TWI872951B - Flavonoids medicament for treatment, prevention, or reduction lung function damage - Google Patents

Abstract

Flavonoids medicament for treatment, prevention, or reduction lung function damage is provided and includes flavonols, flavanonols, or pharmaceutically acceptable salts thereof. The flavonoids medicament is used for lung inflammation or pulmonary fibrosis caused by exposure of mammals alveolar epithelial cells to copper. The flavonoids medicament reduce expression of pulmonary fibrosis-related proteins or molecular markers, thereby inhibiting epithelial-to-mesenchymal transition of the alveolar epithelial cells.

Description

Flavonoids for treating, preventing or reducing lung function impairment

The present invention relates to a flavonoid drug for treating, preventing or alleviating copper-induced lung function damage, and in particular to a flavonoid drug for lung function damage caused by inhibiting autophagy-induced epithelial-mesenchymal transition.

Heavy metal copper is a common heavy metal in industry. Steel has a wide range of uses, such as semiconductors, pipes, fertilizers, algaecides, etc. In addition to occupational exposure, agricultural use also increases the chance of copper exposure in the environment. Heavy metals in the environment can be ingested by the human body through drinking water, food (such as deep-sea fish), and even on suspended particles PM2.5 in the air, increasing the risk of exposed people and affecting human health. Therefore, copper toxicity is considered to be an increasingly serious problem facing human health. In some industrial areas, local residents and workers are exposed to copper-polluted environments for a long time. Excessive accumulation of copper in the body increases the risk of disease. Animal experiments have shown that administering copper to mice can cause lung toxicity and pulmonary fibrosis.

However, the drugs used to treat lung damage and pulmonary fibrosis (e.g., Pirfenidone or Nintedanib) have high side effects, causing diarrhea, nausea, abnormal liver function and liver failure. After drug treatment, patients still cannot reverse the lesions of pulmonary fibrosis, causing permanent damage and unable to achieve a healing effect. Heavy metal chelating agents have been used to treat heavy metal poisoning in the past, but the side effects include renal failure, hypocalcemia, hypoglycemia, incomplete coagulation, congestive heart failure, liver failure, etc., and there are still concerns about safety.

In view of this, in order to solve the problems existing in the above-mentioned prior art, the present invention provides a flavonoid drug for preparing a drug for treating, preventing or alleviating lung function damage, wherein the flavonoid drug comprises flavonols, flavanone alcohols or their pharmaceutically acceptable salts, so as to solve the problem that the known drugs have many side effects and cannot heal lung damage.

Preferably, the flavonol is kaempferol and the flavanone alcohol is 3,3',4',7-tetrahydroxyflavanone.

Preferably, the impairment of lung function is caused by exposure of mammalian alveolar epithelial cells to copper.

Preferably, the impairment of lung function is caused by lung inflammation or lung fibrosis.

Preferably, pulmonary fibrosis is caused by induced autophagy or epithelial-mesenchymal transition of alveolar epithelial cells in mammals.

Preferably, flavonoid drugs inhibit the epithelial-mesenchymal transition of alveolar epithelial cells by reducing the expression of pulmonary fibrosis-related proteins or molecular markers.

Preferably, the pulmonary fibrosis-related protein or molecular marker is a cell autophagy marker protein or an epithelial-mesenchymal transition marker protein, the cell autophagy marker protein is PINKI or LC3, and the epithelial-mesenchymal transition marker protein is Snail or N-Cadherin.

Preferably, the flavonoid drug is administered to alveolar epithelial cells of a mammal.

Preferably, the administration route of the flavonoid drug includes oral, nasal, enteral or parenteral administration.

The inventors of the present invention have found that flavonoids have protective effects on lung inflammation and lung fibrosis induced by heavy metal copper (including copper oxide nanoparticles and copper sulfate). When alveolar epithelial cells (A549) are exposed to copper sulfate, they induce cell autophagy and epithelial-mesenchymal transition (EMT), as well as fibrosis-related proteins including COL1A1, MMP-7, LOXL2 and N-Cadherin. By screening natural substances that can inhibit the above changes among flavonoid compounds, the results show that flavonols and flavanonols can effectively inhibit the above mechanism. Examples of flavonols include kaempferol, and examples of flavanonols include 3,3',4',7-tetrahydroxyflavanone (fustin). The present invention further uses mice for animal experiments, using copper oxide nanoparticles as a source of copper exposure. First, copper oxide nanoparticles are administered nasally to cause lung inflammation (2 weeks after exposure) and lung fibrosis (4 weeks after exposure). Then, 10 mg/kg bw/day of kaempferol is orally fed to mice, which can effectively improve the lung immune cell infiltration and lung fibrosis caused by nasal exposure to copper oxide nanoparticles. Therefore, the flavonoid drugs of the present invention can be used to treat, prevent or reduce lung damage caused by heavy metal copper pollution.

The technical features of the present invention will be described in detail below with specific embodiments and accompanying drawings so that a person skilled in the art can easily understand the purpose, technical features, and advantages of the present invention.

The advantages, features and technical methods achieved by the present invention will be described in more detail with reference to exemplary embodiments and the attached drawings so as to be easier to understand, and the present invention can be implemented in different forms, so it should not be understood to be limited to the embodiments described herein. On the contrary, for those with ordinary knowledge in the relevant technical field, the provided embodiments will make the present disclosure more thorough and comprehensive and completely convey the scope of the present invention, and the present invention will only be defined by the scope of the attached patent application.

Unless otherwise defined, all terms (including technical and scientific terms) used in the present invention have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. It will be further understood that those terms, such as those defined in commonly used dictionaries, should be interpreted as having definitions consistent with their meanings in the context of the relevant art and the present invention, and will not be interpreted as idealized or overly formal meanings unless expressly defined as such herein.

Flavonoids are a type of natural product, including flavanones, flavanols, isoflavones, and flavanoids. They are abundant in vegetables and fruits and have a wide range of biological activities.

The present invention discloses the therapeutic effects and mechanisms of several flavonoids on copper-induced lung epithelial damage and determines the association between urinary copper concentration and lung fibrosis changes in non-smoking individuals.

Materials and Methods:

Cell Culture, Drugs, and Reagents:

Human alveolar type II epithelial cells (A549 cells) were purchased from ATCC (Cat. No. CCL-185) and cultured in DMEM containing 10% FBS, sodium pyruvate, and 1% penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO _2. CuSO 4 , chloroquine, N-acetylcysteine, fusti, apigenin, kaempferol, and genkwanin were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell viability assay (CCK-8 assay):

A549 cells were exposed to different flavonoid drugs at 1, 5 or 10uM for 24 hours, and the cell survival rate was determined by CCK-8 colorimetric assay. The optical density (OD) of absorbed light was detected at a wavelength of 450nm using a microplate reader, and the fold change of absorbance was calculated by normalization with the optical density of the control group.

Western Blotting:

For protein expression analysis after treatment, cells were washed twice with ice-cold PBS and immediately lysed by adding RIPA buffer containing protease inhibitors (cOmplete™, Mini, EDTA-free protease inhibitor cocktail, Sigma-Aldrich, St. Louis, MO, USA) and phosphatase inhibitors (PhosStop™, Sigma-Aldrich, St. Louis, MO, USA) directly to the culture plate, and protein lysates were harvested by centrifugation at 14,000 rpm for 15 minutes at 4°C to pellet cell debris. Protein concentrations were determined using the BCA Dual Range BCA Protein Assay Kit (Visual Protein, Neihu, Taiwan). A total of 10 μg of protein lysate was separated in 6-13% SDS-PAGE and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). After blocking with 5% skim milk at room temperature for 1 hour, the PVDF membrane was incubated with the first antibody at 4°C overnight, washed three times with 0.1% TBST, and then incubated with HRP-conjugated anti-rabbit IgG or anti-goat second antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). The PVDF membrane was reacted with a chemiluminescent HRP substrate (Merck, Darmstadt, Germany) and visualized using a ChemiDoc-It810 imager (Ultra-Violet Products, Upland, CA, USA) to capture protein bands. The antibodies used in the present invention include N-Cadherin (GTX127345, Genetex, Hsinchu, Taiwan), E-Cadherin (GTX100443, Genetex, Hsinchu, Taiwan), GAPDH (GTX100118, GeneTex, Genetex, HsinChu), PINK1 (Cell signaling #6946, USA), LC3 (GTX127375, GeneTex, Hsinchu, Taiwan), p62 (abcam), and Snail (GTX100754, Genetex, Hsinchu, Taiwan).

Transwell cell migration assay:

A549 cells were exposed to flavonoid drugs for 2 hours and then to CuSO ₄ for 24 hours. Cells were detached by trypsin (Millipore, Freehold, NJ, USA) and seeded at a density of 4×10 ⁴ cells in the upper chamber of a transwell with a pore size of 8 μm (BD Biosciences, Franklin Lakes, NJ, USA) and 10 ⁴ cells in 200 uL serum-free DMEM. The lower chamber was filled with complete medium containing drugs. After incubation for 24 hours, cells were fixed with methanol for 10 minutes at room temperature and then stained with 0.2% crystal violet for 10 minutes at room temperature. Cells on the upper side of the chamber were removed with a cotton swab. Migrated cells were identified by counting the number of cells in five randomly selected areas.

RNA extraction and semi-quantitative real-time fluorescent quantitative PCR:

A549 cells grown in 12-well plates were treated with 0, 1, 10, and 100 μM CuSO ₄ for 48 h. Total RNA was isolated using GENEzolTM TriRNA Pure Kit (Geneaid). Complementary DNA was synthesized using SuperScriptTM III reverse transcriptase (ThermoFisher Scientific, MA, USA). Gene expression was detected using the StepOne system (ABI) using Fast SYBR Green MasterMix (Applied Biosystems). The gene accessions and primers used for real-time fluorescent quantitative PCR analysis are shown in Table 1. [Table 1] Target gene Positive Reverse Serial Number COL1A1 (NM_000088) 5t-CCAGAAGAACTGG TACATCAGCA-3t 5t-CGCCATACTCGAACTGGAATC-3t Sequence Number (ID): 1-2 MMP-7 (NM_002423) 5t-TCGGAGGAGATGCTCACTTCGA-3t 5t-GGATCAGAGGAATGTCC CATACC-3t Sequence Number (ID): 3-4 LOXL2 (NM_002318) 5t-TGACTGCAAGCACACGGAGGAT-3t 5t-TCCGAATGTCCTCCACCTGGAT-3t Sequence Number (ID): 5-6 N-Cadherin (NM_001792) 5t-CCTCCAGAGTTTACTGCCATGAC-3t 5t-GTAG GATCTCCGCCACTGATTC-3t Sequence Number (ID): 7-8

Immunofluorescence:

Cells plated on coverslips were treated with CuSO ₄ for different times and doses. After treatment, cells were fixed with 10% formalin for 30 min at room temperature. After washing three times with PBS, cells were permeabilized with ice-cold 100% methanol for 10 min, followed by three PBS washes, and cells were incubated with LC3 antibody (1:200, Genetex, Hsinchu, Taiwan) at 4°C overnight. Nonspecific binding was washed with 0.02% PBST, followed by incubation with goat anti-rabbit Alexa488 (Jackson) for 2 h at room temperature. Cells were then washed with 0.02% PBST and incubated with Alexa647-Tomm20 (1:100) overnight. After washing with PBS, the cells were mounted with DAPI mounting solution, and the cells were analyzed and imaged using a laser confocal microscope (FV1000, OLYMPUS IX-81).

Participant recruitment for clinical analysis statistics:

A general population health survey and lung cancer screening were conducted in Dalinpu in 2016 and 2018. Dalinpu is located in Xiaogang District, Kaohsiung City, southern Taiwan, surrounded by Linyuan, Linhai Petrochemical Complex and refinery park. Individuals who had lived in Dalinpu for more than 2 years were recruited through advertisements. A self-administered questionnaire (diabetes, hypertension, history of hyperlipidemia, education level, physical activity and history of air purifier use) and anthropometric measurements (including weight, height, smoking, history of pneumonia or lung cancer), urine copper and creatinine, and low-dose computed tomography (LDCT) were performed on all participants. Physical activity was measured by asking, "Do you do at least 150 minutes of moderate-intensity aerobic exercise per week or 75 minutes of vigorous aerobic exercise per week, or an equivalent combination?" Lung cancer subjects who had undergone lobectomy or had a history of pneumonia (n = 23) and a history of smoking (n = 283) were excluded from this statistical analysis. Due to WHO's recommendation to prevent urine samples from being too diluted or too concentrated, subjects with urine creatinine concentrations <30 mg/dL and creatinine concentrations >300 mg/dL were excluded (n = 88). A total of 1,458 subjects were included in this statistical analysis, which was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (No. KMUHIRB-G(II)-20190011).

Urine copper analysis, blood biochemical analysis, and LDCT image acquisition and interpretation:

Blood samples were diluted 10-fold with 9 mL of 1% (v/v) nitric acid (J.T. Baker Chemical Company, Phillipsburg, NJ, USA) in 15 mL polypropylene tubes. The samples were analyzed by coupled plasma mass spectrometry (ICP-MS, 7700 Series; Agilent Technologies, Inc., Santa Clara, CA, USA). Quality control and method detection limits were based on National Environmental Laboratory standards.

A hematology analyzer (XE-2100; Sysmex, Kobe, Japan) was used. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), fasting blood glucose, and HbA1c levels were measured using a chemical system (Advia 1800; Siemens Healthcare GmbH, Erlangen, Germany). Serum total IgE levels were measured using ImmunoCAP ^® (Phadia, Uppsala, Sweden).

All scans were performed on a thin-section scanner (Toshiba Aquilion One 640 Slice CT tomography scanner, Japan) with 320 detectors from the lung apex to the lung base without contrast enhancement. In addition, all scans were acquired using a low-dose protocol and the following machine settings: tube voltage, 120 kVp; tube current, 9 (15 mA/0.6 s) or 21 (35 mA/0.6 s) Ma; variable pitch ratio, 1.5:1; and rotation time, 0.35 s. The effective radiation dose ranged from 0.3 to 0.8 mSv. Pulmonary fibrotic changes included parenchymal bands of pulmonary fibrosis patterns, which were defined as the presence of curvilinear or linear densities, streaks, or plate opacities in specific lobes on LDCT images. All images were clinically reviewed and reported by trained radiologists with 10–30 years of experience.

Statistical analysis:

Pulmonary fibrosis changes, sex, alcohol and betel nut consumption, diabetes, and hypertension were considered as nominal variables. Fisher's exact test or chi-square test was used to determine the differences between pulmonary fibrosis changes. Mann-Whitney U test was used to examine the differences between pulmonary fibrosis and non-pulmonary fibrosis changes in terms of urinary copper and creatinine levels and blood biochemical levels. Spearman correlation analysis was used to analyze the associations of urinary copper, age, BMI, and serum biochemical levels. We performed multivariate logistic regression analysis to estimate the odds ratio (OR) and 95% confidence interval (CI) (continuous variables) of pulmonary fibrosis changes associated with urinary copper levels. We included urine creatinine level as a covariate in the regression model to account for dilution-dependent sample variations in urine concentration. Variables (1) were associated with changes in lung fibrosis, (2) were associated with urine copper levels, and (3) were selected based on the literature. Both AST and ALT were significantly associated with urine copper levels, and we selected AST as a confounding factor to prevent multicollinearity. All analyses were performed using SPSS software (version 22; IBM Corp., Armonk, NY, USA). Statistical significance was set at p < 0.05. Statistical analyses were performed using GraphPad Prism for both in vitro and in vivo studies. Statistical significance between the two groups was assessed using Student's t test and one-way analysis of variance with Dunnett's post hoc multiple comparisons. Data are presented as mean + SEM, and the p value was set at 0.05.

result:

In order to clarify whether flavonoid drugs can be developed as a treatment for copper poisoning, different types of flavonoid drugs were studied. Flavonoid compounds are natural compounds that are abundant in vegetables and fruits and have a wide range of biological activities. Flavonoid compounds include: flavonoids, flavonols and flavanone alcohols. Flavonoids include apigenin and genkwanin (apigenin 7-methyl ether); flavonols include kaempferol; and flavanone alcohols include 3,3',4',7-tetrahydroxyflavanone (fustin).

Please refer to Figure 1, which is a schematic diagram of the results of the flavonoid drugs of the present invention for measuring the survival rate of alveolar epithelial cells, wherein API is apigenin, KAE is kaempferol, GEN is genkwanin, and FUS is 3,3',4',7-tetrahydroxyflavanone (fustin).

Treatment of alveolar epithelial cells (A549) with flavonoids at 1 μM, 5 μM, and 10 μM for 24 hours had no effect on cell survival compared to untreated cells.

In addition, since alveolar epithelial cells, in addition to being a physical barrier against external insults, are also crucial in coordinating the fibrosis process, epithelial cells transform into mesenchymal type cells under stimulation, a process called epithelial-mesenchymal transition (EMT). Cells lose their epithelial phenotype and acquire a mesenchymal phenotype, including: increased cell motility, cytoskeleton reorganization, and increased invasiveness of the extracellular matrix. Transformed mesenchymal cells contribute a large proportion of activated fibroblasts, and the released pro-fibrotic cells will further activate other fibroblasts and excessive accumulation of extracellular matrix proteins, which is a characteristic of tissue fibrosis. EMT is a reversible process, and EMT is considered to be the culprit. Targeting EMT is one of the therapeutic targets for fibrotic diseases.

In order to elucidate whether the flavonoid drugs of the present invention can inhibit the copper-induced epithelial-mesenchymal transition of alveolar epithelial cells, the following measurements were performed on different classes of flavonoid drugs.

Please refer to Figure 2A and Figure 2B, which are schematic diagrams showing copper-induced mitochondrial autophagy in alveolar epithelial cells and the results of the flavonoid drugs of the present invention inhibiting the expression of copper-induced pulmonary fibrosis-related proteins or molecular markers, wherein API is apigenin, KAE is kaempferol, GEN is genkwanin, and FUS is 3,3',4',7-tetrahydroxyflavanone (fustin).

As shown in Figure 2A, in order to determine the effect of copper on mitochondrial autophagy in alveolar epithelial cells, alveolar epithelial cells (A549) were treated with 10 μM CuSO ₄ to simulate copper poisoning. The co-localization of LC3 (green) representing autophagosomes and Tomm20 (red) representing mitochondria is shown in yellow (as indicated by arrows). As the time of CuSO ₄ treatment of alveolar epithelial cells (A549) increases, the expression of LC3 increases. It can be seen that CuSO ₄ treatment induces mitochondrial autophagy in alveolar epithelial cells.

As shown in Figure 2B, 10 μM CuSO ₄ was applied to alveolar epithelial cells (A549) for 3 hours. The results also found that CuSO ₄ induced autophagy in alveolar epithelial cells and increased the expression of autophagy-related proteins (LC3 and PINK1). During the process of autophagy, LC3 gradually converted from the cytosolic (LC3-I, 17 kDa) form to the membrane-bound lipidated (LC3-II, 15 kDa) form. PINK1 is a mitochondrial autophagy-specific marker in cells, PTEN-induced kinase 1.

Compared with cells treated with CuSO ₄ , pretreatment with flavonoids at 5 μM and 10 μM for 2 hours inhibited the expression of copper-induced cell autophagy marker proteins (PINK1, LC3) and epithelial-mesenchymal transition marker protein (Snail), among which apigenin and kaempferol had an inhibitory effect on the expression of the above marker proteins and had a dose-responsive effect.

Please refer to Figure 3A and Figure 3B, which are respectively a schematic diagram and a quantitative schematic diagram of the results of the flavonoid drugs of the present invention inhibiting copper-induced alveolar epithelial cell crawling. In Figure 3A, API is apigenin, KAE is kaempferol, GEN is genkwanin, and FUS is 3,3',4',7-tetrahydroxyflavanone (fustin). In Figure 3B, a indicates that it is significantly significant compared to the control group, and b indicates that it is significantly significant compared to the group only administered with CuSO ₄ .

After pretreatment of alveolar epithelial cells (A549) with 10μM of each flavonoid drug for 2 hours, 10μM CuSO ₄ was administered and incubated for 24 hours. The results showed that copper induced alveolar epithelial cells (A549) to migrate from the upper well to the lower well, and pretreatment with flavonoid drugs could inhibit copper-induced cell migration, especially kaempferol and 3,3',4',7-tetrahydroxyflavanone, which had a more significant effect.

Please refer to Figures 4A and 4B, which are schematic diagrams and quantitative diagrams of the expression of proteins or molecular markers related to copper-induced epithelial-mesenchymal transition (EMT). In Figure 4A, API is apigenin, KAE is kaempferol, GEN is genkwanin, and FUS is 3,3',4',7-tetrahydroxyflavanone (fustin).

As shown in the quantified results in Figure 4B, exposure to 10 μM CuSO ₄ increased the epithelial-mesenchymal transition (EMT)-related protein N-cadherin and decreased E-cadherin expression compared to untreated cells.

Compared with cells treated with CuSO ₄ , pretreatment with flavonoids at 10 μM for 2 hours reversed the expression of copper-induced mesenchymal transition marker proteins (N-cadherin and E-cadherin), namely, reducing the expression of epithelial-mesenchymal transition (EMT)-related protein N-cadherin and increasing the expression of E-cadherin, among which kaempferol and 3,3',4',7-tetrahydroxyflavanone (fustin) had more significant effects.

As shown in Figures 2 to 4, the flavonoid drugs of the present invention inhibit copper-induced autophagy of alveolar epithelial cells and epithelial-mesenchymal transition (EMT) induced by autophagy.

Please refer to Figure 5, which is a schematic diagram showing the results of the flavonoid drugs of the present invention inhibiting the expression of copper-induced pulmonary fibrosis-related proteins or molecular markers.

Real-time fluorescent quantitative PCR was used to measure the expression of multiple lung fibrosis markers in alveolar epithelial cells (A549) treated with 1μM, 10μM, and 100μM CuSO ₄ for 48 hours. The results showed that CuSO ₄ promoted the expression of COL1A1, MMP7, LOXL2, and N-Cadherin. These results indicate that CuSO ₄ treatment induces fibrosis of lung epithelial cells.

Please refer to FIG. 6 , which is a schematic diagram showing the results of the flavonoid drugs of the present invention inhibiting the expression of copper-induced epithelial-mesenchymal transition (EMT)-related proteins (N-Cadherin), wherein KAE is kaempferol and FUS is 3,3',4',7-tetrahydroxyflavanone (fustin).

Real-time fluorescence quantitative PCR was used to measure the expression of epithelial-mesenchymal transition (EMT)-related protein (N-Cadherin) in alveolar epithelial cells (A549) pretreated with 5μM and 10μM kaempferol and 3,3',4',7-tetrahydroxyflavanone (fustin) for 2 hours and then treated with CuSO ₄ for 24 hours. The results showed that kaempferol and 3,3',4',7-tetrahydroxyflavanone (fustin) inhibited the copper-induced N-Cadherin expression. * indicates p value less than 0.05; ** indicates p value less than 0.01; *** indicates p value less than 0.001; **** indicates p value less than 0.0001.

Please refer to FIG. 7A and FIG. 7B , which are schematic diagrams and quantitative diagrams showing the results of the flavonoid drugs of the present invention inhibiting the expression of copper-induced epithelial-mesenchymal transition-related proteins or molecular markers (vimentin), wherein CuONP is copper oxide nanoparticles.

Another copper compound (0, 0.5μM, 1μM and 5μM copper oxide nanoparticles) was administered to alveolar epithelial cells (A549) for 2 hours to simulate copper poisoning. The results showed that copper induced alveolar epithelial cells to express a large amount of vimentin. Vimentin is an intermediate filament protein that plays a key role in the epithelial-mesenchymal transition of cells from adhesion to movement. Therefore, it is an epithelial-mesenchymal transition-related protein or molecular marker. The results showed that copper induced epithelial-mesenchymal transition of lung epithelial cells.

As shown in the quantification results in Figure 7B, treatment with 1 μM or 5 μM CuO nanoparticles increased vimentin expression in a dose-responsive manner compared to untreated cells.

Compared with cells treated with CuO nanoparticles, pretreatment with 5 μM kaempferol for 2 hours before copper treatment inhibited copper-induced vimentin expression in alveolar epithelial cells in a dose-responsive manner.

Please refer to FIG. 8A and FIG. 8B , which are respectively a schematic diagram of the results of the flavonoid drugs of the present invention inhibiting the crawling of alveolar epithelial cells induced by copper and a schematic diagram of the corresponding results of the cell survival rate, wherein CuONP is copper oxide nanoparticles.

As shown in Figure 8A, alveolar epithelial cells (A549) were pretreated with 2μM and 5μM kaempferol for 2 hours, and then incubated with 0.2μM or 0.5μM copper oxide nanoparticles (CuONP) for 24 hours. The results show that copper induces alveolar epithelial cells (A549) to migrate from the upper well to the lower well, and pretreatment with kaempferol can inhibit copper-induced cell migration.

As shown in Figure 8B, simultaneous treatment of alveolar epithelial cells (A549) with kaempferol (2μM or 5μM) and CuO nanoparticles (0.2μM or 0.5μM) did not affect cell survival, which is sufficient to prove that the above-mentioned cell migration amount is not caused by the cell survival amount.

Please refer to Figure 9, which is a schematic diagram showing the results of the flavonoid drugs of the present invention inhibiting the expression of copper-induced pulmonary fibrosis-related proteins or molecular markers in the lungs of mice.

In order to demonstrate the efficacy of the flavonoid drugs of the present invention in copper-induced lung injury in mice, the present invention further conducted animal experiments using kaempferol as an example, but the experiment is not limited to this. Control group: mice were not administered CuONP and flavonoid drugs; CuONP group: mice were administered 2 mg/kg of copper oxide nanoparticles through the nasal cavity to simulate the air pollution of heavy metal copper, and the mice were observed to see whether the lungs of the mice had inflammation and fibrosis; CuONP+kaempferol group: mice were orally administered 2 mg/kg of CuONP and 10 mg/kg of the flavonoid drugs of the present invention (kaempferol) at the same time, and the mice were observed to see whether the lung inflammation and pulmonary fibrosis were alleviated or cured; Kaempferol group: Observe whether kaempferol is toxic to the lungs of mice. After 28 days, the mice in the above groups were sacrificed to obtain lung tissues for analysis of the expression of pulmonary fibrosis-related proteins or molecular markers.

The results showed that after mice inhaled CuONP, the lungs induced a large amount of expression of pulmonary fibrosis-related proteins or molecular markers a-SMA and N-Cadherin. Compared with mice given CuONP+kaempferol at the same time, kaempferol could inhibit the copper-induced a-SMA and N-Cadherin expression, and the same phenomenon was observed in the lung tissues of different mice.

Please refer to Figure 10, which is a schematic diagram showing the results of the flavonoid drugs of the present invention inhibiting copper-induced pulmonary fibrosis and lung inflammation in mouse lungs.

The lungs of the mice were sectioned and analyzed for lung inflammation and fibrosis using Masson's trichrome staining. The results showed that nasal administration of copper oxide nanoparticles caused inflammation and fibrosis in the lungs of the mice, while oral administration of CuONPs and the flavonoid drug of the present invention (kaempferol) inhibited copper-induced lung inflammation and fibrosis. Kaempferol alone did not cause toxicity to the lungs of mice, indicating that kaempferol is effective in treating, preventing or alleviating lung damage caused by copper.

In order to clarify the relationship between the level of urinary copper in humans and changes in lung fibrosis, a clinical statistical analysis was conducted, and a total of 1458 people were collected, with the mean ± SD of age and BMI being 57.7 ± 11.2 years and 25.0 ± 3.8 Kg/m ² , respectively. Male gender (32.6%), history of diabetes (11.2%), history of hypertension (27.6%), chewing of betel nut (0.3%), drinking (16%), education level (≤ junior high school: 51.9%, high school: 30.1%, ≥ college: 18.0%), use of air purifiers (84.5%), and changes in lung fibrosis (36.2%) were determined for all subjects.

As shown in Table 2, the number of pulmonary fibrosis changes was higher in the older age group (＞60 years old: 82.6% vs. 62.3%, p ＜ 0.001), and the proportion of hypertension (32.8% vs. 24.6%, p = 0.001), hyperlipidemia (3.4% vs. 1.2%, p = 0.004) and the use of air purifiers (88.8% vs. 82.0%, p = 0.001). In addition, individuals with pulmonary fibrosis had significantly higher AST levels (IU/mL) (28.5 ± 11.7 vs. 27.8 ± 10.8, p < 0.001), higher fasting blood glucose values (mg/dL) (101.2 ± 28.7 vs. 100.4 ± 27.4, p = 0.026), higher HbA1c levels (%) (5.93 ± 0.92 vs. 5.89 ± 0.98, p = 0.019), and lower platelet counts (256.5 ± 68.868. 0.001). [Table 2] Features Non-fibrotic lung Pulmonary fibrosis p (n = 930) (n = 528) Categorical variables, n (%) gender 0.383 female 619 (66.6) 364 (68.9) male 311 (33.4) 164 (31.1) Age (years) <0.001 ≤ 60 351 (37.7) 92 (17.4) >60 579 (62.3) 436 (82.6) BMI (kg/m ² ) 0.543 ≤24 379 (40.8) 224 (42.4) >24 551 (59.2) 304 (57.6) Education <0.001 ≤Junior high school 431 (46.3) 325 (61.6) high school 297 (31.9) 142 (26.9) ≥University 202 (21.7) 61 (11.6) diabetes 0.344 no 831 (89.4) 463 (87.7) yes 99 (10.6) 65 (12.3) High blood pressure 0.001 no 701 (75.4) 355 (67.2) yes 229 (24.6) 173 (32.8) Hyperlipidemia 0.004 no 919 (98.8) 510 (96.6) yes 11 (1.2) 18 (3.4) alcoholism 0.882 no 780 (83.9) 445 (84.3) yes 150 (16.1) 83 (15.7) Physical activity 0.083 no 234 (25.2) 111 (21.0) yes 696 (74.8) 417 (79.0) Air Purifier 0.001 no 167 (18.0) 59 (11.2) yes 763 (82.0) 469 (88.8) Serum, continuous variable, mean ± SD (range) White blood cells × 10 ³ (/μL) 5.9 ± 1.5 (2.4, 14.0) 5.8 ± 1.5 (2.5, 12.2) 0.226 Platelets × 10 ³ (/μL) 260.2 ± 68.6 (60.0, 794.0) 256.5 ± 68.8 (117.0, 672.0) 0.001 Oxytoacetic transaminase (IU/mL) 27.8 ± 10.8 (12.0, 102.0) 28.5 ± 11.7 (14.0, 177.0) <0.001 Alanine aminotransferase (IU/mL) 25.9 ± 18.7 (3.0, 190.0) 25.8 ± 18.5 (4.0, 185.0) 0.852 Creatinine (mg/dL) 0.9 ± 0.3 (0.5, 5.0) 0.9 ± 0.3 (0.6, 6.8) 0.071 Cholesterol (mg/dL) 200.3 ± 36.5 (91.0, 420.0) 200.6 ± 36.8 (100.0, 386.0) 0.912 Triglycerides (mg/dL) 121.2 ± 80.3 (25.0, 1129.0) 121.5 ± 79.6 (29.0, 952.0) 0.705 HDL cholesterol (mg/dL) 53.6 ± 13.6 (25.0, 100.2) 53.5 ± 13.5 (24.4, 94.3) 0.917 LDL cholesterol (mg/dL) 119.6 ± 32.7 (25.0, 306.0) 119.6 ± 33.0 (34.0, 276.0) 0.978 Fasting blood glucose (mg/dL) 100.4 ± 27.4 (57.0, 325.0) 101.2 ± 28.7 (72.0, 348.0) 0.026 Glycated hemoglobin (%) 5.89 ± 0.98 (4.20, 12.30) 5.93 ± 0.92 (4.20, 12.90) 0.019 Eosinophils (/μL) 134.6 ± 124.3 (0.1, 1010.0) 127.6 ± 128.9 (0.1, 1070.0) 0.090 Immunoglobulin E (IU/mL) 76.5 ± 235.1 (0.1, 4439.4) 115.2 ± 518.5 (0.1, 7618.3) 0.128

As shown in Table 3, the urinary copper level (μg/dL) of subjects with pulmonary fibrosis was significantly higher than that of subjects without pulmonary fibrosis (geometric mean: 1.45 vs. 1.39, p=0.081). [Table 3] Project average value Standard Deviation GM Minimum ^25th Median ^75th Maximum p Urine Copper(µg/dL) 0.081 Non-fibrotic lung 1.55 0.78 1.39 0.05 1.05 1.43 1.86 8.25 Pulmonary fibrosis 1.69 0.99 1.45 0.05 1.11 1.48 1.90 9.94 Creatinine (mg/dL) 0.107 Non-fibrotic lung 130.13 59.31 115.92 30.20 83.60 124.55 171.83 296.60 Pulmonary fibrosis 124.68 56.85 111.25 30.40 80.63 116.70 160.95 297.50

As shown in Table 4, urine copper levels were significantly correlated with age (r = 0.17, p < 0.001), BMI (r = 0.11, p < 0.001), WBC (r = 0.11, p < 0.001), AST (r = 0.16, p < 0.001), ALT (r = 0.10, p < 0.001), serum creatinine (r = 0.13, p < 0.001), triglyceride (r = 0.13, p < 0.001), fasting blood glucose (r = 0.17, p < 0.001), HbA1c (r = 0.17, p < 0.001), and eosinophil count (r = 0.10, p < 0.001). Urinary copper level was significantly negatively correlated with platelet count (r = -0.07, p = 0.010) and HDL-C (r = -0.14, p ＜ 0.001). Male subjects (mean ± SD, μg/dL) (male: 1.64 ± 0.89 vs. female: 1.57 ± 0.85, p = 0.014), older age (＞60 years: 1.66 ± 0.92 vs. ≤60 years, p ＜ 0.001), higher BMI (kg/m2) (＞24: 1.65 ± 0.91 vs. ≤24: 1.52 ± 0.79, p ＜ 0.001), lower education level (≥ college: 1.39 ± 0.57 vs. ≤ junior high school: 1.68 ± 0.96, p ＜ 0.001), diabetes (yes: 2.12 ± 1.23 vs. no: 1.53 ± 0.78, p ＜ 0.001), hypertension (yes: 1.80 ± 1.07 vs. no: 1.51 ± 0.75, p ＜ 0.01) and use of air purifier (yes: 1.62 ± 0.88 vs. no: 1.47 ± 0.73, p = 0.016). [Table 4] Project Copper Age BMI WBC PLT AST ALT Cr CHOL TG HDL-C LDL-C blood sugar HbA1c Eosinophilic globules IgE Copper 1 Age 0.17 ^** 1 BMI 0.11 ^** 0.13 ^** 1 WBC 0.11 ^** -0.05 ^* 0.25 ^** 1 PLT 0.01 ^** -0.34 ^** 0.01 0.30 ^** 1 AST 0.16 ^** 0.26 ^** 0.17 ^** 0.06 ^* -0.16 ^** 1 ALT 0.10 ^** 0.11 ^** 0.35 ^** 0.14 ^** -0.13 ^** 0.68 ^** 1 Cr 0.13 ^** 0.25 ^** 0.13 ^** 0.09 ^** -0.21 ^** 0.15 ^** 0.12 ^** 1 CHOL -0.04 0.03 -0.03 0.01 0.10 ^** 0.01 0.01 -0.06 ^* 1 TG 0.13 ^** 0.14 ^** 0.33 0.27 ^** 0.07 ^** 0.14 ^** 0.27 ^** 0.13 ^** 0.18 ^** 1 HDL-C -0.14 ^** -0.12 ^** -0.31 ^** -0.20 ^** 0.06 ^* -0.05 ^* -0.19 ^** -0.26 ^** 0.34 ^** -0.51 ^** 1 LDL-C -0.003 0.01 0.07 ^* 0.07 ^* 0.09 ^** -0.003 0.05 -0.01 0.84 ^** 0.15 ^** 0.04 1 blood sugar 0.17 ^** 0.34 ^** 0.32 ^** 0.15 ^** -0.09 ^** 0.16 ^** 0.22 ^** 0.11 ^** -0.02 0.26 ^** -0.22 ^** 0.01 1 HbA1c 0.17 ^** 0.34 ^** 0.29 ^** 0.22 ^** -0.02 0.19 ^** 0.19 ^** 0.08 ^** -0.01 0.28 ^** -0.23 ^** 0.01 0.63 ^** 1 Eosinophilic globules 0.10 ^** -0.07 ^** 0.10 ^** 0.24 ^** 0.08 ^** 0.08 ^** 0.06 ^* 0.13 ^** 0.001 0.12 ^** -0.11 ^** 0.04 0.03 0.15 ^** 1 IgE 0.04 0.01 0.09 ^** 0.06 ^* 0.01 -0.02 0.03 0.08 ^** -0.08 ^** -0.01 -0.06 ^** -0.04 0.05 0.05 0.13 ^** 1

Abbreviations: PLT: platelet count, AST: aspartate aminotransferase, ALT: alanine aminotransferase, Cr: creatine 9, TG: triglyceride. * p < 0.05, ** p < 0.01.

As shown in Table 5, after adjusting for urine creatinine, age, sex, BMI, AST, HbA1c, triglycerides, HDL-C, eosinophil count, white blood cells, platelets, serum creatinine, education level, and use of air purifiers, a one-unit increase in urine copper concentration was found to be significantly associated with an increased risk of lung fibrosis (odds ratio [OR] = 1.17, 95% confidence interval [CI] = 1.01–1.36, p = 0.038). [Table 5] Urine copper (ug/dL) OR 95% CI p Pulmonary fibrosis Model 1 1.27 (1.10, 1.46) 0.001 Model 2 1.17 (1.01, 1.35) 0.031 Model 3 1.19 (1.03, 1.39) 0.021 Model 4 1.17 (1.01, 1.36) 0.038

Model 1: Adjusted for urine creatinine. Model 2: Model 1 adjusted in addition to age, sex, and BMI. Model 3: Model 2 adjusted in addition to AST, HbA1c, triglycerides, HDL-C, eosinophil count, WBC, platelets, and serum creatinine. Model 4: Model 3 adjusted in addition to education and air purifier use.

In summary, the results obtained from the above tests clearly show that the flavonoid drugs of the present invention have the effect of treating, preventing or alleviating copper-induced lung function damage. Therefore, giving drugs or health foods containing flavonoid drugs to patients with copper-induced lung function damage can significantly improve their lung function damage, especially pulmonary fibrosis, with almost no side effects.

The flavonoid drugs of the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers, including multiple excipients and multiple adjuvants, which help to process the multiple active ingredients into multiple preparations that can be used pharmaceutically. The appropriate formulation depends on the selected route of administration.

The administration routes of the flavonoid drugs of the present invention include oral, nasal, enteral or parenteral administration, and the dosage forms of the flavonoid drugs of the present invention are not particularly limited. For example, liquid solutions, suspensions, emulsions, tablets, pills, capsules, sustained-release preparations, powders, suppositories, liposomes, microparticles, microcapsules, isotonic sterile aqueous buffer solutions, etc. are all considered to be suitable dosage forms.

The flavonoid drug of the present invention may include one or more suitable diluents, fillers, salts, disintegrants, binders, lubricants, glidants, wetting agents, controlled release matrices, colorants, flavoring agents, carriers, formulators, buffers, stabilizers, solubilizers, commercially available adjuvants and/or other additives known in the art.

The term "treating" as used herein refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and/or causing a reduction, alleviation or suppression of a pathology. Those skilled in the art will appreciate that various methods and assays may be used to obtain the development of a pathology, and similarly, various methods and assays may be used to obtain a reduction, alleviation or suppression of a pathology. According to various specific embodiments, treating includes increasing survival.

The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and at least one of the compounds of the present invention or its stereoisomers, tautomers, pharmaceutically acceptable salts or solvents.

As used herein, "pharmaceutically acceptable salts" refer to derivatives of the compounds of the present invention in which the parent compound has been modified by preparing its acid or base salts. The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing a basic or acidic moiety by known chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of such compounds with a stoichiometric amount of an appropriate base or acid in water or in an organic solvent or in a mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. A list of suitable salts is found in Remington's Pharmaceutical Sciences 18th edition, Mack Publishing Company, Easton, PA (1990), the disclosure of which is hereby incorporated by reference.

The above description is for illustrative purposes only and is not intended to be limiting. Any equivalent modifications or changes made to the invention without departing from the spirit and scope of the invention shall be included in the scope of the attached patent application.

In order to more clearly explain the technical scheme of the embodiment of the present invention, the figures required for the description of the embodiment of the present invention will be briefly introduced below. Obviously, the figures described below are only some embodiments of the present invention. For those with ordinary knowledge in the relevant technical field, other figures can be obtained based on these figures. Figure 1 is a schematic diagram of the results of the flavonoid drug of the present invention for the survival rate of alveolar epithelial cells. Figures 2A and 2B are schematic diagrams of the results of copper-induced mitochondrial autophagy in alveolar epithelial cells and the inhibition of copper-induced pulmonary fibrosis-related protein or molecular marker expression by the flavonoid drugs of the present invention. Figures 3A and 3B are schematic diagrams and quantitative diagrams of the results of the flavonoid drugs of the present invention inhibiting copper-induced alveolar epithelial cell crawling. Figures 4A and 4B are schematic diagrams and quantitative diagrams of the expression of copper-induced pulmonary fibrosis-related protein or molecular marker expression. Figure 5 is a schematic diagram of the results of the flavonoid drugs of the present invention inhibiting copper-induced pulmonary fibrosis-related protein or molecular marker expression. Figure 6 is a schematic diagram showing the results of the flavonoid drugs of the present invention inhibiting the expression of copper-induced pulmonary fibrosis-related proteins (N-Cadherin). Figures 7A and 7B are respectively a schematic diagram and a quantitative schematic diagram showing the results of the flavonoid drugs of the present invention inhibiting the expression of copper-induced epithelial-mesenchymal transition-related proteins or molecular markers (vimentin). Figures 8A and 8B are respectively a schematic diagram showing the results of the flavonoid drugs of the present invention inhibiting copper-induced alveolar epithelial cell crawling and a schematic diagram showing the corresponding cell survival rate. Figure 9 is a schematic diagram showing the results of the flavonoid drugs of the present invention inhibiting the expression of copper-induced pulmonary fibrosis-related proteins or molecular markers in mouse lungs. Figure 10 is a schematic diagram of the slices showing that the flavonoid drug of the present invention inhibits copper-induced lung inflammation and pulmonary fibrosis in mouse lungs.

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Production Date: 2024-02-09
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Current application / IP Office: TW
Current application / Applicant file reference: 112006-TW
Applicant name: Kaohsiung Medical University
Applicant name / Language: zh
Applicant name / Name Latin: Kaohsiung Medical University
Invention title: Flavonoid drugs for treating, preventing or alleviating lung function impairment (zh)
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Claims (7)

A flavonoid drug is used to inhibit the expression of cell autophagy marker protein PINK1 to prevent or reduce lung function damage, wherein the flavonoid drug is selected from kaempferol, 3,3',4',7-tetrahydroxyflavanone or a pharmaceutically acceptable salt thereof; and the dosage of the flavonoid drug is 5μM. A flavonoid drug is used to inhibit the expression of epithelial-mesenchymal transition marker protein Snail to prevent or reduce lung function damage, wherein the flavonoid drug is selected from kaempferol or a pharmaceutically acceptable salt thereof; and the dosage of the flavonoid drug is 10 μM. The use as described in claim 1 or claim 2, wherein the lung function impairment is caused by exposure of alveolar epithelial cells of mammals to copper. The use as described in claim 1 or claim 2, wherein the lung function impairment is caused by lung inflammation or lung fibrosis. The use as described in claim 4, wherein the pulmonary fibrosis is caused by induced cell autophagy or epithelial-mesenchymal transition of alveolar epithelial cells of mammals. The use as described in claim 1 or claim 2, wherein the flavonoid drug is administered to alveolar epithelial cells of a mammal. The use as described in claim 6, wherein the administration route includes oral, nasal, enteral or parenteral administration.