CN116785284A - Application of dehydrocorydaline in preparation of medicine for treating idiopathic pulmonary fibrosis - Google Patents

Abstract

The application belongs to the field of medicines, and particularly relates to application of dehydrocorydaline in preparation of a medicine for treating idiopathic pulmonary fibrosis. According to the application, experiments prove that the dehydrocorydaline can reduce the effect of bleomycin-induced pulmonary fibrosis of mice, and can obviously reduce the expression level of fibronectin and smooth muscle actin in fibroblasts; the activation of TGF-beta induced fibroblasts can be inhibited by inhibiting the activation of Smad2, smad3 and Akt signals, thereby inhibiting the progression of pulmonary fibrosis. The application discloses a dehydrocorydaline which has potential value as a novel anti-pulmonary fibrosis drug and can be applied to preparation of the anti-pulmonary fibrosis drug.

Description

Application of dehydrocorydaline in preparation of medicine for treating idiopathic pulmonary fibrosis

Technical Field

The application belongs to the field of medicines, and in particular relates to application of dehydrocorydaline in preparing medicines for delaying and treating idiopathic pulmonary fibrosis.

Background

Idiopathic pulmonary fibrosis (Idiopathic pulmonary fibrosis, IPF) is a chronic progressive interstitial lung disease of unknown, irreversible and fatal etiology, characterized by diffuse alveolitis, alveolar structural disturbance, eventually progressing to pulmonary interstitial fibrosis, characterized clinically by progressive dyspnea, hypoxia with or without dry cough, eventually progressing to respiratory failure leading to death. Unfortunately, the treatment options for IPF patients are very limited, and the two drugs currently approved by the FDA, pirfenidone (Pirfenidone) and nindaanib (Nintedanib), while capable of alleviating the rate of decline of patient Forced Vital Capacity (FVC), slowing the progression of IPF disease, do not successfully reverse the natural progression of IPF and its final end-point. Therefore, the IPF prognosis is extremely poor, the average survival time after diagnosis is only 2.8 years, the survival rate of 5 years is less than 40%, the IPF prognosis has a great threat to human health, and no effective treatment means is available for patients with terminal-stage idiopathic pulmonary fibrosis except for lung transplantation. So that research and treatment of IPF are very important in all countries of the world.

The current research shows that IPF occurs due to injury of alveolar epithelial cells (alveolar epithelial cells, AECs) which in turn cause AECs repair dysfunction, and secrete a large number of cytokines such as transforming growth factor- β (transforming growth factor- β, TGF- β), chemokines, etc., to activate the transdifferentiation of fibroblasts into myofibroblasts, and thus secrete a large number of extracellular matrices, ultimately leading to lung tissue remodeling and scar lung formation. The damage of epithelial cells is the initiating link in the occurrence of IPF, whereas fibroblasts are the final effector link in the formation of IPF fibrosis, and in this process anti-inflammatory and pro-fibrotic factors such as TGF- β play an important role. Therefore, the research on pathogenesis of pulmonary fibrosis and the search of effective targets for treating the pulmonary fibrosis have important scientific values, and are the problems to be solved urgently.

Disclosure of Invention

In one aspect, the application provides the use of dehydrocorydaline or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of pulmonary fibrosis.

In one aspect, the application provides the use of dehydrocorydaline or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of idiopathic pulmonary fibrosis.

In one aspect, the application provides the use of dehydrocorydaline or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of bleomycin-induced pulmonary fibrosis in mice.

In some embodiments, the dehydrocorydaline has a structure represented by formula I:

dehydrocorydaline (DHC) is also called Dehydrocorydaline, and Dehydrocorydaline, belonging to the class of isoquinoline derivatives, and is a quaternary amine alkaloid extracted from corydalis tuber of corydalis genus plant of the Papaveraceae family. It has the main actions of activating blood, promoting qi circulation and relieving pain, and is clinically used for pain due to qi and blood stasis. The alkaloid identified in rhizoma corydalis has reached more than 20, and DHC is one of the active ingredients. The modern pharmacological research proves that DHC has the functions of resisting tumor, resisting bacteria, relieving pain and calming, protecting heart vessels and the like, and the research on the mechanism of DHC is continuously strengthened due to the evaluation of effective treatment and good oral bioavailability of DHC in arrhythmia diseases, gastric and duodenal ulcers and dysmenorrheal in the current world, and the research on the aspect of resisting pulmonary fibrosis by dehydrocorydaline has no related report and patent report.

In some embodiments, the disease is selected from idiopathic pulmonary fibrosis.

In some embodiments, the dehydrocorydaline or pharmaceutically acceptable salt thereof treats idiopathic pulmonary fibrosis by inhibiting activation of Smad and/or Akt signals.

In some embodiments, the dosage form of the drug is selected from any one of the pharmaceutically acceptable dosage forms.

In some embodiments, the dosage form is selected from one or more of a tablet, a granule, a capsule, a pill, an oral liquid, an injection, a mixture, a liposome.

In some embodiments, the mode of administration of the drug is selected from one or more of oral administration, topical administration, intranasal administration, systemic administration, intravenous administration, subcutaneous administration, intramuscular administration, intraventricular administration, intrathecal administration, or transdermal administration.

In some embodiments, the treatment is delaying or preventing the formation of pulmonary fibrosis.

In some embodiments, the treatment is to delay or reduce or prevent idiopathic pulmonary fibrosis.

In some embodiments, the dosage of dehydrocorydaline is 5-10mg/kg.

In some embodiments of the application, the dehydrocorydaline can reduce bleomycin-induced pulmonary fibrosis of mice, can be used for preparing anti-idiopathic pulmonary fibrosis drugs, and has good application prospects. Specifically, dehydrocorydaline can inhibit the activation of fibroblasts and the secretion of extracellular matrix, and specific mechanisms of dehydrocorydaline can inhibit the activation of TGF-beta induced fibroblasts by inhibiting the activation of Smad2, smad3 and Akt signals, thereby inhibiting the progress of pulmonary fibrosis.

In one aspect, the application provides the use of dehydrocorydaline, or a pharmaceutically acceptable salt thereof, as or in the preparation of a Smad inhibitor.

In one aspect, the application provides the use of dehydrocorydaline, or a pharmaceutically acceptable salt thereof, as or in the preparation of an Akt inhibitor.

In one aspect, the application provides a Smad inhibitor comprising dehydrocorydaline or a pharmaceutically acceptable salt thereof.

In some embodiments, the Smad is selected from at least any one of Smad2, smad 3.

In one aspect, the application provides an Akt inhibitor comprising dehydrocorydaline or a pharmaceutically acceptable salt thereof.

In one aspect, the application provides a method of inhibiting activation of lung fibroblasts and/or secretion of extracellular matrix, comprising providing to the lung fibroblasts dehydrocorydaline or a pharmaceutically acceptable salt thereof.

In one aspect, the present application provides a therapeutic system for treating pulmonary fibrosis, the therapeutic system comprising:

1) A signal receiving means configured to receive and display a signal that the patient is a pulmonary fibrosis subject or that the patient is not a pulmonary fibrosis subject;

2) An administration member configured to administer a drug comprising dehydrocorydaline or a pharmaceutically acceptable salt thereof to a patient suffering from pulmonary fibrosis in accordance with a result of the signal receiving member indicating that the patient is a subject of pulmonary fibrosis.

In some embodiments, the dehydrocorydaline has a structure represented by formula I:

in some embodiments, the dosage form of the drug is selected from any one of the pharmaceutically acceptable dosage forms;

in some embodiments, the dosage form is selected from one or more of a tablet, a granule, a capsule, a pill, an oral liquid, an injection, a mixture, a liposome;

in some embodiments, the mode of administration of the drug is selected from one or more of oral administration, topical administration, intranasal administration, systemic administration, intravenous administration, subcutaneous administration, intramuscular administration, intraventricular administration, intrathecal administration, or transdermal administration.

In some embodiments, the pulmonary fibrosis is selected from idiopathic pulmonary fibrosis;

in some embodiments, the pulmonary fibrosis is selected from bleomycin-induced pulmonary fibrosis in mice.

According to the application, experiments prove that the dehydrocorydaline can reduce the effect of bleomycin-induced pulmonary fibrosis of mice, and can obviously reduce the expression level of fibronectin and smooth muscle actin in fibroblasts; dehydrocorydaline can inhibit TGF- β -induced fibroblast activation by inhibiting activation of Smad2, smad3 and Akt signals, thereby inhibiting the progression of pulmonary fibrosis.

Drawings

FIG. 1 shows lung tissue pathology in mice after 21 days of treatment with (A) control and bleomycin, H & E staining, sirius red staining, pinus massoniana staining, x 100; (B) Real-time fluorescent quantitative PCR results of mRNA levels of fibrosis index in lung tissue of each group of mice, fibronectin, collagen i, type one Collagen, < P0.05.

FIG. 2 shows immunofluorescent staining of lung tissue fibrosis index of mice in each group, x 400, α -SMA for α -smooth muscle actin for fibronectin and Collagen I for Collagen type I.

FIG. 3 shows (A) Western blot detection of expression levels of fibronectin and alpha-SMA in human lung primary fibroblasts after TGF-beta 1 stimulation; (B) Immunofluorescence detection of expression level of Collagen I in human lung primary fibroblasts after TGF- β1 stimulation, alpha-SMA was alpha-smooth muscle actin, fibronectin, collagen I was Collagen type I.

FIG. 4 shows Western blot detection of expression levels of p-Smad2, p-Smad3, total Smad2/3, p-Akt and Akt in primary human lung fibroblasts following TGF- β1 stimulation.

Detailed Description

The technical solution of the present application is further illustrated by the following specific examples, which do not represent limitations on the scope of the present application. Some insubstantial modifications and adaptations of the application based on the inventive concept by others remain within the scope of the application.

Experimental materials and methods

Experimental mice

All experimental mice are wild C57BL/6 mice, 9-10 weeks old and male, purchased from Jiangsu Ji-kang biotechnology Co., ltd (SCXK 2018-0008), and animal feeding and experiments are completed in the national institutes of science and research building of the university of China. All animal experiment related procedures strictly follow the relevant guidelines prescribed by the national institutes of health, and have been approved by the institutional animal care committee of the same hospital at the university of science and technology in China.

Human lung tissue

According to the IPF diagnosis standard provided by ATS/ERS, collecting a sample of a patient subjected to lung tissue surgical excision (clinical diagnosis of lung tumor property to be checked, imaging of no fibrosis, postoperative pathological diagnosis of benign lesions, and leaving normal lung tissue with the far end of the lesion position larger than 5 cm) in a hospital after the patient and family members agree. The study was approved by the ethical committee of the same hospital and informed consent was obtained from the patient.

Animal model

Wild type mice of 9 to 10 weeks old were anesthetized with 1% sodium pentobarbital and then intratracheal injection of Bleomycin (BLM) (2 mg/kg, dissolved in saline) and mice of the control group were given the same dose of saline. The mice were randomly divided into 4 groups (DMSO group, blm+dmso group, DHC group, dhc+blm group) of 6 mice each. The blm+dmso group and dhc+blm group were treated with BLM on day 0, while the remaining two groups were treated with physiological saline, mice were intraperitoneally injected with dehydrocorydaline at a dose of 5mg/kg on days 12, 14, 16, 18, 20, and dhc+blm groups, while the remaining two groups were treated with DMSO solvent, and lung tissue was left by killing all mice on day 21.

Experimental cell

The human lung tissue specimen is placed in an aseptic 1.5ml EP tube, tracheal tissue is peeled off by scissors and forceps, the lung tissue is sheared and spread in a 10cm cell dish, the lung tissue specimen is attached to a culture dish, and a high-sugar DMEM culture medium containing 10% fetal calf serum is used for culturing. The fibroblast is attached to the culture dish for growth on days 3-4 after the lung tissue block is attached. And taking cells of 3 to 6 generations after passage for subsequent experiments.

Experimental materials and instruments

High sugar DMEM medium, fetal bovine serum, purchased from Gibco company, usa; pancreatin was purchased from marhan family rayleigh limited; trizol, reverse transcription and real-time fluorescent quantitative PCR kit were purchased from Takara corporation, japan; RIPA lysate was purchased from Beyotime company, shanghai; PVDF membranes were purchased from Milipore, usa; ECL luminescence was purchased from google biology inc; the incubator is purchased from thermo fisher company, usa; biological microscopes were purchased from Olympus corporation, japan; real-time fluorescent quantitative PCR instrument was purchased from Bio-Rad company, usa; nanoDrop microspectrometer was purchased from thermo fisher, usa. Bleomycin was purchased from MCE company; recombinant human TGF-. Beta.1 was purchased from PeproTech, inc. of America.

Tissue section staining

Left lung was infused intratracheally with 4% paraformaldehyde for 24h followed by paraffin embedding and slicing to 5 μm thickness. Following dewaxing, the sections were HE stained, sirius red stained and Masson stained using established techniques. For immunofluorescent staining, sections were permeabilized and blocked after deparaffinization, then section tissues were incubated with either a fibrinectin antibody or a Collagen i antibody or an a-SMA antibody, then fluorescent secondary antibodies were incubated, and photographs were taken using an olympus microscope.

Real-time quantitative PCR experiments

Lung tissue and cellular RNA was extracted with Trizol reagent and RNA concentration and quality was determined using NanoDrop spectrophotometry. Real-time fluorescent quantitative PCR is performed according to the instructions of the reverse transcription and real-time fluorescent quantitative PCR kitAnd (3) reacting. Primers were purchased from Nanjing Optimum Biotechnology Co., ltd., 2 ^-ΔΔCt The method calculates the relative expression of each target gene. Primer sequences are shown in Table 1:

TABLE 1

Note that: fibronectin; collagen I is collagen I, and all of these are mouse-derived genes.

Western blot assay

Western blot analysis was performed according to standard protocols. One antibody includes collagen I, fibronectin, alpha-smooth muscle actin (alpha-SMA) (Proteintech, 1:1000), GAPDH (SantaCruz 1:1000), p-Smad2, p-Smad3, total Smad2/3, p-AKT, AKT (CST, 1:1000). Detection was performed using a chemiluminescent substrate system.

Statistical analysis

All experiments in this study were repeated at least 3 times, the experimental data are expressed as + -s, student's t test was performed using GraphPad Prism (version 8.0) software, and P <0.05 was statistically significant.

Experimental results

Dehydrocorydaline can relieve bleomycin-induced pulmonary fibrosis in mice

The use of bleomycin airway injection to induce pulmonary fibrosis in mice is internationally accepted and most commonly used animal model modeling method for pulmonary fibrosis. We used this method to investigate the effect of dehydrocorydaline on pulmonary fibrosis in mice by intraperitoneal administration of dehydrocorydaline to mice starting at 5mg/kg dose on day 12 of induction with DMSO-containing solution as a control after induction of pulmonary fibrosis in mice.

As shown in fig. 1A, we found that dhc+blm group with dehydrocorydaline intervention had reduced pulmonary fibrosis compared to dmso+blm group, which were both free of pulmonary fibrosis, after normal case staining (H & E staining) and collagen staining (sirius scarlet staining and masson staining) of the lung tissue sections of mice.

Similarly, as shown in FIG. 1B, detection of the mRNA levels of the fibrosis markers (fibronectin and Collagen I) in lung tissue also showed that dehydrocorydaline has a significant inhibitory effect on bleomycin-induced pulmonary fibrosis in mice.

As shown in FIG. 2, immunofluorescent staining of tissue sections (fibronectin, collagen I, and smooth muscle actin. Alpha. -SMA) showed the same results. The above results indicate that dehydrocorydaline can alleviate bleomycin-induced pulmonary fibrosis in mice.

Dehydrocorydaline inhibits fibroblast activation in vitro

Fibroblasts are the primary producer of the extracellular matrix deposited in pulmonary fibrosis, one of the most important effector cells in pulmonary fibrosis. To explore the cause of the inhibitory effect of dehydrocorydaline on lung fibers, we studied its effect on fibroblast activation capacity in primary human lung fibroblasts using dehydrocorydaline.

As shown in fig. 3A, western blot results demonstrate that dehydrocorydaline significantly reduced the expression levels of fibronectin and smooth muscle actin in fibroblasts following TGF- β1 stimulation (P-value < 0.05);

as shown in fig. 3B, immunofluorescent staining showed a significant decrease in collagen type one expression levels following the use of dehydrocorydaline. These results suggest that dehydrocorydaline inhibits fibroblast activation and secretion of extracellular matrix.

Dehydrocorydaline inhibits activation of Smad2, smad3 and Akt signals

Phosphorylation is an essential component of intracellular signaling. Smad pathways (including Smad2, smad 3) are one of the important pathways downstream of TGF- β, with phosphorylated Smad2, smad3 being its active form.

We explored the signaling pathway that dehydrocorydaline acts to inhibit the activation of fibroblasts using primary human lung fibroblasts, as shown in figure 4, and found that dehydrocorydaline can reduce p-Smad2 and p-Smad3 levels after one hour of TGF- β induction.

Protein kinase B, akt, also known as PKB or Rac, plays an important role in cell survival and apoptosis, and we have also found that dehydrocorydaline can reduce p-Akt levels after one hour of TGF- β induction. It is thus known that dehydrocorydaline can inhibit the activation of TGF- β -induced fibroblasts by inhibiting the activation of Smad2, smad3 and Akt signals, thereby inhibiting the progression of pulmonary fibrosis.

Other parts not described in detail are prior art. Although the foregoing embodiments have been described in some, but not all, embodiments of the application, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the application.

Claims (10)

1. Use of dehydrocorydaline or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of pulmonary fibrosis.

2. Use of dehydrocorydaline or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of idiopathic pulmonary fibrosis.

3. Use of dehydrocorydaline or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of bleomycin-induced pulmonary fibrosis in mice.

4. Use according to any one of claims 1 to 3, wherein the dehydrocorydaline has the structure according to formula I:

5. the use according to any one of claims 1 to 3, wherein the dehydrocorydaline or a pharmaceutically acceptable salt thereof treats idiopathic pulmonary fibrosis by inhibiting activation of Smad and/or Akt signals.

6. A use according to any one of claims 1 to 3, wherein the dosage form of the medicament is selected from any one of the pharmaceutically acceptable dosage forms;

preferably, the dosage form is selected from one or more of tablets, granules, capsules, pills, oral liquids, injection, mixture and liposome.

7. The use according to any one of claims 1 to 3, wherein the mode of administration of the medicament is selected from one or more of oral administration, topical administration, intranasal administration, systemic administration, intravenous administration, subcutaneous administration, intramuscular administration, intraventricular administration, intrathecal administration or transdermal administration.

8. A Smad inhibitor or Akt inhibitor, comprising dehydrocorydaline or a pharmaceutically acceptable salt thereof;

preferably, the Smad is at least any one selected from Smad2 and Smad 3.

9. A method of inhibiting activation and/or extracellular matrix secretion of a lung fibroblast, comprising providing to the lung fibroblast dehydrocorydaline or a pharmaceutically acceptable salt thereof.

10. A therapeutic system for treating pulmonary fibrosis, the therapeutic system comprising:

1) A signal receiving means configured to receive and display a signal that the patient is a pulmonary fibrosis subject or that the patient is not a pulmonary fibrosis subject;

2) An administration member configured to administer a drug comprising dehydrocorydaline or a pharmaceutically acceptable salt thereof to a patient suffering from pulmonary fibrosis in accordance with a result of the signal receiving member indicating that the patient is a subject of pulmonary fibrosis.