Application of Huperzine A in the Preparation of Drugs for the Prevention and / or Treatment of Pulmonary Fibrosis

Abstract

This invention provides the application of huperzine A in the preparation of drugs for the prevention and / or treatment of pulmonary fibrosis. Using a bleomycin-induced in vitro pulmonary fibrosis model, this invention demonstrates that huperzine A can act as an aryl hydrocarbon receptor agonist, increasing the intracellular expression of aryl hydrocarbon receptors; using a bleomycin-induced mouse pulmonary fibrosis model, this invention demonstrates that huperzine A possesses anti-pulmonary fibrosis pharmacological activity. By studying the in vivo and in vitro bioactivity of huperzine A in the treatment of pulmonary fibrosis, this invention provides new ideas and solutions for the research and development and production of anti-fibrotic drugs.

Description

Technical Field

[0001] This invention relates to the use of huperzine A in the preparation of drugs for the prevention and / or treatment of pulmonary fibrosis, and belongs to the field of pharmaceutical technology. Background Technology

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive pulmonary fibrosis disease characterized by gradual fibrosis and scarring of lung tissue, destruction of alveolar structure, decreased lung compliance, impaired gas exchange, and ultimately respiratory failure and death, with a mortality rate as high as 50% to 70%. The pathogenesis of IPF mainly involves accelerated aging of lung epithelial cells and repeated subclinical epithelial cell damage, leading to abnormal repair of damaged alveoli and interstitial fibrosis deposition induced by myofibroblasts, causing lung tissue structural reconstruction and ultimately respiratory failure. Many factors can trigger IPF, including environmental, occupational, physical, chemical, and disease-related factors. Epidemiological statistics show that in Europe and North America, there are 3-18 new cases per 100,000 people annually, while in Asia and South America, the incidence is lower, with 0.45-4.2 new cases per 100,000 people annually. IPF patients are more common in men, and the age of onset is rarely less than 50 years old. The median age at diagnosis is about 65 years old, and the survival period after diagnosis is only 2-4 years.

[0003] To date, the main clinical treatments for pulmonary fibrosis (IPF) are pirfenidone and nintedanib. While pirfenidone and nintedanib can slow the decline in lung function and improve progression-free survival in IPF, they do not significantly improve overall mortality or acute exacerbations in IPF patients. Furthermore, pirfenidone and nintedanib have various gastrointestinal and hepatobiliary adverse reactions.

[0004] The pathogenesis of pulmonary fibrosis is complex. Numerous studies have confirmed that the TGF-β / Smad signaling pathway plays a crucial role in the development of pulmonary fibrosis. Transforming growth factor-β1 (TGF-β1) is currently recognized as an important factor inducing fibrotic changes in tissues. The TGF-β1 / Smad signaling pathway mediated by TGF-β1 can regulate the transcription and protein expression of target genes, thereby inducing pulmonary fibrosis.

[0005] Previous studies have shown that the aryl hydrocarbon receptor (AhR) is a highly conserved environmentally responsive transcription factor that integrates dietary, microbial, environmental, and metabolic cues to regulate complex transcriptional programs in a ligand-specific and cell-type-specific manner. It consists of an N-terminal bHLH domain required for DNA binding, followed by two PER-ARNT-SIM (PAS) domains (A and B) and a C-terminal transactivation domain (TAD). In the cytoplasm, AhR forms a stable complex with HSP90, AhR-interacting proteins (HBV X-related protein 2), the common chaperone p23, and the protein kinase SRC. Upon ligand activation, binding of the ligand to the PAS domain of AhR leads to a conformational change, exposing the nuclear localization sequence. AhR dissociates from the complex and enters the nucleus. In the nucleus, AhR forms a dimer with the AhR nuclear translocation factor (ARNT), which regulates the transcription of downstream genes; phosphorylation of AhR enhances its biological activity. Both AhR and ARNT can recruit a co-activating transcriptional pathway that then initiates AhR target genes, including CYP1A1 and CYP1B1, which are the most common genes and encode CYP1 family enzymes. Literature indicates that AhR signaling is also associated with the activation of other pathways such as transforming growth factor-β / Smad (TGF-β1 / Smad) and matrix metalloproteinases (MMPs).

[0006] Huperzine A (HA) is a sesquiterpene alkaloid extracted from Huperzine aegyptiacus. It is a highly effective, selective, and reversible centrally acting acetylcholinesterase inhibitor, possessing properties that enhance cholinergic function, combat oxidative stress, protect against ischemic brain injury, and improve the secretion of inflammatory factors by microglia in the brain. Clinically, it is commonly used to treat age-related memory decline, cognitive and behavioral impairments. Currently, there are no reports on the use of huperzine A in the preparation of drugs for the prevention and / or treatment of pulmonary fibrosis. Summary of the Invention

[0007] In a first aspect, the present invention provides the use of huperzine A in the preparation of drugs for the prevention and / or treatment of pulmonary fibrosis.

[0008] Secondly, the present invention provides the use of huperzine A as an aryl hydrocarbon receptor agonist in the preparation of drugs for the prevention and / or treatment of pulmonary fibrosis.

[0009] Thirdly, the present invention provides the use of pharmaceutical compositions containing huperzine A in the preparation of drugs for the prevention and / or treatment of pulmonary fibrosis.

[0010] Preferably, the pharmaceutical composition comprises: (i) an effective amount of huperzine A, its hydrate, solvate or pharmaceutically acceptable salt; and (ii) a pharmaceutically acceptable carrier and / or excipient.

[0011] Fourthly, the present invention provides a method for significantly downregulating the expression of pulmonary fibrosis marker proteins TGF-β1 and α-SMA in lung tissue by administering an effective amount of huperzine A, its hydrate, solvate, or pharmaceutically acceptable salt to the subject.

[0012] Fifthly, the present invention provides a method for significantly downregulating the expression of pulmonary fibrosis marker proteins TGF-β1 and α-SMA in lung tissue by administering an effective amount of a pharmaceutical composition containing huperzine A to the subject.

[0013] Preferably, the pharmaceutical composition comprises: (i) an effective amount of huperzine A, its hydrate, solvate or pharmaceutically acceptable salt; and (ii) a pharmaceutically acceptable carrier and / or excipient. Attached Figure Description

[0014] Figure 1 The changes in body weight of mice with bleomycin-induced pulmonary fibrosis were characterized by huperzine A in Example 2.

[0015] Figure 2 The effect of huperzine A on the lung coefficient of bleomycin-induced pulmonary fibrosis mice in Example 2 was characterized.

[0016] Figure 3 The effect of huperzine A on the penh value of bleomycin-induced pulmonary fibrosis mice in Example 2 was characterized. The bar charts from left to right are: control group, model group, pirfenidone group (300 mg / kg), huperzine A (0.0375 mg / kg), huperzine A (0.075 mg / kg), and huperzine A (0.15 mg / kg).

[0017] Figure 4 Characterization of huperzine A in bleomycin-induced pulmonary fibrosis mice in Example 2 50 The influence of values. The bar charts from left to right are: control group, model group, pirfenidone group (300 mg / kg), huperzine A (0.0375 mg / kg), huperzine A (0.075 mg / kg), and huperzine A (0.15 mg / kg).

[0018] Figure 5 The effect of huperzine A in Example 2 on the rotarod retention time in bleomycin-induced pulmonary fibrosis mice was characterized.

[0019] Figure 6 HE staining results of paraffin sections of lung tissue from mice with bleomycin-induced pulmonary fibrosis, characterized by huperzine A in Example 2. The scale bar in the figure is in units of 200 μm.

[0020] Figure 7The Ashcroft score of lung tissue in mice with bleomycin-induced pulmonary fibrosis was characterized by the effect of huperzine A on lung tissue in Example 2.

[0021] Figure 8 The results of Sirius red staining of paraffin sections of lung tissue from mice with bleomycin-induced pulmonary fibrosis, as characterized by huperzine A in Example 2, are shown. The scale bar in the figure is in units of 200 μm.

[0022] Figure 9 Characterize the percentage of lung fibrosis in mice with bleomycin-induced pulmonary fibrosis induced by huperzine A in Example 2.

[0023] Figure 10 Characterize the expression of AhR, TGF-β1 and α-SMA in lung tissue of bleomycin-induced pulmonary fibrosis mice in Example 2 using huperzine A. Detailed Implementation

[0024] The present invention is further illustrated by the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0025] This invention discloses the use of huperzine A in the preparation of drugs for the prevention and / or treatment of pulmonary fibrosis.

[0026] The term "prevention" refers, for example, to preventing the development of clinical symptoms of a disease in mammals that may be exposed to or pretreated with the disease but have not yet experienced or shown symptoms of the disease.

[0027] The term "treatment" can refer to suppressing a disease, such as preventing or reducing the progression of a disease or its clinical symptoms, or alleviating a disease, such as causing the disease or its clinical symptoms to regress. As an example, the term "treatment" refers to reducing or alleviating the progression, severity, and / or duration of a disease (e.g., a proliferative disease) or improving one or more symptoms of the disease (preferably one or more perceptible symptoms) due to the application of one or more therapies.

[0028] Huperzine A is a monomeric compound derived from traditional Chinese medicine, and its structure is shown in the following formula. For simplicity, huperzine A may also be referred to as the compound of this invention or the active compound below.

[0029]

[0030] Huperzine A is a treatment for Alzheimer's disease that has been used clinically for many years. Its safety and side effects have been studied extensively, making it more readily accepted by the human body with fewer side effects. This invention, by studying the in vitro and in vivo bioactivity of huperzine A in combating pulmonary fibrosis, provides new ideas and solutions for the research and development and production of anti-fibrotic drugs.

[0031] Specifically, this invention discloses the use of huperzine A as an aryl hydrocarbon receptor agonist in the preparation of drugs for the prevention and / or treatment of pulmonary fibrosis.

[0032] In specific implementation methods, bleomycin-induced in vitro pulmonary fibrosis models and pulmonary fibrosis animal models were used to demonstrate that huperzine A, as an aryl hydrocarbon receptor agonist, has pharmacological activity against pulmonary fibrosis.

[0033] Using a bleomycin-induced in vitro pulmonary fibrosis model, it was demonstrated that huperzine A can act as an aryl hydrocarbon receptor agonist to increase the intracellular expression of aryl hydrocarbon receptors, and its EC50... 50 The concentration was 8.04 μM. Using a bleomycin-induced mouse pulmonary fibrosis model, huperzine A was demonstrated to possess anti-pulmonary fibrosis pharmacological activity. Specifically, huperzine A effectively and dose-dependently improved bleomycin-induced pulmonary fibrosis damage in mice, reduced lung coefficient, enhanced lung compliance, decreased lung resistance, and improved respiratory index and cardiopulmonary function.

[0034] Further mechanistic studies have demonstrated that huperzine A, as an aryl hydrocarbon receptor agonist, can significantly downregulate the production of pulmonary fibrosis marker proteins TGF-β1 and α-SMA in lung tissue. In other words, huperzine A, as an aryl hydrocarbon receptor agonist, can significantly downregulate the expression of pulmonary fibrosis marker proteins TGF-β1 and α-SMA in lung tissue, inhibit collagen fiber formation and differentiation of lung fibroblasts into myofibroblasts, and promote collagen degradation. This results in the improvement of bleomycin-induced pulmonary fibrosis damage in mice, lung coefficient, enhanced lung compliance, reduced lung resistance, improved respiratory index and cardiopulmonary function, thereby exerting a pharmacological effect against pulmonary fibrosis.

[0035] The present invention also discloses the use of pharmaceutical compositions containing huperzine A in the preparation of drugs for the prevention and / or treatment of pulmonary fibrosis.

[0036] The pharmaceutical composition comprises: (i) an effective amount of huperzine A or its hydrate, solvate, or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition contains huperzine A or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient or carrier within a safe and effective range. "Safe and effective range" means that the amount of the compound is sufficient to significantly improve the condition without causing serious side effects.

[0037] The term "pharmaceutically acceptable salt" refers to a salt formed by the compounds of the present invention with an acid or base that is suitable for use as a medicine. Pharmaceutically acceptable salts include both inorganic and organic salts. A preferred class of salts are those formed by the compounds of the present invention with an acid. Acids suitable for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, and benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.

[0038] The term "pharmaceutically acceptable carrier" refers to a carrier that can be used to prepare a pharmaceutical composition. These carriers are generally safe, non-toxic, and not biologically or otherwise undesirable, and include carriers that are pharmaceutically acceptable to animals and humans. As used in the specification and claims, "pharmaceutically acceptable carrier" includes one or more of these carriers.

[0039] Specifically, "pharmaceutically acceptable carriers" can refer to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here refers to the ability of the components in the composition to interact with and be mixed with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as... Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0040] This invention also discloses a method for significantly downregulating the expression of pulmonary fibrosis marker proteins TGF-β1 and α-SMA in lung tissue. An effective amount of huperzine A or its hydrate, solvate, or a pharmaceutically acceptable salt thereof is administered to the subject.

[0041] "Administration" means the physical introduction of a composition containing a therapeutic agent into an individual using any of the various methods and delivery systems known to those skilled in the art. Subjects of administration include any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In some embodiments, the individual is a human. The terms "individual" and "patient" are used interchangeably herein.

[0042] There are no particular restrictions on the method of administration. Representative methods of administration include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and local administration.

[0043] Preferred routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes, such as by injection or infusion. "Parenteral administration" refers to a mode of administration other than enteral and local administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intralesional, intracystic, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, epidermal, intra-articular, subcystic, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as intracorporeal electroporation. Other parenteral routes include local, epidermal, or mucosal administration routes, such as intranasal, vaginal, rectal, sublingual, or local administration. Administration may also be performed, for example, once, multiple times, and / or over one or more extended periods.

[0044] Using selective pharmaceutically acceptable carriers or excipients, drugs or drug compositions with huperzine A as the main active ingredient can be prepared into any pharmaceutically acceptable dosage form.

[0045] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. Sprays, tablets, capsules, and syrups are preferred for oral administration. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[0046] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0047] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0048] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0049] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0050] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0051] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0052] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0053] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) requiring treatment, wherein the dose administered is the pharmaceutically considered effective dose. As an example, but not a limitation, for a person weighing 60 kg, the daily dose is typically 0.1–0.9 mg, preferably 0.2–0.9 mg. Of course, the specific dosage should also consider factors such as the route of administration and the patient's health condition, which are within the scope of a skilled physician's expertise.

[0054] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0055] Example 1: Study on the effect of huperzine A in alleviating bleomycin-induced in vitro pulmonary fibrosis model

[0056] 1. Materials and Instruments

[0057] 1.1 Cells

[0058] Human embryonic lung fibroblast cell line (MRC-5) was purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0059] 1.2 Instruments and Reagents

[0060] High-glucose DMEM medium (Gibco, USA, batch number: 2646156), fetal bovine serum (Gibco, USA, batch number: 1891605), penicillin and streptomycin (Gibco, USA, batch number: 2041564), PBS phosphate buffer (Beyotime, batch number: 052223230618), trypsin cell digestion solution (0.25% trypsin, m / v) (Beyotime, batch number: 032823230403), bleomycin (manufacturer: Aladdin (USA; Batch No.: D2312011), Huperzine A (Manufacturer: Shanghai Bid Pharmaceutical Technology Co., Ltd. - China; Batch No.: CKU030), SDS-PAGE Gel Preparation Kit (Manufacturer: Wuhan Sewell Biotechnology - China; Batch No.: CR2211110), 0.22 PVDF Membrane (Manufacturer: Wuhan Sewell Biotechnology - China; Batch No.: CR2209046), General ECL Chemiluminescence Kit (Manufacturer: Wuhan Sewell Biotechnology - China; Batch No.: ECL) Solution A: CR2302076-1; Solution B: CR2302076-2), GAPDH(14C10) Rabbit mAb (Manufacturer: CST-Canada; Lot No.: 14), Ah Receptor antibody (Manufacturer: StanCruz Biotechnology-USA; Lot No.: L1321), Horseradish peroxidase-labeled goat anti-rabbit IgG (Manufacturer: Wuhan Sewell Biotechnology-China; Lot No.: CR2201040), BCA protein concentration assay kit (Manufacturer: Beyotime-China; Lot No.: 111922230120), Skim milk powder (Manufacturer: Servicebio; Lot No.: CR2201115).

[0061] 2. Preparation of main reagents

[0062] High-glucose DMEM complete medium: Take an appropriate amount of high-glucose DMDM ​​medium, add 10% (v / v) fetal bovine serum inactivated at 56℃ and 1% (v / v) antibiotics, filter through a 0.22μm filter membrane, and store at 4℃ for later use.

[0063] 3. Experimental Methods

[0064] MRC-5 cells in logarithmic growth phase were harvested and their concentration adjusted to 1.5 × 10⁻⁶ cells. 5 / mL, 2mL was seeded into 6-well plates. After 24h of sample preparation, the culture medium was removed and replaced with serum-free culture medium containing different concentrations of drug. Blank control group, model group, huperzine A 100μM group, huperzine A 50μM group, huperzine A 25μM group, huperzine A 12.5μM group, huperzine A 6.25μM group, and huperzine A 3.125μM group were established. Each drug treatment group was given the corresponding concentration of drug solution, while the blank control group and model group were given an equal volume of culture medium. Except for the blank control group, all other groups were stimulated with bleomycin (final concentration 15μM) 30min after drug administration. After 48h of culture, cells were collected, and proteins were extracted according to the RIPA lysis buffer instructions. Protein concentration was determined by the BCA method, loading buffer was added to adjust the protein concentration, and the proteins were denatured by boiling in a metal bath at 96℃ for 10min. Separation was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane using wet transfer, blocked with 5% skim milk powder (m / v), and washed with PBST. Primary antibodies (GAPDH(14C10) Rabbit mAb, 1:1000; Ah Receptor, 1:100) were added and incubated overnight at 4°C, followed by washing with PBST. Goat anti-rabbit secondary antibody (1:3000) was added and incubated at room temperature for 1 h. Exposure and imaging were performed using a fully automated chemiluminescence imaging system. ImageJ analysis software was used to analyze the grayscale values ​​of the target protein bands and calculate EC50. 50 .

[0065] 4. Experimental Results

[0066] The experimental results are shown in Table 1.

[0067] Table 1. Effects of different huperzine A contents on the relative expression level of intracellular AhR in bleomycin-stimulated MRC-5 cells.

[0068]

[0069] In this embodiment, the AhR levels in bleomycin-stimulated MRC-5 cells increased with increasing loading concentration at different concentrations of huperzine A (Table 1), EC 50 The concentration was 8.04 μM. This is because AhR signaling is related to the activation of the TGF-β1 / Smad signaling pathway, and activation of the aryl hydrocarbon receptor can inhibit the activation of the TGF-β1 / Smad signaling pathway, thus playing a role in improving pulmonary fibrosis. This indicates that huperzine A acts as an agonist for AhR in the bleomycin-induced MRC-5 cell pulmonary fibrosis model.

[0070] Example 2: Study on the effect of huperzine A on bleomycin-induced mouse pulmonary fibrosis model

[0071] 1. Materials and Instruments

[0072] 1.1 Animals

[0073] Male C57BL / 6J mice, 8–10 weeks old, 22±2g, were purchased from Shanghai Silex Laboratory Animal Co., Ltd. Mice were housed in an SPF environment with a room temperature maintained at 20–25℃ and humidity controlled at 50–60%. Mice were fed normally, and the light cycle was regulated to a 12-hour diurnal pattern. Mice underwent one week of acclimatization to the environment before the experiments. All animal experiments were conducted in accordance with the animal care regulations and guidelines of Shanghai Medicilon Biomedical Co., Ltd., and were approved by the Animal Committee of Shanghai Medicilon Biomedical Co., Ltd.

[0074] 1.2 Experimental Materials

[0075] Bleomycin (Manufacturer: Aladdin - USA; Batch No.: D2312011), Huperzine A (Manufacturer: Shanghai Bid Pharmaceutical Technology Co., Ltd. - China; Batch No.: CKU030), Physiological Saline (Manufacturer: Zhejiang Tianrui Pharmaceutical Co., Ltd. - China; Batch No.: 12310203), Isoflurane (Manufacturer: Shandong Ante Animal Husbandry Technology Co., Ltd. - China; Batch No.: 20230529), SDS-PAGE Gel Preparation Kit (Manufacturer: Wuhan Sewell Biotechnology - China; Batch No.: CR2211110), 0.22 PVDF Membrane (Manufacturer: Wuhan Sewell Biotechnology - China; Batch No.: CR2209046), General ECL Chemiluminescence Kit (Manufacturer: Wuhan Sewell Biotechnology - China; Batch No.: ECL A solution CR2302076-1; ECL B solution CR2302076-2), Anti-alpha Smooth Muscle Actin Antibody (Manufacturer: Abcam - UK; Batch No.: 1009584), Anti-TGF Beta1 antibody (manufacturer: Abcam - UK; lot number: 1007290), GAPDH (14C10) Rabbit mAb (manufacturer: CST - Canada; lot number: 14), Ah Receptor antibody (manufacturer: Stan Cruz) Biotechnology (USA; Batch No.: L1321), Horseradish Peroxidase-Labeled Goat Anti-Rabbit IgG (Manufacturer: Wuhan Sewell Biotechnology (China); Batch No.: CR2201040), BCA Protein Concentration Detection Kit (Manufacturer: Beyotime (China); Batch No.: 111922230120), Small Animal Anesthesia Machine - Universal Type (Manufacturer: Shenzhen Ruiwode Life Technology Co., Ltd.; Model: R500IE), Handheld Liquid Aerosol Lung Delivery Device (Manufacturer: Beijing Huilaihe Technology Co., Ltd.; Model: HRH-MAG4), WBP Small Animal Whole Body Volume Saturation System (Manufacturer: Shanghai Tawang Intelligent Technology Co., Ltd.; Model: WBP-8MR), Rotary Fatigue Meter (Manufacturer: Huaibei Zhenghua Bio-Instrument Equipment Co., Ltd.; Model: YLS-4C), Digital Display Constant Voltage and Current Electrophoresis System (Manufacturer: Tanon (Shanghai, China); Model: EPS-300), Microplate Reader (Manufacturer: MolecularDevices (USA); Model: Versa Max), Surgical Forceps, Surgical Plate.

[0076] 1.3 Preparation of main reagents

[0077] Bleomycin sulfate solution: Bleomycin sulfate solution is prepared with physiological saline to a concentration of 1 mg / mL and should be prepared and used immediately.

[0078] 2. Experimental Methods

[0079] 2.1 Methods for establishing a pulmonary fibrosis model

[0080] After weighing, mice were anesthetized with isoflurane inhalation. Once the righting reflex disappeared, the mice were suspended with their upper incisors facing upwards and fixed to a surgical board, ensuring sufficient light to visualize the vocal cords. Using sterile, padded surgical forceps, the tongue was gently pulled towards the corner of the mouth, exposing the vocal cords towards the mandible. A delivery device containing 20 μL of bleomycin was inserted through the glottis, penetrating 1-2 cm, and the bleomycin nebulizer solution was rapidly sprayed. The delivery device was then removed, and the animals were returned to their cages after waking. Control group animals received an equal volume of sterile saline. At the experimental endpoint, animals were anesthetized with deep CO2 inhalation. Blood was drawn from the heart, and pulmonary lavage fluid was collected. Both lungs were dissected and removed. The left lung was preserved in formalin for histopathological section staining, and the right lung was cryopreserved for later use.

[0081] 2.2 Animal grouping and administration methods

[0082] Six days after bleomycin modeling (Day-0), animals were evenly divided into groups based on their respiratory index: a blank control group, a model group, a positive control group (pirfenidone), and low, medium, and high dose groups of huperzine A, with eight animals in each group. One week after modeling, the animals in each group were administered the drugs orally via gavage at the doses listed in the table below, once daily for 15 consecutive days.

[0083] Table 2. Dosage and administration method for each group of animals

[0084]

[0085] 2.3 Indicator Assessment

[0086] 2.3.1 General Observation

[0087] Observe the mice's condition daily, including their fur, mental state, and weight.

[0088] 2.3.2 Respiratory Index

[0089] Respiratory indices were collected from mice in each group on days 0, 7, and 15 of drug administration using the WBP small animal whole-body volume plethysmography system.

[0090] 2.3.3 Fatigue test of the rotating rod

[0091] On day 14 after drug administration, the rotarod retention time of mice in each group was examined using a fatigue rotarod apparatus.

[0092] 2.3.4 Collection of materials

[0093] At the experimental endpoint, the body weight of mice in each group was recorded. Mice were deeply anesthetized with excessive CO2, and blood was collected from the heart. The obtained whole blood was allowed to stand at 4°C for 2 hours, centrifuged at 3500 r / min for 15 minutes, and the supernatant serum was collected and frozen at -80°C for later testing. Mice were euthanized after blood collection, and the lung tissue was dissected and removed.

[0094] 2.3.5 Lung coefficient

[0095] The clean lung tissue obtained from the dissection was weighed, and the lung coefficient of the mouse was calculated according to the formula: Lung coefficient = Lung mass (mg) / Mouse body weight before the last administration (g).

[0096] 2.3.6 HE staining

[0097] After weighing the lung tissue, the left lung of each group of mice was taken, fixed in formalin solution for 72 hours, dehydrated, embedded in paraffin, sectioned (4 μm thick), and stained with hematoxylin and eosin (HE). The pathological morphology of the lung tissue was observed and images were acquired using an optical microscope (×100). Pulmonary fibrosis was scored according to the Ashcroft scoring system, with higher scores indicating more severe pulmonary fibrosis. The Ashcroft scoring system is as follows: 0 points: normal lung tissue; 1 point: slight thickening of the alveolar or bronchial walls; 3 points: moderate thickening of the alveolar or bronchial walls, but no significant destruction of alveolar structure; 5 points: formation of cord-like fibrous bands or small fibrotic foci, with significant destruction of alveolar structure; 7 points: severe deformation of alveolar structure, widespread formation of fibrotic foci, presenting as "honeycomb lung"; 8 points: full-field fibrosis of the lung tissue, with scores 2, 4, and 6 falling between these values.

[0098] 2.3.7 Sirius Red Staining

[0099] After weighing the lung tissue, the left lung of each group of mice was taken, fixed in formalin solution for 72 hours, dehydrated, embedded in paraffin, sectioned (4 μm thick), and stained with Sirius red. Sirius red dye is strongly acidic and readily binds to the basic groups in collagen molecules. Collagen was stained red, and muscle fibers were stained yellow. The pathological morphology of the lung tissue was observed and images were acquired using an optical microscope (×100). The area of ​​the red collagen region was acquired using ImageJ software to obtain the fibrosis ratio. The formula for calculating the fibrosis ratio is: area of ​​red collagen region / area of ​​lung tissue × 100%.

[0100] 2.3.8 Relative expression levels of AhR, TGF-β1, and α-SMA in lung tissue

[0101] A measured amount of right lung tissue was weighed, homogenized with protein lysis buffer, and the supernatant was obtained. Protein concentration was determined using the BCA method. Loading buffer was added to adjust the protein concentration, and the protein was denatured by boiling in a metal bath at 96°C for 10 min. Separation was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane using wet transfer, blocked with 5% skim milk powder, and washed with PBST. Primary antibodies (Anti-alpha smooth muscle Actin, 1:2500; Anti-TGF beta1 antibody, 1:1000; GAPDH(14C10) Rabbit mAb, 1:1000; Ah Receptor, 1:100) were added and incubated overnight at 4°C. The membrane was washed with PBST. Goat anti-rabbit secondary antibody (1:3000) was added and incubated at room temperature for 1 h. The membrane was exposed and photographed using a fully automated chemiluminescence imaging system, and the target protein bands were analyzed for grayscale values ​​using ImageJ analysis software.

[0102] 3. Data Processing

[0103] Statistical analysis was performed using SPSS version 22.0 software. Quantitative data were expressed as mean ± standard deviation. The results are indicated below. One-way ANOVA was used for comparisons between groups. The LSD test was used when the variances were homogeneous, and Dunnett's T3 test was used when the variances were unequal. (Note: Compared with the model group, *P<0.05, **P<0.01; compared with the control group, #P<0.05, ##P<0.01).

[0104] 4. Experimental Results

[0105] 4.1 Observation of the general condition of mice with bleomycin-induced pulmonary fibrosis after using huperzine A

[0106] Throughout the experiment, the control group mice exhibited bright eyes, soft fur, no abnormal breathing sounds, and a steady increase in weight. Four days after modeling, the model group mice showed significantly slower responses, huddled together, trembling, cyanosis of the extremities, and wrinkled skin. Regarding weight, as... Figure 1 As shown, on Day 0, the body weight of mice with pulmonary fibrosis was significantly lower than that of the control group (p<0.01). On Day 3, the body weight of mice in all treatment groups began to recover, with the pirfenidone group and the high-dose huperzine A group showing the greatest recovery compared to the model group. On Day 15, the body weight of mice in the high-dose huperzine A group was significantly higher than that of the model group (p<0.05), approaching that of the normal group.

[0107] 4.2 Effect of huperzine A on lung coefficient in bleomycin-induced pulmonary fibrosis mice

[0108] An increase in the lung coefficient may be related to abnormal remodeling of lung tissue structure. Under the catalysis of numerous cytokines, lung fibroblasts transform into myofibroblasts, promoting collagen synthesis and secretion, and increasing extracellular matrix deposition, thereby increasing lung weight and the lung coefficient. Therefore, measuring the lung coefficient can indirectly reflect the degree of lung fibrosis. Observations of the lung coefficient in mouse lungs, such as... Figure 2 As shown in the figure, the lung organ coefficient in the model group was significantly increased compared with the normal group (p<0.05); compared with the model group, the lung organ coefficient in the high-dose huperzine A group was significantly decreased (p<0.05), and the lung organ coefficient in the positive control drug pirfenidone group was also decreased, but no statistical difference was shown. This indicates that huperzine A can effectively and in a dose-dependent manner improve the lung organ coefficient in bleomycin-induced pulmonary fibrosis mice.

[0109] 4.3 Effects of huperzine A on respiratory index in bleomycin-induced pulmonary fibrosis mice

[0110] Dyspnea is the most common symptom of pulmonary fibrosis. With the continuous development of respiratory pharmacology and respiratory physiology, pulmonary function-related parameters have become the main indicators for diagnosing respiratory diseases and evaluating treatment efficacy. WBP (Waist-Body Pressure) is a respiratory index assessment performed non-invasively and in a conscious state in mice, avoiding the influence of anesthetic drugs on pulmonary function parameters. It is also repeatable and suitable for follow-up studies. The airway narrowing index (Penh value) is an indicator of airway resistance, representing the degree of bronchial constriction, and is widely used in the evaluation of airway resistance. In the pathogenesis of pulmonary fibrosis, increased airway resistance is one of the main causes of dyspnea. The Penh value is not simply a measurement of airway resistance; it also comprehensively considers parameters such as respiratory rate, maximum expiratory volume, tidal volume, and apnea. It is calculated by plotting a respiratory curve based on changes in gas pressure within the chamber, and is more accurately described as an indirect indicator of airway responsiveness. Furthermore, airway hyperresponsiveness refers to an overreaction of the airway to certain stimuli, exhibiting sensitive and excessive bronchial smooth muscle contraction, causing airway narrowing and increased airway resistance, thereby triggering symptoms such as cough, chest tightness, dyspnea, and wheezing. In clinical practice, lung diffusion capacity is usually monitored in patients with pulmonary fibrosis, but pulmonary ventilation function is not tested. However, recent experimental studies have shown that bleomycin-induced pulmonary fibrosis animal models exhibit increased airway responsiveness in vitro, and the maximum expiratory flow rate at 50% vital capacity (EF50) is one of the main indicators for evaluating small airway function. Figure 3 and Figure 4 As shown, compared with the control group, the model group mice had higher Penh and EF values ​​on Day-0, Day-7, and Day-15. 50The values ​​were significantly increased (p<0.05) compared to the model group. The penh and EF values ​​of mice in the pirfenidone group and the high-dose huperzine A group were significantly increased on Day-7 and Day-15. 50 All values ​​were significantly reduced (p<0.05). This indicates that huperzine A can effectively, in a dose-dependent manner, improve lung resistance and respiratory index in bleomycin-induced pulmonary fibrosis mice.

[0111] 4.4 Effects of Huperzine A on Cardiopulmonary Function in Bleomycin-Induced Pulmonary Fibrosis Mice

[0112] Pulmonary fibrosis eventually leads to complete loss of cardiopulmonary function. Clinically, the six-minute walk test is a simple, safe, and convenient exercise test that reflects the cardiopulmonary function status of patients with pulmonary fibrosis by assessing their exercise capacity. Due to its simplicity, low cost, good repeatability, and patient acceptance, it has become a supplement to traditional cardiopulmonary tests (pulmonary function tests, blood gas analysis, etc.) to monitor various lung diseases, and the six-minute walk test is increasingly valued as a method for assessing lung function. Studies have shown that in comparative tests for pulmonary fibrosis, the six-minute walk distance is significantly reduced. The rotarod test is used to assess the coordination and balance of sustained movement in rodents. The animal must maintain balance on a rotating bar to avoid falling. The instrument records the time (delay) required for the animal to fall from a bar rotating at different speeds or continuously accelerating (e.g., from 4-40 rpm), the speed of the bar at the time of fall, and the distance the animal travels, indirectly reflecting the cardiopulmonary function of mice. Figure 5 As shown, compared with the control group, the fatigue rotarod retention time of mice in the model group was significantly reduced (p<0.01); compared with the model group, the fatigue rotarod retention time of mice in the high-dose huperzine A group was significantly increased (p<0.05), indicating that huperzine A can effectively and dose-dependently improve cardiopulmonary function in bleomycin-induced pulmonary fibrosis mice.

[0113] 4.5 Effects of huperzine A on pathological sections of bleomycin-induced pulmonary fibrosis in mice

[0114] To further observe the lung tissue damage in mice in detail, histopathological examination of the mouse lung tissue was performed in this embodiment. After HE staining, the tissue was observed under a light microscope. The results are as follows: Figure 6 and 7As shown, the control group had a generally normal tissue structure with clear alveolar structure. After bleomycin stimulation, the lung tissue structure of mice was significantly disordered, with disappearance of alveolar structure, thickening of alveolar septa, obvious vascular congestion, a significant increase in inflammatory cells with diffuse distribution and infiltration, lung tissue consolidation, and obvious fibrosis. Fifteen days after administration of high-dose huperzine A, the lung tissue consolidation in mice was reduced. Referring to the Ashcroft score, compared with the control group, the Ashcroft score of the model group mice was significantly increased (p<0.01); compared with the model group, the neutral and high-dose huperzine A groups significantly improved the Ashcroft score of the lung tissue in mice with pulmonary fibrosis (p<0.05, p<0.01). Sirius red staining of mouse lung tissue showed the following results: Figure 8 and 9 As shown, no obvious collagen deposition was observed in the lung tissue sections of the control group mice, and the alveolar structure was relatively clear, with slight collagen deposition visible around the bronchi. After bleomycin stimulation, obvious collagen deposition was observed in the lung tissue sections of the mice, with a diffuse distribution. Compared with the control group, the proportion of pulmonary fibrosis was significantly increased (p<0.01). After administration of high doses of huperzine A, collagen deposition was significantly reduced, and the alveolar structure was relatively normal and clear, but slight collagen deposition was still visible around the bronchi. Compared with the model group, the proportion of fibrosis in the lung tissue of animals in the high-dose huperzine A group was significantly reduced (p<0.05). Pathological sections showed that the high-dose huperzine A group could reduce the degree of lung inflammation and fibrosis in mice with pulmonary fibrosis and reduce collagen fiber deposition in the lungs of fibrotic mice.

[0115] 4.6 Relative expression levels of AhR, TGF-β1, and α-SMA in lung tissue of bleomycin-induced pulmonary fibrosis mice by huperzine A

[0116] The TGF-β1 / SMA signaling pathway is one of the main pathogenic mechanisms in the development of pulmonary fibrosis. During pulmonary fibrosis, the increase in TGF-β1 and α-SMA is positively correlated with pulmonary fibrosis; therefore, TGF-β1 and α-SMA are key markers of pulmonary fibroblast activation. AhR signaling is also related to the activation of the TGF-β1 / Smad signaling pathway. Activation of aryl hydrocarbon receptors can inhibit the activation of the TGF-β1 / Smad signaling pathway, thus playing a role in improving pulmonary fibrosis. In this example, compared with the control group, the expression level of AhR in the lung tissue of the model group mice was significantly decreased (p<0.05), while the expression levels of TGF-β1 and α-SMA were significantly increased (p<0.05); compared with the model group, the expression level of AhR in the lung tissue of the high-dose huperzine A group mice was significantly increased (p<0.01), while the expression levels of TGF-β1 and α-SMA were significantly decreased (p<0.05). The above results indicate that huperzine A can act as an aryl hydrocarbon receptor agonist to regulate the TGF-β1 / SMA signaling pathway, thereby playing a role in improving pulmonary fibrosis.

[0117] In conclusion, huperzine A can act as an aryl hydrocarbon receptor agonist to regulate the TGF-β1 / SMA signaling pathway and exert an anti-pulmonary fibrosis effect.

[0118] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. The use of huperzine A or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention and / or treatment of pulmonary fibrosis.

2. The use of huperzine A or its pharmaceutically acceptable salts as aryl hydrocarbon receptor agonists in the preparation of drugs for the prevention and / or treatment of pulmonary fibrosis.

3. The use of pharmaceutical compositions containing huperzine A or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention and / or treatment of pulmonary fibrosis.

4. The application according to claim 3, characterized in that, The pharmaceutical composition comprises: (i) an effective amount of huperzine A or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier.

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