Application of peimisine in preparation of anti-fibrosis drugs

Abstract

The invention discloses application of peimisine in preparation of an anti-fibrosis medicine, and belongs to the technical field of biological medicines. According to the application, mouse pulmonary interstitial fibrosis is induced through bleomycin, the treatment effect of peimine on pulmonary fibrosis is observed, and the influence of peimine on the pulmonary fibrosis process is explored. Results show that peimisine can reduce mouse lung index increase caused by bleomycin, improve damage of pulmonary alveolar structures of mice with pulmonary fibrosis, reduce aggregation of inflammatory cells, reduce serum fibrosis factors TGF-beta1 and inflammatory factors IL-2 of the mice with pulmonary fibrosis, and reduce the number of macrophages and neutrophils in BALF of the mice with pulmonary fibrosis. The peimisine has an obvious regulating effect on MRC-5 cell abnormal proliferation induced by TGF-beta1, can improve in-vivo pulmonary fibrosis induced by bleomycin by regulating the process of extracellular matrix and intercellular matrix, has an obvious treatment effect on mouse pulmonary fibrosis induced by bleomycin, is beneficial to development and utilization of peimisine, and has a good application prospect. And an effective method is provided for clinical treatment of pulmonary fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to the application of fritillaria in the preparation of antifibrotic drugs. Background Technology

[0002] Pulmonary fibrosis is a chronic, progressive interstitial lung disease that severely reduces patients' quality of life. Clinically, pirfenidone and nintedanib are the two main marketed drugs, offering good treatment results, but both have certain side effects. Furthermore, there is no definitive cure for pulmonary fibrosis; lung damage cannot be repaired, and currently, only symptom relief is possible. Research indicates that in the field of pulmonary fibrosis, highly contractile myofibroblasts resist apoptosis and excessively synthesize collagen, leading to abnormal ECM deposition and excessive EMT transformation. Currently, Western medicine primarily uses hormone and immunosuppressant therapy to treat pulmonary fibrosis, but the treatment effects are not entirely satisfactory and certain side effects exist. Pulmonary fibrosis is not named in traditional Chinese medicine literature; its symptoms can be categorized as "lung obstruction," "lung atrophy," and "cough," among others. Clinical practice shows that traditional Chinese medicine has certain advantages in treating pulmonary fibrosis. Veratrum-type isosteroidal alkaloids, as a major pharmacologically active component of the Fritillaria genus, possess excellent biological activity; these alkaloids are abundant in Fritillaria cirrhosa.

[0003] The main pathological change in fibrosis is the proliferation of extracellular matrix (ECM) to repair damaged tissue, but this proliferation lacks the structure and function of parenchymal cells. The continued development of fibrosis leads to structural damage and functional loss of organs. Pulmonary fibrosis is a chronic, progressive interstitial lung disease with a complex mechanism. Clinically, it involves alveolar epithelial cell damage, excessive proliferation of pulmonary fibroblasts (FB), massive ECM deposition, and destruction of lung tissue structure, ultimately leading to respiratory failure and decreased lung function. In current TCM prevention and treatment of pulmonary fibrosis, its related mechanisms include: anti-free radical damage, regulation of the body's immune function, inhibition of inflammatory factor release, reduction of stress response intensity, interference with collagen metabolism, and reduction of collagen formation. These mechanisms include TGF-β / Smad, PI3K / Akt, Nrf2 / Akt, SphK1 / S1P, and CaSR / PLC-γ1, and these signaling pathways are interconnected and mutually influential. The progression of pulmonary fibrosis also involves oxidative stress, inflammation, and immune processes. The cause of this disease remains unclear, and therefore there are currently no available treatments or medications.

[0004] Fritillaria cirrhosa is an isosteroidal alkaloid that has therapeutic effects on cough, asthma, and acute lung injury, and can also inhibit liver fibrosis in rats. However, there are no reports on whether Fritillaria cirrhosa has an inhibitory effect on pulmonary fibrosis, nor are there any reports on the application of Veratrum-type isosteroidal alkaloids derived from the Fritillaria genus in the prevention and treatment of pulmonary fibrosis. Therefore, this invention contributes to the development and utilization of Fritillaria cirrhosa, providing an effective method for the clinical treatment of pulmonary fibrosis. Summary of the Invention

[0005] To address the problems mentioned in the background section, this invention establishes a bleomycin-induced mouse model of pulmonary interstitial fibrosis and observes the therapeutic effect of fritillaria on pulmonary fibrosis. This also contributes to the development and utilization of fritillaria, providing an effective method for the clinical treatment of pulmonary fibrosis.

[0006] The purpose of this invention is to provide the application of fritillaria in the preparation of antifibrotic drugs.

[0007] Furthermore, the fibrosis is pulmonary fibrosis or liver fibrosis.

[0008] Furthermore, the antifibrotic drug uses fritillaria as its active ingredient and also contains pharmaceutically acceptable excipients; the dosage forms of the drug include: injections, tablets, capsules, granules, syrups, oral liquids, suppositories, pills, and powders.

[0009] Furthermore, the injection is an intraperitoneal injection or an intravenous injection; when the injection is an intraperitoneal injection, the concentration of fritillaria in the injection is 0.4 mg / mL.

[0010] Furthermore, the dosage of fritillaria in the antifibrotic drug is 2-6 mg per kilogram of body weight in animals.

[0011] Furthermore, the fritillary cin exerts its anti-fibrotic effect by reducing the production of α-SMA, vimentin, and type I collagen, and by decreasing the mRNA expression level of fibrin or vimentin.

[0012] Furthermore, the fritillaria cirrhosa exerts its anti-fibrotic effect by regulating the abnormal proliferation of MRC-5 cells induced by TGF-β1.

[0013] Furthermore, the fritillaria cirrhosa exerts its anti-fibrotic effect by increasing the mRNA expression level of E-cadherin.

[0014] Furthermore, the fritillaria cirrhosa exerts its anti-fibrotic effect by increasing the mRNA expression level of E-cadherin.

[0015] Furthermore, the fritillaria exerts its anti-fibrotic effect by reducing the number of macrophages or neutrophils in bronchoalveolar lavage fluid.

[0016] The beneficial effects of this invention are: 1. This invention found that mice with pulmonary fibrosis exhibited obvious symptoms of pulmonary fibrosis. Compared with mice with pulmonary fibrosis, administration of Fritillaria cirrhosa alleviated the decrease in body weight and the increase in lung index, especially after treatment with high doses of Fritillaria cirrhosa. The accumulation of inflammatory factors, increased collagen content, and significant damage to alveolar structure in the model group are important pathological features of pulmonary fibrosis. After intervention with different doses of Fritillaria cirrhosa, lung damage and inflammation in mice with pulmonary fibrosis were significantly reduced, and collagen content decreased. The treatment effect of medium doses was comparable to that of high doses.

[0017] 2. This invention reveals that fritillaria cirrhosa can improve bleomycin-induced pulmonary fibrosis in vivo by regulating the processes of the extracellular matrix and intercellular matrix. Under different doses of fritillaria cirrhosa intervention, lung injury and inflammation in pulmonary fibrosis mice were significantly reduced, and the levels of liver fibrosis factor (TGF-β1) and inflammatory cytokines (IL-2) and collagen content in mice were decreased. The therapeutic effect of the medium dose was comparable to that of the high dose, indicating that fritillaria cirrhosa can inhibit TGF-β1-induced proliferation of myofibroblasts in vitro and inhibit the transformation of fibroblasts into myofibroblasts. Attached Figure Description

[0018] Figure 1 shows the effects of fritillaria cirrhosa on body weight and lung index in mice with pulmonary fibrosis; among which... Figure 1 Figure 1B shows the changes in body weight of mice in each group over 21 days. Figure 2 shows the effects of fritillaria cirrhosa on the lungs of mice with pulmonary fibrosis; among them, Figure 2 A is a histological image of a mouse lung stained with HE (×100). Figure 2 B is a histological image of a Masson-stained mouse lung (×100). Figure 2 C represents the pulmonary fibrosis score. Figure 2 D represents the collagen fiber region; Figure 3 shows the effect of fritillaria cirrhosa on cytokine levels in mice with pulmonary fibrosis; among them, Figure 3 A represents the TGF-β1 level. Figure 3 B represents the IL-2 level; Figure 4 The effect of fritillaria cirrhosa on the fibrosis index in mice with pulmonary fibrosis; among which... Figure 4 A represents the level of type I collagen expression. Figure 4 B represents α-SMA level expression. Figure 4 C represents the level of fibronectin expression. Figure 4 D represents the level of vimentin expression. Figure 4 E represents the level of E-cadherin expression; Figure 5 shows the effect of fritillaria cirrhosa on inflammatory cells in mice with pulmonary fibrosis; among them, Figure 5 A represents the results of macrophage flow cytometry. Figure 5 B represents the flow cytometry results of neutrophils. Figure 5 C represents macrophage-level expression. Figure 5 D represents neutrophil level expression; Figure 6 shows the effects of fritillaria on myofibroblasts; among them, Figure 6 A represents the cytotoxicity results of fritillaria cirrhosae. Figure 6 B represents the inhibitory effect of fritillaria on myofibroblasts. Detailed Implementation

[0019] To better understand the content of this invention, the following detailed description is provided in conjunction with specific implementation methods. However, the scope of protection of this invention is not limited to the following embodiments.

[0020] The reagents used in this invention are all commercially available products and can be purchased on the market.

[0021] I. Experimental Materials 1. Laboratory animals Sixty male C57BL / 6 mice, weighing 24-25g, were purchased from the Laboratory Animal Center of Southern Medical University. All mice were housed under specific pathogen-free conditions in a room with a temperature of 25±2°C, humidity of 50±5%, and a 12 / 12-hour light / dark cycle. All mice had free access to food and water. This experiment was approved by the Laboratory Animal Ethics Committee of Southern Medical University.

[0022] 2. Medicines and reagents Fritillaria cirrhosa (>99.2% purity) was purchased from Maclean Biochemical Co., Ltd. (Shanghai, China). Pirfenidone (>98.9% purity) was purchased from Beijing Contini Pharmaceutical Co., Ltd. (Beijing, China). Bleomycin for injection was manufactured by Hanhui Pharmaceutical Co., Ltd. (Hangzhou, China). MRC-5 cells were obtained from KGI Biotechnology Co., Ltd. (Nanjing, China). Masson staining kits were purchased from Gibco Company (USA). Enzyme-linked immunosorbent assay (ELISA) kits for estimating lung levels of TGF-β1 and IL-2 were purchased from Wuhan Elite Biotechnology Co., Ltd. (Wuhan, China). Reverse transcription-polymerase chain reaction (RT-PCR) kits for type I collagen, α-SMA, fibrin, and vimentin were purchased from Toyobo Co., Ltd. (Japan).

[0023] Dissolve 10 mg of fritillaria thiocyanate in 0.3 mL of dimethyl sulfoxide (DMSO), dilute with physiological saline, and bring the volume to 25 mL, resulting in a concentration of 0.4 mg / mL.

[0024] II. Experimental Methods 1. Animal grouping and modeling Male C57BL / 6 mice were randomly divided into 6 groups, with 10 mice in each group: Control group: Intratracheal infusion of normal saline and intraperitoneal injection of normal saline; Model group: Bleomycin 5 mg / kg was infused into the trachea (to induce pulmonary fibrosis), and normal saline was injected into the peritoneum; Pirfenidone group: After bleomycin modeling, pirfenidone 300 mg / kg was injected intraperitoneally; Low-dose group of Fritillaria: After bleomycin modeling, Fritillaria 2 mg / kg was injected intraperitoneally; Medium-dose group of Fritillaria: After bleomycin modeling, Fritillaria 4 mg / kg was injected intraperitoneally; High-dose group of Fritillaria: After bleomycin modeling, Fritillaria 6 mg / kg was injected intraperitoneally.

[0025] Each group received the medication continuously for 21 days, once a day.

[0026] MRC-5 cells were seeded in 96 mL plates and randomly divided into a control group, a model group, a pirfenidone group, and a fritillaria cirrhosa group. Specifically, the control group consisted of normally cultured MRC-5 cells, the model group (treated with 5 ng / ml TGF-β1), the pirfenidone group (treated with 2 μmol / L pirfenidone), and the fritillaria cirrhosa group (156.25 μg / ml).

[0027] III. Sample Set 0.8 ml of blood was collected from the abdominal aorta of anesthetized mice and centrifuged at 4000 rpm for 15 minutes, retaining the supernatant. The serum was frozen at -80°C. The right lung was used for molecular biology experiments, and the left lung was fixed with 4% paraformaldehyde for histopathological analysis. Bronchoalveolar lavage fluid (BALF) was used for flow cytometry analysis to determine the number of inflammatory cells.

[0028] 1. Histopathological analysis The left lung was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), stained with Masson's stain, and sealed. The lung tissue was scored using Ashcroft and the expression of type I collagen was observed under a microscope.

[0029] 2. RT-PCR method for detecting mRNA expression of related genes Total RNA was extracted from cells using the triple sol method, and RNA concentration was determined. RT reactions were performed according to the manufacturer's instructions. cDNA was synthesized and then amplified by PCR. Amplification was performed according to the instructions of Toyobo's PowerSYBR Green PCR Master Mix kit. The reaction conditions were: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, and 60℃ denaturation for 34 s, for 40 cycles. The ΔΔCt value and RQ value were calculated for each group, and the mRNA expression levels of each group were compared.

[0030] 3. Measurement of serum TGF-β1 and interleukin-2 The serum was thawed in a water bath at room temperature. The levels of TGF-β1 and IL-2 in the serum were measured using an ELISA kit according to the kit instructions.

[0031] 4. Measurement of BALF macrophages and neutrophils Centrifuge 500 μL of BALF, precipitate, resuspend in 200 μL of PBS, and place on ice. Dissolve mouse antibodies CD11b, F4 / 80, and LY6G in PBS containing 0.5% BSA. Add 1 μL of each antibody to each tube and stain on ice for 20 minutes. Finally, transfer to flow tubes and run as soon as possible in the dark.

[0032] 5. CCK8 assay for MRC-5 cell viability MRC-5 cells were seeded in 96-cell plates and cultured at 37°C for 24 hours. The experimental groups were divided into a control group (normally cultured MRC-5 cells), a model group (treated with 5 ng / ml TGF-β1), a pirfenidone group (treated with 2 μmol / L pirfenidone), and a fritillaria group (156.25 μg / ml). After 48 hours of culture, each group of MRC-5 cells was incubated with CCK-8 solution for 2 hours, and absorbance was measured. Cell viability was calculated as [(OD treatment group - OD control group / (OD model group - OD control group)] × 100%.

[0033] IV. Statistical Methods Data analysis was performed using Graphpad Prism 8.3.0 software. Data with normal distribution and homogeneous variance were analyzed using one-way ANOVA and multiple comparisons using the least significant difference (LSD) t-test. A p-value < 0.05 was considered statistically significant.

[0034] V. Results Analysis 1. Effects of Fritillaria cirrhosa on body weight and lung index in mice with pulmonary fibrosis After establishing a mouse model of pulmonary fibrosis, the mice exhibited lung damage, decreased appetite and mental state, and gradual weight loss (Figure 1A). Changes in the lung index are a hallmark of pulmonary fibrosis. Following bleomycin modeling, the lung index of the model group mice gradually increased, indicating progressively worsening pulmonary fibrosis. Administration of fritillaria (2 mg / kg, 4 mg / kg, 6 mg / kg) resulted in a dose-dependent decrease in the lung index of the pulmonary fibrosis mice (Figure 1B).

[0035] 2. Effects of Fritillaria cirrhosa on pathological changes in lung tissue HE staining showed that the alveolar structure in the control group was basically normal, with most lung tissue showing good structure and no obvious inflammatory cell infiltration. Compared with the control group, the alveolar septa in the model group were significantly thickened and edematous, with obvious structural damage, impaired alveolar capillary structure, and a large number of inflammatory cells. Compared with the model group, after treatment with fritillaria cirrhosa, the alveolar structural damage and inflammatory cell accumulation in mice were reduced. The therapeutic effect of low-dose fritillaria cirrhosa was weak, while the therapeutic effect of high-dose fritillaria cirrhosa was similar to that of pirfenidone. Figure 2 A).

[0036] As shown in Figure 2, Masson staining revealed that, compared to the control group, the model group had a large number of blue-stained collagen fibers in the peribronchial, perivascular, and alveolar cavities. After treatment with fritillaria cirrhosa, the area of ​​collagen fibers decreased in a dose-dependent manner. Figure 2 B). Fibrosis grade (Fig. 2C) and type I collagen expression (Fig. 2D).

[0037] 3. Effects of Fritillaria cirrhosa on cytokine levels in mice with pulmonary fibrosis As shown in Figure 3, compared with the control group, the serum levels of TGF-β1 and IL-2 in the model group were significantly increased. After administration of different doses of fritillaria cirrhosa, the levels of TGF-β1 and IL-2 decreased. The therapeutic effect of high-dose fritillaria cirrhosa was better than that of low-dose fritillaria cirrhosa, and similar to that of pirfenidone (Figure 3A-B).

[0038] 4. Effects of Fritillaria cirrhosa on fibrosis markers in pulmonary fibrosis mice like Figure 4 As shown, compared with the control group, the mRNA levels of type I collagen, α-SMA, fibrin, and vimentin were significantly increased in the model group, while the mRNA expression level of E-cadherin was decreased. After treatment with different concentrations of fritillaria cirrhosae, compared with the model group, the mRNA levels of type I collagen, α-SMA, fibrin, and vimentin were significantly reduced. Although the high-dose fritillaria cirrhosae group showed a significant therapeutic effect, the pirfenidone group showed a better therapeutic effect (Figures 4A-D). The high-dose group showed an increase in the expression level of E-cadherin mRNA (Figure 4E).

[0039] 5. Effects of Fritillaria cirrhosa on inflammatory cells in mice with pulmonary fibrosis As shown in Figure 5, compared with the control group, the number of macrophages and neutrophils in the model group was significantly increased. Treatment with different concentrations of fritillaria cirrhosae resulted in a dose-dependent decrease in the number of macrophages and neutrophils (Figures 5A-D). 6. Effects of Fritillaria cirrhosa on TGF-β1-induced abnormal proliferation of MRC-5 cells Figure 6 shows the effect of fritillaria cirrhosa on myofibroblasts. First, the safe dosage of fritillaria cirrhosa was explored. The results, shown in Figure 6A, demonstrate the cytotoxicity of fritillaria cirrhosa. In subsequent experiments, 156.25 ug / ml was selected as the dosage for the fritillaria cirrhosa group. Compared to the control group, the cell number in the model group was significantly increased. Compared to the model group, after fritillaria cirrhosa treatment, combined with TGF-β group 1, the cell number in both the pirfenidone group and the fritillaria cirrhosa group was significantly reduced (Figure 6B).

[0040] The experimental results above show that this invention has found that mice with pulmonary fibrosis exhibit obvious symptoms of pulmonary fibrosis. Compared with mice with pulmonary fibrosis, the decrease in body weight and the increase in lung index were alleviated after administration of Fritillaria cirrhosa, especially after treatment with high doses of Fritillaria cirrhosa. The accumulation of inflammatory factors, increased collagen content, and significant damage to alveolar structure in the model group are important pathological features of pulmonary fibrosis. After intervention with different doses of Fritillaria cirrhosa, lung damage and inflammation in mice with pulmonary fibrosis were significantly reduced, and collagen content decreased. The treatment effect of medium doses was comparable to that of high doses.

[0041] TGF-β1 is considered the most important fibrotic factor. It is closely related to immune responses, inflammation, and matrix synthesis. In vitro, TGF-β1-inducing myofibroblasts proliferate very rapidly. In the Fritillaria cirrhosa group of this invention, Fritillaria cirrhosa inhibits the transformation of fibroblasts into myofibroblasts. After bleomycin modeling, the levels of fibrotic factor (TGF-β1) and inflammatory cytokine (IL-2) in the model group mice significantly increased. After Fritillaria cirrhosa intervention, the levels of liver fibrotic factor (TGF-β1) and inflammatory cytokine (IL-2) in mice decreased, with high-dose treatment showing better efficacy than low-dose treatment, similar to pirfenidone.

[0042] ECM remodeling and EMT transformation are the main pathological processes of fibrosis. α-SMA is a typical marker of myofibroblasts. Vimentin is the most important intermediate fibrin protein in interstitial cells. Type I collagen is an essential protein in connective tissue. α-SMA, vimentin, and type I collagen are key effectors in the ECM process. E-cadherin is involved in mediating contact, adhesion, and proliferation between epithelial cells. Fibrin is the main cell adhesion molecule, and both are typical epithelial cell markers of EMT. In the model group, the mRNA levels of type I collagen, α-SMA, fibrin, and vimentin were significantly increased, while the mRNA expression level of E-cadherin was decreased. In this invention, after intervention with different doses of fritillaria, the mRNA levels of type I collagen, α-SMA, fibrin, and vimentin decreased, while the mRNA level of E-cadherin increased. This indicates that fritillaria can regulate the fibrotic process in mice with pulmonary fibrosis. However, the pirfenidone group showed better therapeutic effects.

[0043] Neutrophils, the most numerous immune cells, can be recruited to infection sites, accumulating in large numbers in the lungs and releasing inflammatory mediators such as oxygen free radicals and proteases, leading to inflammatory damage to alveolar epithelial cells and pulmonary capillary endothelial cells. Macrophages are indispensable frontline indicators of host defense. Both are important markers of inflammatory phenotypes. The model group showed a significant increase in the content of both neutrophils and macrophages. After intervention with different doses of fritillaria cirrhosae, the content of both cell types decreased in a dose-dependent manner, and inflammation was suppressed to a certain extent.

[0044] Therefore, this invention experimentally demonstrates that Fritillaria cirrhosa can effectively inhibit the development of bleomycin-induced pulmonary fibrosis in mice, and its mechanism is related to the regulation of ECM and EMT processes. It can inhibit neutrophils and macrophages, alleviating lung damage and inflammation in mice with pulmonary fibrosis. It reduces the production of α-SMA, vimentin, and type I collagen, thus decreasing collagen content. Fritillaria cirrhosa has a significant regulatory effect on TGF-β1-induced abnormal proliferation of MRC-5 cells. Fritillaria cirrhosa has a significant therapeutic effect on bleomycin-induced pulmonary fibrosis in mice.

[0045] In summary, fritillaria can improve bleomycin-induced pulmonary fibrosis in vivo by regulating the processes of the extracellular matrix and intercellular matrix. Under different doses of fritillaria intervention, lung injury and inflammation in pulmonary fibrosis mice were significantly reduced, and the levels of liver fibrosis factor-β1 (TGF-β1) and inflammatory cytokines (IL-2), as well as collagen content, were decreased. The therapeutic effect of the medium dose was comparable to that of the high dose, indicating that fritillaria can inhibit TGF-β1-induced proliferation of myofibroblasts in vitro and inhibit the transformation of fibroblasts into myofibroblasts.

Claims

1. Peimisine for use in the preparation of an anti-fibrosis drug.

2. Use according to claim 1, characterized in that, The fibrosis is pulmonary fibrosis or liver fibrosis.

3. Use according to claim 1, characterized in that, The anti-fibrosis drug takes peimisine as an active ingredient and further comprises pharmaceutically acceptable adjuvants; the dosage form of the drug includes injection, tablet, capsule, granule, syrup, oral liquid, suppository, pill and powder.

4. Use according to claim 3, characterized in that, The injection is intraperitoneal injection or intravenous injection; when the injection is intraperitoneal injection, the concentration of peimisine in the injection is 0.4 mg / mL.

5. The use according to claim 1, characterized in that, The dosage of peimisine in the anti-fibrosis drug is 2 mg-6 mg per kilogram of body weight of animals.

6. The use according to claim 1, characterized in that, Peimisine exerts an anti-fibrosis effect by reducing the generation of α-SMA, vimentin and type I collagen and reducing the mRNA expression level of fibrous protein or vimentin.

7. The use according to claim 1, characterized in that, Peimisine exerts an anti-fibrosis effect by regulating the abnormal proliferation of MRC-5 cells induced by TGF-β1.

8. The use according to claim 1, characterized in that, Peimisine exerts an anti-fibrosis effect by increasing the mRNA expression level of E-cadherin.

9. The use according to claim 1, characterized in that, Peimisine exerts an anti-fibrosis effect by increasing the mRNA expression level of E-cadherin.

10. The use according to claim 1, characterized in that, Peimisine exerts an anti-fibrosis effect by reducing the number of macrophages or neutrophils in bronchoalveolar lavage fluid.

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