Application of chrysophanol in preparation of Wnt / beta-catenin signaling pathway activator and medicine for preventing and treating emphysema and COPD

Abstract

The invention relates to the technical field of medicines, in particular to application of chrysophanol in preparation of a Wnt / beta-catenin signaling pathway activator and a medicine for preventing and treating emphysema and COPD (chronic obstructive pulmonary disease). Tests find that chrysophanol has a remarkable activation effect on a Wnt / beta-catenin signal channel, and activation of the Wnt / beta-catenin signal channel can prevent or reduce ATII cell damage caused by smoke exposure, promote alveolar organ and epithelial cell proliferation, prevent emphysema or relieve emphysema symptoms; meanwhile, the chrysophanol can reduce the alveolar diameter of a mouse COPD model and improve the lung function index, so that the chrysophanol can prevent and improve the clinical symptoms of short breath and dyspnea, and can further prevent the occurrence of emphysema or relieve the symptoms of emphysema.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to application of chrysophanol in preparing Wnt / β-catenin signaling pathway activators and drugs for preventing and treating emphysema and COPD. Background Art

[0002] Chronic obstructive pulmonary disease (COPD), also known as COPD, is characterized by persistent airflow limitation and is the most common chronic disease of the respiratory system. It is related to the chronic abnormal inflammatory response of the airways and lungs to toxic particles or harmful gases. The main symptoms of COPD are shortness of breath or dyspnea, which appear during exertion in the early stage and then gradually worsen, so that they occur in daily life or even during rest. COPD can also further develop into common chronic diseases such as cor pulmonale and respiratory failure.

[0003] COPD usually refers to two diseases: chronic bronchitis and emphysema, and the coexistence of the two is very common. Emphysema specifically refers to the pathological state of decreased airway elasticity in the distal end of the respiratory bronchioles of the lungs, including respiratory bronchioles, alveolar ducts, alveolar sacs and alveoli, with persistent abnormal excessive gas content, over-expansion, inflation and increased lung capacity, or accompanied by airway wall destruction. It is a serious lung disease that not only causes symptoms such as dyspnea in patients, but also causes complications such as spontaneous pneumothorax, subcutaneous emphysema, respiratory failure, and chronic cor pulmonale, which seriously threatens the patient's physical and mental health.

[0004] Therefore, there is a need to develop more drugs that can be used to treat COPD, especially emphysema. Summary of the invention

[0005] In view of the above technical problems, the present invention provides the use of chrysophanol in preparing Wnt / β-catenin signaling pathway activators and drugs for preventing and treating emphysema and COPD. The present invention has found through experimental research that chrysophanol can activate the Wnt / β-catenin signaling pathway, and can play the role of reducing alveolar diameter and improving lung function for COPD, so it can be used to prepare drugs for preventing and treating emphysema and COPD.

[0006] In order to achieve the above-mentioned object of the invention, the embodiment of the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides the use of chrysophanol in preparing a Wnt / β-catenin signaling pathway activator.

[0008] The Wnt signal transduction pathway is a highly conservative signal pathway that is widely present in invertebrates and vertebrates. β-catenin is a key regulatory factor in the Wnt signal pathway. Studies have found that in lung tissue, Wnt signals are actively expressed in alveolar type II epithelial (ATⅡ) cells. When ATⅡ cells are damaged, the reduced activity of the Wnt signals they secrete will cause ATⅡ cells to lose self-renewal, while continuous Wnt signal secretion will maintain the stemness of ATⅡ cells and prevent them from transdifferentiating into ATⅠ cells, thereby maintaining their own cell populations through cell proliferation, and further maintaining the secretion of pulmonary surfactant and gas exchange function. The present invention has found through research that chrysophanol has a significant activation effect on the Wnt / β-catenin signal pathway, which can prevent or reduce the damage to ATⅡ cells caused by smoke exposure, maintain their normal structure and function, and thus prevent the occurrence of emphysema or alleviate the symptoms of emphysema. Moreover, the activation effect of chrysophanol on the Wnt / β-catenin signaling pathway is better than that of rhein, rhubarb, chrysophanol methyl ether and rhubarb extract, and the difference is significant.

[0009] In addition, studies have found that activating the Wnt / β-catenin signaling can induce the death of glioma stem cells (GSCs). Therefore, chrysophanol is also expected to treat GSCs by activating the Wnt / β-catenin signaling pathway.

[0010] In combination with the first aspect, the Wnt / β-catenin signaling pathway activator also includes rhein and / or rhein methyl ether. Rhein and rhein methyl ether can also produce a certain activation effect on the Wnt / β-catenin signaling pathway, and are significantly better than rhein and rhubarb extract.

[0011] In a second aspect, the present invention provides the use of chrysophanol in the preparation of a medicine for preventing and treating emphysema, wherein the active ingredient of the medicine includes chrysophanol.

[0012] The present invention finds through research that chrysophanol can prevent the occurrence of emphysema and alleviate the symptoms of emphysema by activating the Wnt / β-catenin signaling pathway; and the present invention also finds through a mouse COPD model that chrysophanol has an excellent effect in reducing the average diameter of alveoli, and is significantly better than rhein, rhubarb, physophanol methyl ether and rhubarb extract and has a significant difference; at the same time, chrysophanol can also effectively reduce the maximum inspiratory volume and lung compliance of the mouse COPD model, improve the lung ventilation function of emphysema mice, and thus effectively prevent and improve the symptoms of emphysema. Therefore, chrysophanol can be used to prepare drugs for preventing and treating emphysema.

[0013] Preferably, the drug is in the form of an injection or oral preparation. Injections include commonly used clinical injection forms, such as injection solutions, lyophilized powder injections, etc.; oral preparations include commonly used clinical oral forms, such as tablets, capsules, granules, oral liquids, etc.

[0014] Optionally, the drug may further include pharmaceutically acceptable excipients and additives, which may be conventionally selected according to dosage form requirements. Chrysophanol and pharmaceutically acceptable excipients and additives may be prepared into the desired preparation by conventional methods, and the present invention is not limited thereto.

[0015] In a third aspect, the present invention provides the use of chrysophanol in the preparation of a drug for preventing and treating chronic obstructive pulmonary disease, wherein the active ingredient of the drug includes chrysophanol.

[0016] The present invention has found through research that chrysophanol can activate the Wnt / β-catenin signaling pathway, reduce the alveolar diameter of the COPD model, significantly reduce the maximum inspiratory volume and lung compliance, and improve the lung ventilation function. The above effects enable it to prevent and improve the clinical symptoms of shortness of breath and dyspnea, and therefore can be used to prepare drugs for the prevention and treatment of chronic obstructive pulmonary disease.

[0017] Preferably, the drug is in the form of an injection or oral preparation. Injections include commonly used clinical injection forms, such as injection solutions, lyophilized powder injections, etc.; oral preparations include commonly used clinical oral forms, such as tablets, capsules, granules, oral liquids, etc.

[0018] Optionally, the drug may further include pharmaceutically acceptable excipients and additives, which may be conventionally selected according to dosage form requirements. Chrysophanol and pharmaceutically acceptable excipients and additives may be prepared into the desired preparation by conventional methods, and the present invention is not limited thereto.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention first discovered that chrysophanol has a significant activation effect on the Wnt / β-catenin signaling pathway, and the activation of the Wnt / β-catenin signaling pathway can prevent or reduce the damage of ATⅡ cells caused by smoke exposure, promote the proliferation of alveolar organoids and epithelial cells, prevent the occurrence of emphysema or alleviate the symptoms of emphysema; the present invention also first discovered that chrysophanol can reduce the average alveolar diameter and improve lung function indicators through a COPD model of mice induced by smoke combined with elastase, thereby preventing and improving the clinical symptoms of shortness of breath and dyspnea, and further preventing the occurrence of emphysema or alleviating the symptoms of emphysema. The above-mentioned effects of chrysophanol enable it to be used to prepare Wnt / β-catenin signaling pathway activators, drugs for preventing and treating emphysema, and drugs for preventing and treating COPD. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effect of rhubarb and the compound in Example 1 of the present invention on cell survival activity (** indicates p < 0.01 compared with the Control group);

[0021] Figure 2 The activation effect of rhubarb and the compound in Example 1 of the present invention on the luciferase reporter gene (** indicates p < 0.01 compared with the Control group; * indicates p < 0.05 compared with the Control group; ## indicates p < 0.01 compared with the chrysophanol 10 μM group; # indicates p < 0.05 compared with the chrysophanol 10 μM group);

[0022] Figure 3 The cell viability activity of different concentrations of chrysophanol (CH) and biotin chrysophanol (bio-CH) in Example 2 of the present invention ( Figure 3 A) and luciferase activity ( Figure 3 B) (** indicates p < 0.01 compared with the Control group; * indicates p < 0.05 compared with the Control group;

[0023] Figure 4 is the binding property of CH and WWP2 in Example 2 of the present invention;

[0024] Figure 5 The effects of siRNA 1, 2 and 3 on knocking down WWP2 in Example 2 of the present invention;

[0025] Figure 6 The effect of siRNA 2 and 3 on down-regulating β-catenin in Example 2 of the present invention;

[0026] Figure 7 The effect of CH and siRNA3 on the signal intensity of β-catenin in Example 2 of the present invention;

[0027] Figure 8 The effect of CH and siRNA3 on Dvl-2 level in Example 3 of the present invention, wherein Figure 8 AB are the results of co-immunoprecipitation experiments. Figure 8 C is the PLA experimental result;

[0028] Fig. 9 The effect of CH on the diameter and number of alveolar organoids in Example 4 of the present invention;

[0029] Fig.10 The effect of CH on β-catenin protein level and Ki67 in Example 4 of the present invention [+] Effect of MLE-12 cell number;

[0030] Fig.11The inhibitory effect of niclosamide on the proliferation of CH-activated lung epithelial cells in Example 4 of the present invention;

[0031] Fig.12 HE staining of lung tissues of mice in each group in Example 5 of the present invention;

[0032] Fig.13 is the average diameter of the alveoli of each group of mice in Example 5 of the present invention (## indicates p<0.01 compared with the Control group; ** indicates p<0.01 compared with the model group);

[0033] Fig.14 This is the lung function of mice in each group in Example 5 (**p<0.01; *p<0.05). DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] COPD is the most common chronic disease of the respiratory system, with the main symptoms being shortness of breath or difficulty breathing. Chronic bronchitis and emphysema are two symptoms of COPD, which often coexist. Among them, emphysema is a serious lung disease that seriously threatens the patient's physical and mental health. Therefore, it is necessary to develop more drugs that can be used to treat COPD, especially emphysema.

[0036] Studies have found that chrysophanol has an anti-inflammatory effect on acute lung injury and can promote the repair of lung injury in the middle and late stages of acute lung injury. However, there are great differences between acute lung injury and COPD: the causes of acute lung injury include pneumonia, sepsis, inhalation of toxic substances, severe trauma, shock or other factors that can cause pulmonary edema or hypoxic respiratory failure, while the cause of COPD is mainly exposure to toxic particles and harmful gases; the pathological characteristics of acute lung injury mainly include alveolar cavity edema, fibrin and collagen infiltration, neutrophil aggregation, pulmonary vascular congestion, and pulmonary interstitial edema, while the pathological characteristics of COPD mainly include fixed airway obstruction, airway wall structural remodeling, alveolar septum destruction, and emphysema. Therefore, in clinical medication, drugs for the treatment of lung injury may not necessarily treat COPD, let alone emphysema.

[0037] The present invention has found for the first time through research that chrysophanol has a significant activating effect on the Wnt / β-catenin signaling pathway, and has the effect of reducing the average alveolar diameter and improving lung function indicators in the COPD model of mice induced by smoke combined with elastase, and the above effects enable it to be used to prevent the occurrence of emphysema or alleviate the symptoms of emphysema, and prevent and improve the clinical symptoms of shortness of breath and dyspnea of ​​COPD. Based on the above research results, the embodiment of the present invention provides the use of chrysophanol in the preparation of Wnt / β-catenin signaling pathway activators and in drugs for preventing and treating emphysema and COPD. In clinical application, chrysophanol can be used alone or in combination according to the above effects of chrysophanol and the clinical symptoms of patients.

[0038] The scheme of the present invention is described below through specific embodiments.

[0039] Unless otherwise specified, other reagents and drugs used in the following examples are commonly used laboratory reagents and drugs obtained from commercial channels.

[0040] Example 1

[0041] This example provides the activation effect of chrysophanol on the Wnt / β-catenin signaling pathway.

[0042] 1. Test materials

[0043] 1.1 Test sample

[0044] Rhubarb powder, provided by Yiling Pharmaceutical Chinese Medicine Branch;

[0045] Monomeric compounds: rhein, chrysophanol (CH), rhein, and physophanol methyl ether, all of which were standard products, purchased from Chengdu Munster, and stored at 4°C.

[0046] 1.2 Solvent

[0047] DMSO.

[0048] 1.3 Cell vector

[0049] TCF / LEF-Luc2P MDA-MB-231stable cell line.

[0050] 1.4 Main instruments

[0051] ME104E Mettler Precision Balance, Mettler-Toledo Instruments (Shanghai) Co., Ltd.

[0052] 2. Test methods

[0053] 2.1. Grouping

[0054] The mice were divided into control group (Control), rhein-administered group, chrysophanol-administered group, rhubarb-administered group, physophanol-administered group and rhubarb-administered group.

[0055] 2.2 Dosage

[0056] According to literature data and cytotoxicity tests, the dosage of the rhubarb administration group was set at 0.05 mg / ml, and the low, medium and high dosages of each monomer compound administration group were set at 0.1 μM, 1 μM and 10 μM, respectively.

[0057] 2.3 Preparation and storage of test products

[0058] Preparation method of test substance stock solution: Taking the dosage of chrysophanol as 10 μM as an example, prepare 1000 times the stock solution of 10 mM, weigh the test substance: 1.27 mg, dissolve it in 500 μL DMSO, and vortex to mix evenly.

[0059] The preparation of other test samples can refer to the above method and adjust the liquid volume according to the required amount of the test.

[0060] 2.4 Administration

[0061] 24 hours after cell plating (96-well plate), the culture medium was aspirated and culture medium containing the corresponding concentration of drug solution was added to dilute the drug to the corresponding concentration. Incubation was continued for 24 hours.

[0062] 2.5 Observation indicators, time and content

[0063] 24 hours after dosing, refer to ONE-Glo TM Luciferase Assay System(ONE-Glo TM Luciferase assay system (Promega, E6110) was used for detection.

[0064] 2.6 Data processing

[0065] The data results were obtained using It means that the normal distribution test was first performed using SPSS statistical software, and the one-way analysis of variance was used for those that met the normal distribution, and the non-parametric test was used for those that did not meet the normal distribution.

[0066] 3. Results

[0067] 3.1 Cell viability

[0068] When the drug concentration was 10 μM, rhein had a significant inhibitory effect on cell activity, while other compounds had no significant inhibitory effect. Figure 1 .

[0069] 3.2 Activation of Wnt / β-catenin signaling pathway

[0070] Chrysophanol, chrysophanol, and chrysophanol methyl ether can significantly activate the Wnt reporter gene, and chrysophanol is dose-dependent. Figure 2 .

[0071] 4. Conclusion

[0072] Under the experimental conditions of this study, chrysophanol significantly activated the Wnt / β-catenin signaling pathway in a dose-dependent manner.

[0073] Example 2

[0074] This example studies the effect of chrysophanol (CH) on the target of Wnt / β-catenin signaling pathway.

[0075] 1. Experimental methods

[0076] 1.1 Preparation of biotinylated chrysophanol (bio-CH):

[0077]

[0078] In order to explore the potential molecular targets of CH, biotin-labeled CH (bio-CH, DH-P1 / P2) was synthesized: Compound A4 (300 mg, 0.79 mmol), K2CO3 (163 mg, 1.19 mmol) and CH (A5, 201 mg, 0.79 mmol) were dissolved in DMF (6 mL). The mixture was heated to 55-60°C and stirred for 18 hours. The reaction progress was monitored using LC-MS. After cooling to room temperature, the mixture was diluted with water (12 mL) and DCM (12 mL), and the pH was adjusted to 5-6 with 1N HCl. The organic layer was separated, dried over sodium sulfate, concentrated, and the residue was chromatographed to obtain a crude product. Further purification by pre-HPLC gave 25 mg of bio-CH (a mixture of DH-P1 and DH-P2), which was confirmed by LC-MS, m / z 553 [M+H] + .

[0079] 1.2 Target recognition of chrysophanol

[0080] Human Proteome Microarray V4.0 (HuProt TM )To study the potential associated targets of chrysophanol.

[0081] (1) Storage of protein chips

[0082] Store the plastic chip box containing the HuProtV4.0 protein chip in a -80° refrigerator or on dry ice. After the chip is taken out, it should be directly placed in the blocking solution, and the side with the barcode is the front of the chip.

[0083] Note: The chip should be stored in an ultra-low temperature, dry environment, and there should be no condensation on the surface before use.

[0084] (2) Closed

[0085] 1% BSA (w / v) was added to TBS-T (G2150-1L, Wuhan Saiweier Biotechnology Co., Ltd.) as a blocking solution. 3.0 mL of blocking solution was added to each well of the 4-well plate. The chip was carefully removed from the plastic box on dry ice with flat-tipped tweezers and immediately placed in the 4-well plate with the blocking solution added, facing up, and incubated at room temperature for 1.5 hours, gently shaking on an oscillator (60 rpm).

[0086] Note: In the following experiments, the 4-well plate containing the HuProtV4.0 chip should be covered with aluminum foil to protect it from light.

[0087] (3) Sample preparation

[0088] Bio-CH and CH were diluted to 50 μM with blocking buffer, and biotin was diluted to 10 μM with blocking buffer as blank control, and kept on ice for later use.

[0089] (4) Hybridization

[0090] Aspirate the blocking solution from the corner of the 4-well plate and carefully add the prepared sample (3.0 mL) with the chip facing up.

[0091] Note: The pipette tip must not touch the chip surface. Incubate on a shaker at room temperature for 1 hour (60 rpm).

[0092] (5) Film development I

[0093] After the incubation, remove the liquid from the corner of the 4-well plate, add 3.0 mL TBS-T to each well, and rinse quickly three times. Then add 3.0 mL TBS-T to each well, shake and wash at room temperature for 5 minutes (60 rpm), and repeat three times. The chip must not be allowed to dry out during the experiment.

[0094] (6) Secondary antibody incubation

[0095] Cy3-streptavidin was diluted 1:1000 with blocking solution, 3.0 mL was added to each well of a 4-well plate, and incubated at room temperature with shaking (60 rpm) for 1.5 hr.

[0096] (7) Film development II

[0097] The solution was aspirated, 3.0 ml TBS-T was added, and the cells were quickly rinsed three times. Then 3.0 mL TBS-T was added to each well, and the cells were shaken at room temperature for 5 minutes (60 rpm), and repeated three times.

[0098] (8) Dry slice

[0099] Place absorbent paper on the bottom of the slide box. You can also use a 50ml centrifuge tube. Each slide box can hold multiple chips, but each centrifuge tube can only hold one chip. Use flat-tipped tweezers to remove the chip from the four-well plate, place it vertically on the absorbent paper, absorb the residual water from the edge of the chip, place the chip vertically in the slide box or 50ml centrifuge tube, and centrifuge at 800rpm for 3 minutes. After centrifugation, transfer the chip to a new clean slide box.

[0100] (9) Scan and save

[0101] Scan the dried chip immediately or store it in a light-proof slide box at -20°C. Note: The chip must be scanned and data collected within three days after the experiment.

[0102] 1.3 Biolayer interferometry (BLI) binding assay

[0103] The binding affinity between chrysophanol and WWP2 was measured using BLI and the Octet Red 96 instrument (Gottingen, ForteBio).

[0104] 1) Divide 4 NTA sensors into 2 groups and pre-wet them in PBST for more than 10 minutes. Two sensors are cured, and the other two sensors are used as reference sensors, which do not need to be cured. Add PBST to the first column of a 96-well sample plate and 50 μg / ml WWP2 to the second column. Set baseline 60s, loading 3000s, so that the curing height reaches more than 4nm, and then baseline 60s for equilibrium.

[0105] 2) Take a new 96-well plate and set up the Buffer and Sample wells. Add PBST+0.1% DMSO buffer to the Buffer well and CH to the Sample well. The drug concentrations are 1.56, 3.125, 6.25, 12.5, 25, and 50 μM from left to right.

[0106] 3) After solidification, first set a relatively long equilibration step (more than 10 minutes), and then enter the baseline / association / dissociation cycle. The Assay Definition settings are as follows: baseline 60 seconds, association 120 seconds, dissociation 360 seconds.

[0107] 4) After setting the curing sensor cycle, add a set of reference sensor cycles, which should be consistent with the curing sensor cycle program.

[0108] 5) After setting, set the position of the sensor in Sensor Assignment. The sensors are divided into two columns, one group of solidification sensors and one group of reference sensors, with 2 sensors in each column. After Review Experiment is checked, set the save path, file name, temperature, etc. in Run Experiment and click GO to run.

[0109] 6) Use Octet analysis software Data Analysis software to perform kinetic and steady-state analysis on the raw data

[0110] 1.4 Short interfering RNA transfection

[0111] WWP2 short interfering RNA (siRNA) was synthesized by Anhui General Biotechnology Co., Ltd. The siRNA sequence is shown in Table 1. MLE12 cells in logarithmic growth phase were obtained and digested with trypsin at 1×10 5 The cells were seeded at a density of 100 μM / mL in 6-well plates. After the cells adhered, they were divided into control group, CH group (10 μM CH), siRNA group (50 nmol / L siRNA), and siRNA+CH (50 nmol / L siRNA+10 μM CH) group. The transfection was performed according to the instructions of the jet PRIME transfection kit (Polyplus, France). The protein expression level of the cells in each group was evaluated after 48 h.

[0112] Table 1. siRNA sequences

[0113]

[0114] 1.5 Data Analysis

[0115] All numerical data are expressed as mean ± standard deviation (SD), and all statistical analyses and plots were performed using SPSS 20.0 (SPSS, Armonk, USA). T-test was used to assess the statistical significance of differences between the two groups. One-way analysis of variance (ANOVA) was used to determine differences between groups, and post hoc comparisons were performed using Tukey's test. p < 0.05 was considered a significant difference.

[0116] 2. Experimental results

[0117] like Figure 3 A and Figure 3As shown in B, bio-CH (DH-P1 / P2) was proven to be non-toxic and has Wnt / β-catenin pathway activation properties at a concentration of 10 μM. The results of HuProt chip incubation showed that WWP2 (an E3 ubiquitin ligase) had the highest signal-to-noise ratio (SNR) among the candidate proteins ( Figure 4 A). To demonstrate the binding efficiency of chrysophanol to WWP2, biolayer interferometry (BLI) analysis was performed, and the results showed that chrysophanol specifically bound to WWP2 with a dissociation constant (KD) of 1.158 μM ( Figure 4 B). Next, to confirm that CH regulates β-catenin through WWP2, we used three short interfering RNAs (siRNA1 / 2 / 3) to knock down WWP2 protein in MLE-12 cells. The results showed that only siRNA2 and siRNA3 showed a knockdown trend or effect of WWP2 ( Figure 5 AB). After siRNA3 down-regulated WWP2 protein expression, Western Blot results showed that the protein level of β-catenin decreased ( Figure 6 AB). In addition, immunofluorescence staining results of β-catenin showed that downregulation of WWP2 interfered with CH-dependent accumulation of β-catenin ( Figure 7 AB). Taken together, these results suggest that WWP2 is a potential protein target of CH and that CH regulates β-catenin levels through WWP2.

[0118] Example 3

[0119] This example studies the activation of Wnt / β-catenin signaling by chrysophanol through the WWP2-Dlv2-β-catenin axis.

[0120] 1. Experimental methods

[0121] 1.1 Co-immunoprecipitation (Co-IP) assay

[0122] Take logarithmically growing MLE-12 cells and, when the cell density reaches 70%, plate them at 1×10 5 The cells were inoculated into a 10 cm culture dish at a density of 100 cells / mL and cultured in an incubator for 24 h. After the cells grew to 70% adherent, they were divided into 4 groups, namely blank control group, CH group (10uM CH), siRNA-WWP2 (50uM siRNA3), siRNA-WWP2+CH group (cells were treated with 50uM siRNA3 for 24 h, then 10uM CH was given and incubated with the cells for 24 h). After 24 h of cell culture, COIP test was performed.

[0123] The specific detection method is:

[0124] (1) Collect cell lysate, centrifuge at 12000 rpm, 4°C for 10 min, collect supernatant, and then determine protein concentration using BCA method. The total protein concentration is selected in the range of 0.5-1ug / ml.

[0125] (2) Preparation of magnetic beads: Wash Protein A / G magnetic beads three times using washing buffer.

[0126] (3) Removal of non-specific binding: Add the sample to the washed magnetic beads, incubate with shaking at 4°C for 30 min, place on a magnetic stand for 10 s, and take the supernatant for subsequent immunoprecipitation (divide into 3 tubes: input group, CO-IP group, and IgG group).

[0127] (4) Immunoprecipitation: Add 0.2-2ug antibody solution / Mouse / Rabbit IgG to the washed magnetic beads, incubate at room temperature for 30 min, place on a magnetic stand for 10 s, and discard the supernatant.

[0128] (5) Incubate the protein supernatant from step 3 and the magnetic beads from step 4 overnight, separate them on a magnetic rack, collect the precipitate and wash it.

[0129] (6) Add 50 μl of 1× Loding buffer, gently resuspend the magnetic bead-Ab-Ag complex, and heat at 70°C for 10 min.

[0130] (7) Place the EP tube on a magnetic rack and take the supernatant as a sample for SDS-PAGE electrophoresis and WB detection.

[0131] 1.2 Proximity Ligation Assay (PLA)

[0132] Take logarithmically growing MLE-12 cells and, when the cell density reaches 70%, plate them at 1×10 5Cells were seeded into 35 mm glass-bottomed culture dishes at a density of 10 μM / mL and placed in an incubator for 24 h. After the cells grew to 70% adherent, they were divided into the control group and the CH group, which were given DMEM / F12 basal medium and DMEM / F12 basal medium containing 10 μM CH, respectively. After incubation for 24 h, the detection was performed. Proximity ligation assay (PLA) was performed using a commercial kit (DUO92101; Sigma-Aldrich) according to the manufacturer's instructions. The method first incubated the sample with DVL2 and WWP2 antibodies. Thereafter, a secondary antibody labeled with an oligonucleotide (PLA probe) was applied to lay the foundation for ligation and rolling circle amplification reactions. The resulting products were visualized using a Zeiss confocal microscope (Oberkochen, Germany) and analyzed with the help of Zeiss imaging software to ensure accurate representation of protein interactions.

[0133] 1.3 Data Analysis

[0134] All numerical data are expressed as mean ± standard deviation (Mean ± SD), and all statistical analyses and plots were performed using SPSS 20.0 (SPSS, Armonk, USA). T-test was used to assess the statistical significance of differences between the two groups. One-way analysis of variance (ANOVA) was used to determine differences between groups, and post hoc comparisons were performed using Tukey's test, and p < 0.05 was considered a significant difference.

[0135] 2. Experimental results

[0136] The cell lysates collected from the blank control group, CH group, siRNA-WWP2 group, and siRNA-WWP2+CH group were co-immunoprecipitated with Dvl-2 antibody, and then Western blotting analysis was performed using WWP2 antibody ( Figure 8 AB), the results showed that WWP2 may form a complex with Dvl2, and CH up-regulated the protein level of WWP2 and enhanced the binding ability of WWP2 with Dvl2 ( Figure 8 A), thereby stabilizing the protein level of β-catenin.

[0137] PLA results showed that the interaction between WWP2 and Dvl2 was enhanced after CH treatment ( Figure 8 C). It has been reported that the β-catenin degradation complex can be trapped by WPP2-Dvl2-associated phase separation, and thus β-catenin can be released from the degradation complex. Collectively, these results suggest that CH treatment activates the Wnt / β-catenin signaling pathway by mediating WWP2-Dvl2 interaction, which may further disrupt the degradation complex, thereby releasing β-catenin for degradation.

[0138] Example 4

[0139] This example provides an experiment on the effect of chrysophanol on the proliferation of alveolar organoids and alveolar epithelial cells.

[0140] 1. Experimental methods

[0141] 1.1 Alveolar organoid culture

[0142] Human ATII (HT2-280[+]) cells were seeded in PneumaCult TM Then, on the second or third day of culture, the medium was changed to complete PneumaCult TM AvOE medium was used and the medium was changed every 3-4 days to obtain mature ATII organoids. On day 10, alveolar organoids can be passaged or differentiated. Control group organoids were cultured using PneumaCult TM AvOE medium was completely replaced every 3 to 4 days. CH group organoids were grown in PneumaCult TM CH is added to the AvOD complete medium and the medium is replaced every 3 to 4 days. After the 10-day differentiation experiment, the differentiated ATI organoids can be analyzed.

[0143] 1.2 Immunofluorescence staining

[0144] Take logarithmically growing cells and when the cell density reaches 70%, plate MLE-12 cells at 1×10 5The cells were inoculated into 35 mm glass-bottomed culture dishes at a density of 100 cells / mL and placed in an incubator for 24 hours. After the cells grew to 70% adherent to the wall, they were divided into the Control group and the CH group, and given DMEM / F12 basal medium and basal medium containing 10uM CH, respectively. After incubation for 24 hours, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 20 minutes, and washed twice with PBS. Permeabilization was performed with 0.5% Triton X-100 at room temperature for 10 minutes, and washed twice with PBS. The cells were blocked with blocking solution at room temperature for 1 hour. The cells were incubated with β-catenin (ab32572, 1:250, Abcam) and Ki67 (ab279653, 1:50, Abcam) antibodies at 4°C overnight, washed twice with PBS, and then incubated with fluorescent secondary antibodies labeled with Alexa Fluor 488 (1:1000; P0176; Beyotime) and Alexa Fluor 555 (1:1000; P0179; Beyotime) at 37°C for 1 h. The nuclei were stained with DAPI (S2110, Solarbio), and the samples were imaged using a Zeiss confocal microscope (Oberkochen, Germany).

[0145] 2. Experimental results

[0146] 2.1CH increases the diameter and number of alveolar organoids

[0147] CH was applied in vitro to human ATII (HT2-280 [+] ) cells and then quantified the size and number of alveoli ( Fig. 9 AC). Quantitative results showed that the diameter of organoids after CH treatment ( Fig. 9 B) and quantity ( Fig. 9 C) increased significantly.

[0148] 2.2CH upregulates β-catenin protein levels and increases Ki67 [+] MLE-12 cell ratio

[0149] Immunofluorescence staining was performed using antibodies against β-catenin and Ki67. Quantitative results showed that CH treatment upregulated the levels of β-catenin protein and Ki67. [+] The increase in the proportion of MLE-12 cells ( Fig.10 AC). It is noteworthy that the addition of 1 μM niclosamide (Frizzled inhibitor that blocks Wnt / β-catenin signaling) to the CH group inhibited the proliferation of CH-activated MLE-12 cells ( Fig.11). The above results demonstrate that CH has the potential to activate the proliferation of alveolar organoids and lung epithelial cells by activating the Wnt / β-catenin signaling pathway.

[0150] Example 5

[0151] This example provides a pharmacodynamic test of chrysophanol on COPD in mice induced by smoke combined with elastase.

[0152] 1. Test materials

[0153] 1.1 Test sample

[0154] Rhubarb powder, provided by Yiling Pharmaceutical Chinese Medicine Branch;

[0155] Monomeric compounds: rhein, chrysophanol (CH), rhein, and physophanol methyl ether, all of which were standard products, purchased from Chengdu Munster, and stored at 4°C.

[0156] 1.2 Solvent

[0157] 10% v / v DMSO, 10% v / v Tween 80, 80% v / v normal saline.

[0158] 1.3 Experimental animals

[0159] SPF male C57BL / 6j mice, 4–6 weeks old, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., license number: SCXK(Beijing)2021-0011.

[0160] The animals were adaptively fed for 3 days before the experiment, during which time there were no abnormalities in their water intake, food intake, and health status, and no signs of disease or death were found.

[0161] 1.4 Main instruments

[0162] ME104E Mettler precision balance, Mettler-Toledo Instruments (Shanghai) Co., Ltd., DSI pulmonary function tester (PFT).

[0163] 2. Test methods

[0164] 2.1 Grouping

[0165] The mice were divided into normal group, model group, rhein-administered group, chrysophanol-administered group (divided into low-, medium- and high-dose administration groups), rhubarb-administered group, physophanol-administered group and rhubarb-administered group according to their body weight, with 7 mice in each group and marked with ear tags.

[0166] 2.2 Dosage

[0167] According to literature data, the mice in the rhein, chrysophanol, rhein, and physophanol methyl ether administration groups were administered by intraperitoneal injection, with the dose set at 0.1-10 mg / kg, and the dose for the rhubarb administration group was set at 0.1 g of crude drug / kg.

[0168] 2.3 Preparation and storage of test products

[0169] Preparation method of the test substance: Taking the dosage of chrysophanol as 10 mg / kg as an example, the concentration to be prepared is

[0170] 1.67mg / ml, weigh the test sample: 16.7mg, dissolve it with 1ml DMSO, then add 1ml Tween 80 to dissolve it, finally make up to 10ml with physiological saline and mix well.

[0171] The preparation of other test samples can refer to the above method and adjust the liquid volume according to the required amount of the test.

[0172] 2.4 Administration

[0173] The administration volume was 6 ml / kg, and the corresponding test substance was drawn with a syringe for administration.

[0174] Except for the normal group, the mice in the other groups were fumigated with cigarette smoke for 2 months (5 days / week, 1 time / day, 2 hours / time, ventilation once every 40 minutes, ventilation for 10 minutes each time, the amount of smoke was 15 cigarettes each time, and 15 cigarettes were re-lit after ventilation, a total of 45 cigarettes / day). After the end of the fumigation, the mice were given the prescribed administration method and dosage for 3 consecutive days, once a day, and then 4.5U / kg of porcine pancreatic elastase (elastase) was instilled into the trachea, 50μl per mouse, and then the drug was continuously administered for 7 consecutive days, once a day. The normal group was instilled with an equal amount of normal saline in the trachea and injected with normal saline intraperitoneally.

[0175] 2.5 Observation indicators, time and content

[0176] 24 hours after the last administration, the lung function of the mice was measured, lung tissues were taken for HE staining, and the mean alveolar diameter (MLI) was calculated.

[0177] 2.6 Data processing

[0178] The data results were expressed as x±SD. SPSS statistical software was used to perform a normal distribution test. If the data conformed to the normal distribution, one-way analysis of variance was used, and if the data did not conform to the normal distribution, a non-parametric test was used.

[0179] 3. Results

[0180] 3.1 HE staining of lung tissue to calculate the average diameter of alveoli

[0181] Compared with the model control group, chrysophanol could significantly reduce the average diameter of alveoli. Fig.12 and Fig.13 .

[0182] 3.2 Lung function

[0183] Compared with the model control group, chrysophanol can significantly reduce the maximum inspiratory capacity (IC) and lung compliance (dynamic lung compliance Cdyn and static lung compliance Cchord) of COPD model mice, and improve the lung ventilation function FEV20 / FVC of emphysema mice. CH improves the lung function of mice in a dose-dependent manner. Fig.14

[0184] In addition, from Fig.12 Model group slices, Fig.13 The diameter of the alveoli and Fig.14 The lung function of the model group was improved, and the mice in the model group showed alveolar expansion and lung function damage, indicating that the COPD model obtained by smoke fumigation combined with tracheal instillation of elastase was not an acute lung injury, and symptoms of emphysema appeared in this experiment.

[0185] 4. Conclusion

[0186] Under the experimental conditions, chrysophanol had drug-related effects on the COPD model of mice induced by smoke combined with elastase, which was manifested by reducing the mean alveolar diameter and improving lung function indicators.

[0187] Example 6

[0188] This embodiment provides a granule for preventing and treating emphysema.

[0189] Mix chrysophanol with microcrystalline cellulose, pregelatinized starch and cross-linked polyvinyl pyrrolidone, use 10%wt starch slurry as a binder to granulate, add magnesium stearate after granulation, mix, and pack into granule packaging bags to obtain the product.

[0190] Example 7

[0191] This embodiment provides an injection for preventing and treating emphysema.

[0192] After dissolving chrysophanol in DMSO, mix it with physiological saline (the volume ratio of DMSO to physiological saline is 1:9), add lecithin to homogenize, decolorize with 1% activated carbon, filter, sterilize, and aseptically package to obtain the product.

[0193] Example 8

[0194] This embodiment provides a tablet for preventing and treating COPD.

[0195] The chrysophanol is mixed with microcrystalline cellulose, pregelatinized starch and cross-linked polyvinyl pyrrolidone, granulated with 5% PVP as a binder, talcum powder is added after granulation, mixed, and tableted to obtain the product.

[0196] Example 9

[0197] This embodiment provides an injection for preventing and treating COPD.

[0198] After dissolving chrysophanol in DMSO, mix it with physiological saline (the volume ratio of DMSO to physiological saline is 1:9), add poloxamer 188 for homogenization, decolorize with 1% activated carbon, filter, sterilize, and aseptically package to obtain the product.

[0199] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of chrysophanol in the preparation of Wnt / β-catenin signaling pathway activators.

2. The use according to claim 1, characterized in that: The Wnt / β-catenin signaling pathway activator also includes rhamnosin and / or rhamnosin methyl ether.

3. The use of chrysophanol in the preparation of a drug for preventing and treating emphysema, characterized in that: The active ingredient of the drug includes chrysophanol.

4. The use according to claim 3, characterized in that: The dosage form of the medicine is injection or oral preparation.

5. The use according to claim 4, characterized in that: The medicine also includes pharmaceutically acceptable excipients and additives.

6. Use of chrysophanol in the preparation of a drug for preventing and treating chronic obstructive pulmonary disease, characterized in that: The active ingredient of the drug includes chrysophanol.

7. The use according to claim 6, characterized in that: The dosage form of the medicine is injection or oral preparation.

8. The use according to claim 7, characterized in that: The medicine also includes pharmaceutically acceptable excipients and additives.

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