preferred embodiments, the aforementioned ciliary dysfunction-related diseases or disorders are ciliary damage caused by acute or chronic bacterial infections.

[0039] In some preferred embodiments, the aforementioned ciliary dysfunction-related disease or disorder is the loss of respiratory cilia due to bacterial infection.

[0040] In some specific implementations, the bacteria are Gram-negative bacteria.

[0041] In some embodiments, the aforementioned drug, drug composition or drug combination is a topical administration formulation, an inhaled administration formulation or an oral administration formulation.

[0042] In some preferred embodiments, the aforementioned drug, pharmaceutical composition, or combination of drugs is a tablet, lozenge, capsule, pill, solution, suspension, syrup, injection, suppository, inhalation solution, inhalation powder, aerosol, or spray.

[0043] The beneficial effects of the present invention include at least the following: In this invention, kaempferol monotherapy has a significant ciliary repair effect. Its effect on promoting ciliary growth is concentration-dependent and can widely repair ciliary defects caused by various reasons (such as viral damage, IFT system damage, and CEP290 deficiency).

[0044] Ambroxol monotherapy has limited efficacy in treating ciliary shortening caused by IFT system damage or CEP290 deficiency. However, the combined use of kaempferol and ambroxol exhibits a synergistic effect: the combined treatment not only salvages viral ciliary damage but also demonstrates superior repair efficacy against ciliary damage caused by IFT system damage or CEP290 deficiency compared to kaempferol monotherapy, especially at higher concentrations where the synergistic effect is more pronounced. Attached Figure Description

[0045] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this specification and, together with the description, serve to explain the principles of this specification.

[0046] Figure 1 The effects of kaempferol on ciliary formation and ciliary length are shown.

[0047] Figure 2 This study demonstrates the restorative effect of kaempferol on ciliary loss caused by viral infection.

[0048] Figure 3 This study demonstrates the restorative effect of kaempferol on ciliary loss in damaged cells of the IFT system.

[0049] Figure 4 This study demonstrates the restorative effect of kaempferol on various hereditary ciliary loss caused by CEP290 reduction (siCEP290).

[0050] Figure 5 The effects of combined use of kaempferol and ambroxol on the number and length of cilia were shown.

[0051] Figure 6 This study demonstrates the restorative effect of the combined use of kaempferol and ambroxol on ciliary loss caused by viral infection.

[0052] Figure 7 This study demonstrates the restorative effect of the combined use of kaempferol and ambroxol on the loss of cilia in cells damaged by the IFT system.

[0053] Figure 8 This study demonstrates the restorative effect of the combined use of kaempferol and ambroxol on various hereditary ciliary loss caused by CEP290 reduction (siCEP290).

[0054] Figure 9 This study demonstrates the restorative effect of kaempferol on respiratory cilia damage in a bacterial infection model (lipopolysaccharide-induced). Detailed Implementation

[0055] I. Definition In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0056] As used herein and unless otherwise stated, the terms “comprising,” “including,” “having,” and “containing,” including their grammatical equivalents, should generally be understood as open-ended and non-restrictive, e.g., not excluding other unlisted elements or steps.

[0057] In this invention, the disclosure of all ranges should be regarded as the disclosure of all sub-ranges and all point values ​​within the range. For example, the disclosure of 1-1000 should be regarded as the disclosure of ranges such as 1-200 and 200-300, as well as point values ​​such as 200, 300, 400, 500, 600, 700, 800, 900, and 1000.

[0058] The compounds can exist in pharmaceutical compositions as pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to a salt of the compounds of this invention, prepared by reacting a compound having specific substituents discovered in this invention with a relatively non-toxic acid or base. When the compounds of this invention contain relatively acidic functional groups, base addition salts can be obtained by contacting a neutral form of such compound with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts or similar salts. When the compounds of this invention contain relatively basic functional groups, acid addition salts can be obtained by contacting a neutral form of such compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; as well as salts of amino acids (such as arginine) and salts of organic acids such as glucuronic acid. Certain compounds of the present invention contain both basic and acidic functional groups, and thus can be converted into either a base or an acid addition salt.

[0059] The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing acid radicals or bases by conventional chemical methods. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture thereof.

[0060] In addition to the salt form, the compounds provided by this invention also exist in prodrug form. The prodrugs of the compounds described herein readily undergo chemical changes under physiological conditions to be converted into the compounds of this invention. Furthermore, the prodrugs can be converted into the compounds of this invention in the in vivo environment by chemical or biochemical methods. For example, when the prodrug is placed in a transdermal patch reservoir containing suitable enzymes or chemical reagents, the prodrug can be slowly converted into the compounds of this invention.

[0061] Some compounds of this invention may exist in non-solventized or solvated forms, including hydrated forms. Generally, solvated and non-solventized forms are equivalent and both are included within the scope of this invention. Solventized forms are generally equivalent to non-solventized forms and should be included within the scope of this invention. Some compounds of this invention may exist in polymorphic or amorphous forms. Generally, all physical forms are equivalent for the applications contemplated by this invention and should be included within the scope of this invention.

[0062] As used herein, the term "kaempferol derivative" refers to kaempferol (chemical formula C30), a natural flavonoid compound. 15 H 10Using O6 as the parent nucleus, structural analogs are formed through chemical modification (such as glycosylation, methylation, acetylation, isopentenylation, etc.) or biotransformation. Exemplary examples of kaempferol derivatives include, but are not limited to, kaempferol-3-glucoside, kaempferol-3-glucanol, 6-hydroxykaempferol-3-glucoside, 6-hydroxykaempferol-3,6-diglucoside, 6-hydroxykaempferol-3,6,7-triglucoside, kaempferol-3,7-dirhamnoside, 7-rhamnosylkaempferol-3-glucoside, 7-gluco-kaempferol-3-rhamnoside, kaempferol-4'-glucoside, 3-acetylkaempferol-7-glucoside, kaempferol-7-glucoside, 4'-acetylkaempferol-7-glucoside, 3-hydroxyethylkaempferol- 7-Rhamnose, kaempferol-7,4'-dirhamnoglucoside, kaempferol-5-methyl ether-7-glucoside, kaempferol-3-rhamnose, kaempferol-3-arabinoside, kaempferol-3-galactosylglycoside, 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, etc.

[0063] As used herein, the terms “promote” or “inhibit” are used relative to a control. Those skilled in the art will readily determine the appropriate control for each experiment. For example, an elevated or decreased response in a subject or cell treated with the compound is compared to a response in a subject or cell not treated with the compound.

[0064] As used herein, the term "combination" or "co-administration" is a method of administration that includes various situations in which two drugs are administered simultaneously, concurrently, sequentially, continuously, alternately, or separately. "Simultaneously" as used herein includes administration within the same dosing cycle, such as administering the two drugs over two days or one day. "Sequentially," "continuously," "alternatingly," or "separately" as used herein includes administration within different dosing cycles. The "combination" method of administration includes simultaneous administration, independent formulation and co-administration, or independent formulation and sequential administration. All of the above methods of administration fall under the category of combined administration as described in this invention.

[0065] As used herein, the term "effective dose" or "therapeutic effective dose" includes a dose sufficient to improve or prevent the symptoms or condition of a medical condition. An effective dose also means a dose sufficient to allow or facilitate diagnosis. The effective dose for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective dose can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0066] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0067] As used herein, the term "pharmaceutically acceptable carrier" refers to non-toxic solid, semi-solid, or liquid fillers, diluents, encapsulating materials, formulation adjuvants, or carriers conventionally used in the art for use with therapeutic agents, together constituting a "pharmaceutical composition" for individual administration. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dose and concentration used and is compatible with other components of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation used. Suitable carriers are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, liposomes, polymer micelles, or inorganic nanocarriers, etc.

[0068] As used herein, the term "pharmaceutical composition" refers to a mixture of one or more components. The purpose of a pharmaceutical composition is to facilitate the administration of the disclosed compounds to an organism. The pharmaceutical compositions of this disclosure may include one or more pharmaceutically acceptable salts, pharmaceutically acceptable carriers, and / or adjuvants, such as antioxidants, preservatives, wetting agents, emulsifiers, and dispersants.

[0069] The "pharmaceutical composition" disclosed herein can also be administered to patients or subjects requiring such treatment via any suitable route of administration, such as oral, intravenous, parenteral, rectal, pulmonary, or local administration. When intended for oral administration, the pharmaceutical composition can be formulated into oral dosage forms, such as oral solid dosage forms, like tablets, capsules, pills, granules, etc.; or oral liquid dosage forms, such as oral solutions, oral suspensions, syrups, etc. When formulated into oral dosage forms, the pharmaceutical dosage forms may also contain suitable fillers, binders, disintegrants, lubricants, etc.

[0070] The pharmaceutical compositions disclosed herein can be formulated into any pharmaceutically acceptable dosage form for oral, nasal, topical (including oral cavity and sublingual), rectal, vaginal and / or parenteral administration, for example, they can be formulated into tablets, lozenges, capsules, pills, solutions, suspensions, syrups, injections, suppositories, inhalation solutions, inhalation powders, aerosols or sprays.

[0071] As used in this article, the term damaged ciliated cells or “ciliated damaged cells” refers to cells whose ciliary structure is disrupted (e.g., abnormal arrangement of axonal microtubules, matrix misalignment) or whose function is impaired (e.g., reduced beating frequency, signal transduction failure) due to genetic mutations, infections (e.g., bacteria, viruses), environmental stimuli (e.g., pollutants, chemical toxins) or disease states (e.g., chronic inflammation). The core characteristics are shortened ciliary length, reduced number, or loss of function, which in turn affects cell movement, signal perception, and tissue homeostasis.

[0072] As used in this article, the term "ciliary dysfunction-related diseases or disorders" refers to a class of diseases or disorders caused by abnormalities in the structure or function of cilia, mainly manifested as impaired cilia clearance, movement or signal transduction, which in turn leads to multi-system dysfunction.

[0073] As used herein, the term "bacterial infection-induced ciliary damage" refers to the pathological process in which pathogenic toxins (such as lipopolysaccharide LPS, hemolysin, etc.) or their metabolites, along with the host's subsequent excessive immune inflammatory response, directly or indirectly cause structural damage (manifested as ciliary shortening, shedding, or disordered arrangement) and functional dysfunction (such as decreased beating frequency and abnormal rhythm) of the cilia of respiratory mucosal epithelial cells. This weakens the defense function of the mucociliary clearance system and further exacerbates the vicious cycle of pathogen colonization and persistent inflammation. Bacterial infection can be used to construct experimental models of ciliary damage. For example, in some embodiments of this disclosure, a secondary ciliary dysfunction model was established by administering lipopolysaccharide (LPS) to simulate Gram-negative bacterial infection. This model is classified under respiratory diseases and mucus clearance disorders and was used to verify the restorative effect of kaempferol on this type of dysfunction.

[0074] II. Detailed Implementation Plan For the purpose of clarity and concise description, features are described herein as part of some identical or separate embodiments; however, it will be understood that the scope of this disclosure may include some embodiments having combinations of all or some of the described features. The following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the following examples, they are performed according to techniques or conditions described in the literature in the art or according to product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0075] Example Example 1: Kaempferol regulates ciliary length in a concentration-dependent manner Human retinal pigment epithelial (ARPE19) cells (purchased from Wuhan Pronosai Biotechnology Co., Ltd. and identified by STR) were used to verify whether kaempferol regulates ciliogenesis and growth. These cells undergo ciliogenesis during starvation (serum removal).

[0076] Cells were routinely cultured in DMEM / F12 medium containing 10% fetal bovine serum at 37°C and 5% CO2. To induce ciliation, when the cells reached approximately 90% confluence, the medium was replaced with serum-free medium, and serum starvation culture was continued for 24 hours. Subsequently, different final concentrations of kaempferol (1 µM, 3 µM, and 5 µM) were added to the starved cells for 24 hours. Cells were then fixed, nuclei were stained with DAPI, and cilia were stained with the ciliary membrane marker ARL13B. Immunofluorescence microscopy was used to image and analyze the cilia.

[0077] The staining process began with cell fixation using 4% paraformaldehyde for 15 minutes, followed by washing twice with PBS and permeabilization with 0.2% Triton X-100 solution for 20 minutes. Cells were then blocked with 10% goat serum. Antibody incubation followed: cells were incubated overnight at 4°C with rabbit anti-ARL13B and mouse anti-ACTUB primary antibodies; the next day, they were incubated for 2 hours at room temperature in the dark with the corresponding Alexa Fluor 488 and Alexa Fluor 568 labeled secondary antibodies. Finally, cells were mounted using a DAPI-containing anti-quenching mounting medium. Confocal microscopy was used to observe and image the stained cells, and the fluorescence signals of ARL13B and DAPI were analyzed to image and statistically determine the occurrence and growth of cilia. Primary antibodies included rabbit anti-ARL13B (1:1000; 17711-1-AP, Proteintech) and mouse anti-ACTUB (1:2000; T6793, Sigma). The secondary antibodies include goat anti-rabbit IgG-Alexa Fluor 488 conjugate antibody (1:1000; A11036, Invitrogen) and goat anti-mouse IgG-Alexa Fluor 568 conjugate antibody (1:1000; A11004, Invitrogen).

[0078] The results are as follows Figure 1As shown, Figure A is a fluorescence image of ciliated cells after treatment with kaempferol. Cilia are labeled red with ARL13B, and cell nuclei are labeled 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).

[0079] 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).

[0080] The results are as follows Figure 1 As shown in Figures E, F, and G, the effects of kaempferol on the expression of ciliary-associated 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-associated 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.

[0081] These results indicate that kaempferol promotes an increase in ciliary length in a concentration-dependent manner.

[0082] Example 2: Kaempferol restores the number and length of cilia in cells damaged by viral cilia.

[0083] A viral ciliary loss model was established by transfecting ARPE19 cells with the SARS-CoV-2 structural protein NSP13, resulting in extensive loss of cellular cilia. This study aimed to verify whether kaempferol could restore ciliary damage induced by the virus. Transfection was performed using Starvio transfection reagent (T21003, Shanghai Xingqi Baidai Biotechnology Co., Ltd.) after cells reached 80% confluency. The transfection system is shown in Table 1. After serum removal, the ciliated ARPE19 cells were treated with 1 µM, 3 µM, and 5 µM kaempferol for 24 hours, respectively. Cilia were then imaged and analyzed using immunofluorescence microscopy, with the immunofluorescence staining method identical to that in Example 1. The primary antibody used further included mouse anti-FLAG (1:1000; 66008-3-1g, MBL).

[0084] Table 1. Starvio Transfection Reagent Transfection System Formulation The results are as follows Figure 2 As shown, Figure A is a fluorescent image of viral ciliary loss restored by kaempferol. Cilia are marked in red with ARL13B, and virus-infected cells are marked in green with FLAG. 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. The mean length of cilia and the percentage of ciliated cells in cells transfected with the SARS-CoV-2 structural protein NSP13 were significantly reduced. However, when cells transfected with NSP13 were treated with 1 µM, 3 µM, and 5 µM kaempferol, respectively, the ciliary length defect and the reduction in ciliary number were rescued, with the ciliary length being higher than the control level (**). p <0.01 compared with the blank control group, # p < 0.05, ## p< 0.01 and #### p Values ​​<0.001 are compared with the NSP13 virus infection model group. All values ​​in the figure are mean ± standard error, n ≥ 3; data are shown in Table 2.

[0085] Table 2 In addition, the expression of ciliary-related proteins (ACTUB and ARL13B) in starved ARPE19 cells treated with 5 µM kaempferol for 24 hours after NSP13 transfection was detected by Western blotting, and semi-quantitative analysis was performed. The results are as follows: Figure 2As shown in Figures E, F, and G, kaempferol affects the expression of ciliary-related proteins (ACTUB and ARL13B) in viral infection-induced ciliary loss. E represents representative Western Blot results, and F and G show the quantitative analyses of ACTUB and ARL13B, respectively. Compared to the control group, the expression of ciliary protein markers ACTUB and ARL13B was significantly reduced after transfection with the SARS-CoV-2 NSP13 plasmid. However, compared to the NSP13 transfection group, the expression of ciliary-related proteins (ACTUB and ARL13B) was significantly upregulated in the kaempferol-treated group, showing statistically significant differences. p <0.05 and **** p <0.001 is compared with the blank control group, ## p< 0.01 and #### p Values ​​<0.001 are compared with the NSP13 virus infection model group. All values ​​in the figure are mean ± standard error, n ≥ 3.

[0086] These results indicate that kaempferol rescues cilia development and cilia length defects in cells damaged by viral cilia.

[0087] Example 3: Kaempferol restores the ciliary length of damaged cells in the IFT system.

[0088] A model of cilia reduction and shortening was constructed using the small-molecule cilia inhibitor Ciliobrevin D (which inhibits the function of the dynein-2 complex, an important component of the IFT system), to verify whether kaempferol could restore cilia shortening caused by IFT system damage. ARPE19 cells were treated for 24 hours 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 under serum-free conditions. Cilia were then immunofluorescently stained with cilia marker proteins (ACTUB and ARL13B), and the cilia were imaged and analyzed using immunofluorescence microscopy.

[0089] The results are as follows Figure 3As shown, Figure A is a fluorescence image of kaempferol-induced restoration of damaged cilia in IFT system cells. Cilia are labeled with ARL13B in red and ACTUB in green, and cell nuclei are labeled in blue; the scale bars in the figures are all 20 µm. Figure B shows the analysis of cilia length; Figure C shows the frequency analysis of different cilia lengths; Figure D shows the quantitative analysis of the percentage of ciliated cells. Compared with the control group, the number and length of cilia in cells treated with 50 µM Ciliobrevin D were significantly reduced. The addition of different concentrations of kaempferol restored cilia length, but did not improve the number of cilia (*). p <0.05 and **** p Compared with the dimethyl sulfoxide group, <0.001, #### p <0.001 Compared with Cilibrevin D group, ns: no statistical significance, all values ​​in the figure are mean ± standard error, n ≥ 3; data are shown in Table 3).

[0090] Table 3 In addition, the expression of ciliate-related proteins (ACTUB and ARL13B) in starved ARPE19 cells after treatment with 5 µM kaempferol for 24 hours following treatment with the ciliate inhibitor Cilibrevin D was detected by Western blotting, and semi-quantitative analysis was performed. The results are as follows: Figure 3 As shown in Figures E, F, and G, kaempferol has an effect on the expression of proteins related to cilia loss in the IFT system treated with Cilibrevin D (ACTUB and ARL13B). E represents representative Western Blot results, and F and G show the quantitative analyses of ACTUB and ARL13B, respectively. Compared to the control group, the expression of both cilia protein markers ACTUB and ARL13B was significantly reduced after Cilibrevin D treatment. However, compared to the Cilibrevin D group, the expression of cilia-related proteins (ACTUB and ARL13B) was significantly upregulated in the kaempferol-treated group, showing statistically significant differences. p <0.005 compared with the blank control group, # p <0.05 compared with Cilibrevin D group, all values ​​in the figure are mean ± standard error, n ≥ 3.

[0091] These results indicate that kaempferol can rescue ciliary shortening caused by damage to the IFT system.

[0092] Example 4: Kaempferol restores ciliary length in siCEP290 cells.

[0093] CEP290 is a protein in the ciliary transition zone, primarily maintaining the normal structure of cilia. To determine the recovery of cilia inhibition caused by kaempferol, CEP290 was knocked down in ARPE19 cells, and then 1 µM, 3 µM, and 5 µM kaempferol were added for 24 hours after serum removal. Cilia were then stained with immunofluorescence using the ciliary marker protein ACTUB and CEP290 antibody, and the cilia were imaged and analyzed by immunofluorescence microscopy. The immunofluorescence staining method was the same as in Example 1; the primary antibody used further included rabbit anti-CEP290 (1:1000; 22490-1-AP, Proteintech).

[0094] The results are as follows Figure 4 As shown, Figure A is a fluorescence image of siCEP290 cell cilia restored by kaempferol. Cilia are labeled green with ACTUB, CEP290 is labeled red, and cell nuclei are labeled blue; the scale bars in the figures are all 20 µm. Figure B shows the analysis of ciliary length; Figure C shows the frequency analysis of different ciliary lengths; Figure D shows the quantitative analysis of the percentage of ciliated cells. Compared with the blank control group, the number and length of cilia in cells knocked down by CEP290 were significantly reduced. After treatment with different concentrations of kaempferol, the ciliary length was restored to some extent, but the number of cilia was not improved (*). p <0.05 and **** p All values ​​<0.001 were compared with the blank control group. p <0.001 and ns were compared with the siCEP290 group. ns: no statistical significance. All values ​​in the figure are mean ± standard error, n ≥ 3; data are shown in Table 4.

[0095] Table 4 In addition, the expression of ciliary-related proteins (ACTUB and ARL13B) in starved ARPE19 cells treated with 5 µM kaempferol for 24 hours after siCEP290 treatment was detected by Western blotting, and semi-quantitative analysis was performed. The results are as follows: Figure 4 As shown in Figures E, F, and G, the effects of kaempferol on the expression of related proteins (ACTUB and ARL13B) in ARPE19 cells after siCEP290 treatment are illustrated. E represents the representative Western Blot results, and F and G show the quantitative analyses of ACTUB and ARL13B, respectively. Compared to the blank control group, the expression of ciliary protein markers ACTUB and ARL13B was significantly reduced after siCEP290 treatment. However, compared to the siCEP290 group, the expression of ciliary-related proteins (ACTUB and ARL13B) in the kaempferol-treated group was significantly upregulated, showing statistically significant differences. p <0.05 compared with the blank control group, #p Compared with the siCEP290 group, values ​​<0.05 are all mean ± standard error in the figure, n ≥ 3.

[0096] These results indicate that kaempferol can rescue ciliary shortening caused by CEP290 deficiency.

[0097] Example 5: Regulating ciliary length with combined use of kaempferol and the commonly used expectorant ambroxol Ambroxol is a novel agonist that thins sputum and enhances airway ciliary pulsation. To determine the effect of combined kaempferol and ambroxol on the number and length of cilia, we treated ARPE19 cells with different concentrations of ambroxol (5 µM, 10 µM, and 50 µM) after serum removal, as well as with 50 µM ambroxol + 1 µM kaempferol, 50 µM ambroxol + 3 µM kaempferol, and 50 µM ambroxol + 5 µM kaempferol for 24 hours. After treatment, the cells were fixed, the nuclei were stained with DAPI, and the cilia were stained with ciliary-associated proteins (ACTUB and ARL13B). The cilia were then imaged and analyzed using immunofluorescence microscopy.

[0098] The results are as follows Figure 5 As shown, Figure A presents fluorescence images of ciliated cells after treatment with a combination of kaempferol and ambroxol. Cilia are labeled with ARL13B in red and ACTUB in green, while cell nuclei are labeled in blue. The scale bars in the figures are all 20 µm. Figure B shows ciliary length analysis; Figure C shows frequency analysis of different ciliary lengths; and Figure D shows quantitative analysis of the percentage of ciliated cells. Compared with the blank control group, the ciliary length of cells treated with 5 µM and 10 µM ambroxol did not change significantly, while the ciliary length of cells treated with 50 µM ambroxol increased significantly. Furthermore, the ciliary length of cells treated with different concentrations of kaempferol showed a concentration-dependent increase. Compared with the 50 µM ambroxol group, ambroxol combined with different concentrations of kaempferol promoted ciliary length growth, with ciliary lengths exceeding those achieved with ambroxol alone. Furthermore, the 50 µM ambroxol combined with 1 µM, 3 µM, and 5 µM kaempferol also resulted in longer ciliary lengths compared to 1 µM, 3 µM, or 5 µM kaempferol alone. p <0.01 and **** p <0.001 was compared with the blank control group, & p <0.05 and &&&& p <0.001 compared to the 50 µM ambroxol group, § p Compared with the 1 µM kaempferol group, <0.05,^^^^ p <0.001 compared to the 3 µM kaempferol group,†p <0.05 compared with the 5 µM kaempferol group, ns: no statistical significance, all values ​​in the figure are mean ± standard error, n ≥ 3; data are shown in Table 5).

[0099] Table 5 These results indicate that the combined use of ambroxol and kaempferol can effectively and synergistically promote ciliary length elongation.

[0100] Example 6: The combined use of kaempferol and the commonly used expectorant ambroxol restored the number and length of cilia in cells damaged by viral cilia.

[0101] The same model as in Example 2 was used to verify whether the combined use of kaempferol and ambroxol could restore ciliary damage caused by the virus. After removing serum, ARPE19 cells with missing cilia were treated 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. The cilia were then imaged and analyzed using immunofluorescence microscopy.

[0102] The results are as follows Figure 6 As shown, Figure A presents fluorescence images of ciliated cells after treatment with a combination of kaempferol and ambroxol. Cilia are labeled red with ARL13B, and virus-infected positive cells are labeled green with FLAG. Cell nuclei are labeled blue. The scale bars in the figures 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 average length of cilia and the percentage of ciliated cells in cells transfected with the SARS-CoV-2 structural protein NSP13 were significantly reduced. However, when NSP13-transfected cells were treated with 50 µM ambroxol, no recovery of ciliary length and number was observed. However, the ciliary length of cells treated with different concentrations of kaempferol increased significantly in a concentration-dependent manner, and the number of cilia was also restored. Compared with the group treated with 50 µM ambroxol alone, treatment of NSP13-transfected cells with ambroxol and different concentrations of kaempferol showed a more significant rescue effect on ciliary length defects and reduced ciliary number; and the combination of high concentration (5 µM) kaempferol and ambroxol was superior to the recovery effect of kaempferol alone. (*) p <0.05 and ** p <0.01 compared with the blank control group, # p <0.05、## p <0.01 and #### p <0.001 compared to NSP13 group, & p <0.05、&& p<0.01, &&& p <0.005 and &&&& 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 6.

[0103] Table 6 These results indicate that the combined use of kaempferol and ambroxol can rescue cilia development and cilia length defects in cells damaged by viral cilia.

[0104] Example 7: Combined use of kaempferol and the commonly used expectorant ambroxol to restore the ciliary length of damaged cells in the IFT system.

[0105] The same model as in Example 3 was used to verify whether the combined use of kaempferol and ambroxol could restore ciliary shortening caused by IFT system damage. ARPE19 cells, under serum-free conditions, 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. Cells were treated with µM kaempferol for 24 hours, and the cilia were stained with immunofluorescence using ciliary marker proteins (ACTUB and ARL13B). The cilia were then imaged and analyzed using immunofluorescence microscopy.

[0106] The results are as follows Figure 7As shown, Figure A is a fluorescence image of ciliated cells after treatment with a combination of kaempferol and ambroxol. Cilia are labeled with ARL13B in red and ACTUB in green, and cell nuclei are labeled in blue; the scale bars in the figures are all 20 µm. Figure B shows the quantitative analysis of the percentage of ciliated cells; Figure C shows the analysis of ciliary length; Figure D shows the frequency analysis of different ciliary lengths. Compared with the blank control group, the number and length of cilia in cells treated with 50 µM Ciliobrevin D were significantly reduced. The addition of 50 µM ambroxol did not significantly restore the number and length of cilia. However, treatment with 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 did not restore the number of cilia, but the length of cilia was restored to some extent. Furthermore, the combined use of higher concentrations (3 µM and 5 µM) of kaempferol and ambroxol showed better recovery effects than ambroxol or kaempferol alone. (***) p <0.005 and **** p <0.001 is compared with the blank control group, ## p <0.01 and #### p <0.001 were compared with the 50 µM Ciliobrevin D group, & p <0.05 and && p <0.01 compared to the 50 µM ambroxol group, ^^ p <0.01 compared to the 3 µM kaempferol 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 7.

[0107] Table 7 These results suggest that ambroxol alone may not be able to restore ciliary shortening caused by damage to the IFT system, while higher concentrations of ambroxol combined with kaempferol can restore ciliary length in cells damaged by the IFT system, and the recovery effect is better than that of ambroxol or kaempferol alone.

[0108] Example 8: Combined use of kaempferol and the commonly used expectorant ambroxol to restore the ciliary length of siCEP290 cells.

[0109] 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.

[0110] 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 kaempferol, 3 µM kaempferol, 5 µM kaempferol, and 50 µM ambroxol combined with different concentrations of 1 µM, 3 µM, and 5 µM kaempferol 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 combined use 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.

[0111] 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.

[0112] Example 9: Kaempferol restores respiratory cilia loss caused by bacterial infection.

[0113] Damage or dysfunction of respiratory cilia is widespread in a variety of acute and chronic respiratory diseases. To determine whether kaempferol can restore respiratory cilia damage induced by acute and chronic bacterial infections, a model of respiratory cilia damage induced by bacterial infection (lipopolysaccharide) was established in mice.

[0114] (1) Animal model construction Six-week-old SPF-grade C57 mice underwent airway surgery after being anesthetized by intraperitoneal injection of tribromoethanol. The chronic irritation group received an intratracheal injection of 0.4 μg / μL LPS (50 μL); the acute irritation group received an intratracheal injection of 1 μg / μL LPS (50 μL); and the control group received an equal volume of physiological saline. The wounds were sutured after surgery, and the mice were returned to their home after regaining consciousness.

[0115] (2) Drug therapy A 10 mg / mL kaempferol suspension was prepared using 0.5% carboxymethyl cellulose sodium (CMC-Na). Mice were divided into three groups: a control group, a model group (0.5% CMC-Na, 10 mL / kg / d), and kaempferol gavage groups (25 mg / kg, 50 mg / kg, and 100 mg / kg), with three replicates per group. Dosing regimen: Chronic model: Kaempferol was administered via gavage 24 hours after LPS injection, once every 12 hours for a total of 24 hours.

[0116] Acute model: Kaempferol was administered by gavage 1 hour after LPS injection, once every 12 hours for a total of 24 hours.

[0117] One hour after the last gavage administration of kaempferol, tracheal tissue was collected from mice and fixed overnight with 4% paraformaldehyde solution. Airway cilia were observed by immunofluorescence staining (using mouse anti-ACTUB primary antibody and Alexa Fluor 568-labeled secondary antibody) and scanning electron microscopy.

[0118] The results are as follows Figure 9As shown, Figure A presents fluorescence images of airway cilia after treatment with different concentrations of kaempferol following acute and chronic bacterial infections. Cilia are labeled green with ACTUB, and cell nuclei are labeled blue. The scale bar in the figures is 10 µm. Figure B presents scanning electron micrographs of airway cilia after treatment with different concentrations of kaempferol following acute and chronic bacterial infections. The scale bar in the figures is 5 µm. Compared with the blank control group, mice injected with lipopolysaccharide (LPS) at 0.4 μg / μL for 24 hours and 1 μg / μL for 1 hour showed significant loss of airway cilia, with sparse cilia arrangement and some exposed epithelial surface. In the chronic stimulation model group injected with LPS at 0.4 μg / μL for 24 hours, treatment with different concentrations of kaempferol resulted in the recovery of airway cilia, with a significant increase in the number of cilia, denser arrangement, and re-covering of the epithelial surface with cilia. The ciliary recovery effect was significantly better with 50 mg / kg kaempferol than with 100 mg / kg kaempferol. In the acute model group injected with LPS at 1 μg / μL for 1 hour, it was observed that the number and density of cilia increased significantly with increasing kaempferol concentration, and the best ciliary recovery effect was observed with 100 mg / kg kaempferol, showing a significant increase in the number of cilia and a more dense arrangement. In summary, kaempferol may be more advantageous in restoring cilia loss caused by chronic bacterial infections.

[0119] These results suggest that certain concentrations of kaempferol can, to some extent, reverse airway ciliary damage caused by acute or chronic bacterial infections.

Claims

1. Use of the pharmaceutical composition in the preparation of a medicament for treating diseases related to ciliary dysfunction; in, The pharmaceutical composition comprises therapeutically effective amounts of ambroxol and kaempferol, wherein the concentration of kaempferol is 1-5 μM and the molar ratio of kaempferol to ambroxol is (1-5): 50; The ciliary dysfunction-related diseases are selected from the following (1) or (2): (1) Ciliopathy, wherein the ciliopathy is polycystic kidney disease, Bardet-Biedl syndrome, Joubert syndrome, Meckel-Gruber syndrome or Leber congenital amaurosis; (2) Ciliary damage caused by SARS-CoV-2 infection.

2. The application according to claim 1, wherein, The molar ratio of kaempferol to ambroxol in the pharmaceutical composition is 1:50, 3:50, or 5:

50.

3. The application according to claim 1, wherein, The concentration of kaempferol in the pharmaceutical composition is 1-3 μM or 3-5 μM.

4. The application according to claim 1, wherein, The concentration of kaempferol in the pharmaceutical composition is 1 μM, 3 μM or 5 μM.

5. The application according to claim 1, wherein, The pharmaceutical composition is a topical, inhaled, or oral administration formulation.

6. The application according to claim 1, wherein, The pharmaceutical composition is in the form of tablets, lozenges, capsules, pills, solutions, suspensions, injections, suppositories, inhaled powders, aerosols, or sprays.

7. The application according to claim 1, wherein, The pharmaceutical composition is an inhalation solution.

8. The application according to claim 1, wherein, The pharmaceutical composition is a syrup.

9. The application according to any one of claims 1-8, wherein, The pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or adjuvant therapeutic agent.

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