Application of 3, 5, 6, 7, 8, 3 ', 4'-heptamethoxyflavone in preparation of medicine for preventing or treating pulmonary fibrosis

Abstract

The invention discloses a preparation method of 3, 5, 6, 7, 8, 9-tetramethyl-3, 5, 6, the invention relates to application of 3, 5, 6, 7, 8, 3 ', 4'-heptamethoxyflavone in preparation of a medicine for preventing or treating pulmonary fibrosis, and test results show that 3, 5, 6, 7, 8, 3 ', 4'-heptamethoxyflavone can inhibit the oxidative stress level of mice with pulmonary fibrosis and activate the autophagy level at the same time, and show that HMF can play a role in inhibiting pulmonary fibrosis by adjusting the oxidative stress and autophagy level, which is the effect of 3, 5, 6, 7, 8, 3 ', 4'-heptamethoxyflavone. The application of the 3, 3 ', 4, 4'-heptamethoxyflavone to clinical treatment of idiopathic pulmonary fibrosis provides a theoretical basis, and develops a new treatment medicine and way.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicines, and in particular to application of 3,5,6,7,8,3',4'-heptamethoxyflavone in preparing medicines for preventing or treating pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis (PF) is a chronic, progressive lung disease characterized by abnormal scarring and collagen deposition in the lung tissue, which leads to thickening and hardening of the lung tissue and eventually loss of lung function. This lesion is caused by a variety of factors, such as environmental exposure, genetics, drug side effects, radiotherapy, and connective tissue diseases. These factors interact with each other, resulting in damage to alveolar epithelial cells, an increase in fibroblasts and their transformation into myofibroblasts, leading to the deposition of extracellular matrix, and ultimately reduced lung elasticity, obstructed gas exchange, decreased lung function, and dyspnea. In severe cases, it can lead to respiratory failure and endanger life.

[0003] Pirfenidone and nintedanib are currently recognized as the best anti-fibrotic drugs with clinical effects, but these drugs can cause some adverse side effects in clinical use, such as nausea and diarrhea. Although these two drugs can slow down the decline of lung function, they cannot reverse the fibrosis that has already occurred. In addition to drug treatment, lung transplantation can effectively prolong the survival of patients, but due to the high cost, high risk and organ donor restrictions of lung transplantation, it is difficult to implement and use clinically. However, due to the high cost and risk of surgery and the limited source of lungs available for transplantation, lung transplantation faces many challenges in clinical practice. In general, current treatments have limited effects and lack a definite and effective treatment method. Therefore, in-depth research on the pathogenesis of pulmonary fibrosis and the development of new treatments have become a hot topic in the research field.

[0004] Traditional Chinese medicine has significant advantages in disease prevention and treatment. The natural chemical components and active ingredients in Chinese medicine provide broad application prospects for disease prevention and treatment due to their multiple targets and low toxicity and side effects. 3,5,6,7,8,3',4'-heptemthoxyflavone (HMF) is a flavonoid compound extracted from dried orange peel and has strong biological activity. Flavonoid compounds have multiple biological activities such as anti-inflammatory, anti-tumor, hypoglycemic, improvement of Alzheimer's disease, and antiviral. They are widely used in the treatment of various diseases due to their low toxicity and side effects and low drug dependence. Current studies have shown that HMF has anti-inflammatory, anti-depressant, immunomodulatory and adipogenic effects. However, there are few studies on the effects and mechanisms of HMF on pulmonary fibrosis. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a new use of 3,5,6,7,8,3',4'-heptamethoxyflavone in the preparation of drugs for preventing or treating pulmonary fibrosis, thereby providing a theoretical basis for its application in the clinical treatment of idiopathic pulmonary fibrosis and exploring new therapeutic drugs and approaches.

[0006] 3,5,6,7,8,3',4'-Heptamethoxyflavone (HMF) is a flavonoid compound isolated from dried orange peel. It has been identified as the main active ingredient in dried orange peel and has strong biological activity. Its structural formula is as follows:

[0007]

[0008] The 3,5,6,7,8,3',4'-heptamethoxyflavone (HMF) of the present invention can be prepared by self-production or purchased from the market.

[0009] The pulmonary fibrosis described in the present invention includes idiopathic pulmonary fibrosis (IPF), secondary pulmonary fibrosis and hereditary pulmonary fibrosis, etc., with idiopathic pulmonary fibrosis being preferred.

[0010] The present invention uses bleomycin (BLM) to induce an in vivo model of pulmonary fibrosis in mice. The results show that HMF has a significant regulatory effect on the abnormal changes of lung tissue morphology, lung function indexes, HE staining, Masson staining and pulmonary fibrosis-related indexes in IPF mice. At the same time, TGF-β1 is used to induce an in vitro pulmonary fibrosis model of mouse embryonic fibroblasts. It is found that after HMF treatment, the vitality of NIH / 3T3 cells is significantly increased, the cell migration rate is significantly reduced, and the expression levels of pulmonary fibrosis-related proteins α-SMA, collagen-I, MMP2 and TIMP1 are also significantly reduced. In vivo and in vitro models, oxidative stress and autophagy levels are detected and found that HMF can increase the expression levels of SOD, GSH-Px, Nrf2 and HO-1, reduce the expression levels of MDA and keap-1, and play an anti-oxidative stress role, both in vivo and in vitro; at the same time, it can also increase the protein expression levels of LC3B and Beclin-1. It is also observed in vitro that HMF can significantly increase the content of autophagosomes in NIH / 3T3 cells and play an activating role in autophagy. In vitro, 3-MA autophagy inhibitor was used to block the level of autophagy flow. The results showed that the addition of 3-MA weakened the expression of fibrotic proteins inhibited by HMF and enhanced the level of oxidative stress. This shows that HMF can inhibit the level of oxidative stress and activate the level of autophagy both in vivo and in vitro, thereby achieving the effect of improving pulmonary fibrosis.

[0011] The medicine of the present invention comprises an effective amount of 3,5,6,7,8,3',4'-heptamethoxyflavone and a pharmaceutically acceptable carrier.

[0012] Preferably, the drug is in the form of tablets, capsules, powders, oral liquids, powders, pills or granules.

[0013] The pharmaceutically acceptable carriers described in the present invention include, but are not limited to, lubricants, fillers, binders, disintegrants, pH regulators, surfactants, antioxidants, etc., and their usage amounts are conventional amounts in the art.

[0014] The pharmaceutical dosage form of the present invention can be prepared according to conventional methods in the art.

[0015] Compared with the prior art, the present invention has the following excellent effects:

[0016] The present invention provides a new use of 3,5,6,7,8,3',4'-heptamethoxyflavone in preparing a drug for preventing or treating pulmonary fibrosis, studies the effect of HMF in idiopathic pulmonary fibrosis through in vivo experiments and in vitro experiments, explores the action mechanism of HMF, and the experimental results show that 3,5,6,7,8,3',4'-heptamethoxyflavone can inhibit the oxidative stress level of mice with pulmonary fibrosis and activate the level of autophagy, indicating that HMF may inhibit pulmonary fibrosis by regulating the levels of oxidative stress and autophagy, which provides a theoretical basis for the application of 3,5,6,7,8,3',4'-heptamethoxyflavone in the clinical treatment of idiopathic pulmonary fibrosis and develops new therapeutic drugs and approaches. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Effects of HMF on organ coefficients in mice with pulmonary fibrosis n=6; compared with the Sham group, ## P<0.01; compared with the BLM group, ** P<0.01);

[0018] Figure 2 This is the appearance of the lungs of mice with pulmonary fibrosis (the black arrow indicates the obvious lesion);

[0019] Figure 3 Effects of HMF on lung function in mice with pulmonary fibrosis ( n=6; A. Tidal volume (TV); B. Dynamic lung compliance (Cdyn); C. Respiratory index (RI); Compared with the Sham group, ## P<0.01; compared with the BLM group, * P<0.05, ** P<0.01);

[0020] Figure 4Effect of HMF on hydroxyproline content in lung tissue of mice with pulmonary fibrosis ( n=6; compared with the Sham group, ## P<0.01; compared with the BLM group, ** P<0.01);

[0021] Figure 5 The effect of HMF on HE staining of mice with pulmonary fibrosis (×100);

[0022] Figure 6 The effect of HMF on Masson staining in mice with pulmonary fibrosis (×100);

[0023] Figure 7 Effect of HMF on the expression level of related fibrin in mice with pulmonary fibrosis ( n=6; AB: expression levels of collagen-I, α-SMA, TGF-β1, MMP2, and TIMP1 proteins; C: Elisa method was used to detect the content of TGF-β1; DE: immunohistochemical staining was used to detect the protein expression of collagen-I; compared with the Sham group, # P<0.05, ## P<0.01; compared with the BLM group, * P<0.05, ** P<0.01);

[0024] Figure 8 Effects of HMF on oxidative damage in lung tissue of mice with pulmonary fibrosis ( n=6; A. GSH level; B. SOD level; C. MDA level; D. ROS level; Compared with the Sham group, ## P<0.01; compared with the BLM group, * P<0.05, ** P<0.01);

[0025] Fig. 9 Effects of HMF on oxidative stress-related proteins in lung tissue of mice with pulmonary fibrosis ( or (P25, P50, P75), n=6; compared with the Sham group, # P<0.05, ## P<0.01; compared with the BLM group, * P<0.05, ** P<0.01);

[0026] Fig.10 Effects of HMF on autophagy-related proteins in lung tissue of mice with pulmonary fibrosis ( or (P25, P50, P75), n=6; AB: protein expression levels of Beclin-1 and LC3B; CD: immunohistochemical staining to detect the protein content of LC3B; compared with the Sham group, # P<0.05, ## P<0.01; compared with the BLM group, * P<0.05, ** P<0.01);

[0027] Fig.11 Effects of HMF on toxicity and viability of lung fibroblasts ( (P25, P50, P75), n=3; A: cytotoxicity; B: cell viability; compared with the Control group, # P<0.05, ## P<0.01; compared with TGF-β1 group, * P<0.05, ** P<0.01);

[0028] Fig.12 Effects of HMF on the migration ability of lung fibroblasts (P25, P50, P75, n = 3; compared with the Control group, # P<0.05; compared with TGF-β1 group, *P<0.05);

[0029] Fig.13 The effect of HMF on the morphology of lung fibroblasts (the red arrows are typical myofibroblasts);

[0030] Fig.14 Effects of HMF on fiber-associated proteins in lung fibroblasts ( n=6; AB: α-SMA, collagen-I, MMP2, TIMP1 protein expression levels; C: α-SMA, collagen-I immunofluorescence images; D: average immunofluorescence of α-SMA; E: average immunofluorescence intensity of collagen-I; compared with the Control group, # P<0.05; compared with TGF-β1 group, * P<0.05);

[0031] Fig.15 Effects of HMF on oxidative damage indicators in lung fibroblasts (P25, P50, P75, n = 3; A: GSH-Px level; B: SOD level; C: MDA level; DE: ROS level; compared with the Control group, # P<0.05; compared with TGF-β1 group, * P<0.05);

[0032] Fig.16 Effects of HMF on oxidative stress-related proteins in lung fibroblasts ( n=6; compared with the Control group, # P<0.05; compared with TGF-β1 group, * P<0.05);

[0033] Fig.17 Effects of HMF on autophagy-related proteins in lung fibroblasts ( n=6; compared with the Control group, # P<0.05; compared with TGF-β1 group, * P<0.05);

[0034] Fig.18 The effect of HMF on the content of autophagosomes in lung fibroblasts (the red arrows are typical autophagosomes);

[0035] Fig.19 HMF inhibits the expression of fibrin in lung fibroblasts through autophagy ( n=6; AB: expression levels of Beclin-1, α-SMA, and LC3B proteins; C: collagen-I immunofluorescence image; D: average immunofluorescence intensity of collagen-I; E: MDC staining; compared with the Control group, # P<0.05; compared with TGF-β1 group, * P<0.05);

[0036] Fig. 20 Effects of HMF on oxidative stress in lung fibroblasts under autophagy inhibition ( n=6; AB: ROS content; CD: Nrf2 and keap1 protein expression levels; compared with the Control group, # P<0.05; compared with TGF-β1 group, * P<0.05). DETAILED DESCRIPTION

[0037] The present invention is further described below by means of specific implementation methods. The following examples are specific implementation methods of the present invention, but the implementation methods of the present invention are not limited to the following examples.

[0038] Example 1: HMF inhibits fibrosis in lung tissue of mice with bleomycin-induced pulmonary fibrosis

[0039] 1. Materials

[0040] 1. Animals

[0041] 40 SPF-grade C57BL / 6 male mice, 6-8 weeks old, weighing 20-24 g, were provided by Guangdong Medical Laboratory Animal Center, with the laboratory animal quality certificate number: No.44007200124034.

[0042] (II) Main drugs and reagents

[0043] The main drugs and reagents used in the embodiments of the present invention are commercially available, among which 3,5,6,7,8,3',4'-heptamethoxyflavone was purchased from Sichuan Cuiyirun Biotechnology Co., Ltd. with the product number CYQ-Q0095210112.

[0044] 2. Experimental Methods

[0045] 1. Animal Grouping

[0046] After one week of adaptive feeding, forty male C57BL / 6 mice aged 6 to 8 weeks were randomly divided into sham operation group (Sham), model group (BLM), low-dose 3,5,6,7,8,3',4'-heptamethoxyflavone group (BLM+HMF-L), high-dose 3,5,6,7,8,3',4'-heptamethoxyflavone group (BLM+HMF-H) and positive drug control group (BLM+PFD) according to their body weight, with 8 mice in each group.

[0047] (II) Establishment of pulmonary fibrosis mouse model

[0048] 1. Preparation before modeling:

[0049] The mice were fasted but not watered for 8 h to prevent feed residues from obstructing the field of vision or falling into the trachea during modeling, causing suffocation of the mice. The mice were anesthetized after being weighed and anesthetized by intraperitoneal injection of 0.075 mL / 10 g 1% sodium pentobarbital. The modeling agent (bleomycin, concentration of 1.5 mg / kg) was prepared. The micro-liquid intratracheal atomization device was assembled to ensure the normal operation of the atomizer, and a tablet computer was used to connect the animal laryngoscope device to ensure stable signals and clear images.

[0050] 2. Modeling process:

[0051] The anesthetized mice were fixed on a special animal operating table; first, the tongue of the mouse was clamped out with tweezers, and the front end of the laryngoscope was inserted into the root of the mouse tongue with the left hand, and the epiglottis cartilage was pressed down to fully expose the glottis (it can be seen opening and closing), and the micro-liquid intratracheal atomization device was held in the right hand. When the glottis was open, the atomizer was quickly inserted into the glottis about 0.5cm, and a quantitative column (50μL) of bleomycin aerosol was quickly pushed to the lungs. Except for the Sham group, which was sprayed with normal saline, the other groups were sprayed with bleomycin. After the spraying, the mouse was removed from the fixator and shaken up and down and left and right 10 times each to make the bleomycin more evenly distributed in the mouse lungs.

[0052] 3. Post-modeling treatment: Place the mouse on its side on the bedding, and after observing that there is no abnormality, return it to the cage and continue to raise it.

[0053] (III) Animal drug administration

[0054] Gavage administration began on the first day after modeling. The Sham group and the BLM group were given ultrapure water by gavage, the BLM+HMF-L group was given 25 mg / (kg·d) HMF by gavage, the BLM+HMF-H group was given 50 mg / (kg·d) HMF by gavage, and the BLM+PFD group was given 156 mg / (kg·d) pirfenidone by gavage. The drugs were administered once a day according to their body weight (10 ml / kg) for 28 consecutive days.

[0055] (IV) Sample collection and preservation

[0056] After the last treatment, the mice were anesthetized, blood was collected, and then the mice were killed by cervical dislocation to obtain the mouse lung tissue. The lung tissue was rinsed with saline and photographed. An appropriate amount of lung tissue was cut from the same position of the left lung and stored in 4% paraformaldehyde for HE staining, Masson staining, etc. The remaining lung tissue was stored at -80°C for subsequent experiments. The collected blood was left at room temperature for 2 hours, centrifuged at 3500 rpm for 10 minutes, and the upper serum was collected and stored at -80°C.

[0057] (V) Determination of lung coefficient

[0058] The body weight of the mice was recorded before sampling on the 28th day. The lungs of the mice were removed and rinsed with saline and then weighed with an electronic balance to obtain the wet weight of the lungs. The lung coefficient = wet weight of lungs / mouse body weight (mg / g).

[0059] 6. Pulmonary function test

[0060] After the mice were intraperitoneally injected with 1% sodium pentobarbital 0.075 mL / 10 g body weight, they were anesthetized, and then their cervical trachea was exposed by dissection and a special cannula for the pulmonary function test was inserted and fixed with silk thread; the mice were placed in a special experimental cavity, and the tidal volume (TV), dynamic lung compliance (Cdyn) and respiratory index (RI) were tested, and the data were saved and exported.

[0061] (VII) Determination of Hydroxyproline Content

[0062] Accurately weigh about 50 mg of tissue wet weight, put it into a test tube, accurately add the hydrolyzate and mix evenly. After covering, hydrolyze in a boiling water bath for 20 minutes. Adjust the pH value to about 6.5, then add double distilled water to 10 ml and mix evenly. Take 3 ml of the diluted hydrolyzate, add an appropriate amount of activated carbon, and the above clear liquid is colorless after centrifugation. Carefully take 1 ml of the supernatant to prepare the test solution, prepare the blank control solution and the standard solution, mix evenly, and soak in 60°C water for 15 minutes. After cooling, take the supernatant, and measure the absorbance value of each tube at a wavelength of 550nm.

[0063] (VIII) HE staining

[0064] The same part of the left lung tissue of each group of mice was fixed in tissue fixative, dehydrated, embedded, and sliced ​​(4 μM). HE staining was used to observe the pathological changes of lung tissue.

[0065] (IX) Masson staining

[0066] The degree of lung fibrosis was observed by Masson staining of paraffin sections.

[0067] (X) Immunohistochemical staining to detect the protein expression of collagen-I

[0068] Paraffin sections were dewaxed to water, tissue sections were placed in a repair box filled with citric acid antigen repair buffer (PH6.0) in a microwave oven for antigen repair, 3% hydrogen peroxide solution was used to block endogenous peroxidase, 3% BSA was added for blocking, and primary antibodies prepared in a certain proportion of PBS were added, incubated overnight at 4°C, incubated with secondary antibodies for 50 minutes, DAB color development, counterstaining, dehydration, and sealing. Images were collected under a microscope.

[0069] (XI) Western Blot to detect related protein expression

[0070] Weigh 30 mg of lung tissue, add lysis buffer to grind, centrifuge, extract tissue protein, load sample after BCA protein determination, electrophoresis, transfer, block PVDF membrane with rapid blocking solution, incubate primary antibody at 4°C overnight, wash membrane, incubate secondary antibody, wash membrane, and finally develop with chemiluminescence for gel image analysis.

[0071] (XII) ELISA method to detect the content of TGF-β1 and ROS in lung tissue of mice with pulmonary fibrosis

[0072] 1. Extraction of Lung Tissue Homogenate

[0073] Weigh 20 mg of right lung tissue and place it in a 2 mL frozen grinding tube, add 200 μL PBS and a 3 mm steel bead, sonicate for 70 s at 4°C using a frozen ultrasonic grinder, place in a 4°C refrigerator for 1-2 h, centrifuge at 10,000 rpm for 15 min at 4°C, take the supernatant, and determine the protein concentration using the BCA protein quantification method.

[0074] 2. Test kit

[0075] The ELISA kit was operated according to the operating instructions, the OD value of the sample was measured at a wavelength of 450 nm, and the content of each indicator in the lung tissue was calculated by establishing a standard curve and substituting it into the corresponding formula.

[0076] (XIII) Detection of MDA, SOD and GSH-Px levels

[0077] Lung tissue homogenate was extracted according to the ELISA method for detecting the content of TGF-β1 and ROS in the lung tissue of mice with pulmonary fibrosis, and then the MDA, SOD and GSH-Px levels were detected according to the instructions.

[0078] 14. Statistical methods

[0079] All data processing in this study was completed by SPSS26.0 software, and GraphPad Prism 9 was used for graphing.

[0080] 3. Experimental Results

[0081] (I) Effect of HMF on the lung coefficient of mice with pulmonary fibrosis

[0082] After 28 days of BLM induction, the lung coefficient of mice in the BLM group was significantly increased compared with the Sham group (P<0.01); compared with the BLM group, the lung coefficient of mice in each drug group was significantly decreased (P<0.01), and the decrease in the BLM+HMF-H group was the most significant. Figure 1 : Compared with the Sham group, the BLM group had a significant difference (P<0.01). Compared with the BLM group, the BLM+HMF-L group, BLM+HMF-H group and BLM+PFD group had a significant difference (P<0.01).

[0083] (II) Effects of HMF on the appearance of lung tissue in mice with pulmonary fibrosis

[0084] Observation of the lung tissues of each group showed that compared with the Sham group, the lung tissues of the mice in the BLM group were dark red, swollen, and lost their luster and elasticity; compared with the BLM group, the color, texture, morphology, and elasticity of the lung tissues of the mice in the BLM+HMF-L group, BLM+HMF-H group, and BLM+PFD group were significantly improved. Figure 2 .

[0085] 3. Effect of HMF on lung function in mice with pulmonary fibrosis

[0086] The lung function of mice in each group was tested. Compared with the Sham group, the TV and Cdyn of the BLM group were decreased (P<0.01), while the RI was increased (P<0.01). Compared with the BLM group, the TV and Cdyn of the BLM+HMF-L group, BLM+HMF-H group and BLM+PFD group were increased (P<0.01, P<0.01), while the RI was decreased (P<0.05). The changes of the BLM+HMF-H group and the BLM+PFD group were similar. Figure 3 .

[0087] (IV) Effect of HMF on Hydroxyproline Content in Lung Tissue of Mice with Pulmonary Fibrosis

[0088] HYP is a unique amino acid in collagen, so the detection of hydroxyproline content can determine whether collagen is deposited and the severity of pulmonary fibrosis. Compared with the Sham group, the HYP content in the lung tissue of mice in the BLM group was significantly increased (P<0.01). Compared with the BLM group, the HYP content in the lung tissue of each drug-treated group was significantly decreased (P<0.01). Figure 4 .

[0089] (V) Observation of lung tissue pathology sections

[0090] 1. HE staining

[0091] The lung tissue structure of mice in the sham group was normal, and the alveolar structure was intact; the alveolar structure of the lung tissue of mice in the BLM group was severely damaged, and inflammatory infiltration and cell necrosis were observed; compared with the BLM group, each drug-treated group had different degrees of improvement and gradually returned to normal. Among them, the improvement of the BLM+HMF-H group and the BLM+PFD group was close and close to the normal group. Results are shown in Figure 5 .

[0092] 2.Masson staining

[0093] Masson staining can be used to determine the content of collagen fibers in the lung tissue of mice, and thus determine the severity of pulmonary fibrosis in mice. Compared with the Sham group, the alveolar structure of the lung tissue of mice in the BLM group was severely damaged, and collagen fibers were deposited; compared with the BLM group, the collagen fiber content of each drug-treated group was reduced, and the improvement of the BLM+HMF-H group and the BLM+PFD group was similar and close to the normal group. Results are shown in Figure 6 .

[0094] (VI) Effect of HMF on the expression level of related fibrin in mice with pulmonary fibrosis

[0095] Compared with the Sham group, the protein expression levels of α-SMA, collagen-I, TGF-β1, MMP2 and TIMP1 in the lung tissue of mice in the BLM group were significantly increased (P<0.01); compared with the BLM group, the expression levels of related fibrin in each drug-treated group were significantly decreased (P<0.05, P<0.01), and there was a certain dose-dependency. Figure 7 The results of Elisa test showed that after HMF treatment, the content of TGF-β1 in the lung tissue of mice was significantly reduced (P<0.05). Figure 7 C. IHC results showed that the protein expression level of collagen-I in the lung tissue of mice in each drug-treated group was significantly decreased (P<0.05), as shown in Figures 7, DE.

[0096] (VII) Effect of HMF on oxidative damage indicators in lung tissue of mice with pulmonary fibrosis

[0097] ROS plays a core role in redox regulation and cell signaling, but excessive ROS production can trigger oxidative stress. The accumulation of ROS stimulates the production of MDA and reduces the activity of SOD and GSH. The results showed that compared with the Sham group, the levels of ROS (P<0.01) and MDA (P<0.01) in the lung tissue of mice in the BLM group were significantly increased, while the levels of SOD (P<0.01) and GSH (P<0.01) were significantly decreased; after HMF treatment, the levels of ROS (P<0.05, P<0.01) and MDA (P<0.05) in each concentration of the drug administration group were significantly decreased, and the levels of SOD (P<0.01) and GSH (P<0.01) were significantly increased. Results are shown in Figure 8 .

[0098] (VIII) Effect of HMF on oxidative stress-related proteins in lung tissue of mice with pulmonary fibrosis

[0099] Compared with the Sham group, the protein expression levels of Nrf2 (P<0.05) and HO-1 (P<0.05) in the lung tissue of the mice in the BLM group were decreased, while the protein expression level of keap-1 (P<0.05) was increased; compared with the BLM group, after HMF treatment, the protein expression levels of Nrf2 (P<0.05) and HO-1 (P<0.05) were significantly increased, while the protein expression level of keap-1 (P<0.05) was significantly decreased. Fig. 9 .

[0100] (IX) Effects of HMF on autophagy-related proteins in lung tissue of mice with pulmonary fibrosis

[0101] Oxidative stress can also regulate autophagy activity. Moderate oxidative stress-induced autophagy activation can promote the clearance of damaged organelles and maintain cell survival, but excessive oxidative stress can cause autophagy dysregulation, leading to lysosomal membrane damage and cell apoptosis. Therefore, in this experiment, by detecting the protein expression levels of Beclin-1 and LC3B in mouse lung tissue, it was found that after HMF treatment, the protein expression levels of Beclin-1 (P<0.05) and LC3B II (P<0.05) were significantly increased. The results are shown in Fig.10 The results of IHC showed that the protein expression level of LC3B (P<0.05) in the lung tissue of mice was significantly increased after HMF treatment. Fig.10 Medium CD.

[0102] In summary, in vivo experiments confirmed that HMF can inhibit the level of oxidative stress in mice with pulmonary fibrosis and activate the level of autophagy, indicating that HMF may inhibit pulmonary fibrosis by regulating the levels of oxidative stress and autophagy.

[0103] Example 2: HMF inhibits lung fibroblast differentiation by regulating oxidative stress and autophagy

[0104] 1. Materials

[0105] (A) Cells: NIH / 3T3 cells were purchased from Shanghai Biopharmaceuticals Co., Ltd.

[0106] (ii) Reagents and antibodies: The main reagents and antibodies used in this example were purchased from the market.

[0107] 2. Experimental Methods

[0108] (I) Cell experiment grouping

[0109] NIH / 3T3 cells: a. control group, TGF-β1 group, TGF-β1+HMF 12.5μM group, TGF-β1+HMF 25μM group; b. control group, TGF-β1 group, TGF-β1+HMF 25μM group, TGF-β1+HMF+3-MA group.

[0110] (II) Drug treatment methods

[0111] 1. HMF mother solution: Weigh 5 mg of HMF powder and dissolve it in 28.9 μL DMSO to a concentration of 400 mM. Use DMSO to dilute to 200, 100, 50, 25 and 12.5 mM in sequence and store in a -20°C refrigerator for later use.

[0112] 2. TGF-β1 group: NIH / 3T3 cells were cultured. When the cell density reached about 50%, the culture medium was replaced and TGF-β1 at a concentration of 10 ng / mL was added to continue culturing for 24 h to induce NIH / 3T3 cell differentiation;

[0113] 3. TGF-β1+HMF 12.5μM group and TGF-β1+HMF 25μM group: NIH / 3T3 cells were cultured. When the cell density reached about 50%, the culture medium was replaced and TGF-β1 at a concentration of 10ng / mL and HMF at concentrations of 12.5 and 25μM were added and cultured for 24h;

[0114] 4. TGF-β1+HMF+3-MA group: NIH / 3T3 cells were cultured. When the cell density reached about 50%, the culture medium was replaced and 3-MA (60 μM) was added for pretreatment for 2 h. Then, 10 ng / mL TGF-β1 and 25 μM HMF were added and co-cultured for 24 h.

[0115] (III) Cell culture

[0116] After NIH / 3T3 cells are revived, transfer them to a cell culture dish, shake them thoroughly, and place them in an incubator for culture. After subculturing, inoculate them into a culture dish at a ratio of 1:3 or 1:4 and continue to culture them in an incubator. Cryopreserve the excess cells for later use.

[0117] (IV) Determination of cell viability

[0118] 1. Cytotoxicity Assay

[0119] (1) Take NIH / 3T3 cells in the logarithmic growth phase and add 7×10 3 / well were inoculated in a 96-well plate and cultured for 24 h;

[0120] (2) Except for the blank group and the control group, which were added with 200 μL of culture medium, all other wells were added with different concentrations of HMF and cultured for 24 h;

[0121] (3) Remove the culture medium, add 10% CCK8 solution (diluted with culture medium) to each well, and incubate for 2 h;

[0122] (4) Use a multifunctional microplate reader to measure the absorbance value of each well at 450 nm, record and analyze the cell viability, and calculate the cell viability value according to the formula.

[0123] 2. Cell Proliferation Assay

[0124] (1) Take NIH / 3T3 cells in the logarithmic growth phase and add 7×10 3 / well were inoculated in a 96-well plate and cultured for 24 h;

[0125] (2) 200 μL of culture medium was added to the blank group and control group, 10 ng / mL of TGF-β1 was added to the TGF-β1 group, and 10 ng / mL of TGF-β1 and different concentrations of HMF were added to each HMF administration group and cultured for 24 h;

[0126] (3) Remove the culture medium, add 10% CCK8 solution (diluted with culture medium) to each well, and incubate for 2 h;

[0127] (4) Use a multifunctional microplate reader to measure the absorbance value of each well at 450 nm, record and analyze the cell viability, and calculate the cell viability value according to the formula. Cell survival rate (%) = [(A 实验孔 -A 空白孔 ) / A 对照孔 -A 空白孔 )]×100%.

[0128] (V) Cell scratch assay

[0129] NIH / 3T3 cells in the logarithmic growth phase were taken and 5×10 5 / well inoculated in a 6-well plate, after the cells adhered to the wall, use a 200 μL gun tip vertically against a ruler to quickly scratch, wash with sterile PBS 3 times, remove the scratched cells, and take pictures under an optical microscope, then add serum-free culture medium, treat the cells according to the experimental design for 24 hours and take pictures again. Calculate the scratch area in imageJ software, and calculate the cell migration rate according to the formula. Cell migration rate (%) = (initial scratch area - final scratch area) / initial scratch area × 100%.

[0130] (VI) Western blotting

[0131] Protein extraction and quantification: After modeling and drug administration of NIH / 3T3 cells according to the experimental design for a certain period of time, the culture medium was removed, and an appropriate amount of Ripa lysis solution was added to lyse on ice for 30 minutes. The cells were centrifuged at 12,000 rpm for 10 minutes, and the supernatant was taken for BCA protein determination. The BCA working solution was prepared according to A:B=50:1, and 2 μL of sample, 18 μL of PBS solution and 200 μL of BCA working solution were added to each well. The cells were incubated at 37°C for 30 minutes, and the absorbance of each well was measured at 562 nm using an ELISA reader. The protein concentration of each sample was calculated according to the equation of the standard curve. Finally, the loading buffer and Ripa solution were added to quantify the cell protein to 3 μg / μL.

[0132] Electrophoresis, membrane transfer; blocking; incubation with primary antibody and secondary antibody; development (same as in Example 1).

[0133] 7. Immunofluorescence

[0134] 1. Cell treatment and incubation with primary antibody

[0135] NIH / 3T3 cells in the logarithmic growth phase were taken and 1×10 4 / well were inoculated in a 24-well plate. After the cells adhered to the wall, the cells were treated for 24 hours according to the experimental design; the culture medium was discarded and the cells were gently washed 3 times with PBS, each time for 10 seconds; the cells were fixed with 4% tissue fixative at room temperature for 20 minutes, and washed 3 times with PBS, each time for 10 seconds; 1% Triton X-100 was permeabilized at room temperature for 20 minutes, and washed 3 times with PBS, each time for 5 minutes; goat serum was used for blocking for 1 hour; after adding the primary antibody, incubated in a 4°C refrigerator overnight.

[0136] 2. Incubate with secondary antibody and take photos

[0137] Wash with PBST three times, 5 min each time; add fluorescent secondary antibody (protected from light) and incubate at room temperature for 1 h; wash with PBST three times, 5 min each time; add DAPI staining solution (1:100, diluted with PBS) and stain for 5 min in the dark; wash once with PBST, observe the fluorescence intensity under a fluorescence microscope and take pictures.

[0138] (VIII) Detection of MDA, SOD and GSH-Px levels

[0139] Add an appropriate amount of Ripa lysis buffer to a 6-well plate and lyse on ice for 30 minutes, centrifuge at 4°C, 12000g for 10 minutes, and take the supernatant for determination. MDA detection kit (same as Example 1); GSH-Px detection kit (same as Example 1); SOD detection kit (same as Example 1).

[0140] (IX) Detection of ROS levels

[0141] NIH / 3T3 cells in the logarithmic growth phase were taken and 1×10 5 / well were inoculated in a 6-well plate. After the cells adhered to the wall, the cells were treated for 24 hours according to the experimental design, the culture medium was removed, DCFH-DA was diluted to 10 μM at a ratio of 1:1000 using serum-free culture medium, 1 mL of DCFH-DA dilution was added to each well, and the plates were incubated at 37°C for 20 min. The cells were washed 3 times with serum-free culture medium to fully remove the DCFH-DA that did not enter the cells, and the cells were collected and the intracellular fluorescence intensity was detected by flow cytometry.

[0142] (10) MDC staining

[0143] NIH / 3T3 cells in the logarithmic growth phase were taken and 1×10 5 / well were inoculated in a 6-well plate. After the cells adhered to the wall, the cells were treated for 24 h according to the experimental design, the culture medium was removed, and the cells were washed once with 1× wash buffer. MDC Stain was diluted with serum-free culture medium at a ratio of 1:9, and 600 μL of MDC Stain dilution was added to each well. The cells were incubated at 37°C in the dark for 40 min, and washed twice with 1× wash buffer. Finally, the cells were observed and photographed under a fluorescence microscope.

[0144] 3. Experimental Results

[0145] (I) Effects of HMF on toxicity and viability of lung fibroblasts

[0146] In order to study the effect of HMF on the differentiation of lung fibroblasts, the CCK-8 method was used to detect the effect of different concentrations of HMF on the activity of NIH / 3T3 cells in order to screen out the appropriate drug treatment concentration. The results showed that compared with the control group, cell viability began to decrease significantly at a HMF concentration of 100 μM, indicating that HMF concentrations above 100 μM may be toxic to cells. Compared with TGF-β1, cell viability began to decrease significantly from a HMF concentration of 12.5 μM. Therefore, HMF concentrations of 12.5 μM and 25 μM were selected for subsequent experiments. Results are shown in Fig.11 .

[0147] (II) Effect of HMF on the migration ability of lung fibroblasts

[0148] TGF-β1 has been shown to induce NIH / 3T3 cells to transform into myofibroblasts, so the scratch test was used to detect the effect of HMF on the proliferation and migration of NIH / 3T3 cells. The results showed that compared with the control group, the cells in the TGF-β1 group migrated to the middle of the scratch after 24 hours of intervention, and the scratch gap was significantly narrowed; compared with the TGF-β1 group, the cell migration speed in each HMF-treated group was significantly slowed down, the scratch gap was widened, and the cell migration rate was significantly decreased. Results are shown in Fig.12 .

[0149] (III) Effect of HMF on the morphology of lung fibroblasts

[0150] The results showed that compared with the control group, the number of myofibroblasts in NIH / 3T3 cells in the TGF-β1 group was significantly increased, while compared with the TGF-β1 group, the number of flat myofibroblasts was significantly reduced after HMF treatment. Fig.13 .

[0151] (IV) Effects of HMF on fibroblast-associated proteins

[0152] In NIH / 3T3 cells induced by TGF-β1, the effect of HMF on the protein expression of α-SMA, collagen-I, MMP2 and TIMP1 was detected by WB and immunofluorescence. The results showed that compared with the Control group, the protein expression levels of α-SMA, collagen-I, MMP2 and TIMP1 in the TGF-β1 group were significantly increased, and compared with the TGF-β1 group, the expression levels of fiber-related proteins in the HMF administration groups at various concentrations were significantly decreased. The results are shown in Fig.14 AB in the middle; immunofluorescence showed that the fluorescence intensity of α-SMA and collagen-I was significantly reduced after HMF treatment, which was consistent with the results of WB. Fig.14 Medium CE.

[0153] (V) Effects of HMF on oxidative damage indicators in lung fibroblasts

[0154] The results showed that compared with the Control group, the expression levels of SOD and GSH-Px in the TGF-β1 group were significantly decreased, and the expression level of MDA was significantly increased. Compared with the TGF-β1 group, after HMF intervention, the expression levels of SOD and GSH-Px were significantly increased, and the expression level of MDA was significantly decreased. Fig.15 .

[0155] (VI) Effects of HMF on oxidative stress-related proteins in lung fibroblasts

[0156] Compared with the Control group, the protein expression levels of Nrf2 and HO-1 in the TGF-β1 group were significantly decreased, and the protein expression level of keap-1 was significantly increased. Compared with the TGF-β1 group, the expression levels of Nrf2 and HO-1 antioxidant proteins in lung fibroblasts were significantly increased, and the expression level of keap-1 protein was significantly decreased after HMF intervention. Fig.16 .

[0157] (VII) Effects of HMF on autophagy-related proteins in lung fibroblasts

[0158] Compared with the Control group, the protein expression levels of LC3B II and Beclin-1 in the TGF-β1 group were significantly decreased, while the protein expression levels of LC3B and Beclin-1 in lung fibroblasts in the HMF different concentrations administration groups were significantly increased compared with the TGF-β1 group. Fig.17 .

[0159] (VIII) Effect of HMF on the content of autophagosomes in lung fibroblasts

[0160] Autophagosomes are the end product of the autophagic process, so detecting the content of autophagosomes can determine the activity of autophagy. In this study, the MDC staining method was used to stain the autophagosomes in lung fibroblasts. The results showed that the content of autophagosomes in the TGF-β1 group was significantly reduced, while compared with the TGF-β1 group, the content of autophagosomes in lung fibroblasts in each HMF administration group was significantly increased. Fig.18 .

[0161] (IX) HMF inhibits the expression of fibrin and the content of autophagosomes in lung fibroblasts through autophagy

[0162] In this experiment, the autophagy inhibitor 3-MA was used to inhibit autophagy activity, and then the effect of HMF on the expression of fibrin in TGF-β1-induced NIH / 3T3 cells was detected. The results showed that compared with the group without 3-MA, the expression levels of LC3B II and Beclin-1 in NIH / 3T3 cells were significantly reduced in the group using 3-MA, and the content of autophagosomes in the C+3-MA group was significantly reduced by MDC staining, indicating that autophagy activity was successfully inhibited. Subsequently, the expression of fibrotic proteins in NIH / 3T3 cells induced by TGF-β1 was detected, and it was found that the expression level of α-SMA was significantly reduced after the use of 3-MA. At the same time, the immunofluorescence results showed that the expression content of collagen-I was also significantly reduced after the use of 3-MA. At the same time, the content of autophagosomes in lung fibroblasts was significantly reduced after the use of 3-MA. The results are shown in Fig.19 This indicates that HMF plays an anti-pulmonary fibrosis role by regulating autophagy.

[0163] (X) Effect of HMF on oxidative stress in lung fibroblasts under autophagy inhibition

[0164] In this experiment, 3-MA autophagy inhibitor was used to inhibit autophagy activity and observe the effect of HMF on oxidative stress. The results showed that after the addition of 3-MA inhibitor, the content of ROS increased significantly, while the protein expression level of Nrf2 decreased significantly and the protein expression level of keap1 increased significantly, indicating that when autophagy is inhibited, the oxidative stress in lung fibroblasts will be aggravated, and HMF regulates oxidative stress by activating autophagy. Results are shown in Fig. 20 .

[0165] In summary, this example studied the role of HMF in the differentiation of lung fibroblasts through in vitro experiments. The results showed that HMF inhibited the differentiation of lung fibroblasts by inhibiting oxidative stress and activating autophagy, and reduced the expression levels of pulmonary fibrosis-related proteins.

Claims

1. Use of 3,5,6,7,8,3',4'-heptamethoxyflavone in the preparation of drugs for preventing or treating pulmonary fibrosis.

2. The use according to claim 1, characterized in that: The pulmonary fibrosis includes idiopathic pulmonary fibrosis, secondary pulmonary fibrosis and hereditary pulmonary fibrosis, preferably idiopathic pulmonary fibrosis.

3. The use according to claim 1, characterized in that: The 3,5,6,7,8,3',4'-heptamethoxyflavone inhibits pulmonary fibrosis by regulating the levels of oxidative stress and autophagy.

4. The use according to claim 3, characterized in that: The 3,5,6,7,8,3',4'-heptamethoxyflavone inhibits oxidative stress and activates autophagy in NIH / 3T3 cells, thereby inhibiting the differentiation of lung fibroblasts and reducing the expression level of pulmonary fibrosis-related proteins, thereby exerting an anti-pulmonary fibrosis effect.

5. The use according to claim 1, characterized in that: The medicine comprises an effective amount of 3,5,6,7,8,3',4'-heptamethoxyflavone and a pharmaceutically acceptable carrier.

6. The use according to claim 5, characterized in that: The dosage form of the medicine is tablet, capsule, powder, oral liquid, powder, pill or granule.

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