New application of cytisine

Abstract

The invention provides a novel application of cytisine, and belongs to the field of medicines. The invention provides application of cytisine in preparation of a medicine for treating pulmonary heart disease. Compared with the cytisine, the cytisine-polypeptide coupling compound (PDC medicine) prepared by coupling the cytisine and the polypeptide has the advantages that the dosage is obviously reduced, the effect is better, the right heart dysfunction can be improved, the myocardial fibrosis is reduced, the right heart remodeling is alleviated, and a new choice is provided for clinically treating the pulmonary heart disease.

Description

[0001] This application is a divisional application of the application with the application number 2024113126285, the application date of September 20, 2024, and the invention title "New Uses of Cytisine, Cytisine-Polypeptide Conjugates and Their Preparation Methods and Uses". Technical Field

[0002] The present invention relates to new uses of cytisine, cytisine-polypeptide conjugates and their preparation methods and uses, and belongs to the field of pharmaceuticals. Background Art

[0003] Pulmonary heart disease (PHD) refers to a type of heart disease caused by lesions in the lung tissue or pulmonary artery and its branches (such as lung diseases, pulmonary vascular diseases, chest deformities, or respiratory center disorders), resulting in increased pulmonary circulation resistance, pulmonary arterial hypertension (PAH), right ventricular hypertrophy, myocardial fibrosis, and ultimately decreased cardiopulmonary function, seriously endangering people's health and increasing the medical burden. Clinically, the treatment of PHD is still very limited, and it is unable to effectively cure cardiopulmonary tissue remodeling and functional recovery. The main treatment plan is to maintain lung function and keep the oxygen saturation level > 90%. PHD mainly includes two aspects: surgical treatment and drug treatment. Surgical treatment includes oxygen therapy, percutaneous pulmonary valvuloplasty, pulmonary thromboendarterectomy, and lung transplantation surgery.

[0004] Currently, the clinical treatment of PHD is limited. The main first-line drugs include cardiotonic agents, diuretics, antibiotics, vasodilators, expectorants, and anti-asthmatic agents, etc., aiming to delay the progression of underlying diseases and improve the quality of life of patients, but they cannot repair the existing tissue damage in the heart and lungs. Therefore, there is an urgent need to find new targets and new drugs that can reverse the process of cardiopulmonary remodeling and effectively treat pulmonary heart disease.

[0005] Cytisine, also known as baptitoxine or Cytisine, is an alkaloid mainly isolated from leguminous plants. It has a crystalline solid form ranging from off-white to yellowish-brown, with specific chemical properties, such as the molecular formula C 11 H 14 N2O, a molecular weight of 190.242, and a melting point of 154 - 156 °C. Cytisine has various biological activities in pharmacology, including smoking cessation, reducing alcohol consumption behavior, anti-tumor, cardiovascular protection, blood glucose regulation, neuroprotection, and prevention and treatment of osteoporosis, etc. It also has effects such as anti-arrhythmia, anti-microbial infection, anti-ulcer, and increasing white blood cells. In addition, cytisine has certain toxicity. For example, in acute toxicity tests, the lethal doses for different animals are reported in relevant literature.

[0006] There is currently no literature reporting the use of cytisine in the treatment of cor pulmonale. Summary of the Invention

[0007] The technical solution of the present invention provides a new use of cytisine, specifically, its use in the preparation of a drug for the treatment of cor pulmonale. The present invention also provides a cytisine-polypeptide conjugate, its preparation method and use.

[0008] The present invention provides the use of cytisine in the preparation of a drug for the treatment of cor pulmonale.

[0009] The present invention provides a cytisine-polypeptide conjugate, which is formed by connecting cytisine and a polypeptide through cathepsin B.

[0010] Among them, the C-terminal 5 peptides of the polypeptide form a cyclic peptide, and the sequence is as follows:

[0011] Cys-Thr-Arg-Gln-Arg-Tyr-Cys;

[0012] The substrate sequences of the cathepsin B include: Phe-Lys, Val-Cit, Glu-Val-Cit, GGFG, Phe-Arg, Val-Ala, Ala-Ala, cBU-Cit, Abz-Gn VR↓AK(Dnp)-OH, Z-VR-AMC, Z-ER-AMC.

[0013] Further preferably, the substrate sequence of the cathepsin B is Val-Cit, and the structural formula of the cytisine-polypeptide conjugate is: \n

[0014]

[0015] The present invention provides a preparation method of the cytisine-polypeptide conjugate, which is synthesized by solid-phase synthesis.

[0016] Specifically, the preparation method of the present invention includes the following steps:

[0017] a. Place the resin in a solid-phase reactor, soak it with dichloromethane, add a piperidine / DMF solution to the reactor, soak it, react with nitrogen bubbling, and then wash it with DMF;

[0018] b. Add cysteine, threonine, arginine, glutamine, arginine, and tyrosine in sequence;

[0019] c. Cut the polypeptide from the resin;

[0020] d. Couple cytisine, cleave the polypeptide, and purify to obtain the cytisine-polypeptide conjugate.

[0021] Among them, the cutting reagents described in step c are TFE and DCM, and the volume ratio is 1:4; the cracking reagents are TFA, water, EDT, and Tis, and the volume ratio is 95:1:2:2.

[0022] The present invention also provides the use of the sparteine-polypeptide conjugate compound in the preparation of a drug for treating cor pulmonale.

[0023] The present invention also provides a pharmaceutical composition for treating cor pulmonale, which is prepared from the sparteine-polypeptide conjugate compound as an active ingredient and added with pharmaceutically acceptable excipients or auxiliary ingredients into a pharmaceutically common preparation.

[0024] Among them, the preparation is an oral preparation.

[0025] The beneficial effects of the present invention are as follows:

[0026] Pharmacodynamic experiments prove that the sparteine of the present invention has the effect of treating cor pulmonale and can inhibit the proliferation of human lung fibroblasts MRC-5. By conjugating sparteine with polypeptides, the prepared sparteine-polypeptide conjugate compound (PDC drug), compared with sparteine, has a significantly reduced dosage and better effects, can improve right heart dysfunction, reduce myocardial fibrosis, and alleviate right heart remodeling, providing a new option for the clinical treatment of cor pulmonale. Description of the Drawings

[0027] Figure 1 HPLC chromatogram;

[0028] Figure 2 MS spectrum;

[0029] Figure 3 EdU experiments confirm that PDC can inhibit the proliferation of pulmonary myofibroblasts in a dose-dependent manner;

[0030] Figure 4 PDC can inhibit the migration of human lung fibroblasts (MRC-5);

[0031] Figure 5 Ultrasound results show that the PDC drug can dose-dependently reduce right heart dysfunction in rats;

[0032] Figure 6 Masson staining results show that the PDC drug can dose-dependently reduce right heart fibrosis in rats. Detailed Embodiments

[0033] Example 1 Preparation of the sparteine-polypeptide conjugate compound PDC of the present invention

[0034] 1. Synthesize the polypeptide conjugate compound by solid-phase synthesis method

[0035] 1.1 Experimental equipment: ten-thousandth electronic balance; vertical reactor (20*250mm, No. 1 sintered glass filter); 50ml centrifuge tube; centrifuge, one semi-preparative HPLC, 2 cm C18 column, disposable pipette; nitrogen; circulating water vacuum pump; wash bottle; test tube; long-necked pipette; constant temperature heater.

[0036] 1.2 Experimental raw materials and reagents: 2-chlorotrityl chloride resin; anhydrous DCM, DIC, DIEA, methanol, industrial grade DMF; analytical grade DMF; 20% piperidine / DMF, detection reagent A (5g ninhydrin - 100mL absolute ethanol), B (analytical grade pyridine), HOBT, acetonitrile, ether, TFA, TFE, EDT, TIS, cytisine;

[0037] Cytisine is a type of quinolizidine alkaloid with a tricyclic fused structure extracted from the seeds of leguminous plants: molecular formula C 11 H 14 N2O; molecular weight 190.24.

[0038]

[0039] Amino acids required:

[0040] FMOC-CIT-OH, FMNOC-VAL-OH, FMOC-ARG(PBF)-OH, FMOC-THR(TBU)-OH, FMOC-CYS(TRT)-OH, FMOC-GLN(TRT)-OH, FMOC-TYR(TBU)-OH.

[0041] 1.3 Select a reactor (20*250mm, No. 1 sintered glass filter), and write the name of the polypeptide on the reactor as a label. Weigh 0.5g of 2-chlorotrityl chloride resin with an electronic balance and put it into the reactor. Add DCM to soak for 30 minutes to remove Fmoc: Add 20% piperidine / DMF solution to the reactor with a wash bottle. The volume of the reagent is about 3 times the volume of the resin to completely soak the resin. Bubble with nitrogen for 20 min, and then wash with DMF 5 times.

[0042] 1.4 Weigh 0.15 mmol of FMOC-CIT-OH (C-terminal) and HOBT and add them to a centrifuge tube. Then add 10 mL of anhydrous DCM and 0.5 mmol of DIEA, and shake well. Use a disposable pipette to suck the solution and add it to the reactor in the previous step. Bubble with nitrogen for 90 min. After the reaction, add 2 mL of methanol + 6 mL of DCM and react for 20 min.

[0043] 1.5 Washing: After pumping the liquid in the reactor dry with a circulating water vacuum pump, add industrial-grade DMF to the reactor with a wash bottle. The volume of the reagent is about 3 times the volume of the resin, so that the resin is completely immersed in the solution. Wash for 30 s, then pump the liquid in the reactor dry with a circulating water vacuum pump. The drying time is about 30 s. Repeat this operation 4 times.

[0044] 1.6 Deprotection of Fmoc: Add 20% piperidine / DMF solution to the reactor with a wash bottle. The volume of the reagent is about 3 times the volume of the resin, so that the resin is completely immersed in the solution. React with nitrogen bubbling for 20 min.

[0045] 1.7 Washing: Refer to 1.5, and replace the industrial-grade DMF with analytical grade DMF during the fifth washing process;

[0046] 1.8 Resin detection: Use a long-necked pipette to take 10 - 20 resin beads from the reactor and place them at the bottom of the test tube. Then use a dropper to add two drops of detection reagents A and B to the test tube respectively, so that the resin can fully contact with the detection reagents. Then place the test tube in a constant temperature of 100 °C and heat for 2 min. Observe the color of the resin. If the resin shows color, it means that the deprotection of Fmoc in 1.6 is successful; if not, repeat the operations of 1.6 - 1.8.

[0047] 1.9 Condensation (Coupling): Weigh 0.5 mmol of FMOC-VAL-OH (the second position at the C-terminus) and 0.5 mmol of HOBT into a centrifuge tube. After fully dissolving with 5 ml of DMF; then add 0.5 mmol of DIC, mix for 1 min, and add it to the dried resin. React with nitrogen bubbling for 1 h. It is strictly prohibited to directly add DIC to the reactor; when activating AA in advance, the generated crystals are not added to the reactor. If the solution in the reactor cannot make the resin shake evenly during the reaction process, a little DMF can be appropriately added;

[0048] 1.10 Resin detection: Refer to 1.8, observe the color of the resin. If it is colorless, it indicates that the connection is complete, and proceed to 1.11 operation; if it has color, repeat 1.9.

[0049] 1.11 Washing, same as 1.5.

[0050] 1.12 Repeat the operations of 1.6 - 1.11, connect the amino acid raw materials in sequence according to the polypeptide sequence until the peptide coupling is completed and finally do not remove Fmoc. Then carry out washing (see 1.5), add 0.5 mmol of BOC anhydride and 0.5 mmol of Diea, react with DCM as the solvent for 20 min, and then carry out washing (see 1.5).

[0051] The polypeptide sequence is: A cyclic peptide composed of the C-terminal 5 peptides of NPY: Cys-Thr Arg Gln Arg Tyr-Cys.

[0052] 1.13 Resin drying: Wash the resin 3 times with methanol, then filter and dry by suction.

[0053] 2. Cleavage of polypeptide from the resin

[0054] 2.1 Preparation of cleavage reagent

[0055] Taking 100 mL of cleavage reagent as an example, the formula for preparing the cleavage reagent is: 20 mL of TFE + 80 mL of DCM. Set aside. Generally, the preparation amount is 20 mL of cleavage reagent for 1 g of resin.

[0056] 2.2 Cleavage of polypeptide

[0057] Weigh the resin after suction drying, add the cleavage solution and react for 2 h. Then filter and rotary evaporate the filtrate to obtain the crude peptide.

[0058] 2.3 Coupling of small molecule

[0059] Mix the fully protected peptide, HOBT and small molecule in equimolar amounts, dissolve them in DMF, add equimolar DIC and stir for 2 h, then rotary evaporate and freeze-dry.

[0060] 2.4 Polypeptide cleavage

[0061] Taking 100 ml of cleavage reagent as an example, the formula for preparing the cleavage reagent is: 95 mL of TFA + 1 mL of water + 2 mL of EDT + 2 mL of Tis. Set aside. Generally, the preparation amount is 10 mL of cleavage reagent for 1 g of resin. Wash 1 mL of cleavage reagent with 10 mL of ether and centrifuge to obtain the crude product.

[0062] 3. Polypeptide purification

[0063] Chromatographic column: 20 * 250 mm diagesol 8 μm

[0064] Mobile phase: A: 0.1% TFA in water; B: 0.1% TFA in acetonitrile

[0065] Flow rate: 10 mL / min

[0066] Load the sample onto the column through pump A, then run with 10% acetonitrile for 5 min to balance and start running the gradient.

[0067]

[0068] Prepare, collect and detect the sample peak. Analyze the purity to be greater than 80%, and collect it into a beaker.

[0069] Preparation of disulfide bond:

[0070] Add water with the same volume as the sample in the beaker, adjust the pH to 8 with ammonium bicarbonate, and stir overnight to form disulfide bonds.

[0071] Load the sample onto the column using pump A, and perform preparation and purification using the same preparation method as above. Prepare and collect the sample peak for detection, and analyze the purity to be greater than 95%.

[0072] 4. Freeze-dry the sample

[0073] Transfer the sample into a freeze-drying bottle and place it in a freeze dryer for 24 h to obtain the pure product.

[0074] Structural characterization of Cys-Thr Arg Gln Arg Tyr-Cys-Val-Cit-Cytisine:

[0075] The HPLC results are shown in Figure 1 , and the MS results are shown in Figure 2 .

[0076] The structural formula is:

[0077]

[0078] Select Val-Cit as the linker to connect the polypeptide and cytisine, denoted as Cys-Thr Arg Gln Arg Tyr-Cys-Val-Cit-Cytisine.

[0079] The beneficial effects of the present invention are demonstrated by the following pharmacodynamic experiments.

[0080] Experimental Example 1 In vitro experiments of cytisine and cytisine-polypeptide conjugate of the present invention

[0081] 1. Cell culture, grouping and drug administration

[0082] Human lung fibroblasts MRC-5 are cultured in a culture medium containing 10% fetal bovine serum, adherently cultured at 37 °C and 5% CO2. When the cell confluence reaches about 90% of the bottle wall area, digest and passage the cells with 0.25% trypsin. The cell culture experiment is divided into 6 groups: Ctrl group, TGF-β + PBS group, TGF-β + cytisine CYT (20 μM) group, TGF-β + PDC-L (2.5 μM) group, TGF-β + PDC-M (5 μM) group, and TGF-β + PDC-H (10 μM) group. Among them, the PDC group is the cytisine-polypeptide conjugate prepared in Example 1.

[0083] 2. Detect the proliferation of MRC-5 stimulated by TGF-β (5 ng / mL) with the EDU experiment using PDC

[0084] (1) Cell culture and drug treatment

[0085] Inoculate the MRC-5 cell suspension (cell suspension density is 5×10 4 cells / mL, 100 μL / well) in a 96-well cell culture plate. After the cells are cultured overnight and restored to the normal state, pretreat with low (2.5 μM), medium (5 μM), and high-dose (10 μM) of cytisine CYT and PDC for 2 h, and then stimulate with TGF-β (5 ng / mL) for 24 h. The specific groups are as follows: Ctrl group, TGF-β + PBS group, TGF-β + cytisine CYT group, TGF-β + PDC-L group, TGF-β + PDC-M group, and TGF-β + PDC-H group.

[0086] (2) EdU labeling

[0087] According to the results of the preliminary experiment, the final concentration of EdU used is set at 10 μM (1×), and the EdU (10 mM) is diluted 1 / 500 with cell culture medium to obtain a 2× EdU working solution (20 μM). Add an equal volume of the pre-warmed EdU working solution to the 96-well plate to make the final concentration of EdU in the 96-well plate 10 μM, and continue to incubate the cells for 2 h.

[0088] (3) Cell fixation and permeabilization

[0089] a. After the EdU-labeled cells are completed, remove the culture medium, wash with PBS 1 - 2 times, 5 min each time. Then add 50 μL of 4% paraformaldehyde to each well and fix at room temperature for 15 min;

[0090] b. Remove the fixing solution, add 50 μL of 2 mg / mL glycine to each well, incubate on a shaker for 5 min, and then discard the glycine solution;

[0091] c. Add 100 μL of PBS to each well to wash the cells 3 times, 3 - 5 min each time;

[0092] d. Remove the PBS, add 50 μL of permeabilization solution (PBS containing 0.3% Triton X-100) to each well, and incubate at room temperature for 10 - 15 min;

[0093] e. Remove the permeabilization solution, add 100 μL of PBS to each well to wash the cells 1 - 2 times, 3 - 5 min each time.

[0094] (4) Apollo staining:

[0095] a. Prepare the Apollo staining reaction solution according to the kit instructions;

[0096] b. Remove the washing solution from the previous step, add 50 μL of Apollo staining reaction solution to each well, gently shake the culture plate to ensure that the reaction mixture can evenly cover the sample, and incubate at room temperature in the dark for 30 min;

[0097] c. Aspirate the Apollo staining reaction solution and wash 3 times with PBS for 3 - 5 minutes each time.

[0098] (4) Nucleus staining:

[0099] a. After aspirating the washing solution, add 50 μL of ready - to - use DAPI staining solution to each well and incubate in the dark at room temperature for 10 minutes.

[0100] b. Aspirate the DAPI staining solution and wash 3 times with PBS for 3 - 5 minutes each time.

[0101] Subsequently, a high - content cell drug analysis system can be used for fluorescence detection and analysis of the proportion of EdU - positive cells; DAPI emits blue fluorescence with a maximum excitation wavelength of 346 nm and a maximum emission wavelength of 460 nm; Apollo 643 emits red fluorescence with a maximum excitation wavelength of 653 nm and a maximum emission wavelength of 667 nm.

[0102] 3. Cell migration (Transwell) experiment to detect the migratory effect of PDC on MRC - 5 stimulated by TGF - β (5 ng / mL). The cell scratch assay is to scratch the cell monolayer and capture images regularly under the microscope to study cell migration, repair ability, and cell - cell interaction.

[0103] The specific steps of the scratch assay are as follows:

[0104] (1) Use a marker pen to evenly draw horizontal lines on the back of the 6 - well plate (with the help of a ruler), crossing the wells, and draw 3 horizontal lines for each well.

[0105] (2) Inoculate the cell suspension (the density of the MRC - 5 cell suspension is 1×10 5 cells / mL, 2 mL / well; the density of the HPMEC cell suspension is 2×10 5 cells / mL, 2 mL / well) into the 6 - well plate. After the cells adhere overnight, observe the cell confluence rate under the microscope. When it reaches 100%, scratching can be carried out.

[0106] (3) Use a 200 - μL pipette tip against the ruler to scratch vertically across the horizontal lines on the back. The pipette tip should be perpendicular to the bottom of the plate and draw the lines quickly with a uniform force. Draw 2 vertical lines for each well. Discard the culture medium, wash twice with PBS to remove the scratched - off cells, add serum - free medium and perform drug treatment.

[0107] (4) Take pictures under a 4 - fold microscope. At 0 h, take 1 picture above and below the intersection of the scratch and the horizontal line for each well, a total of 12 pictures per well; ensure that the scratch is centered and vertical, and pay attention to the consistent background. After taking the pictures, continue to culture the cells in the incubator routinely. Take samples and take pictures again after 24 h.

[0108] (5) The scratch area was analyzed using ImageJ software with the find - edges method. The specific steps are as follows:

[0109] a. Open the picture in ImageJ software. Click on Image and select the picture type as 8 - bit.

[0110] b. Select Process - Enhance Contrast, check Normalize, and generally select a value of 0.5%; just make the boundary between the cell edges at the scratch and the background obvious.

[0111] c. Select Process - Find Edges, which will greatly enhance the contrast between cells and the background through binary processing.

[0112] d. Click on Image - Adjust - Threshold in sequence. By adjusting the threshold, make the mask fill the scratch; then click Apply for binary processing, and as a result, the picture becomes black - and - white.

[0113] e. After binary processing, the background color is uniform. You can directly use the Magic Wand tool to select the scratch, create a selection area, and select the actual scratch area.

[0114] f. Click Analyze - Measure to calculate the area of the selection area (i.e., the scratch area) of this picture.

[0115] Data processing was carried out using Excel. The formula for calculating the cell migration rate is: Cell migration rate (scratch healing rate)=(Initial scratch area - Scratch area at time t) / Initial scratch area×100%

[0116] 4. Results of in vitro experiments: Treatment with the PDC drug can inhibit the proliferation and migration of TGF - β - stimulated MRC - 5. The specific results are as follows:

[0117] 4.1. Results of the EDU experiment showed that compared with the PBS group, treatment with 20 μM cytisine (CYT) could reduce the number of EDU - positive MRC - 5 cells, and PDC at 2.5, 5, and 10 μM could dose - dependently reduce the number of EDU - positive MRC - 5 cells, indicating inhibition of the proliferation of MRC - 5 cells. Moreover, the effect of 10 μM PDC was better than that of 20 μM cytisine, and the differences were all statistically significant (P < 0.05), as Figure 3 。

[0118] 4.2. Results of cell migration experiments showed that compared with the PBS group, the administration of 20 μM cytisine (CYT) could increase the distance of Transwell tubules, and 2.5, 5, and 10 μM of PDC could dose-dependently increase the distance of Transwell tubules, suggesting the inhibition of the migration of MRC-5 cells. Moreover, the effect of 10 μM PDC was better than that of 20 μM cytisine, and the differences were statistically significant (P < 0.05), as Figure 4 .

[0119] The above experiments demonstrated that compared with cytisine alone, PDC had less dosage and better effects.

[0120] Experimental Example 2 In vivo experiments of the cytisine-polypeptide conjugate of the present invention

[0121] 1. Animal model establishment, grouping, and administration

[0122] SPF-grade male SD rats (150 - 180 g) were used. Monocrotaline (MCT) was dissolved in anhydrous ethanol solution and then diluted with normal saline (anhydrous ethanol: normal saline = 2:8) to a 10 mg / ml MCT suspension. The suspension was ultrasonically dissolved in an ultrasonic cleaner for 1 hour, shaken overnight at 37 °C in a shaker for sufficient dissolution, and then filtered through a 0.22 μm sterile filter. Rats in the model group (PH group) were injected with MCT solution (50 mg / kg) into the abdomen, and rats in the control group (Sham group) were injected with the same volume of normal saline. The animals after model establishment were raised under normal oxygen and normal pressure conditions for 14 days. All operations on experimental animals were carried out in accordance with the Guide for the Care and Use of Laboratory Animals promulgated by the National Institutes of Health of the United States (National Institutes of Health publication No. 85 - 23, revised in 1996). The PDC drug was prepared with 0.9% normal saline. Fourteen days after model establishment, the rats in the PH group were randomly divided into a normal saline group, a low-dose PDC group (5 mg / kg), a medium-dose PDC group (10 mg / kg), and a high-dose PDC group (20 mg / kg), and were administered by gavage every day. The animals continued to be raised under normal oxygen and normal pressure conditions for 14 days.

[0123] 2. Detection of rat heart function by animal ultrasound

[0124] After the animals were raised under normal oxygen and normal pressure conditions for 28 days, the heart function of rats was detected using a high-resolution small animal ultrasound imaging system. Rats were anesthetized with 3% inhaled isoflurane, and the anesthesia was maintained with 1.5% isoflurane, keeping spontaneous respiration and maintaining a heart rate of 300 - 350 beats per minute. The rats were fixed in the supine position on the operating table, and a cleaning operation was performed. First, the chest surface hair was removed with a hair clipper, and then the remaining hair was removed with hair removal cream. Transthoracic echocardiography was performed using a Vevo3100 small animal ultrasound imaging system equipped with a 20-MHz probe.

[0125] Right heart function ultrasound is mainly completed by collecting the pulmonary artery section, right heart long-axis section, and four-chamber heart section. The probe positioning of the short-axis section of the pulmonary artery is based on the standard parasternal short-axis section. The probe is moved towards the aorta until the aortic valve comes into view. The short-axis section of the pulmonary artery can obtain ultrasonic data such as the pulmonary artery diameter and pulmonary artery blood flow through the B-mode and pulsed Doppler modes, and calculate the pulmonary artery acceleration time (PAT) and pulmonary artery ejection time (PET). The probe positioning of the right heart long-axis section is to translate to the right heart direction on the basis of the parasternal long-axis section until the RV view appears. Under the right heart long-axis section, RVFW and the right heart inner diameter (RVID) can be obtained through the M-mode. The probe positioning of the four-chamber heart section is based on the parasternal short-axis section. The animal's head is tilted towards the lower left corner, and at the same time, the probe angle is rotated and tilted to obtain the cardiac coronal section view cut from the apex of the heart. Under the four-chamber heart section, the RV area and tricuspid annular plane systolic excursion (TAPSE) can be obtained through the B-mode and M-mode.

[0126] 3. Masson staining

[0127] After the ultrasound examination, the rats were anesthetized by intraperitoneal injection of 4% tribromoethanol solution (350 - 400 mg / kg). The hearts were taken to detect the cardiac fibrosis by Masson staining. The chest cavity was exposed with surgical scissors, and the heart tissue was taken out. The residual blood was rinsed with pre-cooled 1×PBS and fixed in 4% paraformaldehyde solution for 24 hours. After the tissue was trimmed flat, it was dehydrated in a dehydrator with gradient alcohol in turn. The trimmed tissue block was infiltrated with wax and then embedded in an embedding machine. After further trimming the wax block, it was sectioned (4 μm) with a paraffin slicer. After the sections were dried in an oven at 60°C to remove moisture, the sections were taken out and stored at room temperature for standby.

[0128] The paraffin sections were dewaxed in xylene and washed with gradient alcohol until hydrated. The sections were immersed in Masson A solution overnight and rinsed with clear water; then continued to be immersed in the equal ratio mixture of Masson B and C for 1 minute and rinsed with clear water; differentiated with 1% hydrochloric acid alcohol and rinsed with clear water; then immersed in Masson D solution for 6 minutes and rinsed with clear water; immersed in Masson E solution for 1 minute, slightly drained and directly put into Masson F solution for 2 - 30 seconds. The sections were rinsed and differentiated with 1% glacial acetic acid, dehydrated, cleared and sealed with anhydrous ethanol. The sections were placed under a microscope for observation, and image acquisition and data analysis were carried out.

[0129] 4. Experimental results

[0130] After 14 days of PDC treatment, it can dose-dependently improve right heart function disorder, reduce myocardial fibrosis, and alleviate right heart remodeling. The specific results are as follows:

[0131] 1. The ultrasound results showed that the indicators related to pulmonary artery and right heart function, such as the pulmonary artery acceleration time (PAT), PAT / pulmonary artery ejection time (PET), right ventricular free wall (RVFW), and tricuspid annular plane systolic excursion (TAPSE) in the rats of the normal saline group were significantly decreased, accompanied by an increase in RVFW, suggesting that the right heart of PH rats was under pressure and volume load states and accompanied by right heart insufficiency. However, administration of the PDC drug could dose-dependently increase PAT, PAT / PET, and TAPSE while decreasing RVFW, and the differences were all statistically significant (P<0.05), as Figure 5 .

[0132] 2. The results of Masson staining showed that compared with the normal saline group, the PDC drug dose-dependently improved the tissue fibrosis of the RV in PH rats, and the differences were all statistically significant (P<0.05), as Figure 6 .

Claims

1. Use of cytisine in the preparation of a medicament for treating cor pulmonale.

Images