Application of lindley eupatorium lactone A in preparation of medicine for preventing or treating pulmonary fibrosis

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of lindley eupatorium lactone A in preparation of medicine for preventing or treating pulmonary fibrosis. According to the research, a lung fibroblast differentiation model induced by a transforming growth factor beta and a mouse pulmonary fibrosis model established by instilling bleomycin into a trachea are adopted, and lindley eupatorium lactone A serving as a potential therapeutic agent is explored for the first time. In the two models, key biochemical indexes and pathological changes are systematically monitored, so that the curative effect of lindley eupatorium lactone A on pulmonary fibrosis is evaluated, and a basis is provided for clinical application of lindley eupatorium lactone A in treating pulmonary fibrosis diseases. The medicine disclosed by the invention comprises an effective component lindley eupatorium lactone A and a pharmaceutically acceptable carrier.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to the application of yemachalide lactone A in preparing a drug for preventing or treating pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis is an interstitial lung disease with unknown causes, diffuse alveolitis and alveolar structural disorder, leading to alveolar and pulmonary interstitial fibrosis, and eventually respiratory failure. At present, its pathogenesis is still unclear. Its pathological characteristics are damage to alveolar epithelial cells and abnormal tissue repair, which leads to the destruction of normal lung tissue structure, replaced by abnormal proliferation of lung fibroblasts and a large number of lung myofibroblasts transformed from lung fibroblasts, forming the so-called fibroblast foci. These cells secrete a large amount of extracellular matrix and synthesize collagen fibers, which eventually lead to the formation of pulmonary fibrosis. Since fibroblast foci are important factors in the progression and poor prognosis of pulmonary fibrosis, the source of fibroblast foci, abnormal transformation of lung fibroblasts, is the key driving factor for the onset of pulmonary fibrosis and the only drug target for the treatment of pulmonary fibrosis. Ultimately, the abnormal transformation of lung fibroblasts is inhibited to prevent the pathological process of pulmonary fibrosis.

[0003] Collagen fibers are important components of the extracellular matrix. Their biochemical components are collagen (collagen, referred to as collagen). Hydroxyproline (HYP) is one of the main components of collagen in the body, accounting for about 13.4% of its total amino acids. The HYP content in tissues can be used as an important indicator of collagen tissue metabolism, measuring collagen content and reflecting interstitial collagen fiber deposition.

[0004] Currently, there are only two therapeutic drugs in clinical practice - nintedanib and pirfenidone, which delay the progression of pulmonary fibrosis by inhibiting the activation and transformation of lung fibroblasts. However, they have more adverse reactions and can only slow down the progression of pulmonary fibrosis in a short period of time, which makes the discovery of new anti-pulmonary fibrosis drugs an urgent need.

[0005] The crude extract of the Chinese medicine Herba Eupatoriae has been made into tablets, granules, syrups, etc. for the treatment of respiratory diseases. It has been confirmed in the early stage that the Herba Eupatoriae sesquiterpenoid extract has anti-pulmonary fibrosis activity, but the anti-pulmonary fibrosis activity of the specific sesquiterpenoid compounds in Herba Eupatoriae is still unclear, which hinders the internationalization of the traditional Chinese medicine Herba Eupatoriae as a scientific modern medicine. Secondly, although the Herba Eupatoriae sesquiterpenoid compounds belong to the same class of compounds with similar parent nuclei, the activities of different compounds vary greatly. In order to discover new structures of pulmonary fibrosis drugs with international potential, the activity of the Herba Eupatoriae sesquiterpenoid lactone monomer compounds was studied on the basis of the previous patents. Summary of the invention

[0006] In order to solve the above-mentioned technical problems, this application provides the following technical solutions:

[0007] The present invention provides application of yemachulide A in preparing a medicine for preventing or treating pulmonary fibrosis.

[0008] Preferably, the phymachus lactone A also includes a pharmaceutically acceptable salt thereof.

[0009] Preferably, the purity of the yamalide lactone A (CAS#: 877822-41-8) is ≥ 98%.

[0010] Preferably, the pharmaceutically acceptable salt is safflower lactone A sodium salt and / or safflower lactone A sulfonate.

[0011] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0012] Preferably, the application in the preparation of a drug for preventing or treating pulmonary fibrosis refers to the preparation of a drug for preventing or treating pulmonary fibrosis, wherein the drug contains a therapeutically effective amount of yamachulide A and a pharmaceutically acceptable carrier. The drug of the present invention can be prepared into tablets, capsules, pills, powders, suppositories, ointments, solutions and suspensions according to methods known in the art. Among them, capsules and tablets suitable for gastrointestinal administration are preferred. When preparing capsules, tablets, pills and powders suitable for oral administration, sucrose, lactose, corn starch, microcrystalline cellulose, carboxymethyl cellulose can be used as carriers or excipients.

[0013] In addition, the medicine of the present invention can also be made into solutions and suspensions suitable for oral administration using methods and auxiliary components known in the pharmaceutical industry. In order to prepare solutions or suspensions suitable for parenteral administration, distilled water, water for injection, isotonic sodium chloride solution or glucose solution, or low concentration (e.g., 1-100 mM) phosphate buffered saline (PBS) can be used as carriers or diluents. One or more other auxiliary components or additives can be added to these parenteral preparations, such as ascorbic acid can be used as an antioxidant, sodium benzoate, etc. can be used as a preservative. Other suitable solubilizers, disintegrants, lubricants, coloring agents, dispersants, or surfactants can also be contained in these dosage forms.

[0014] Furthermore, the drug is used to inhibit TGF-β1-induced lung fibroblast transformation.

[0015] Furthermore, the drug is used to reduce the level of cell myofilament formation in lung fibroblasts.

[0016] Furthermore, the drug is used to increase the gel area of ​​lung fibroblast collagen.

[0017] Furthermore, the drug is used to reduce the expression level of type I collagen in lung fibroblasts.

[0018] Furthermore, the drug is used to reduce the content of hydroxyproline in lung tissue.

[0019] Furthermore, the drug is used to reduce lung tissue inflammation and collagen deposition.

[0020] The present invention also provides a pharmaceutical composition for preventing or treating pulmonary fibrosis, comprising yamachulide A.

[0021] Furthermore, the dosage of the drug is 1-80 mg / kg / d.

[0022] However, the exact dosage should be determined by the clinician based on factors such as the nature and severity of the disease or condition to be treated, the age and weight of the patient, the sensitivity of the patient to the drug to be treated, and the route of administration.

[0023] Furthermore, the pharmaceutical composition is administered by one or more of oral administration, intraperitoneal injection, subcutaneous injection, intravenous injection and intramuscular injection.

[0024] The present invention investigated the anti-pulmonary fibrosis activity of three compounds in safflower, namely, safflower lactone A, safflower lactone G, and safflower lactone I, by using a TGF-β1-induced MRC-5 lung fibroblast model. Figure 7 As shown in the figure above, under the same dosage conditions, yemachulide A can significantly reduce the expression level of α-SMA protein in MRC-5 cells and effectively inhibit the transformation of lung fibroblasts, which is significantly better than the effects of yemachulide G and yemachulide I. In order to illustrate the particularity of the activity of sesquiterpene lactone compounds, the present invention further compares the anti-pulmonary fibrosis activity of yemachulide A with those of ginkgolide A (GA), ginkgolide B (GB), ginkgolide C (GC), and bilobalide (BB). Figure 7 As shown in the figure below, although these compounds from Ginkgo biloba have shown significant biological activity in multiple fields such as anti-inflammation and neuroprotection, no obvious anti-pulmonary fibrosis activity was observed even when the dose was increased to 5 times the dose of Yesamachulide A. This further confirms the advantages of Yesamachulide A in the prevention and treatment of pulmonary fibrosis, and it has a new structural lead compound that is different from existing pulmonary fibrosis drugs.

[0025] Based on the above research findings, the present invention specifically applies for a method for preventing and treating pulmonary fibrosis with yemachalide lactone A.

[0026] The technical solution of the present invention has the following advantages over the prior art:

[0027] 1. The present invention is the first to apply the sesquiterpene compound of the traditional Chinese medicine Herba Cynoglossi, Herba Cynoglossi lactone A, to the classic and widely used pulmonary fibrosis cell and animal models to conduct experimental research on the prevention and treatment of pulmonary fibrosis. The experimental results suggest that Herba Cynoglossi lactone A has anti-pulmonary fibrosis effects.

[0028] 2. The present invention found that the mechanism of action of yemachain lactone A in preventing and treating pulmonary fibrosis is related to its significant inhibition of pulmonary fibroblast transformation.

[0029] The medicine based on the phytosalis lactone A of the present invention has a single component, definite therapeutic effect, and is easy to use, and can be widely used in preparing medicines for preventing and treating pulmonary fibrosis and related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a diagram showing the effect of yemachain lactone A on transforming growth factor-induced lung fibroblast differentiation.

[0031] Figure 2 This is a diagram showing the effect of yamachulide A on cell myofilament formation in lung fibroblasts induced by transforming growth factor.

[0032] Figure 3 Figure 3 Effect of yamachulide A on transforming growth factor-induced contraction of lung fibroblast collagen.

[0033] Figure 4 Figure 3 Effects of yamachulide A on type I collagen production in transforming growth factor-induced lung fibroblasts.

[0034] Figure 5 Effect of yemachain lactone A on the morphology of lung tissue in bleomycin-treated mice (HE staining).

[0035] Figure 6 Effects of yemachain lactone A on the morphology of lung tissue in bleomycin-treated mice (Masson staining).

[0036] Figure 7 To compare the activities of inhibiting TGF-β1-induced lung fibroblast transformation; the upper figure is a comparison of the anti-pulmonary fibrosis activity of sesquiterpene lactone compounds of Mustard, and the lower figure is a comparison of the anti-pulmonary fibrosis activity of Mustard lactone A and other lactone compounds. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0038] Example 1

[0039] 1.1 Experimental Purpose

[0040] To observe the effect of yemachailactone A on transforming growth factor-induced transformation of lung fibroblasts.

[0041] 1.2 Experimental Materials

[0042] Experimental cells: Human lung fibroblast MRC-5, provided by ATCC Culture Collection Center, USA.

[0043] Experimental drug: Eupalinolide A (EA); molecular formula: C 24 H 30 O 9； CAS number: 877822-40-7, purity ≥98%; transforming growth factor, provided by R&D Biotechnology Company, USA.

[0044] Reagents and instruments: α-smooth muscle actin (α-SMA) (ab124694), GAPDH (ab181602) were purchased from Abcam (Cambridge, UK); electrophoresis and membrane transfer apparatus were purchased from Bio-rad, USA; chemiluminescence imager was purchased from Shanghai Tianneng Biotechnology Co., Ltd.

[0045] 1.3 Experimental methods

[0046] Cell culture and administration Human embryonic lung fibroblast MRC-5 cell line was cultured in MEM medium containing 10% FBS at 37°C in a cell culture incubator containing 5% CO2. MRC-5 cells in the logarithmic growth phase were taken to prepare a single cell suspension at a cell density of 2×10 per ml. 4 100 cells were inoculated into 6-well plates, and 2 mL of single cell suspension was added to each well. The cells were starved with MEM medium containing 0.5% FBS every other day. After starvation for 24 h, different treatments were given, including solvent control group, model group (TGF-β12 ng·mL -1 ) and yamarin lactone group A (1 μM), and the cells were harvested at the indicated times.

[0047] 1.4 Protein immunoblotting analysis

[0048] After the cell culture is completed, the cells are placed on an ice box and collected into a centrifuge tube, and then ultrasonically lysed with a cell ultrasonic disruptor in ice water, and the supernatant is taken after centrifugation. The crude protein extract is heated in a 95.0℃ metal bath for 5 minutes to denature the protein, which is the protein sample. The sample is separated using 10% SDS-PAGE electrophoresis and then transferred to a polyvinylidene fluoride membrane (PVDF membrane). The PVDF membrane is blocked with 5% milk for 1 hour and then incubated with the corresponding primary antibody overnight. The next day, the secondary antibody is incubated for 1 hour, and the image is scanned and photographed using the Tianneng Chemical Imaging System. Western blot images are used for relative quantitative analysis of the grayscale of protein bands using Image J.

[0049] 1.5 Experimental Results

[0050] α-Smooth muscle actin (α-SMA) is one of the key markers for the transformation of lung fibroblasts into lung myofibroblasts. The present invention uses Western blot to detect the expression level of α-SMA in lung fibroblasts treated in different ways. Figure 1 As shown in the figure, compared with the control group, the expression level of α-SMA in the lung fibroblast differentiation model induced by transforming growth factor was significantly increased. The expression level of α-SMA in the lung fibroblasts of the yamachulide A group was significantly lower than that of the model group ( Figure 1 These experimental results indicate that yamachulide A can inhibit TGF-β1-induced lung fibroblast transformation and has anti-pulmonary fibrosis activity.

[0051] Example 2

[0052] 2.1 Experimental Purpose

[0053] Effects of yamachulide A on myofilament formation in lung fibroblasts induced by transforming growth factor

[0054] 2.2 Experimental Materials

[0055] Experimental cells: Human lung fibroblast MRC-5, provided by ATCC Culture Collection Center, USA.

[0056] Experimental drug: Eupalinolide A (EA); molecular formula: C24H30O9; CAS number: 877822-40-7, purity ≥98%; transforming growth factor, provided by R&D Biotechnology Company of the United States.

[0057] Reagents and instruments: Rhodamine phalloidin (PHDR1) was purchased from Cytoskeleton (Denver, CO); Olympus X-51 fluorescence inverted microscope was purchased from Olympus Microscopy Co., Ltd., Japan.

[0058] 2.3 Experimental methods

[0059] Cell culture and administration Human embryonic lung fibroblast MRC-5 cell line was cultured in MEM medium containing 10% FBS at 37°C in a cell culture incubator containing 5% CO2. MRC-5 cells in the logarithmic growth phase were taken to prepare a single cell suspension at a cell density of 2×10 per ml. 4 100 cells were inoculated into 6-well plates, and 2 mL of single cell suspension was added to each well. The cells were starved with MEM medium containing 0.5% FBS every other day. After starvation for 24 h, different treatments were given, including solvent control group, model group (TGF-β12 ng·mL -1 ) and yamasulide group A (1 μM), MRC-5 cells were induced with TGF-β1 (2 ng / mL) for 48 h, and then the cells were rinsed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde solution, and then permeabilized with 0.1% Triton-100 and incubated with 100 nM working concentration of rhodamine phalloidin, and placed in the dark at room temperature for 30 min, and then counterstained with 100 nM concentration of 4',6-diamidino-2-phenylindole (DAPI) for 30 s. Finally, the cells were rinsed three times with PBS and photographed using an Olympus X-51 microscope.

[0060] 2.4 Experimental Results

[0061] Myofilament formation is an important cell phenotype in the transformation of lung fibroblasts into lung myofibroblasts. Figure 2 As shown, compared with the control group, in the pulmonary fibrosis cell model induced by transforming growth factor-induced lung fibroblast differentiation, cell myofilament formation was significantly increased (F-actin; red). The level of cell myofilament formation in lung fibroblasts in the nomachulide A group was significantly lower than that in the model group. These experimental results indicate that nomachulide A has anti-pulmonary fibrosis activity.

[0062] Example 3

[0063] 3.1 Experimental Purpose

[0064] Observation of the effect of yemachain lactone A on the contraction of collagen gel of lung fibroblasts induced by transforming growth factor 3.2 Experimental materials

[0065] Experimental cells: Human lung fibroblast MRC-5, provided by ATCC Culture Collection Center, USA.

[0066] Experimental drug: Eupalinolide A (EA); molecular formula: C24H30O9; CAS number: 877822-40-7, purity ≥98%; transforming growth factor, provided by R&D Biotechnology Company of the United States.

[0067] Reagents and instruments: Rat tail collagen (sc-136157) was purchased from Santa Cruz (Dallas, TA); Olympus microscope was purchased from Olympus Microscopy Co., Ltd., Japan.

[0068] 3.3 Experimental methods

[0069] Cell culture and administration: Human embryonic lung fibroblast MRC-5 cell line was cultured in MEM medium containing 10% FBS at 37°C in a cell culture incubator containing 5% CO2. MRC-5 cells in the logarithmic growth phase were taken to prepare a single cell suspension. First, 10×10 4 / mL density of MRC-5 cell suspension and rat tail collagen were mixed in a ratio of 2:1 (cell suspension: collagen), and then the cell suspension was inoculated into a 24-well cell culture plate. After solidification at room temperature for 30 minutes, the gel was dissociated from the plate with a sterilized pipette tip and allowed to rotate easily in the well to confirm that it did not adhere to the bottom of the well. Then 500 μL of MEM culture medium containing 0.5% FBS was added and the cells were placed in a cell culture incubator at 37°C, 5% CO2 for continued culture. On the next day, the gel was stimulated with 2.5 ng / mL of TGF-β1 and 1 μM group A of tadalafil for 48 hours. Finally, the changes in the gel were observed and the gel images were taken. The surface area of ​​each gel was measured using Image J software.

[0070] 3.4 Experimental Results

[0071] After the lung fibroblasts are transformed into lung myofibroblasts in pulmonary fibrosis, their contraction ability is significantly improved. In this example, the present invention uses the contraction of collagen gel containing MRC-5 cells to detect the anti-pulmonary fibrosis activity of yamachulide A. Figure 3 As shown in the figure, compared with the control group, the surface area of ​​the collagen gel containing lung fibroblasts was significantly reduced after treatment with transforming growth factor, while the collagen gel area of ​​lung fibroblasts in the yemachaylactone A group was significantly larger than that in the model group. These experimental results indicate that yemachaylactone A has anti-pulmonary fibrosis activity.

[0072] Example 4

[0073] 4.1 Experimental Purpose

[0074] To observe the effect of yamachulide A on type I collagen production in lung fibroblasts induced by transforming growth factor.

[0075] 4.2 Experimental Materials

[0076] Experimental cells: Human lung fibroblast MRC-5, provided by ATCC Culture Collection Center, USA.

[0077] Experimental drug: Eupalinolide A (EA); molecular formula: C24H30O9; CAS number: 877822-40-7, purity ≥98%; transforming growth factor, provided by R&D Biotechnology Company of the United States.

[0078] Reagents and instruments: collagen I (ab138492) and GAPDH (ab181602) were purchased from Abcam (Cambridge, UK); electrophoresis and membrane transfer apparatus were purchased from Bio-rad, USA; chemiluminescence imager was purchased from Shanghai Tianneng Biotechnology Co., Ltd.

[0079] 4.3 Experimental methods

[0080] Cell culture and administration: Human embryonic lung fibroblast MRC-5 cell line was cultured in MEM medium containing 10% FBS at 37°C in a cell culture incubator containing 5% CO2. MRC-5 cells in the logarithmic growth phase were taken to prepare a single cell suspension at a cell density of 2×10 per ml. 4 100 cells were inoculated into 6-well plates, and 2 mL of single cell suspension was added to each well. The cells were starved with MEM medium containing 0.5% FBS every other day. After starvation for 24 h, different treatments were given, including solvent control group, model group (TGF-β12 ng·mL -1 ) and yamazolide group A (1 μM), and the cells were harvested at the indicated times.

[0081] After the cell culture was completed, the cells were placed on an ice box and collected into a centrifuge tube. Then, the cells were ultrasonically lysed in ice water using a cell ultrasonic disruptor and the supernatant was collected after centrifugation. The crude protein extract was heated in a 95.0°C metal bath for 5 minutes to denature the protein, which was the protein sample. The sample was separated using 10% SDS-PAGE electrophoresis and then transferred to a polyvinylidene fluoride membrane (PVDF membrane). The PVDF membrane was blocked with 5% milk for 1 hour and then incubated with the corresponding primary antibody overnight. The next day, the secondary antibody was incubated for 1 hour, and the Odyssey near-infrared dual-color laser imaging system was used to scan and photograph the image. Western blot images were used for relative quantitative analysis of the grayscale of protein bands using Image J.

[0082] 4.5 Experimental Results

[0083] Collagen is the main component of newly synthesized extracellular matrix in fibrotic tissue, and type I collagen accounts for about 80% of the total collagen product. Therefore, the present invention uses Western blot to detect the expression level of type I collagen to further illustrate the anti-pulmonary fibrosis activity of yamachulide A. Figure 4As shown in the figure, compared with the control group, the expression level of type I collagen was significantly increased in the lung fibroblast differentiation model induced by transforming growth factor. The expression level of type I collagen in lung fibroblasts in the yamachulide A group was significantly lower than that in the model group. These experimental results indicate that yamachulide A can inhibit the production of extracellular matrix proteins in lung fibroblasts induced by TGF-β1 and has anti-pulmonary fibrosis activity.

[0084] Example 5

[0085] 5.1 Experimental Purpose

[0086] To observe the effect of yemachalide lactone A on the hydroxyproline content in lung tissue of mice with bleomycin-induced pulmonary fibrosis.

[0087] 5.2 Experimental Materials

[0088] Experimental animals: 30 C57 mice, male, weighing 18-22 g, SPF grade, provided by the Animal Experiment Center of Soochow University. Laboratory Animal Production License Number: SCXK(Su)2021-0007, Laboratory Animal Use License Number: SYXK(Su)2021-0035.

[0089] Experimental drugs: Eupalinolide A (EA); molecular formula: C24H30O9; CAS number: 877822-40-7, purity ≥98%; bleomycin, white lyophilized powder, 15 mg / vial, produced by Nippon Kayaku Co., Ltd.; 4% chloral hydrate; pirfenidone, Beijing Solebao Biotechnology Co., Ltd.

[0090] Reagents and instruments: Hydroxyproline (HYP) kit (lot number: 20211019) was purchased from Nanjing Jiancheng Biotechnology Research Institute. UV-visible spectrophotometer, T6 New Century model, produced by Beijing Puxi General Instrument Co., Ltd.; high-speed low-temperature refrigerated centrifuge, model 5417R, produced by Germany Eppendorf AG; electric constant temperature water bath, model HHS6, produced by Shanghai Rongfeng Scientific Instrument Co., Ltd.

[0091] 5.3 Experimental methods

[0092] Animal grouping: 30 male C57 mice were weighed, numbered, and randomly divided into 5 groups, namely, normal group, model group, pirfenidone group (200 mg / kg), high-dose safflower lactone A group (80 mg / kg), and low-dose safflower lactone A group (40 mg / kg), with 6 mice in each group.

[0093] Model preparation: Bleomycin was used to replicate the animal pulmonary fibrosis model in the model group and the mice in each treatment group. The specific operation steps are as follows: During the experiment, the mice were anesthetized by intraperitoneal injection of 4% chloral hydrate (0.01mL / g), lying on the operating table, fixing the head and limbs, and administering bleomycin (5mg / kg) through tracheal high-pressure spray. The normal control group was given an equal volume of saline under the same conditions. Immediately after the injection, the mice were upright and rotated left and right along the longitudinal axis of the body for 3 minutes to evenly distribute the drug solution in the lungs on both sides. After the mice woke up, they were sent to the observation room for routine feeding.

[0094] Dosage scheme: After the animals in each group were modeled with bleomycin, the mice were weighed every other day starting from the seventh day, and the drugs were administered by gavage according to the corresponding body weight. The normal group and the model group were given an equal volume of normal saline, the positive control group was given pirfenidone, and each treatment group was given different doses of yamachulide A. The drugs were administered continuously for 21 days.

[0095] Hydroxyproline determination: On the day of animal sacrifice, about 25 mg of the middle lobe of the right lung was taken and placed in a 10 mL glass centrifuge tube with a scale and stored in a -20°C refrigerator for the determination of the hydroxyproline content in the lung tissue of mice in each experimental group; the determination method is as follows: 0.5 mL of hydrolysis solution was accurately added to the scaled glass test tube of the frozen small piece of lung tissue, mixed, and then covered and hydrolyzed in a boiling water bath for 20 minutes, of which 10 minutes were mixed once. After that, the pH value of the test tube liquid was adjusted to about 6.5, an appropriate amount of activated carbon was added, mixed, transferred to a 1.5 mL centrifuge tube, centrifuged at 3500 r / min for 10 minutes, 0.5 mL of the supernatant was taken, and three reaction reagents were added in sequence, mixed, 60°C water bath for 15 minutes, cooled and centrifuged at 3500 r / min for 10 minutes, and the absorbance of each tube was measured at 550nm and 1cm. The hydroxyproline content in the lung tissue was calculated according to the formula in the instruction manual.

[0096] 5.5 Experimental Results

[0097] Compared with the normal control group, the model group mice significantly lost weight, the hydroxyproline content in lung tissue increased significantly, and the lung coefficient was significantly higher than that of the normal group; the weight of mice in each dose group of Yemazhui lactone A increased significantly, the hydroxyproline content in lung tissue of mice was significantly lower than that of the model group, and the lung coefficient was significantly lower than that of the model group. The hydroxyproline content in lung tissue of mice in the positive control drug pirfenidone group was significantly lower than that of the model group, and the lung coefficient was significantly decreased, as shown in Table 1.

[0098] Table 1 Effects of yemachai lactone A on hydroxyproline in lung tissue of mice with pulmonary fibrosis (n=6)

[0099]

[0100] 5.6 Conclusion

[0101] After 28 days of modeling, the hydroxyproline content in the lung tissue of mice in the model group increased significantly, and the lung coefficient increased significantly. After 21 days of administration, yemachuide lactone A can significantly reduce the hydroxyproline content in the lung tissue of mice. The positive control drug pirfenidone can also reduce the hydroxyproline content in the lung tissue of mice with pulmonary fibrosis. It suggests that yemachuide lactone A can reduce the production of alveolar interstitial collagen by reducing the content of hydroxyproline in lung tissue, thereby exerting an anti-fibrotic effect.

[0102] Example 6

[0103] 6.1 Experimental Purpose

[0104] To observe the effect of yemachalide A on the pathological morphology of lung tissue in mice with bleomycin-induced pulmonary fibrosis.

[0105] 6.2 Experimental Materials

[0106] Experimental animals: 30 C57 mice, male, weighing 18-22 g, SPF grade, provided by the Animal Experiment Center of Soochow University. Laboratory Animal Production License Number: SCXK(Su)2021-0007, Laboratory Animal Use License Number: SYXK(Su)2021-0035.

[0107] Experimental drugs: Eupalinolide A (EA); molecular formula: C24H30O9; CAS number: 877822-40-7, purity ≥98%; bleomycin, white lyophilized powder, 15 mg / vial, produced by Nippon Kayaku Co., Ltd.; 4% chloral hydrate; pirfenidone, Beijing Solebao Biotechnology Co., Ltd.

[0108] Experimental instrument: optical microscope, OLYMPUS CX31.

[0109] 6.3 Experimental methods

[0110] Animal grouping: 30 male C57 mice were weighed, numbered, and randomly divided into 5 groups, namely, normal group, model group, pirfenidone group (200 mg / kg), high-dose safflower lactone A group (80 mg / kg), and low-dose safflower lactone A group (40 mg / kg), with 6 mice in each group.

[0111] Model preparation: The model group and each treatment group mice were anesthetized with bleomycin by intraperitoneal injection of 4% chloral hydrate (0.01mL / g), lying on the operating table, fixing the head and limbs, and administering bleomycin (5mg / kg) through tracheal high-pressure spray. The normal control group was given an equal volume of saline under the same conditions. Immediately after the injection, the mice were upright and rotated left and right along the longitudinal axis of the body for 3 minutes to evenly distribute the drug solution in both lungs. After the mice woke up, they were sent to the observation room for routine feeding.

[0112] 6.4 Dosage Regimen

[0113] After the animals in each group were modeled with bleomycin, the mice were weighed every other day starting from the seventh day, and the drugs were administered by gavage according to the corresponding body weight. The normal group and the model group were given an equal volume of normal saline, the positive control group was given pirfenidone, and each treatment group was given different doses of yamachulide A. The drugs were administered continuously for 21 days.

[0114] Staining of lung tissue pathological sections: The right lung was taken and fixed, embedded and sliced ​​according to conventional pathological methods. The pathological sections were stained with HE, and then the degree of alveolitis and pulmonary fibrosis was divided into four levels with reference to the method of Szaopiel et al., which was slightly modified: Level 0, no obvious pathological changes, clear alveolar structure, translucent alveoli, normal alveolar septa; very little inflammatory cell infiltration, very little fibroblast production; no collagen fiber production; Level 1, mild pathological changes, slightly disordered alveolar structure, widened alveolar septa due to inflammatory cell infiltration and fibroblast proliferation; less inflammatory cell infiltration, less fibroblast proliferation; less collagen fiber production. The lesions range from 20% to 50% of the whole lung. The alveolar structure is disordered, and the alveolar septa are significantly widened. There is a small amount of inflammatory cell infiltration, a small amount of fibroblast proliferation, and a small amount of collagen fiber production. The lesions range from 20% to 50% of the whole lung. The lesions range from 30% to 50% of the whole lung. The lesions range from 20% to 50% of the whole lung. The lesions range from 30% to 50% of the whole lung. There is a large amount of inflammatory cell infiltration, a large amount of fibroblast proliferation, and a large amount of collagen fiber deposition. Obvious pulmonary fibrosis lesions can be seen, and the lesions range from 50% to 50% of the whole lung.

[0115] 6.5 Experimental Results

[0116] After 28 days of modeling, compared with the normal control group, the degree of alveolitis and pulmonary fibrosis in the lung tissue of the mice in the model group was significantly aggravated, showing pulmonary fibrosis parenchymal lesions; compared with the model group, the alveolar structure, alveolar septal inflammatory cell infiltration, collagen deposition and fibroblast proliferation of the lung tissue of the mice in each dose group of Yemazhui lactone A were improved to varying degrees, and the degree of alveolitis and pulmonary fibrosis were significantly reduced; the degree of alveolitis and pulmonary fibrosis in the lung tissue of the mice in the positive control drug pirfenidone group was significantly reduced compared with the model group mice, as shown in Figure 2. Figure 5 , Figure 6 .

[0117] 6.6 Conclusion

[0118] After 28 days of modeling, the degree of alveolitis and pulmonary fibrosis in the lung tissue of mice in the model group was significantly aggravated, showing typical pulmonary fibrosis parenchymal lesions. After 21 days of administration, the lung coefficient of mice in each dose group of Yemazhui lactone A was significantly reduced, and the degree of alveolitis and pulmonary fibrosis in the lung tissue was significantly alleviated. This indicates that the mouse pulmonary fibrosis model was successfully replicated under the experimental conditions. Yemazhui lactone A can significantly reduce the degree of alveolitis and pulmonary fibrosis in mice with pulmonary fibrosis, and has the effect of preventing and treating pulmonary fibrosis.

[0119] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. Application of yemachaylactone A in the preparation of drugs for preventing or treating pulmonary fibrosis.

2. The use according to claim 1, It is characterized in that The yamachus lactone A also includes pharmaceutically acceptable salts thereof.

3. The use according to claim 2, It is characterized in that The pharmaceutically acceptable salt is selected from the sodium salt of safflower lactone A and / or safflower lactone A sulfonate.

4. The use according to claim 1, It is characterized in that The medicament also includes a pharmaceutically acceptable carrier.

5. The use according to claim 4, It is characterized in that The carrier is selected from one or more of tablets, capsules, pills, powders, suppositories, ointments, solutions and suspensions.

6. The use according to claim 5, It is characterized in that The capsules, tablets, pills and powders are selected from one or more of sucrose, lactose, corn starch, microcrystalline cellulose and carboxymethyl cellulose.

7. The use according to claim 5, It is characterized in that The solution and suspension are selected from one or more of water, sodium chloride solution, glucose solution and phosphate buffer.

8. The use according to claim 1, It is characterized in that The medicine further comprises an additive; the additive is one or more of an antioxidant, a preservative, a solubilizer, a disintegrant, a lubricant, a colorant, a dispersant and a surfactant.

9. A pharmaceutical composition for preventing or treating pulmonary fibrosis, It is characterized in that Including phytonadione A.

10. The pharmaceutical composition according to claim 9, It is characterized in that The dosage of the drug is 1-80 mg / kg / d.

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